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DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO Ph.D

DR ANTHONY MELVIN CRASTO, Born in Mumbai in 1964 and graduated from Mumbai University, Completed his Ph.D from ICT, 1991,Matunga, Mumbai, India, in Organic Chemistry, The thesis topic was Synthesis of Novel Pyrethroid Analogues, Currently he is working with GLENMARK PHARMACEUTICALS LTD, Research Centre as Principal Scientist, Process Research (bulk actives) at Mahape, Navi Mumbai, India. Total Industry exp 29 plus yrs, Prior to joining Glenmark, he has worked with major multinationals like Hoechst Marion Roussel, now Sanofi, Searle India Ltd, now RPG lifesciences, etc. He has worked with notable scientists like Dr K Nagarajan, Dr Ralph Stapel, Prof S Seshadri etc, He did custom synthesis for major multinationals in his career like BASF, Novartis, Sanofi, etc., He has worked in Discovery, Natural products, Bulk drugs, Generics, Intermediates, Fine chemicals, Neutraceuticals, GMP, Scaleups, etc, he is now helping millions, has 9 million plus hits on Google on all Organic chemistry websites. His friends call him worlddrugtracker. His New Drug Approvals, Green Chemistry International, All about drugs, Eurekamoments, Organic spectroscopy international, etc in organic chemistry are some most read blogs He has hands on experience in initiation and developing novel routes for drug molecules and implementation them on commercial scale over a 29 year tenure till date Aug 2016, Around 30 plus products in his career. He has good knowledge of IPM, GMP, Regulatory aspects, he has several International patents published worldwide . He has good proficiency in Technology transfer, Spectroscopy, Stereochemistry, Synthesis, Polymorphism etc., He suffered a paralytic stroke/ Acute Transverse mylitis in Dec 2007 and is 90 %Paralysed, He is bound to a wheelchair, this seems to have injected feul in him to help chemists all around the world, he is more active than before and is pushing boundaries, He has 9 million plus hits on Google, 2.5 lakh plus connections on all networking sites, 25 Lakh plus views on dozen plus blogs, He makes himself available to all, contact him on +91 9323115463, email amcrasto@gmail.com, Twitter, @amcrasto , He lives and will die for his family, 90% paralysis cannot kill his soul., Notably he has 13 lakh plus views on New Drug Approvals Blog in 212 countries......https://newdrugapprovals.wordpress.com/ , He appreciates the help he gets from one and all, Friends, Family, Glenmark, Readers, Wellwishers, Doctors, Drug authorities, His Contacts, Physiotherapist, etc

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Pridopidine.svg

Pridopidine

  • Molecular Formula C15H23NO2S
  • Average mass 281.414 Da
346688-38-8  CAS FREE FORM
882737-42-0 (hydrochloride)
1440284-30-9 HBr
4-[3-(Methylsulfonyl)phenyl]-1-propylpiperidin
4- (3 -Methanesulfonyl-phenyl ) – 1-propyl -piperidine
ACR16
Huntexil
UNII-HD4TW8S2VK;
4-[3-(Methylsulfonyl)phenyl]-1-propylpiperidine
ACR 16
  • ASP 2314
FR 310826

Huntingtons chorea

Dopamine D2 receptor antagonist; Opioid receptor sigma agonist 1

Neurosearch INNOVATORS, In 2012, the product was acquired by Teva

In January 2017, pridopidine was reported to be in phase 3 clinical development,  pridopidine for treating or improving cognitive functions and Alzheimer’s disease.

Teva Pharmaceutical Industries, following an asset acquisition from NeuroSearch, is developing pridopidine, a fast-off dopamine D2 receptor antagonist that strengthens glutamate function, for treating HD.
The drug holds orphan drug designation in the U.S. and the E.U. for the treatment of Huntington’s disease

PRIDOPIDINE.png

About Huntington Disease

HD is a fatal neurodegenerative disease for which there is no known cure or prevention. People who suffer from HD will likely have a variety of steadily-worsening symptoms, including uncoordinated and uncontrolled movements, cognition and memory deterioration and a range of behavioral and psychological problems. HD symptoms typically start in middle age, but the disease may also manifest itself in childhood and in old age. Disease progression is characterized by a gradual decline in motor control, cognition and mental stability, and generally results in death within 15 to 25 years of clinical diagnosis. Current treatment is limited to managing the symptoms of HD, as there are no treatments that have been shown to alter the progression of HD. Studies estimate that HD affects about 13 to 15 people per 100,000 in Caucasians, and for every affected person there are approximately three to five people who may carry the mutation but are not yet ill.

Image result for Pridopidine

Pridopidine, also known as ACR16, is a dopamine stabilizer, which improves motor performance and shows neuroprotective effects in Huntington disease R6/2 mouse model. Huntington disease (HD) is a neurodegenerative disorder for which new treatments are urgently needed. Pridopidine is a new dopaminergic stabilizer, recently developed for the treatment of motor symptoms associated with HD.

Figure

Dopamine D2 ligands. Dopamine D2 receptor agonists dopamine (1) and apomorphine (2), classical antagonists haloperidol (3) and olanzapine (4), partial agonists (−)-3-(3-hydroxyphenyl)-Nn-propylpiperidine (5), bifeprunox (6), aripiprazole (7), and 3-(1-benzylpiperidin-4-yl)phenol (9a), and dopaminergic stabilizers S-(−)-OSU6162 (8) and pridopidine (12b).

Dopamine is a neurotransmitter in the brain. Since this discovery, made in the 1950s, the function of dopa-mine in the brain has been intensely explored. To date, it is well established that dopamine is essential in several aspects of brain function including motor, cognitive, sensory, emotional and autonomous (e.g. regulation of appetite, body temperature, sleep) functions. Thus, modulation of dopaminergic function may be beneficial in the treatment of a wide range of disorders affecting brain functions. In fact, both neurologic and psychiatric disorders are treated with medications based on interactions with dopamine systems and dopamine receptors in the brain.
Drugs that act, directly or indirectly, at central dopamine receptors are commonly used in the treatment of neurologic and psychiatric disorders, e.g. Parkinson’s disease and schizophrenia. Currently available dopaminer-gic pharmaceuticals have severe side effects, such as ex-trapyramidal side effects and tardive dyskinesia in dopaminergic antagonists used as antipsychotic agents, and dyskinesias and psychoses in dopaminergic agonists used as anti -Parkinson ‘ s agents. Therapeutic effects are un-satisfactory in many respects. To improve efficacy and reduce side effects of dopaminergic pharmaceuticals, novel dopamine receptor ligands with selectivity at specific dopamine receptor subtypes or regional selectivity are sought for. In this context, also partial dopamine receptor agonists, i.e. dopamine receptor ligands with some but not full intrinsic activity at dopamine receptors, are being developed to achieve an optimal degree of stimulation at dopamine receptors, avoiding excessive do-pamine receptor blockade or excessive stimulation.
Compounds belonging to the class of substituted 4- (phenyl-N-alkyl) -piperazine and substituted 4-(phenyl-N-alkyl) -piperidines have been previously reported. Among these compounds, some are inactive in the CNS, some dis-play serotonergic or mixed serotonergic/dopaminergic pharmacological profiles while some are full or partial dopamine receptor agonists or antagonists with high affinity for dopamine receptors.
A number of 4-phenylpiperazines and 4 -phenyl -piperidine derivatives are known and described, for example Costall et al . European J. Pharm. 31, 94, (1975), Mewshaw et al . Bioorg. Med. Chem. Lett., 8, 295, (1998). The reported compounds are substituted 4 -phenyl -piperazine ‘ s, most of them being 2-, 3- or 4 -OH phenyl substituted and displaying DA autoreceptor agonist properties .
Fuller R. W. et al , J. Pharmacol. Exp . Therapeut . 218, 636, (1981) disclose substituted piperazines (e.g. 1- (m-trifluoro-methylphenyl) piperazine) which reportedly act as serotonin agonists and inhibit serotonin uptake.

Fuller R. W. et al , Res. Commun. Chem. Pathol . Pharmacol. 17, 551, (1977) disclose the comparative effects on the 3 , 4-dihydroxy-phenylacetic acid and Res. Commun. Chem. Pathol. Pharmacol. 29, 201, (1980) disclose the compara-tive effects on the 5-hydroxyindole acetic acid concentration in rat brain by 1- (p-chlorophenol) -piperazine .
Boissier J. et al Chem Abstr. 61:10691c, disclose disubstituted piperazines. The compounds are reportedly adrenolytics, antihypertensives , potentiators of barbitu-rates, and depressants of the central nervous system.
A number of different substituted piperazines have been published as ligands at 5-HT1A receptors, for example Glennon R.A. et al J. Med. Chem., 31, 1968, (1988), van Steen B.J., J. Med. Chem., 36, 2751, (1993), Mokrosz, J. et al, Arch. Pharm. (Weinheim) 328, 143-148 (1995), and Dukat M.-L., J. Med. Chem., 39, 4017, (1996). Glennon R. A. discloses, in international patent applications WO93/00313 and WO 91/09594 various amines, among them substituted piperazines, as sigma receptor ligands. Clinical studies investigating the properties of sigma receptor ligands in schizophrenic patients have not generated evi-dence of antipsychotic activity, or activity in any other CNS disorder. Two of the most extensively studied selective sigma receptor antagonists, BW234U (rimcazole) and BMY14802, have both failed in clinical studies in schizophrenic patients (Borison et al , 1991, Psychopharmacol Bull 27(2): 103-106; Gewirtz et al , 1994, Neuropsycho-pharmacology 10:37-40) .
Further, WO 93/04684 and GB 2027703 also describe specific substituted piperazines useful in the treatment of CNS disorders

Pridopidine (Huntexil, formerly ACR16) is an experimental drug candidate belonging to a class of agents known as dopidines, which act as dopaminergic stabilizers in the central nervous system. These compounds may counteract the effects of excessive or insufficient dopaminergic transmission,[1][2] and are therefore under investigation for application in neurological and psychiatric disorders characterized by altered dopaminergic transmission, such as Huntington’s disease (HD).

Pridopidine is in late-stage development by Teva Pharmaceutical Industries who acquired the rights to the product from its original developer NeuroSearch in 2012. In April 2010, NeuroSearch announced results from the largest European phase 3 study in HD carried out to date (MermaiHD). The MermaiHD study examined the effects of pridopidine in patients with HD and the results showed after six months of treatment, pridopidine improved total motor symptoms, although the primary endpoint of the study was not met. Pridopidine was well tolerated and had an adverse event profile similar to placebo.[3]

The US Food and Drug Administration (FDA) and European Medicines Agency (EMA) have both indicated they will not issue approval for pridopidine to be used in human patients on the basis of the MermaiHD and HART trials, and a further, positive phase 3 trial is required for approval.[4][5]

Image result for Pridopidine

Dopidines

Dopidines, a new class of pharmaceutical compounds, act as dopaminergic stabilizers, enhancing or counteracting dopaminergic effects in the central nervous system.[1][2] They have a dual mechanism of action, displaying functional antagonism of subcortical dopamine type 2 (D2) receptors, as well as strengthening of cortical glutamate and dopamine transmission.[6] Dopidines are, therefore, able to regulate both hypoactive and hyperactive functioning in areas of the brain that receive dopaminergic input (i.e. cortical and subcortical regions). This potential ability to restore the cortical–subcortical circuitry to normal suggests dopidines may have the potential to improve symptoms associated with several neurological and psychiatric disorders, including HD.

SYNTHESIS

Figure

aReagents and conditions: (a) n-butyllithium, 1-Boc-4-piperidone, THF; (b) trifluoroacetic acid, CH2Cl2, Δ; (c) triethylamine, methyl chloroformate, CH2Cl2; (d) m-CPBA, CH2Cl2; (e) Pd/C, H2, MeOH, HCl; (f) HCl, EtOH, Δ; (g) RX, K2CO3, acetonitrile, Δ.

Pharmacology

In vitro studies demonstrate pridopidine exerts its effects by functional antagonism of D2 receptors. However, pridopidine possesses a number of characteristics[1][2][6][7] that differentiate it from traditional D2 receptor antagonists (agents that block receptor responses).

  • Lower affinity for D2 receptors than traditional D2 ligands[8]
  • Preferential binding to activated D2 (D2high) receptors (i.e. dopamine-bound D2 receptors)[8]
  • Rapid dissociation (fast ‘off-rate’) from D2 receptors
  • D2 receptor antagonism that is surmountable by dopamine
  • Rapid recovery of D2-receptor-mediated responses after washout[1][2][6][7]

Pridopidine is less likely to produce extrapyramidal symptoms, such as akinesia (inability to initiate movement) and akathisia (inability to remain motionless), than dopamine antagonists (such as antipsychotics).[9] Furthermore, pridopidine displays no detectable intrinsic activity,[9][10] differentiating it from D2 receptor agonists and partial agonists (agents that stimulate receptor responses). Pridopidine, therefore, differs from D2 receptor antagonists, agonists and partial agonists.[6]

As a dopaminergic stabilizer, pridopidine can be considered to be a dual-acting agent, displaying functional antagonism of subcortical dopaminergic transmission and strengthening of cortical glutamate transmission.

Clinical development

The MermaiHD study

In 2009, NeuroSearch completed the largest European HD trial to date, the Multinational EuRopean Multicentre ACR16 study In Huntington’s Disease (MermaiHD) study.

This six-month, phase 3, randomized, double-blind, placebo-controlled trial recruited patients from Austria, Belgium, France, Germany, Italy, Portugal, Spain and the UK, and compared two different pridopidine dose regimens with placebo. Patients were randomly allocated to receive pridopidine (45 mg once daily or 45 mg twice daily) or placebo. During weeks 1–4, patients received once-daily treatment (as a morning dose). Thereafter, patients took two doses (one morning and one afternoon dose) until the end of the treatment period. The study had a target recruitment of 420 patients; recruitment was finalized in April 2009 with 437 patients enrolled.[14]

The purpose of the study was to assess the effects of pridopidine on a specific subset of HD motor symptoms defined in the modified motor score (mMS).[14] The mMS comprises 10 items relating to voluntary motor function from the Unified Huntington’s Disease Rating Scale Total Motor Score (UHDRS—TMS).[14] Other study endpoints included the UHDRS—TMS, submotor items, cognitive function, behaviour and symptoms of depression and anxiety.

After six months of treatment, patients who received pridopidine 45 mg twice daily showed significant improvements in motor function, as measured by the UHDRS-TMS, compared with placebo. For the mMS, which was the primary endpoint of the study, a strong trend in treatment effect was seen, although statistical significance was not reached. Pridopidine was also very well tolerated, had an adverse event profile similar to placebo and gave no indication of treatment-associated worsening of symptoms.[3]

The MermaiHD study – open-label extension

Patients who completed the six-month, randomized phase of the MermaiHD study could choose to enter the MermaiHD open-label extension study and receive pridopidine 45 mg twice daily for six months. In total, 357 patients were enrolled into the MermaiHD open-label extension study and of these, 305 patients completed the entire 12-month treatment period.[15]

The objective of this study was to evaluate the long-term safety and tolerability profile of pridopidine and to collect efficacy data after a 12-month treatment period to support the safety evaluation. Safety and tolerability assessments included the incidence and severity of adverse events, routine laboratory parameters, vital signs and electrocardiogram measurements.[15]

Results from the MermaiHD open-label extension study showed treatment with pridopidine for up to 12 months (up to 45 mg twice daily for the first six months; 45 mg twice daily for the last six months) was well tolerated and demonstrated a good safety profile.[3][15]

The HART study

In October 2010, NeuroSearch reported results from their three-month, phase 2b, randomized, double-blind, placebo-controlled study carried out in Canada and the USA – Huntington’s disease ACR16 Randomized Trial (HART). This study was conducted in 28 centres and enrolled a total of 227 patients, who were randomly allocated to receive pridopidine 10 mg, 22.5 mg or 45 mg twice daily) or placebo.[14][16] During weeks 1–4, patients received once-daily treatment (as a morning dose). Thereafter, patients took two treatment doses (one morning and one afternoon dose) until the end of the treatment period. Study endpoints were the same as those for the MermaiHD study.

Results from the HART study were consistent with findings from the larger MermaiHD study. After 12 weeks of treatment with pridopidine 45 mg twice daily, total motor function significantly improved, as measured by the UHDRS–TMS. The primary endpoint, improvement in the mMS, was not met.[16]

In both studies, the effects on the UHDRS–TMS and the mMS were driven by significant improvements in motor symptoms such as gait and balance, and hand movements, deemed by the authors to be “clinically relevant”. However, the magnitude of the improvements was small. Pridopdiine demonstrated a favourable tolerability and safety profile, including no observations of treatment-related disadvantages in terms of worsening of other disease signs or symptoms.[15][16]

Compassionate use programme and open-ended, open-label study

To meet requests from patients and healthcare professionals for continued treatment with pridopidine, NeuroSearch has established a compassionate use programme in Europe to ensure continued access to pridopidine for patients who have completed treatment in the MermaiHD open-label extension study. The programme is active in all of the eight European countries where the MermaiHD study was conducted.

NeuroSearch has initiated an open-ended, open-label clinical study in the USA and Canada, called the Open HART study. In this study, all patients who have completed treatment in the HART study are offered the chance to restart treatment with pridopidine until either marketing approval has been obtained in the countries in question, or the drug’s development is discontinued. The first patients were enrolled in March 2011.[3]

Regulatory agency advice

The results of the MermaiHD and HART trials were presented to the American and European regulatory agencies: the FDA in March 2011 and EMA in May, 2011. Both agencies indicated insufficient evidence had been produced to allow approval in human patients, and a further phase 3 trial would be required for approval.[4][5]

PATENT

WO 2001046145

Example 6: 4- (3 -Methanesulfonyl-phenyl ) – 1-propyl -piperidine
m.p. 200°C (HCl) MS m/z (relative intensity, 70 eV) 281 (M+, 5), 252 (bp) , 129 (20), 115 (20), 70 (25.

PAPER

Journal of Medicinal Chemistry (2010), 53(6), 2510-2520.

Synthesis and Evaluation of a Set of 4-Phenylpiperidines and 4-Phenylpiperazines as D2 Receptor Ligands and the Discovery of the Dopaminergic Stabilizer 4-[3-(Methylsulfonyl)phenyl]-1-propylpiperidine (Huntexil, Pridopidine, ACR16)

NeuroSearch Sweden AB, Arvid Wallgrens Backe 20, S-413 46 Göteborg, Sweden
J. Med. Chem., 2010, 53 (6), pp 2510–2520
DOI: 10.1021/jm901689v
*To whom correspondence should be addressed. Phone: +(46) 31 7727710. Fax: +(46) 31 7727701. E-mail: fredrik.pettersson@neurosearch.se.

Abstract

Abstract Image

Modification of the partial dopamine type 2 receptor (D2) agonist 3-(1-benzylpiperidin-4-yl)phenol (9a) generated a series of novel functional D2 antagonists with fast-off kinetic properties. A representative of this series, pridopidine (4-[3-(methylsulfonyl)phenyl]-1-propylpiperidine; ACR16, 12b), bound competitively with low affinity to D2 in vitro, without displaying properties essential for interaction with D2 in the inactive state, thereby allowing receptors to rapidly regain responsiveness. In vivo, neurochemical effects of 12b were similar to those of D2 antagonists, and in a model of locomotor hyperactivity, 12b dose-dependently reduced activity. In contrast to classic D2 antagonists, 12b increased spontaneous locomotor activity in partly habituated animals. The “agonist-like” kinetic profile of 12b, combined with its lack of intrinsic activity, induces a functional state-dependent D2 antagonism that can vary with local, real-time dopamine concentration fluctuations around distinct receptor populations. These properties may contribute to its unique “dopaminergic stabilizer” characteristics, differentiating 12b from D2 antagonists and partial D2agonists.

4-[3-(Methylsulfonyl)phenyl]-1-propylpiperidine (12b)

Purification with flash chromatography using CH2Cl2/MeOH [1:1 (v/v)] as eluent afforded pure 12b (3.28 g, 79%).
MS m/z (relative intensity, 70 eV) 281 (M+, 5), 252 (bp), 129 (20), 115 (20), 70 (25).
1H NMR (300 MHz, CDCl3) δ ppm 0.96 (t, J = 7.3 Hz, 3 H), 1.53−1.64 (m, 2 H), 1.89 (dd, J = 9.6, 3.54 Hz, 4 H), 2.03−2.14 (m, 2 H), 2.31−2.41 (m, 2 H), 2.64 (ddd, J = 15.4, 5.7, 5.5 Hz, 1 H), 3.06−3.15 (m, 5 H), 7.51−7.58 (m, 2 H), 7.78−7.86 (m, 2 H).
13C NMR (75 MHz, CDCl3) δ ppm 11.98, 20.18, 33.29, 42.59, 44.43, 54.06, 60.93, 124.99, 125.74, 129.39, 132.04, 148.28.
The amine was converted to the HCl salt and recrystallized in EtOH/diethyl ether: mp 212−214 °C. Anal. (C15H24ClNO2S) C, H, N.

PATENT

WO-2017015609

Pridopidine (Huntexil®) is a unique compound developed for the treatment of patients with motor symptoms associated with Huntington’s disease. The chemical name of pridopidine is 4-(3-(Methylsulfonyl)phenyl)-l-propylpiperidine, and its Chemical Registry Number is CAS 346688-38-8 (CSED:7971505, 2016). The Chemical Registry number of pridopidine hydrochloride is 882737-42-0 (CSID:25948790 2016). Processes of synthesis of pridopidine and a pharmaceutically acceptable salt thereof are disclosed in U.S. Patent No. 7,923,459. U.S. Patent No. 6,903,120 claims pridopidine for the treatment of Parkinson’s disease, dyskinesias, dystonias, Tourette’s disease, iatrogenic and non-iatrogenic psychoses and hallucinoses, mood and anxiety disorders, sleep disorder, autism spectrum disorder, ADHD, Huntington’s disease, age-related cognitive impairment, and disorders related to alcohol abuse and narcotic substance abuse.

US Patent Application Publication Nos. 20140378508 and 20150202302, describe methods of treatment with high doses of pridopidine and modified release formulations of pridopidine, respectively.

EXAMPLES

Example 1: Pridopidine-HCl synthesis

An initial process for synthesizing pridopidine HC1 shown in Scheme 1 and is a modification of the process disclosed in US Patent No. 7,923,459.

The synthesis of Compound 9 started with the halogen-lithium exchange of 3-bromothioanisole (3BTA) in THF employing n-hexyllithium (HexLi) in hexane as the lithium source. Li-thioanisole (3LTA) intermediate thus formed was coupled with 1 -propyl-4-piperidone (1P4P) forming a Li-Compound 9. These two reactions require low (cryogenic) temperature. The quenching of Li-Compound 9 was done in water HCl/MTBE resulting in precipitation of Compound 9-HCl salt. A cryogenic batch mode process for this step was developed and optimized. The 3BTA and THF were cooled to less than -70°C. A solution of HexLi in n-hexane (33%) was added at a temperature below -70°C and the reaction is stirred for more than 1 hour. An in-process control sample was taken and analyzed for completion of halogen exchange, l-propyl-4-piperidone (1P4P) was then added to the reaction at about -70°C letting the reaction mixture to reach -40°C and further stirred at this temperature for about 1 hour. An in-process sample was analyzed to monitor the conversion according to the acceptance criteria (Compound 9 not less than 83% purity). The reaction mixture was added to a mixture of 5N hydrochloric acid (HC1) and methyl teri-butyl ether (MTBE). The resulting precipitate was filtered and washed with MTBE to give the hydrochloric salt of Compound 9 (Compound 9-HCl) wet.

Batch mode technique for step 1 requires an expensive and high energy-consuming cryogenic system that cools the reactor with a methanol heat exchange, in which the methanol is circulated in counter current liquid nitrogen. This process also brings about additional problems originated from the workup procedure. The work-up starts when the reaction mixture is added into a mixture of MTBE and aqueous HC1. This gives three phases: (1) an organic phase that contains the organic solvents MTBE, THF and hexane along with other organic related materials such as thioanisole (TA), hexyl-bromide,

3-hexylthioanisole and other organic side reaction impurities (2) an aqueous phase containing inorganic salts (LiOH and LiBr), and (3) a solid phase which is mostly Compound 9-HCl but also remainders of 1P4P as an HC1 salt.

The isolation of Compound 9-HCl from the three phase work-up mixture is by filtration followed by MTBE washings. A major problem with this work-up is the difficulty of the filtration which resulted in a long filtration and washing operations. The time it takes to complete a centrifugation and washing cycle is by far beyond the normal duration of such a manufacturing operation. The second problem is the inevitable low and non-reproducible assay (purity of -90% on dry basis) of Compound 9-HCl due to the residues of the other two phases. It should be noted that a high assay is important in the next step in order to control the amount of reagents. The third problem is the existence of THF in the wet Compound 9-HCl salt which is responsible for the Compound 3 impurity that is discussed below.

Example 6.2: Pridopidine crude – work-up development

After the reduction, pridopidine HC1 is precipitated by adding HC1/IPA to the solution of pridopidine free base in ΓΡΑ in the process of Example 1. Prior to that, a solvent swap from toluene to ΓΡΑ is completed by 3 consecutive vacuum distillations. The amount of toluene in the ΓΡΑ solution affects the yield and it was set to be not more than 3% (IPC by GC method). The spontaneous precipitation produces fine crystals with wide PSD. In order to narrow the PSD, Example 1 accomplishes HC1/IPA addition in two cycles with cooling/warming profile.

The updated process is advantageous for crystallizing pridopidine free base over the procedure in Example 1 for two reasons.

First, it simplifies the work-up of the crude because the swap from toluene to PA is not required. The pridopidine free base is crystallized from toluene/n-heptanes system. Only one vacuum distillation of toluene is needed (compared to three in the work-up of Example 1) to remove water and to increase yield.

Second, in order to control pridopidine-HCl physical properties. Pridopidine free base is a much better starting material for the final crystallization step compared to the pridopidine HC1 salt because it is easily dissolved in ΓΡΑ which enables a mild absolute (0.2μ) filtration required in the final step of API manufacturing.

Crystallization of pridopidine free base in toluene/n-heptane system

First, crystallization of pridopidine free base in toluene/n-heptane mixture was tested in order to find the right ratio to maximize the yield. In order to obtain pridopidine free base, pridopidine-HCl in water/toluene system was basified with NaOH(aq) to pH>12. Two more water washes of the toluene phase brought the pH of the aqueous phase to <10. Addition of n-heptane into the toluene solution

resulted in pridopidine free base precipitation. Table 21 shows data from the toluene/n-heptane crystallization experiments.

Example 7: Development of the procedure for the purification of Compound 1 in pridopidine free base.

The present example describes lowering Compound 1 levels in pridopidine free base. This procedure involves dissolving pridopidine FB in 5 Vol of toluene at 20-30°C, 5 Vol of water are added and after the mixing phases are separated and the organic phase is washed three times with 5 Vol water. The toluene mixture is then distilled up to 2.5 Vol in the reactor and 4 Vol of heptane are added for crystallization. Experiment No. 2501 was completed using this procedure. Table 24 summarizes the results.

Example 8: Step 4 in Scheme 2: Pridopidine Hydrochloride process

This example discusses the step used to formulate pridopidine-HCl from pridopidine crude. The corresponding stage in Example 1 was part of the last (third) stage in which pridopidine-HCl was obtained directly from Compound 8 without isolation of pridopidine crude. In order to better control pridopidine-HCl physical properties, it is preferable to start with well-defined pridopidine free base which enables control on the exact amount of HC1 and IPA.

Pridopidine-HCl preparation – present procedure

Pridopidine-HCl was prepared according to the following procedure: Solid pridopidine crude was charged into the first reactor followed by 8 Vol of IPA (not more than (NMT) 0.8% water by KF) and the mixture is heated to Tr =40-45°C (dissolution at Tr = 25-28°C). The mixture was then filtered through a 0.2 μιη filter and transferred into the second (crystallizing) reactor. The first hot reactor was washed with 3.8 Vol of IPA. The wash was transferred through the filter to the second reactor. The temperature was raised to 65-67°C and 1.1 eq of IPA/HCl are added to the mixture (1.1 eq of HC1, from IPA/HCl 5N solution, 0.78 v/w). The addition of EPA HCl into the free base is exothermic; therefore, it was performed slowly, and the temperature maintained at Tr = 60-67°C. After the addition, the mixture was stirred for 15 min and pH is measured (pH<4). If pH adjustment is needed,

0.2 eq of HCl (from IPA/HC1 5 N solution) is optional. At the end of the addition, the mixture was stirred for 1 hour at Tr = 66°C to start sedimentation. If sedimentation does not start, seeding with 0.07% pridopidine hydrochloride crystals is optional at this temperature. Breeding of the crystals was performed by stirring for 2.5 h at Tr =64-67°C. The addition HCl line was washed with 0.4 Vol of ΓΡΑ to give~13 Vol solution. The mixture was cooled to Tr =0°C The solid is filtered and washed with cooled 4.6 Vol ΓΡΑ at LT 5°C. Drying as performed under vacuum (P< ) at 30-60°C to constant weight: Dried pridopidine-HCl was obtained as a white solid.

Purification of Compound 4 during pridopidine-HCl process

A relationship between high temperature in the reduction reaction and high levels of Compound 4 impurity have been observed. A reduction in 50°C leads to 0.25% of Compound 4. For that reason the process of Example 1 limits the reduction reaction temperature to 30±5°C since this is the final step and Compound 4 level should be not more than 0.15%. The present process has another crystallization stage by which Compound 4 can be purified.

PATENT

https://www.google.ch/patents/US20130150406

Pridopidine, i.e. 4-(3-methanesulfonyl-phenyl)-1-propyl-piperidine, is a drug substance currently in clinical development for the treatment of Huntington’s disease. The hydrochloride salt of 4-(3-methanesulfonyl-phenyl)-1-propyl-piperidine and a method for its synthesis is described in WO 01/46145. In WO 2006/040155 an alternative method for the synthesis of 4-(3-methanesulfonyl-phenyl)-1-propyl-piperidine is described. In WO 2008/127188 N-oxide and/or di-N-oxide derivatives of certain dopamine receptor stabilizers/modulators are reported, including the 4-(3-methanesulfonyl-phenyl)-1-propyl-piperidine-1-oxide.

1H NMR PREDICTIONS

ACTUAL VALUES

1H NMR (300 MHz, CDCl3) δ ppm 0.96 (t, J = 7.3 Hz, 3 H), 1.53−1.64 (m, 2 H), 1.89 (dd, J = 9.6, 3.54 Hz, 4 H), 2.03−2.14 (m, 2 H), 2.31−2.41 (m, 2 H), 2.64 (ddd, J = 15.4, 5.7, 5.5 Hz, 1 H), 3.06−3.15 (m, 5 H), 7.51−7.58 (m, 2 H), 7.78−7.86 (m, 2 H).
 
13C NMR (75 MHz, CDCl3) δ ppm 11.98, 20.18, 33.29, 42.59, 44.43, 54.06, 60.93, 124.99, 125.74, 129.39, 132.04, 148.28.

13C NMR PREDICTIONS

References

  1.  Seeman P, Tokita K, Matsumoto M, Matsuo A, Sasamata M, Miyata K (October 2009). “The dopaminergic stabilizer ASP2314/ACR16 selectively interacts with D2(High) receptors”. Synapse. 63 (10): 930–4. doi:10.1002/syn.20663. PMID 19588469.
  2.  Rung JP, Rung E, Helgeson L, et al. (June 2008). “Effects of (-)-OSU6162 and ACR16 on motor activity in rats, indicating a unique mechanism of dopaminergic stabilization”. Journal of Neural Transmission. 115 (6): 899–908. doi:10.1007/s00702-008-0038-3. PMID 18351286.
  3. “NeuroSearch A/S announces the results of additional assessment and analysis of data from the Phase III MermaiHD study with Huntexil® in Huntington’s disease” (Press release). NeuroSearch. 28 April 2010. Retrieved 2010-04-28.
  4. “NeuroSearch press releases (dated 23.03.2011 and 24.05.2011)”. NeuroSearch “Huntexil update: EMA asks for further trial”. HDBuzz. Retrieved 11 December 2011.
  5.  Ponten, H.; Kullingsjö, J.; Lagerkvist, S.; Martin, P.; Pettersson, F.; Sonesson, C.; Waters, S.; Waters, N. (2003-11-19) [2000-12-22]. “In vivo pharmacology of the dopaminergic stabilizer pridopidine”. European Journal of Pharmacology. 644 (1-3) (1–3): 88–95. doi:10.1016/j.ejphar.2010.07.023. PMID 20667452.
  6. Dyhring T, Nielsen E, Sonesson C, et al. (February 2010). “The dopaminergic stabilizers pridopidine (ACR16) and (-)-OSU6162 display dopamine D(2) receptor antagonism and fast receptor dissociation properties”. European Journal of Pharmacology. 628 (1–3): 19–26. doi:10.1016/j.ejphar.2009.11.025. PMID 19919834.
  7.  Pettersson, F; Pontén, H; Waters N; Waters S; Sonesson C (March 2010). “Synthesis and Evaluation of a Set of 4-Phenylpiperidines and 4-Phenylpiperazines as D2 Receptor Ligands and the Discovery of the Dopaminergic Stabilizer 4-[3-(methylsulfonyl)phenyl]-1-propylpiperidine (Pridopidine; ACR16)”. Journal of Medicinal Chemistry. 53 (6): 2510–2520. doi:10.1021/jm901689v. PMID 20155917.
  8.  Natesan S, Svensson KA, Reckless GE, et al. (August 2006). “The dopamine stabilizers (S)-(-)-(3-methanesulfonyl-phenyl)-1-propyl-piperidine [(-)-OSU6162] and 4-(3-methanesulfonylphenyl)-1-propyl-piperidine (ACR16) show high in vivo D2 receptor occupancy, antipsychotic-like efficacy, and low potential for motor side effects in the rat”. The Journal of Pharmacology and Experimental Therapeutics. 318 (2): 810–8. doi:10.1124/jpet.106.102905. PMID 16648369.
  9.  Tadori Y, Forbes RA, McQuade RD, Kikuchi T (November 2008). “Characterization of aripiprazole partial agonist activity at human dopamine D3 receptors”. European Journal of Pharmacology. 597 (1–3): 27–33. doi:10.1016/j.ejphar.2008.09.008. PMID 18831971.
  10.  Rung JP, Carlsson A, Markinhuhta KR, Carlsson ML (June 2005). “The dopaminergic stabilizers (-)-OSU6162 and ACR16 reverse (+)-MK-801-induced social withdrawal in rats”. Progress in Neuro-psychopharmacology & Biological Psychiatry. 29 (5): 833–9. doi:10.1016/j.pnpbp.2005.03.003. PMID 15913873.
  11.  Nilsson M, Carlsson A, Markinhuhta KR, et al. (July 2004). “The dopaminergic stabiliser ACR16 counteracts the behavioural primitivization induced by the NMDA receptor antagonist MK-801 in mice: implications for cognition”. Progress in Neuro-psychopharmacology & Biological Psychiatry. 28 (4): 677–85. doi:10.1016/j.pnpbp.2004.05.004. PMID 15276693.
  12. Pettersson F, Waters N, Waters ES, Carlsson A, Sonesson C (November 7, 2002). The development of a new class of dopamine stabilizers. Society for Neuroscience Annual Conference. Orlando, FL.
  13.  Tedroff, J.; Krogh, P. Lindskov; Buusman, A.; Rembratt, Å. (2010). “Poster 20: Pridopidine (ACR16) in Huntington’s Disease: An Update on the MermaiHD and HART Studies”. Neurotherapeutics. 7: 144. doi:10.1016/j.nurt.2009.10.004.
  14.  “NeuroSearch announces results from an open-label safety extension to the Phase III MermaiHD study of Huntexil® in patients with Huntington’s disease” (Press release). NeuroSearch. 15 September 2010. Retrieved 2010-09-15.
  15.  “The HART study with Huntexil® shows significant effect on total motor function in patients with Huntington’s disease although it did not meet the primary endpoint after 12 weeks of treatment” (Press release). NeuroSearch. 14 October 2010. Retrieved 2010-10-14.

REFERENCES CITED:

U.S. Patent No. 6,903,120

U.S. Patent No. 7,923,459

U.S. Publication No. US-2013-0267552-A1

CSED:25948790, http://w .chemspider.com/Chernical-Stmcture.25948790.

CSID:7971505, http://ww.chemspider.com/Chermcal-Stmcture.7971505.html

Ebenezer et al, Tetrahedron Letters 55 (2014) 5323-5326.

REFERENCES

1: Squitieri F, de Yebenes JG. Profile of pridopidine and its potential in the treatment of Huntington disease: the evidence to date. Drug Des Devel Ther. 2015 Oct 28;9:5827-33. doi: 10.2147/DDDT.S65738. eCollection 2015. PubMed PMID: 26604684; PubMed Central PMCID: PMC4629959.

2: Rabinovich-Guilatt L, Siegler KE, Schultz A, Halabi A, Rembratt A, Spiegelstein O. The effect of mild and moderate renal impairment on the pharmacokinetics of pridopidine, a new drug for Huntington’s disease. Br J Clin Pharmacol. 2016 Feb;81(2):246-55. doi: 10.1111/bcp.12792. Epub 2015 Nov 25. PubMed PMID: 26407011.

3: Shannon KM, Fraint A. Therapeutic advances in Huntington’s Disease. Mov Disord. 2015 Sep 15;30(11):1539-46. doi: 10.1002/mds.26331. Epub 2015 Jul 30. Review. PubMed PMID: 26226924.

4: Sahlholm K, Sijbesma JW, Maas B, Kwizera C, Marcellino D, Ramakrishnan NK, Dierckx RA, Elsinga PH, van Waarde A. Pridopidine selectively occupies sigma-1 rather than dopamine D2 receptors at behaviorally active doses. Psychopharmacology (Berl). 2015 Sep;232(18):3443-53. doi: 10.1007/s00213-015-3997-8. Epub 2015 Jul 11. PubMed PMID: 26159455; PubMed Central PMCID: PMC4537502.

5: Squitieri F, Di Pardo A, Favellato M, Amico E, Maglione V, Frati L. Pridopidine, a dopamine stabilizer, improves motor performance and shows neuroprotective effects in Huntington disease R6/2 mouse model. J Cell Mol Med. 2015 Nov;19(11):2540-8. doi: 10.1111/jcmm.12604. Epub 2015 Jun 22. PubMed PMID: 26094900; PubMed Central PMCID: PMC4627560.

6: Waters S, Ponten H, Klamer D, Waters N. Co-administration of the Dopaminergic Stabilizer Pridopidine and Tetrabenazine in Rats. J Huntingtons Dis. 2014;3(3):285-98. doi: 10.3233/JHD-140108. PubMed PMID: 25300332.

7: Waters S, Ponten H, Edling M, Svanberg B, Klamer D, Waters N. The dopaminergic stabilizers pridopidine and ordopidine enhance cortico-striatal Arc gene expression. J Neural Transm (Vienna). 2014 Nov;121(11):1337-47. doi: 10.1007/s00702-014-1231-1. Epub 2014 May 11. PubMed PMID: 24817271.

8: Reilmann R. The pridopidine paradox in Huntington’s disease. Mov Disord. 2013 Sep;28(10):1321-4. doi: 10.1002/mds.25559. Epub 2013 Jul 11. PubMed PMID: 23847099.

9: Gronier B, Waters S, Ponten H. The dopaminergic stabilizer pridopidine increases neuronal activity of pyramidal neurons in the prefrontal cortex. J Neural Transm (Vienna). 2013 Sep;120(9):1281-94. doi: 10.1007/s00702-013-1002-4. Epub 2013 Mar 7. PubMed PMID: 23468085.

10: Huntington Study Group HART Investigators. A randomized, double-blind, placebo-controlled trial of pridopidine in Huntington’s disease. Mov Disord. 2013 Sep;28(10):1407-15. doi: 10.1002/mds.25362. Epub 2013 Feb 28. PubMed PMID: 23450660.

11: Squitieri F, Landwehrmeyer B, Reilmann R, Rosser A, de Yebenes JG, Prang A, Ivkovic J, Bright J, Rembratt A. One-year safety and tolerability profile of pridopidine in patients with Huntington disease. Neurology. 2013 Mar 19;80(12):1086-94. doi: 10.1212/WNL.0b013e3182886965. Epub 2013 Feb 27. PubMed PMID: 23446684.

12: Ponten H, Kullingsjö J, Sonesson C, Waters S, Waters N, Tedroff J. The dopaminergic stabilizer pridopidine decreases expression of L-DOPA-induced locomotor sensitisation in the rat unilateral 6-OHDA model. Eur J Pharmacol. 2013 Jan 5;698(1-3):278-85. doi: 10.1016/j.ejphar.2012.10.039. Epub 2012 Nov 2. PubMed PMID: 23127496.

13: Lindskov Krog P, Osterberg O, Gundorf Drewes P, Rembratt Å, Schultz A, Timmer W. Pharmacokinetic and tolerability profile of pridopidine in healthy-volunteer poor and extensive CYP2D6 metabolizers, following single and multiple dosing. Eur J Drug Metab Pharmacokinet. 2013 Mar;38(1):43-51. doi: 10.1007/s13318-012-0100-2. Epub 2012 Sep 5. PubMed PMID: 22948856.

14: Ruiz C, Casarejos MJ, Rubio I, Gines S, Puigdellivol M, Alberch J, Mena MA, de Yebenes JG. The dopaminergic stabilizer, (-)-OSU6162, rescues striatal neurons with normal and expanded polyglutamine chains in huntingtin protein from exposure to free radicals and mitochondrial toxins. Brain Res. 2012 Jun 12;1459:100-12. doi: 10.1016/j.brainres.2012.04.021. Epub 2012 Apr 21. PubMed PMID: 22560595.

15: Helldén A, Panagiotidis G, Johansson P, Waters N, Waters S, Tedroff J, Bertilsson L. The dopaminergic stabilizer pridopidine is to a major extent N-depropylated by CYP2D6 in humans. Eur J Clin Pharmacol. 2012 Sep;68(9):1281-6. doi: 10.1007/s00228-012-1248-z. Epub 2012 Mar 8. PubMed PMID: 22399238.

16: Sahlholm K, Århem P, Fuxe K, Marcellino D. The dopamine stabilizers ACR16 and (-)-OSU6162 display nanomolar affinities at the σ-1 receptor. Mol Psychiatry. 2013 Jan;18(1):12-4. doi: 10.1038/mp.2012.3. Epub 2012 Feb 21. PubMed PMID: 22349783.

17: Neurodegenerative disease: Pridopidine for Huntington disease falls short of primary efficacy end point in phase III trial. Nat Rev Neurol. 2011 Dec 26;8(1):4. doi: 10.1038/nrneurol.2011.208. PubMed PMID: 22198402.

18: de Yebenes JG, Landwehrmeyer B, Squitieri F, Reilmann R, Rosser A, Barker RA, Saft C, Magnet MK, Sword A, Rembratt A, Tedroff J; MermaiHD study investigators. Pridopidine for the treatment of motor function in patients with Huntington’s disease (MermaiHD): a phase 3, randomised, double-blind, placebo-controlled trial. Lancet Neurol. 2011 Dec;10(12):1049-57. doi: 10.1016/S1474-4422(11)70233-2. Epub 2011 Nov 7. PubMed PMID: 22071279.

19: Feigin A. Pridopidine in treatment of Huntington’s disease: beyond chorea? Lancet Neurol. 2011 Dec;10(12):1036-7. doi: 10.1016/S1474-4422(11)70247-2. Epub 2011 Nov 7. PubMed PMID: 22071278.

20: Esmaeilzadeh M, Kullingsjö J, Ullman H, Varrone A, Tedroff J. Regional cerebral glucose metabolism after pridopidine (ACR16) treatment in patients with Huntington disease. Clin Neuropharmacol. 2011 May-Jun;34(3):95-100. doi: 10.1097/WNF.0b013e31821c31d8. PubMed PMID: 21586914.

US6903120 Dec 22, 2000 Jun 7, 2005 A. Carlsson Research Ab Modulators of dopamine neurotransmission
US7417043 Dec 21, 2004 Aug 26, 2008 Neurosearch Sweden Ab Modulators of dopamine neurotransmission
US7923459 Apr 10, 2007 Apr 12, 2011 Nsab, Filial Af Neurosearch Sweden Ab, Sverige Process for the synthesis of 4-(3-methanesulfonylphenyl)-1-N-propyl-piperidine
US20070238879 * Apr 10, 2007 Oct 11, 2007 Gauthier Donald R Process for the synthesis of 4-(3-methanesulfonylphenyl)-1-n-propyl-piperidine
US20100105736 Apr 14, 2008 Apr 29, 2010 Nsab, Filial Af Neurosearch Sweden Ab, Sverige N-oxide and/or di-n-oxide derivatives of dopamine receptor stabilizers/modulators displaying improved cardiovascular side-effects profiles
US20130150406 Dec 7, 2012 Jun 13, 2013 IVAX International GmbH Hydrobromide salt of pridopidine
US20130197031 Aug 31, 2011 Aug 1, 2013 IVAX International GmbH Deuterated analogs of pridopidine useful as dopaminergic stabilizers
US20130267552 Apr 3, 2013 Oct 10, 2013 IVAX International GmbH Pharmaceutical compositions for combination therapy
US20140088140 Sep 27, 2013 Mar 27, 2014 Teva Pharmaceutical Industries, Ltd. Combination of laquinimod and pridopidine for treating neurodegenerative disorders, in particular huntington’s disease
US20140088145 Sep 27, 2013 Mar 27, 2014 Teva Pharmaceutical Industries, Ltd. Combination of rasagiline and pridopidine for treating neurodegenerative disorders, in particular huntington’s disease
CN101056854A Oct 13, 2005 Oct 17, 2007 神经研究瑞典公司 Process for the synthesis of 4-(3-methanesulfonylphenyl)-1-N-propyl-piperidine
WO2001046145A1 Dec 22, 2000 Jun 28, 2001 A. Carlsson Research Ab New modulators of dopamine neurotransmission
WO2006040155A1 Oct 13, 2005 Apr 20, 2006 Neurosearch Sweden Ab Process for the synthesis of 4-(3-methanesulfonylphenyl)-1-n-propyl-piperidine
US9006445 6. Sept. 2012 14. Apr. 2015 IVAX International GmbH Polymorphic form of pridopidine hydrochloride
US9139525 11. Apr. 2008 22. Sept. 2015 Teva Pharmaceuticals International Gmbh N-oxide and/or di-N-oxide derivatives of dopamine receptor stabilizers/modulators displaying improved cardiovascular side-effects profiles
US20100105736 * 14. Apr. 2008 29. Apr. 2010 Nsab, Filial Af Neurosearch Sweden Ab, Sverige N-oxide and/or di-n-oxide derivatives of dopamine receptor stabilizers/modulators displaying improved cardiovascular side-effects profiles
US20160176821 * 18. Dez. 2015 23. Juni 2016 Teva Pharmaceuticals International Gmbh L-tartrate salt of pridopidine
USRE46117 22. Dez. 2000 23. Aug. 2016 Teva Pharmaceuticals International Gmbh Modulators of dopamine neurotransmission
WO2014205229A1 * 19. Juni 2014 24. Dez. 2014 IVAX International GmbH Use of high dose pridopidine for treating huntington’s disease
WO2015112601A1 * 21. Jan. 2015 30. Juli 2015 IVAX International GmbH Modified release formulations of pridopidine
WO2016106142A1 * 18. Dez. 2015 30. Juni 2016 Teva Pharmaceuticals International Gmbh L-tartrate salt of pridopidine
Pridopidine
Pridopidine.svg
Names
IUPAC name

4-(3-(Methylsulfonyl)phenyl)-1-propylpiperidine
Identifiers
346688-38-8 Yes
3D model (Jmol) Interactive image
ChemSpider 7971505 
KEGG D09953 
PubChem 9795739
UNII HD4TW8S2VK Yes
Properties
C15H23NO2S
Molar mass 281.41 g·mol−1
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

/////////pridopidine, PHASE 3, TEVA, 346688-38-8, orphan drug designation, Neurosearch, ACR16, Huntexil, ASP 2314, FR 310826, UNII-HD4TW8S2VK

CCCN1CCC(CC1)c2cccc(c2)S(C)(=O)=O

OXIDE

Example 5 – Preparation Of Compound 5 (4-(3-(methylsulfonyl)phenyl)-l-propylpiperidine 1-oxide)

Pridopidine (50.0g, 178mmol, leq) was dissolved in methanol (250mL) and 33% hydrogen peroxide (20mL, 213mmol, 1.2eq). The reaction mixture was heated and kept at 40°C for 20h. The reaction mixture was then concentrated in a rotavapor to give 71g light-yellow oil. Water (400mL) was added and the suspension was extracted with isopropyl acetate (150mL) which after separation contains unreacted pridopidine while water phase contains 91% area of Compound 5 (HPLC). The product was then washed with dichloromethane (400mL) after adjusting the water phase pH to 9 by sodium hydroxide. After phase separation the water phase was washed again with dichloromethane (200mL) to give 100% area of Compound 5 in the water phase (HPLC). The product was then extracted from the water phase into butanol (lx400mL, 3x200ml) and the butanol phases were combined and concentrated in a rotavapor to give 80g yellow oil (HPLC: 100% area of Compound 5). The oil was washed with water (150mL) to remove salts and the water was extracted with butanol. The organic phases were combined and concentrated in a rotavapor to give 43g of white solid which was suspended in MTBE for lhr, filtered and dried to give 33g solid that was melted when standing on air. After high vacuum drying (2mbar, 60°C, 2.5h) 32.23g pure Compound 5 were obtained (HPLC: 99.5% area, 1H-NMR assay: 97.4%).

NMR Identity Analysis of Compound 5

Compound 5:

The following data in Tables 10 and 11 was determined using a sample of 63.06 mg Compound 5, a solvent of 1.2 ml DMSO-D6, 99.9 atom%D, and the instrument was a Bruker Avance ΙΠ 400 MHz.

Table 10: Assignment of ¾ NMRa,c

a The assignment is based on the coupling pattern of the signals, coupling constants and chemical shifts.

b Weak signal.

c Spectra is calibrated by the solvent residual peak (2.5 ppm).

Table 11: Assignment of 13C NMRa,b

a The assignment is based on the chemical shifts and 1H-13C couplings extracted from HSQC and HMBC experiments.

b Spectra is calibrated by a solvent peak (39.54 ppm)

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016003919&recNum=5&docAn=US2015038349&queryString=EN_ALL:nmr%20AND%20PA:(teva%20pharmaceutical)&maxRec=677#H3

PATENT

http://www.google.bg/patents/WO2013086425A1?cl=en&hl=bg

Preparation of pridopidine HBr

In order to prepare 33 g of pridopidine HBr, 28.5 g of free base was dissolved in 150 ml 99% ethanol at room temperature. 1 .5 equivalents of hydrobromic acid 48% were added. Precipitation occurred spontaneously, and the suspension was left in refrigerator for 2.5 hours. Then the crystals were filtered, followed by washing with 99% ethanol and ether. The crystals were dried over night under vacuum at 40°C: m.p. 196°C. The results of a CHN analysis are presented in Table 2, below.

NMR 1 H NMR (DMSO-d6): 0.93 ( 3H, t), 1 .68-1 .80 ( 2H, m), 1 .99-2.10 ( 4H, m) 2.97-3.14 (5H, m), 3.24 ( 3H, s), 3.57-3.65 ( 2H, d), 7.60-7.68 (2H, m), 7.78-7.86 ( 2H, m) and 9.41 ppm (1 H, bs).

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Plinabulin


Plinabulin.svg

Plinabulin

  • Molecular FormulaC19H20N4O2
  • Average mass336.388 Da
(3Z,6Z)-3-Benzylidène-6-{[4-(2-méthyl-2-propanyl)-1H-imidazol-5-yl]méthylène}-2,5-pipérazinedione
2,5-Piperazinedione, 3-[[5-(1,1-dimethylethyl)-1H-imidazol-4-yl]methylene]-6-(phenylmethylene)-, (3Z,6Z)-
CAS 714272-27-2
NPI 2358
NPI-2358; NPI 2358
UNII:986FY7F8XR
Phase 3 Clinical

Tubulin antagonist

Cancer; Febrile neutropenia; Non-small-cell lung cancer

Plinabulin (chemical structure, BPI-2358, formerly NPI-2358) is a small molecule under development by BeyondSpring Pharmaceuticals, and is in a world-wide Phase 3 clinical trial for non-small cell lung cancer. [1] Plinabulin blocks the polymerization of tubulin in a unique manner, resulting in multi-factorial effects including an enhanced immune-oncology response, [2] activation of the JNK pathway [3] and disruption of the tumor blood supply. Plinabulin is being investigated for the reduction of chemotherapy-induced neutropenia [4] and for anti-cancer effects in combination with immune checkpoint inhibitors [5] [6] and in KRAS mutated tumors. [7]

ChemSpider 2D Image | Plinabulin | C19H20N4O2

Plinabulin is a synthetic analog of diketopiperazine phenylahistin (halimide) discovered from marine and terrestrial Aspergillus sp. Plinabulin is structurally different from colchicine and its combretastatin-like analogs (eg, fosbretabulin) and binds at or near the colchicine binding site on tubulin monomers. Previous studies showed that plinabulin induced vascular endothelial cell tubulin depolymerization and monolayer permeability at low concentrations compared with colchicine and that it induced apoptosis in Jurkat leukemia cells. Studies of plinabulin as a single agent in patients with advanced malignancies (lung, prostate, and colon cancers) showed a favorable pharmacokinetic, pharmacodynamics, and safety profile.

Beyondspring, under license from Nereus (now Triphase, which licensed the program from the Scripps Institute of Oceanography of the University of California San Diego), is developing plinabulin, the lead in the NPI-2350 halimide series of marine Aspergillus-derived, vascular-targeting antimicrotubule agents, for treating cancer, primarily non-small cell lung cancer.

Image result for BeyondSpring Pharmaceuticals

It is thought that a single, universal cellular mechanism controls the regulation of the eukaryotic cell cycle process. See, e.g., Hartwpll, L.H. et al., Science (1989), 246: 629-34. It is also known that when an abnormality arises in the control mechanism of the cell cycle, cancer or an immune disorder may occur. Accordingly, as is also known, antitumor agents and immune suppressors may be among the substances that regulate the cell cycle. Thus, new methods for producing eukaryotic cell cycle inhibitors are needed as antitumor and immune-enhancing compounds, and should be useful in the treatment of human cancer as chemotherapeutic, anti-tumor agents. See, e.g., Roberge, M. et al., Cancer Res. (1994), 54, 6115-21.

Fungi, especially pathogenic fungi and related infections, represent an increasing clinical challenge. Existing antifungal agents are of limited efficacy and toxicity, and the development and/or discovery of strains of pathogenic fungi that are resistant to drags currently available or under development. By way of example, fungi that are pathogenic in humans include among others Candida spp. including C. albicans, C. tropicalis, C. keƒyr, C. krusei and C. galbrata; Aspergillus spp. including A. fumigatus and A. flavus; Cryptococcus neoƒormans; Blastomyces spp. including Blastomyces dermatitidis; Pneumocystis carinii; Coccidioides immitis; Basidiobolus ranarum; Conidiobolus spp.; Histoplasma capsulatum; Rhizopus spp. including R. oryzae and R. microsporus; Cunninghamella spp.; Rhizomucor spp.; Paracoccidioides brasiliensis; Pseudallescheria boydii; Rhinosporidium seeberi; and Sporothrix schenckii (Kwon-Chung, K.J. & Bennett, J.E. 1992 Medical Mycology, Lea and Febiger, Malvern, PA).

Recently, it has been reported that tryprostatins A and B (which are diketopiperazines consisting of proline and isoprenylated tryptophan residues), and five other structurally-related diketopiperazines, inhibited cell cycle progression in the M phase, see Cui, C. et al., 1996 J Antibiotics 49:527-33; Cui, C. et al. 1996 J Antibiotics 49:534-40, and that these compounds also affect the microtubule assembly, see Usui, T. et al. 1998 Biochem J 333:543-48; Kondon, M. et al. 1998 J Antibiotics 51:801-04. Furthermore, natural and synthetic compounds have been reported to inhibit mitosis, thus inhibit the eukaryotic cell cycle, by binding to the colchicine binding-site (CLC-site) on tubulin, which is a macromolecule that consists of two 50 kDa subunits (α- and β-tubulin) and is the major constituent of microtubules. See, e.g., Iwasaki, S., 1993 Med Res Rev 13:183-198; Hamel, E. 1996 Med Res Rev 16:207-31; Weisenberg, R.C. et al., 1969 Biochemistry 7:4466-79. Microtubules are thought to be involved in several essential cell functions, such as axonal transport, cell motility and determination of cell morphology. Therefore, inhibitors of microtubule function may have broad biological activity, and be applicable to medicinal and agrochemical purposes. It is also possible that colchicine (CLC)-site ligands such as CLC, steganacin, see Kupchan, S.M. et al., 1973 J Am Chem Soc 95:1335-36, podophyllotoxin, see Sackett, D.L., 1993 Pharmacol Ther 59:163-228, and combretastatins, see Pettit, G.R. et al., 1995 J Med Chem 38:166-67, may prove to be valuable as eukaryotic cell cycle inhibitors and, thus, may be useful as chemotherapeutic agents.

Although diketopiperazine-type metabolites have been isolated from various fungi as mycotoxins, see Horak R.M. et al., 1981 JCS Chem Comm 1265-67; Ali M. et al., 1898 Toxicology Letters 48:235-41, or as secondary metabolites, see Smedsgaard J. et al., 1996 J Microbiol Meth 25:5-17, little is known about the specific structure of the diketopiperazine-type metabolites or their derivatives and their antitumor activity, particularly in vivo. Not only have these compounds been isolated as mycotoxins, the chemical synthesis of one type of diketopiperazine-type metabolite, phenylahistin, has been described by Hayashi et al. in J. Org. Chem. (2000) 65, page 8402. In the art, one such diketopiperazine-type metabolite derivative, dehydrophenylahistin, has been prepared by enzymatic dehydrogenation of its parent phenylahistin. With the incidences of cancer on the rise, there exists a particular need for chemically producing a class of substantially purified diketopiperazine-type metabolite-derivatives having animal cell-specific proliferation-inhibiting activity and high antitumor activity and selectivity. There is therefore a particular need for an efficient method of synthetically producing substantially purified, and structurally and biologically characterized, diketopiperazine-type metabolite-derivatives.

Also, PCT Publication WO/0153290 (July 26, 2001) describes a non-synthetic method of producing dehydrophenylahistin by exposing phenylahistin or a particular phenylahistin analog to a dehydrogenase obtained from Streptomyces albulus.

Synthesis

Image result for Plinabulin

Image result for (S)-(-)-phenylahistin

PATENT

WO2001053290,

WO 2004054498

PATENT

WO 2005077940

The imidazolecarboxaldehyde may be prepared, for example, according the procedure disclosed in Hayashi et al., 2000 J Organic Chem 65: 8402 as depicted below:

EXAMPLE 2

Synthesis and Physical Characterization of tBu-dehydrophenylahistin Derivatives

[0207] Structural derivatives of dehydrophenylahistin were synthesized according to the following reaction schemes to produce tBu-dehydrophenylahistin. Synthesis by Route

A (see Figure 1) is similar in certain respects to the synthesis of the dehydrophenylahistin synthesized as in Example 1.

Route A:

[0208] N,N’-diacethyl-2,5-piperazinedione 1 was prepared as in Example 1.

1) 1-Acetyl-3-{(Z)-1-[5-tert-butyl-1H-4-imidazolyl]methylidene}]-2,5-piperazinedione (16)

. [0209] To a solution of 5-tert-butylimidazole-4-carboxaldehyde 15 (3.02 g, 19.8. mmol) in DMF (30 mL) was added compound 1 (5.89 g, 29.72 mmol) and the solution was repeatedly evacuated in a short time to remove oxygen and flushed with Ar, followed by the addition of Cs2CO3 (9.7 g, 29.72 mmol) and the evacuation-flushing process was repeated again. The resultant mixture was stirred for 5 h at room temperature. After the solvent was removed by evaporation, the residue was dissolved in the mixture of EtOAc and 10% Na2CO3, and the organic phase was washed with 10% Na2CO3 again and saturated NaCl for three times, dried over Na2SO4 and concentrated in vacuo. The residual oil was purified by column chromatography on silica using CHCl3-MeOH (100:0 to 50:1) as an eluant to give 1.90 g (33 %) of a pale yellow solid 16. 1H NMR (270 MHz, CDCl3) δ 12.14 (d, br-s, 1H), 9.22 (br-s, 1H), 7.57 (s, 1H), 7.18, (s, 1H), 4.47 (s, 2H), 2.65 (s, 3H), 1.47 (s, 9H).

2) t-Bu-dehydrophenylahistin

[0210] To a solution of 1-Acetyl-3-{(Z)-1-[5-tert-butyl-1H-4-imidazolyl]methylidene}]-2,5-piperazinedione (16) (11 mg, 0.038 mmol) in DMF (1.0 mL) was added benzaldehyde (19 μL, 0.19 mmol, 5 eq) and the solution was repeatedly evacuated in a short time to remove oxygen and flushed with Ar, followed by the addition of Cs2CO3 (43 mg, 0.132 mmol, 3.5 eq) and the evacuation-flushing process was repeated again. The resultant mixture was heated for 2.5 h at 80°C. After the solvent was removed by

evaporation, the residue was dissolved in EtOAc, washed with water for two times and saturated NaCl for three times, dried over Na2SO4 and concentrated in vacuo. The resulting residue was dissolved in 90% MeOH aq and applied to reverse-phase HPLC column (YMC-Pack, ODS-AM, 20 × 250 mm) and eluted using a linear gradient from 70 to 74% MeOH in water over 16 min at a flow rate of 12 mL/min, and the desired fraction was collected and concentrated by evaporation to give a 6.4 mg (50%) of yellow colored tert-butyl-dehydrophenylahistin. 1H NMR (270 MHz, CDCl3) δ 12.34 br-s, 1H), 9.18 (br-s, 1H), 8.09 (s, 1H), 7.59 (s, 1H), 7.31 – 7.49 (m, 5H), 7.01 s, 2H), 1.46 (s, 9H).

[0211] The dehydrophenylahistin reaction to produce tBu-dehydrophenylahistin is identical to Example 1.

[0212] The total yield of the tBu-dehydrophenylahistin recovered was 16.5%. Route B:

[0213] N,N’-diacethyl-2,5-piperazinedione 1 was prepared as in Example 1.

1) 1-Acetyl-3-[(Z)-benzylidenel]-2,5-piperazinedione (17)

[0214] To a solution of benzaldehyde 4 (0.54 g, 5.05. mmol) in DMF (5 mL) was added compound 1 (2.0 g, 10.1 mmol) and the solution was repeatedly evacuated in a short time to remove oxygen and flushed with Ar, followed by the addition of Cs2CO3 (1.65 g, 5.05 mmol) and the evacuation-flushing process was repeated again. The resultant mixture was stirred for 3.5 h at room temperature. After the solvent was removed by evaporation, the residue was dissolved in the mixture of EtOAc and 10% Na2CO3, and the organic phase was washed with 10% Na2CO3 again and saturated NaCl for three times, dried over Na2SO4 and concentrated in vacuo. The residual solid was recrystalized from MeOH-ether to obtain a off-white solid of 17; yield 1.95 g (79%).

2) t-Bu-dehydrophenylahistin

[0215] To a solution of 1-Acetyl-3-[(Z)-benzylidenel]-2,5-piperazinedione (17) (48 mg, 0.197 mmol) in DMF (1.0 mL) was added 5-tert-butylimidazole-4-carboxaldehyde 15 (30 mg, 0.197 mmol) and the solution was repeatedly evacuated in a short time to remove oxygen and flushed with Ar, followed by the addition of Cs2CO3 (96 mg, 0.296 mmol) and the evacuation-flushing process was repeated again. The resultant mixture was heated for 14 h at 80°C. After the solvent was removed by evaporation, the residue was dissolved in EtOAc, washed with water for two times and saturated NaCl for three times, dried over Na2SO4 and concentrated in vacuo. The resulting residue was dissolved in 90% MeOH aq and applied to reverse-phase HPLC column (YMC-Pack, ODS-AM, 20 x 250 mm) and eluted using a linear gradient from 70 to 74% MeOH in water over 16 min at a flow rate of 12 mL/min, and the desired fraction was collected and concentrated by evaporation to give a 0.8 mg (1.2%) of yellow colored tert-butyl-dehydrophenylahistin.

[0216] The total yield of the tBu-dehydrophenylahistin recovered was 0.9%.

[0217] The HPLC profile of the crude synthetic tBu-dehyrophenylahistin from Route A and from Route B is depicted in Figure 4.

[0218] Two other tBu-dehydrophenylahistin derivatives were synthesized according to the method of Route A. In the synthesis of the additional tBu-dehydrophenylahistin derivatives, modifications to the benzaldehyde compound 4 were made.

[0219] Figure 4 illustrates the similarities of the HPLC profiles (Column: YMC-Pack ODS-AM (20 × 250mm); Gradient: 65% to 75% in a methanol-water system for 20 min, then 10 min in a 100% methanol system; Flow rate: 12mL/min; O.D. 230 nm) from the synthesized dehydrophenylahistin of Example 1 (Fig 2) and the above exemplified tBu-dehydrophenylahistin compound produced by Route A.

[0220] The sequence of introduction of the aldehydes is a relevant to the yield and is therefore aspect of the synthesis. An analogue of dehydrophenylahistin was synthesized, as a confrol or model, wherein the dimethylallyl group was changed to the tert-butyl group with a similar steric hindrance at the 5-position of the imidazole ring.

[0221] The synthesis of this “tert-butyl (tBu)-dehydrophenylahistin” using “Route A” was as shown above: Particularly, the sequence of infroduction of the aldehyde exactly follows the dehydrophenylahistin synthesis, and exhibited a total yield of 16.5% tBu-dehydrophenylahistin. This yield was similar to that of dehydrophenylahistin (20%). Using “Route B”, where the sequence of introduction of the aldehydes is opposite that of Route “A” for the dehydrophenylahistin synthesis, only a trace amount of the desired tBu-dehydroPLH was obtained with a total yield of 0.9%, although in the introduction of first benzaldehyde 4 gave a 76% yield of the intermediate compound 17. This result indicated that it may be difficult to introduce the highly bulky imidazole-4-carboxaldehydes 15 with a substituting group having a quaternary-carbon on the adjacent 5-position at the imidazole ring into the intermediate compound 17, suggesting that the sequence for introduction of aldehydes is an important aspect for obtaining a high yield of dehydrophenylahistin or an analog of dehydrophenylahistin employing the synthesis disclosed herein:

[0222] From the HPLC analysis of the final crude products, as shown in Figure 4, a very high content of tBu-dehydrophenylahistin and small amount of by-product formations were observed in the crude sample of Route A (left). However, a relatively smaller amount of the desired tBu-dehydrophenylahistin and several other by-products were observed in the sample obtained using Route B (right).

Synthesis oƒ 3-Z-Benzylidene-6-(5″-tert-butyl-1H-imidazol-4″-Z-ylmethylene)-piperazine-2,5-dione (2)

Reagents: g) SO2Cl2; h) H2NCHO, H2O; I)LiAlH4; j) MnO2; k) 1,4-diacetyl-piperazine-2,5-dione, Cs2CO3; 1) benzaldehyde, Cs2CO3

2-Chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester

[0280] Sulfuryl chloride (14.0 ml, 0.17 mol) was added to a cooled (0°) solution of ethyl pivaloylacetate (27.17 g, 0.16 mol) in chloroform (100 ml). The resulting mixture was allowed to warm to room temperature and was stirred for 30 min, after which it was heated under reflux for 2.5 h. After cooling to room temperature, the reaction mixture was diluted with chloroform, then washed with sodium bicarbonate, water then brine.

[0281] The organic phase was dried and evaporated to afford, as a clear oil, 2-chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester (33.1 g, 102%). (Durant et al., “Aminoalkylimidazoles and Process for their Production.” Patent No. GB1341375 (Great Britain, 1973)).

[0282] HPLC (214nm) tR = 8.80 (92.9%) min.

[0283] 1H NMR (400 MHz, CDCl3) δ 1.27 (s, 9H); 1.29 (t, J= 7.2 Hz, 3H); 4.27

(q, J= 7.2 Hz, 2H); 5.22 (s, 1H).

[0284] 13C NMR (100 MHz, CDCl3) δ 13.8, 26.3, 45.1, 54.5, 62.9, 165.1, 203.6.

5-tert-Butyl-3H-imidazole-4-carboxylic acid ethyl ester

[0285] A solution of 2-chloro-4,4-dimethyl-3-oxo-pentanoic acid ethyl ester (25.0 g, 0.12 mol) in formamide (47.5 ml) and water (2.5 ml) was shaken, then dispensed into 15 x 8 ml vials. All vials were sealed and then heated at 150° for 3.5 h. The vials were allowed to cool to room temperature, then water (20 ml) was added and the mixture was exhaustively extracted with chloroform. The chloroform was removed to give a concentrated formamide solution (22.2 g) which was added to a flash silica column (6 cm diameter, 12 cm height) packed in 1% MeOH/1% Et3N in chloroform. Elution of the column with 2.5 L of this mixture followed by 1 L of 2% MeOH/1% Et3N in chloroform gave, in the early fractions, a product suspected of being 5-tert-butyl-oxazole-4-carboxylic acid ethyl ester (6.3 g, 26%).

[0286] HPLC (214nm) tR = 8.77 min.

[0287] 1H NMR (400 MHz, CDCl3) δ 1.41 (t, J= 7.2 Hz, 3H); 1.43 (s, 9H); 4.40

(q, J= 7.2 Hz, 2H); 7.81 (s, 1H).

[0288] 13C NMR (100 MHz, CDCl3) δ 14.1, 28.8, 32.5, 61.3, 136.9, 149.9, 156.4,

158.3.

[0289] ESMS m/z 198.3 [M+H]+, 239.3 [M+CH4CN]+.

[0290] LC/MS tR = 7.97 (198.1 [M+H]+) min.

[0291] Recovered from later fractions was 5-tert-butyl-3H-imidazole-4-carboxylic acid ethyl ester (6.20 g, 26%). (Durant et al., “Aminoalkylimidazoles and Process for their Production.” Patent No. GB 1341375 (Great Britain, 1973)).

[0292] HPLC (214nm) tR = 5.41 (93.7%) min.

[0293] 1H NMR (400 MHz, CDCl3) δ 1.38 (t, J = 7.0 Hz, 3H); 1.47 (s, 9H); 4.36

(q, J= 7.2 Hz, 2H); 7.54 (s, 1H).

[0294] 13C NMR (100 MHz, CDCl3) δ 13 7, 28.8, 32.0, 59.8, 124.2, 133.3, 149.2,

162.6.

[0295] ESMS m/z 197.3 [M+H]+, 238.3 [M+CH4CN]+.

[0296] Further elution of the column with 1L of 5% MeOh/1% Et3N gave a compound suspected of being 5-tert-butyl-3H-imidazole-4-carboxylic acid (0.50 g, 2%).

[0297] HPLC (245nm) tR = 4.68 (83.1%) min.

[0298] 1H NMR (400 MHz, CD3OD) δ 1.36 (s, 9H); 7.69 (s, 1H).

[0299] 1H NMR (400 MHz, CDCl3) δ 1.37 (s, 9H); 7.74 (s, 1H).

[0300] 1H NMR (400 MHz, CD3SO) δ 1.28 (s, 9H); 7.68 (s, 1H).

[0301] ESMS m/z 169.2 [M+H]+, 210.4 [M+CH4CN]+.

(5-tert-Butyl-3H-imidazol-4-yl)-methanol

[0302] A solution of 5-tert-butyl-3-imidazole-4-carboxylic acid ethyl ester (3.30 g, 16.8 mmol) in THF (60 ml) was added dropwise to a suspension of lithium aluminium hydride (95% suspension, 0.89 g, 22.2 mmol) in THF (40 ml) and the mixture was stirred at room temperature for 3 h. Water was added until the evolution of gas ceased, the mixture was stirred for 10 min, then was filtered through a sintered funnel. The precipitate was washed with THF, then with methanol, the filtrate and washings were combined and evaporated. The residue was freeze-dried overnight to afford, as a white solid (5-tert-butyl- 3H-imidazol-4-yl)-methanol (2.71 g, 105%). (Durant et al., “Aminoalkylimidazoles and Process for their Production.” Patent No. GB1341375 (Great Britain, 1973)).

[0303] HPLC (240nm) tR = 3.70 (67.4%) min.

[0304] 1H NMR (400 MHz, CD3OD) δ 1 36 (s, 9H). 4 62 (s, 2H); 7.43 (s, 1H).

[0305] 13C NMR (100 MHz, CD3OD) δ 31.1, 33.0, 57.9, 131.4, 133.9, 140.8.

[0306] LC/MS tR = 3.41 (155.2 [M+H]+) min.

[0307] This material was used without further purification.

5-tert-Butyl-3H-imidazole-4-carbaldehyde

[0308] Manganese dioxide (30 g, 0.35 mol) was added to a heterogeneous solution of (5-tert-butyl-3H-imidazol-4-yl)-methanol (4.97 g, 0.03 mol) in acetone (700 ml) and the resulting mixture was stirred at room temperature for 4 h. The mixture was filtered through a pad of Celite and the pad was washed with acetone. The filfrate and washings were combined and evaporated. The residue was triturated with ether to afford, as a colorless solid, 5-tert-butyl-3H-imidazole-4-carbaldehyde (2.50 g, 51%). (Hayashi, Personal Communication (2000)).

[0309] HPLC (240nm) tR = 3.71 (89.3%) min.

[0310] 1H NMR (400 MHz, CDCl3) δ 1.48 (s, 9H); 7.67 (s, 1H); 10.06 (s, 1H).

[0311] LC/MS tR = 3.38 (153.2 [M+H]+) min.

[0312] Evaporation of the filtrate from the trituration gave additional 5-tert-butyl-3H-imidazole-4-carbaldehyde (1.88 g, 38%).

1-Acetyl-3-(5′-tert-butyl-1H-imdazol-4′-Z-ylmethylene)-piperazine-2,5-dione

[0313] To a solution of 5-tert-butyl-3H-imidazole-4-carbaldehyde (2.50 g, 164.4 mmol) in DMF (50 ml) was added 1,4-diacetyl-piperazine-2,5-dione (6.50 g, 32.8 mmol) and the solution was evacuated, then flushed with argon. The evacuation-flushing process was repeated a further two times, then cesium carbonate (5.35 g, 16.4 mmol) was added. The evacuation-flushing process was repeated a further three times, then the resultant mixture was stirred at room temperature for 5 h. The reaction mixture was partially evaporated (heat and high vacuum) until a small volume remained and the resultant solution was added dropwise to water (100 ml). The yellow precipitate was collected, then freeze-dried to afford 1-acetyl-3-(5′-tert-butyl-1Η-imidazol-4′-Z-ylmethylene)-piperazine-2,5-dione (2.24 g, 47%). (Hayashi, Personal Communication (2000)).

[0314] HPLC (214nm) tR = 5.54 (94.4%) min.

[0315] 1H NMR (400 MHz, CDCl3) δ 1.47 (s, 9H); 2.65 (s, 3H), 4.47 (s, 2H);

7.19 (s, 1H); 7.57 (s, 1H), 9.26 (s, 1H), 12.14 (s, 1H).

[0316] 13C NMR (100 MHz, CDCI3+CD3OD) δ 27.3, 30.8, 32.1, 46.5, 110.0,

123.2, 131.4, 133.2, 141.7, 160.7, 162.8, 173.0

[0317] LC/MS tR = 5.16 (291.2 [M+H]+, 581.6 [2M+H]+) min.

3-Z-Benzylidene-6-(5″-tert-butyl-lH-imidazol-4″-Z-ylmethylene)-piperazine-2,5-dione

[0318] To a solution of 1-acetyl-3-(5′-tert-butyl-1H-imidazol-4′-Z-ylmethylene)-piperazine-2,5-dione (2.43 g, 8.37 mmol) in DMF (55 ml) was added benzaldehyde (4.26 ml, 41.9 mmol) and the solution was evacuated, then flushed with nitrogen. The evacuation-

flushing process was repeated a further two times, then cesium carbonate (4.09 g, 12.6 mmol) was added. The evacuation-flushing process was repeated a further three times, then the resultant mixture was heated under the temperature gradient as shown below. After a total time of 5 h the reaction was allowed to cool to room temperature and the mixture was added to ice-cold water (400 ml). The precipitate was collected, washed with water, then freeze-dried to afford a yellow solid (2.57 g, HPLC (214nm) tR = 6.83 (83.1%) min.). This material was dissolved in chloroform (100 ml) and evaporated to azeofrope remaining water, resulting in a brown oil. This was dissolved in chloroform (20 ml) and cooled in ice. After 90 min the yellow precipitate was collected and air-dried to afford 3-Z-benzylidene-6-(5″-tert-butyl-1H-imidazol-4″-Z-ylmethylene)-piperazine-2,5-dione (1.59 g, 56%). (Hayashi, Personal Communication (2000)).

[0319] HPLC (214nm) tR = 6.38 (2.1%), 6.80 (95.2) min.

[0320] 1H NMR (400 MHz, CDCl3) δ 1.46 (s, pH). 7 01 (s, 1H, -C-C=CH); 7.03

(s, 1H, -C-C=CH); 7.30-7.50 (m, 5H, Ar); 7.60 (s, 1H); 8.09 (bs, NH); 9.51 (bs, NH); 12.40 (bs, NH).

[0321] LC/MS tR = 5.84 (337.4 [M+H]+, E isomer), 6.25 (337.4 [M+H]+, 673.4 [2M+H]+, Z isomer) min.

[0322] ESMS m/z 337.3 [M+H]+, 378.1 [M+OLGNT.

[0323] Evaporation of the chloroform solution gave additional 3-Z-benzylidene-6-(5″-tert-butyl-1H-imidazol-4″-Z-ylmethylene)-piperazine-2,5-dione (0.82 g, 29%). ΗPLC (214nm) tR = 6.82 (70.6%) min.

PAPER

Journal of Medicinal Chemistry (2012), 55(3), 1056-1071

Abstract Image

Plinabulin (11, NPI-2358) is a potent microtubule-targeting agent derived from the natural diketopiperazine “phenylahistin” (1) with a colchicine-like tubulin depolymerization activity. Compound 11 was recently developed as VDA and is now under phase II clinical trials as an anticancer drug. To develop more potent antimicrotubule and cytotoxic derivatives based on the didehydro-DKP skeleton, we performed further modification on the tert-butyl or phenyl groups of 11, and evaluated their cytotoxic and tubulin-binding activities. In the SAR study, we developed more potent derivatives 33 with 2,5-difluorophenyl and 50 with a benzophenone in place of the phenyl group. The anti-HuVEC activity of 33 and 50 exhibited a lowest effective concentration of 2 and 1 nM for microtubule depolymerization, respectively. The values of 33 and 50 were 5 and 10 times more potent than that of CA-4, respectively. These derivatives could be a valuable second-generation derivative with both vascular disrupting and cytotoxic activities.

Synthesis and Structure–Activity Relationship Study of Antimicrotubule Agents Phenylahistin Derivatives with a Didehydropiperazine-2,5-dione Structure

Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Kyoto 607-8412, Japan
§Nereus Pharmaceuticals, San Diego, California 92121, United States
Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Kyoto 607-8414, Japan
Laboratory of Comparative Agricultural Science, Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
# Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Marine Biotechnology Institute Co., Ltd., Kamaishi, Iwate 026-0001, Japan
J. Med. Chem., 2012, 55 (3), pp 1056–1071
DOI: 10.1021/jm2009088
*Tel/fax: +81-42-676-3275. E-mail: yhayashi@toyaku.ac.jp.
3-{(Z)-1-[5-(tert-Butyl)-1H-4-imidazolyl]methylidene}-6-[(Z)-1-phenylmethylidene]-2,5-piperazinedione
Compound 11 as a yellow solid: yield 81%;
mp 160–162 °C (dec);
IR (KBr, cm–1) 3500, 3459, 3390, 3117, 3078, 2963, 2904, 1673, 1636, 1601, 1413, 1371, 1345;
1H NMR (300 MHz, DMSO-d6) δ 12.26 (s, 2H), 10.16 (br s, 1H), 7.86 (s, 1H), 7.53 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.5 Hz 2H), 7.32 (t, J = 7.4 Hz, 1H), 6.86 (s, 1H), 6.75 (s, 1H), 1.38 (s, 9H);
13C NMR (150 MHz, DMSO-d6) 157.2, 156.4, 145.3, 137.4, 134.5, 133.1, 129.1, 128.6, 127.9, 126.4, 113.9, 112.0, 104.5, 37.4, 27.7;
HRMS (EI) m/z 336.1591 (M+) (calcd for C19H20N4O2 336.1586).
Anal. (C19H20N4O2·0.25H2O·CF3COOH) C, H, N. HPLC (method 1) 99.4% (tR = 18.87 min).
str1 str2

PAPER

Chemistry – A European Journal (2011), 17(45), 12587-12590, S12587/1-S12587/13

Abstract

original image

Click for improved solubility: A water-soluble prodrug of plinabulin was designed and synthesized efficiently by using click chemistry in three steps (see scheme). The product was highly water-soluble, and the parent compound could be regenerated by esterase hydrolysis.

PATENT

WO2017011399,  PLINABULIN COMPOSITIONS

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2017011399&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

References

  1.  “Assessment of Docetaxel + Plinabulin Compared to Docetaxel + Placebo in Patients With Advanced NSCLC With at Least One Measurable Lung Lesion (DUBLIN-3)”.
  2.  Lloyd, G.K.; Muller, Ph.; Kashyap, A.; Zippelius, A.; Huang, L. (January 7–9, 2016), Plinabulin: Evidence for an Immune Mediated Mechanism of Action (Philadelphia (PA) AACR 2016 Abstract nr A07), San Diego CA
  3.  Singh, A.V.; Bandi, M.; Raje, N.; Richardson, P.; Palladino, M.A.; Chauhan, D.; Anderson, K. (2011). “A Novel Vascular Disrupting Agent Plinabulin Triggers JNK-Mediated Apoptosis and Inhibits Angiogenesis in Multiple Myeloma Cells”. Blood. 117 (21): 5692–5700.
  4.  Heist, R.S.; Aren, O.R.; Mita, A.C.; Polikoff, J.; Bazhenova, L.; Lloyd, G.K.; Mikrut, W.; Reich, W.; Spear, M.A.; Huang, L. (2014), Randomized Phase 2 Trial of Plinabulin (NPI-2358) Plus Docetaxel in Patients with Advanced Non-Small Lung Cancer (NSCLC) (abstr 8054)
  5.  “Nivolumab and Plinabulin in Treating Patients With Stage IIIB-IV, Recurrent, or Metastatic Non-small Cell Lung Cancer”.
  6.  “Nivolumab in Combination With Plinabulin in Patients With Metastatic Non-Small Cell Lung Cancer (NSCLC)”.
  7.  Lloyd, G.K.; Du, L.; Lee, G.; Dalsing-Hernandez, J.; Kotlarczyk, K.; Gonzalez, K.; Nawrocki, S.; Carew, J.; Huang, L. (October 5–9, 2015), Activity of Plinabulin in Tumor Models with Kras Mutations (Philadelphia (PA) AACR 2015 Abstract nr. 184), Boston MA
Plinabulin
Plinabulin.svg
Names
IUPAC name

(3Z,6Z)-3-Benzylidene-6-{[5-(2-methyl-2-propanyl)-1H-imidazol-4-yl]methylene}-2,5-piperazinedione
Identifiers
714272-27-2 Yes
3D model (Jmol) Interactive image
ChemSpider 8125252
PubChem 9949641
Properties
C19H20N4O2
Molar mass 336.40 g·mol−1
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

////////////Plinabulin, Phase 3,  Clinical, 714272-27-2, NPI 2358, Nereus,  (S)-(-)-phenylahistin,  NPI-2350,  (-)-phenylahistin,  KPU-2, KPU-02, KPU-35

O=C3N\C(=C/c1ncnc1C(C)(C)C)C(=O)N/C3=C\c2ccccc2

NEW PATENT, SUGAMMADEX, WO 2016194001


Image result for patent animation
NEW PATENT, SUGAMMADEX, WO 2016194001
WO2016194001,  PROCESSES FOR PREPARATION OF SUGAMMADEX AND INTERMEDIATES THEREOF
ALAPARTHI, Lakshmi Prasad; (IN).
PAL, Palash; (IN).
GINJUPALLI, Sadasiva Rao; (IN).
SHARMA, Uday; (IN).
CHOWDARY, Talluri Bhushaiah; (IN).
MANTRI, Anand Vijaykumar; (IN).
GADE, Bharath Reddy; (IN).
KULKARNI, Gaurav; (IN)
LINK

Sugammadex (Org 25969, Bridion) is chemically known as Cyclooctakis-(l-→4)-[6-S-(2-carboxyethyl)-6-thio-a-D-glucopyranosyl]. Sugammadex is an agent for reversal of neuromuscular blockade by the neuromuscular blocking agents (NMBAs) rocuronium, vecuronium, pancuronium in general anesthesia. It is the first selective relaxant binding agent (SRBA). SRBAs are a new class of drugs that selectively encapsulates and binds NMBAs.

The word Sugammadex is derived from Su= Sugar and Gamma cyclodex = Cyclodextrin. Sugammadex is inert chemically and does not bind to any receptor. It acts by rapidly encapsulating steroidal NMBDs to form a stable complex at a 1 : 1 ratio and thus decreasing the free concentration of the drug from the plasma. This creates a concentration gradient favoring the movement of the remaining rocuronium molecules from the neuromuscular junction back into the plasma, where they are encapsulated by free Sugammadex molecules. The latter molecules also enter the tissues and form a complex with rocuronium. Therefore, the neuromuscular blockade of rocuronium is terminated rapidly by the diffusion of rocuronium away from the neuromuscular junction back into the plasma.

NMBDs are quaternary ammonium compounds with at least one charged nitrogen atom. Cyclodextrins have a lipophilic center but a hydrophilic outer core, attributable to negatively charged ions on their surface. These negatively charged ions on the surface of Sugammadex attract the positive charges of the quaternary ammonium relaxant, drawing the drug in to the central core of the cyclodextrin. The binding of the guest molecule into the host cyclodextrin occurs because of vander waal’s forces, hydrophobic and electrostatic interactions. The structure of the cyclodextrin is such that all four hydrophobic rings of the steroidal relaxant fit tightly within the concentric doughnut forming an inclusion complex. This has been confirmed by calorimetry and X-ray crystallography. Such a reaction occurs in the plasma not at the neuromuscular junction and the concentration of free rocuronium in the plasma decrease rapidly after Sugammadex administration.

[0004] US 6670340 disclose process for preparation of Sugammadex sodium. The process as disclosed in example 4 of this patent involves reaction of iodo γ-cyclodextrin intermediate with 3-mercapto propionic acid in presence of sodium hydride and DMF to give 6-per-deoxy-6-per-(3-carboxyethyl)thio-Y-cyclodextrin, sodium salt (Sugammadex sodium). The preparation of iodo intermediate, 6-per-deoxy-6-per-iodo-y-cyclodextrin is as given in example 3 which involves reaction of γ-cyclodextrin with iodine in presence of triphenylphosphine (PPh3) and DMF. In practice, and to develop a process that has to be taken from lab scale to manufacturing scale, purity is one of the most important criteria. Since this process involves use of triphenylphosphine reagent there is formation of triphenylphosphine oxide as a by-product. Removal of triphenylphosphine oxide from the reaction mass is very difficult as it requires repeated washing with the solvent, which leads to inconsistency in yield of final product Sugammadex sodium. Furthermore, the product was dialysed for 36 hours to get pure compound. The dialysis purification is expensive and provides product in lower yield and hence such processes are not feasible and economical at industrial scale.

[0005] Another process for preparing the intermediate compound, 6-perdeoxy-6-per-chloro gamma cyclodextrin as disclosed in WO2012025937 involves use of phosphorous halide in particular, phosphorous pentachloride. WO2012025937 also disclose process for preparation of Sugammadex sodium using this intermediate which involves a) reaction of gamma-cyclodextrin with phosphorous pentachloride and dimethylformamide to obtain 6-perdeoxy-6-per-chloro gamma cyclodextrin and b) reaction of 6-perdeoxy-6-per-chloro gamma cyclodextrin with 3-mercapto propionic acid in presence of alkali metal hydrides and an organic solvent to give Sugammadex sodium. Preparation of chloro gamma cyclodextrine intermediate using phosphorous pentachloride is associated with formation of phosphorous impurities during the reaction, which are difficult to remove and also it involves tedious workup procedure.

[0006] WO2014125501 discloses preparation of 6-perdeoxy-6-per-chloro gamma cyclodextrin using phosphorous pentachloride (see example 1). The process as given in example 1 of this patent application was repeated by the present inventors. The first step provided yellow to brown mass which lacked the powder form and the flow properties. The mass was pasty at times and difficult to filter. Thus the process was unclean and tedious. Overall, no consistent product was obtained. WO2014125501 also disclose preparation of Sugammadex sodium using this intermediate which involves reaction of 6-perdeoxy-6-per-halo-gamma-cyclodextrin with 3-mercapto propionic acid in presence of alkali metal alkoxide such as sodium methoxide and organic solvent, the drawback of this this reaction is that it needs anhydrous conditions for completion of the reaction.

[0007] It has been reported that the generation of impurities and obtaining less pure compounds are major concerns with Sugammadex. Applicant Nippon Organon K.K.in their “Report on the Deliberation Results” submitted to Evaluation and Licensing Division, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare, mentions as follows:

For related substances, specifications for 14 different related substances (Related Substance A, Org 48301, Related Substance B, Related Substance D, Related Substance E, Related Substance F, Related Substance G, Related Substance H, Related Substance I, Related Substance J, Related Substance K, Related Substance L, Related Substance M, Related Substance N), other individual related substances, and total related substances have been set. In the course of regulatory review, the specifications limit for 4 different related substances (Related Substance A, Related Substance D, Related Substance F, Related Substance G) have been changed based on the results of batch analyses. For related substances (degradation products), specifications for Related Substance E, Related Substance I, Related Substance C, Related Substance G, Related Substance D, Related Substance K, other individual degradation products, and total degradation products have been established. In the course of regulatory review, a specification for Impurity A which arises in *** (hidden part) step has been newly set and the specification limits for individual degradation products have been changed based on the results of batch analyses and stability studies.

The cause for change of the colour of the drug product (the light yellow-brown colour darkened) was investigated using liquid chromatography -ultraviolet-visible spectrophotometry (LC-UV/VIS) and liquid chromatography-mass spectrometry (LC-MS), which suggested that trace amounts of varieties of unspecified degradation products (unidentified), instead of a single degradation product, were involved and in addition to *** investigated in formulation development, *** and *** content of the drug substance, *** and *** during the manufacture of the drug product, and *** were considered to affect the color of the drug product. Therefore, *** and *** have been included in the drug substance specification and the relevant manufacturing process steps have been improved.

[0008] In view of the above it is clear that Sugammadex is not only prone to degradation but traces of degradation impurities affect and change the colour to yellowish brown and makes it unacceptable in quality. Therefore, it is crucial to carefully select the process to prepare pure Sugammadex sodium.

[0009] The reported purification techniques for Sugammadex sodium employ column chromatographic and membrane dialysis which are costly and not convenient in large scale operations. Therefore, the reported processes for preparation of Sugammadex sodium as discussed herein are time consuming and not economically and industrially viable.

Thus, there exist a need to provide a process of preparation of Sugammadex sodium which is simple, convenient, with easy work up procedure, economically efficient and the one which provides Sugammadex sodium in good yield and high purity.

str0

Figure 2 is 1HNMR of 6-perdeoxy-6-per-chloro gamma cyclodextrin

str0

Figure 6 is 1HNMR of Sugammadex prepared according to example 6

str0

Figure 7 is 13CNMR of Sugammadex prepared according to example 6

str0

Figure 12 is 1HNMR of Sugammadex prepared according to example 8

SEE PATENT PLEASE

Figure 13 is HPLC profile of Sugammadex prepared according to process of example 1 of WO2014125501.

scheme 1.

scheme 2.

the process for preparation of Sugammadex sodium comprising reaction of 6-perdeoxy-6-per-chloro gamma cyclodextrin (Formula II) with 3-mercaptopropionic acid in presence of alkali metal amide selected from lithium amide, sodium amide (sodamide) or potassium amide to get Sugammadex sodium.

Sugammadex Sodium

scheme 4.

the present invention provides process for preparation of Sugammadex comprising reacting the acid of Sugammadex of formula (IV) with sodium hydroxide to form Sugammadex sodium of formula (I).

Formula IV Formula I

Scheme 6

scheme 7.

scheme 8.

scheme 9.

Examples

Example 1

[0079] Preparation of 6-perdeoxy-6-per-chloro gammacyclodextrin

In a four-neck round bottomed flask (2L) equipped with mechanical stirrer, thermometer pocket in a tub charged anhydrous DMF (250ml) under nitrogen atmosphere. Triphosgene (36.5g, 0.123mol) was added to the flask at 0-15°C and the mixture was stirred for lh. Dry gamma cyclodextrin (20g, 0.015mol) was added to the obtained slurry with stirring for 30 min followed by addition of DMF (50ml). The reaction mixture was heated at 65-70°C 16 h. After the completion of reaction, the reaction mixture was cooled and diisopropyl ether (800ml) was charged to the mixture to precipitate out the material. The solvent mixture of DMF and diisopropyl ether was decanted off from the reaction mixture to obtain gummy brown mass. The reaction mass was treated with saturated sodium bicarbonate solution (800ml) which leads to precipitation of the solid. The precipitated solid was filtered, washed with the water (250x3ml) and dried. This compound was used for the next step without any purification.

Yield: 95%, HPLC Purity: 99%

Example 2

[0080] Preparation of 6-perdeoxy-6-per-chloro gamma-cyclodextrin

In a 5L four-necked flask equipped with stirrer, dropping funnel, nitrogen inlet, and thermometer with pocket, oxalyl chloride (293.8g, 198.5ml, 2315mmol) was added to DMF (1200 ml) and maintained the mixture at 0-5°C under nitrogen followed by stirring at 20-25°C for lhr. A solution of gamma-cyclodextrin (lOOg, 77.16mmol) in DMF (500ml) was added to above mixture at 5-10°C under nitrogen. The mixture was stirred at 65-70°C for 14- 16 hr. After the completion of reaction, the reaction mixture was cooled to 20-25°C and diluted with diisopropyl ether (1.2L). The organic layer was decanted and the viscous residue was treated with 10% NaOH solution at 5- 10°C until PH = 8. The resulting slurry was stirred for one hour at 20-25°C. The slurry was filtered under vacuum and the solid was washed with water (3 x 500ml) and dried under vacuum. The crude material was suspended in methanol (750ml), stirred for 30min, filtered under vacuum and washed with diisopropyl ether (500ml). The solid obtained was dried at 55- 60°C in an oven for 12-16hr to afford the titled compound (95g).

Yield: 85%, Purity: 98%, melting point: 226-228°C

lH NMR (400 MHz, DMSO-d6): δ 6.0 (br s., 16 H), 4.99 (m, 8 H), 4.04 (d, J = 10 Hz, 8 H), 3.87

– 3.78 (m, 16H), 3.64 – 3.56 (m, 8 H), 3.46 – 3.34 (m, 16 H) ppm.

13C NMR (100 MHz, DMSO-d6): δ 101.98, 82.93, 72.30, 72.16, 71.11, 44.92 ppm.

Mass: m/z (M+Na)+ calcd for
1463.14; found: 1463.06.

Example 3

[0081] Preparation of 6-perdeoxy-6-per-chloro gamma-cyclodextrin

In a clean, dried 50L glass reactor equipped with stirrer, dropping funnel, nitrogen inlet, and thermometer with pocket was charged anhydrous dimethylformamide (15L, moisture content NMT 0.4%) while maintaining the temperature at 0-5°C (using dry ice acetone bath). Oxalyl chloride (2L, 23635mmol, 30eq) was added slowly over a period 4-5hr (while maintaining the temperature below 5°C) and stirring was continued for lhr at the same temperature. A solution of dry gamma-cyclodextrin (1.0kg, 770.94mmol) dissolved in dimethylformamide (5L) was added slowly into the above reaction mixture. The solution was heated at 65-70°C for 16hr. The reaction was monitored by TLC at regular intervals. After the completion of reaction, the reaction mixture was cooled to room temperature and diisopropyl ether (10L) was added to the reaction mixture with stirring. The gummy solid precipitate out. The upper layer solvent was decanted, the gummy brown material was cooled to 0 to 5°C and was neutralized (pH 8.0) with slow addition of aqueous sodium hydroxide solution (20%, 5L) with stirring. The slurry obtained was stirred for lhr at temperature 0 to 5°C. The precipitate was filtered, washed with the water (3 x 2L) and dried under vacuum. The wet cake was suspended into methanol (10L), stirred, filtered, washed with diisopropyl ether (2L) and dried in oven at 60°C for 14-16hr to give the titled compound (980g). Yield: 87.9%, Purity: 98.1% as measured by HPLC.

Example 4

[0082] Preparation of Sugammadex sodium

In a four-neck round bottomed flask (3L) equipped with mechanical stirrer, thermometer pocket in a tub under the nitrogen atmosphere, anhydrous DMF (300ml) and 3-Mercaptopropionic acid (18.3g, 0.172mol) were charged at 0-5°C followed by addition of sodamide (20g, O.38mol). The reaction mixture was stirred at the same temperature for lh. 6-perdeoxy-6-per-chloro gamma cyclodextrin (25g, 0.017mol, as obtained in example 1) was charged slowly. The reaction mixture was heated at 90-95°C for 16h. After completion of reaction, the reaction mixture was cooled to room temperature and methanol (300ml) was added to it. The mixture was stirred and the precipitated material was filtered off. The precipitated material was dissolved in a mixture of methanol (50ml) and water (50ml) and re-precipitated with the excess addition of methanol (450ml). The solid was filtered and dried. Yield: 76%

The dried solid was purified by the preparative HPLC method using formic acid buffer in mixture of acetonitrile and water (80:20%) followed by lyophilization to get acid of Sugammadex which is further converted to Sugammadex sodium using sodium hydroxide.

Example 5

[0083] Preparation of Sugammadex sodium

In a four-neck round bottomed flask (5L) equipped with mechanical stirrer, thermometer pocket in a tub under the nitrogen atmosphere, anhydrous DMF (1500ml) and 3-mercaptopropionic acid (HOg, 1038mmol) were charged at 0-5°C followed by addition of sodamide (81g, 2077mmol). The mixture was stirred at the same temperature for lh. 6-perdeoxy-6-per-chloro gamma cyclodextrin (lOOg, 69.25mmol, as obtained in example 1) was charged slowly. Extra DMF (500ml) was added to the mixture. The temperature of the mixture was raised to 80-85°C and maintained for 16h. After completion of reaction, the reaction mixture was cooled to room temperature and methanol (1500 ml) was added to it. The mixture was stirred and the precipitated material was filtered off. The precipitated material (wet cake) was dissolved in a mixture of methanol (800ml) and water (800ml). Charcoal (50g) was added and the mixture was stirred for 30mins at 50-55°C. The solution was filtered off through a pad of celite. Methanol (2500ml) was added the solution and precipitated solid was filtered and dried furnishing the titled compound (105g). Yield: 69.6%, Purity: 85.3%.

Example 6

[0084] Preparation of Sugammadex sodium

A clean, dried 10L four neck flask equipped with stirrer, dropping funnel, nitrogen inlet, and thermometer with pocket, was charged with a solution of sodium hydroxide (83g, 2077mmol) dissolved in water (100ml) followed by addition of anhydrous DMF (2L) maintained under inert atmosphere using nitrogen. A solution of 3-mercapto propionic acid (HOg, 1037mmol) in DMF (1L) was added slowly under nitrogen maintaining the temperature between 0-5°C. The mixture was stirred for another lhr at this temperature. A mixture of 6-deoxy-6-chloro gamma cyclodextrin (lOOg, 69mmol) in DMF (1L) was added slowly at 5-10°C. The resulting mixture was heated to 75-80°C for 16-20hr. After the completion of reaction, the reaction mixture was cooled to 25-30°C and methanol (1.5L) was added into the reaction mixture, the resulting precipitate was stirred at 20-25°C, filtered, and dried under vacuum. The dried solid was dissolved in water (1L), treated with activated carbon (50 g, 5%) at 50°C, stirred and filtered through celite. The filtrate was stirred at 60°C and excess methanol (2.5L) was added slowly to the filtrate to get the precipitate. The precipitated material was filtered under vacuum as white solid, washed with methanol (500ml) and dried in oven to give pure Sugammadex sodium (90 g).

Yield: 90 g, Purity: 91.2%.

lU NMR (400 MHz, D20): δ 5.09 (m, 8H); 3.98-3.94 (m, 8H); 3.88-3.83 (m, 8H); 3.58-3.52 (m, 16H); 3.07-3.01 (m, 8H); 2.92-2.87 (m, 8H); 2.78-2.74 (m, 16H); 2.34-2.47 (m, 16H) ppm.

13C NMR (100 MHz, D20): δ 180.18, 100.60, 81.96, 72.14, 71.84, 70.72, 37.24, 32.83, 29.06 ppm. Mass: m/z (M-Na7+H6)+ calcd for C72HnoNa048S8: 2023.12; found: 2023.39.

Example 7

Preparation of Sugammadex acid (Compound of formula IV)

In a clean, dried 5L four neck flask equipped with stirrer, dropping funnel, nitrogen inlet, and thermometer with pocket was charged dimethylformamide (1500ml) followed by addition of potassium hydroxide (194.0 g, 3464mmol) and the mixture maintained at 0-5°C. A solution of 3-mercapto propionic acid (186.35g, 153.0ml, 1756mmol) in DMF (500ml) was added to the reactor over a period of 30 minutes under nitrogen while maintaining the temperature between 0-5°C. The

resulting mixture was stirred at this temperature for 60 minutes. A solution of 6-deoxy-6-chloro gamma cyclodextrin (lOOg, 69.22mmol) in DMF (500ml) was added to the flask. The resulting mixture was heated at 110-120°C for 1.5-2hr while monitoring the progress of the reaction through HPLC. After completion of the reaction, the temperature of the reaction mixture was brought to 40-50°C and methanol (1000ml) was added to the mixture. The resulted precipitate was stirred at 20-25°C for lhr, filtered under vacuum and washed with methanol (500ml). The wet solid was dissolved in water (2000ml) with vigorous stirring and the solution was acidified with concentrated hydrochloric acid to give the white solid precipitate. The precipitated solid was filtered and suspended in ethyl acetate (500 ml), stirred for 30 minutes and filtered. The solid was dried to afford the titled compound (75g).

Yield: 55%, Purity: 95.8% as measured by HPLC.

lH NMR (400 MHz, DMSO-d6): δ 5.94 (br. s, 16H), 3.82-3.73 (m, 8H), 3.63-3.54 (m, 8H), 3.43-3.32 (m, 16H), 3.08-3.02 (m, 8H), 2.89-2.81 (m, 8H), 2.78-2.72 (m, 16H), 2.55-2.43 (m, 16H) ppm.

13C NMR (100 MHz, DMSO-d6): δ 173.00, 102.01, 83.94, 72.45, 72.33, 71.36, 34.53, 33.08, 27.87 ppm.

Mass: m/z (M-H2+K) + calcd for C72Hno048S8K: 2039.24; found: 2039.26.

Example 8

Preparation of Sugammadex Sodium

In a clean, dried 3L four neck flask equipped with stirrer, dropping funnel, nitrogen inlet, and thermometer with pocket, the compound (75g) as obtained in example 4 was dissolved in solution of sodium hydroxide (37.5g, 0.937mol) in water (100ml) and methanol (100ml). The pH of resultant mixture was maintained between 8-10. To this mixture methanol (1.5L) was slowly added at room temperature and the mixture was stirred for additional 30 minutes. The precipitated white solid was filtered off under vacuum and thoroughly washed with methanol (500ml). The solid was dried at 50°C under vacuum oven for 24hr to afford Sugammadex sodium (79g).

Yield: 96.9%, Purity: 95.5% measured by HPLC.

VADADUSTAT


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ChemSpider 2D Image | Vadadustat | C14H11ClN2O4

VADADUSTAT

AKB-6548, PG-1016548
PG1016548, UNII:I60W9520VV, B-506

CAS 1000025-07-9

[5-(3-chlorophenyl)-3-hydroxypyridine-2-carboxamido]acetic acid

N-[[5-(3-Chlorophenyl)-3-hydroxy-2-pyridinyl]carbonyl]glycine

MF C14H11ClN2O4 , 306.0407

Inventors Richard Kawamoto
Original Assignee The Procter & Gamble Company

for Treatment of Anemia associated with Chronic Kidney Disease (CKD)

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  • Originator Procter & Gamble
  • Developer Akebia Therapeutics
  • Class Antianaemics; Chlorophenols; Pyridines; Small molecules
  • Mechanism of Action Hypoxia-inducible factor-proline dioxygenase inhibitors
  • Phase III Anaemia
  • 01 Aug 2016 Akebia Therapeutics initiates the phase III INNO2VATE trial for Anaemia in USA (NCT02865850)
  • 23 May 2016 Interim drug interactions and adverse events data from a phase I trial (In volunteers) Chronic kidney disease released by Akebia
  • 05 May 2016 Akebia completes a clinical trial (ethnobridging study) in Healthy volunteers

Vadadustat (also known as AKB-6548) in anemia secondary to chronic kidney disease (CKD)

We are developing our lead product candidate, vadadustat, to be the potential best-in-class hypoxia inducible factor–prolyl hydroxylase inhibitor for the treatment of anemia secondary to CKD.

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PATENT

CN 105837502

HIF inhibitor Vadadustat (Code AKB-6548) The chemical name N- [5- (3- chlorophenyl) -3-hydroxypyridine-2-carbonyl] glycine,

Vadadustat is a treatment for anemia associated with chronic kidney disease oral HIF inhibitor, is an American biopharmaceutical company Akebia Therapeutics invention in the research of new drugs, has completed Phase II pivotal clinical trial treatment studies, successfully met the researchers set given the level of hemoglobin in vivo target and good security, a significant effect, and phase III clinical trials.

 U.S. Patent Publication US20120309977 synthetic route for preparing a Vadadustat: A 3-chlorophenyl boronic acid and 3,5_-dichloro-2-cyanopyridine as starting materials, by-catalyzed coupling methoxy substituted, cyano hydrolysis and condensation and ester hydrolysis reaction Vadadustat, process route is as follows:

 

Since the entire synthetic route 12 steps long, complicated operation, high cost.U.S. Patent No. 1 2 ^ ¥ disclosed 20070299086 & (^ (Scheme 3 1118 seven seven to 3,5-dichloro-2-cyanopyridine starting material, first-dichloro substituted with benzyloxy, then cyano hydrolysis, condensation, hydrogenation and deprotection trifluorosulfonyl, to give N- [5- trifluoromethanesulfonyloxy-3-hydroxypyridine-2-carbonyl) glycine methyl ester, 3-chlorophenyl and then boronic acid catalyzed coupling reactions, the final ester hydrolysis reaction Vadadustat, process route is as follows:

The synthesis steps long, intermediate products and final products contain more impurities and byproducts, thus purified requires the use of large amounts of solvents, complicated operation, low yield, and because the hydrogenation reaction is a security risk on the production, not conducive to the promotion of industrial production, it is necessary to explore a short process, simple operation, low cost synthetic method whereby industrial production Vadadus tat fit.

Example 1

A) Preparation of N- (3,5_-dichloro-2-carbonyl) glycine methyl ester:

3,5-dichloro-2-pyridinecarboxylic acid (19.2g, 0.10mol) and N, N’_ carbonyldiimidazole (24.3g, 0.15mol) was dissolved in N, N- dimethylformamide (100 mL ), was added glycine methyl ester hydrochloride (15.18,0.12111〇1), 11 was added dropwise diisopropylethylamine (51.7g, 0.40mol), the reaction mixture was stirred 35 ° C for 8 hours, TLC determined the completion of reaction gussets The reaction solution was concentrated by rotary evaporation to dryness, dilute hydrochloric acid was adjusted to neutral by adding ethyl acetate, dried over magnesium sulfate, and concentrated by rotary evaporation to dryness, and recrystallized from methanol to give N- (3,5- dichloro-pyridin-2 – carbonyl) glycine methyl ester, an off-white solid (21.6g), a yield of 82.0%, this reaction step is as follows:

1234567 B) Preparation of N- [5- (3- chlorophenyl) -3-chloropyridine-2-carbonyl] glycine methyl ester: 2

1 (3,5-dichloro-2-carbonyl) glycine methyl ester (20 (^, 〇1 76111111), 3-chlorophenyl boronic acid (13.18, 3 83.7mmol), [l, l’- bis (diphenylphosphino) ferrocene] dichloropalladium (2.8g, 3.8mmol), potassium carbonate (14.2g, 4 0. lmo 1) and N, N- dimethylformamide (75mL) was added The reaction flask, the reaction mixture was heated to 60 ° C for 20 hours the reaction was stirred for 5:00, point TLC plates to determine completion of the reaction, the reaction solution was cooled to room temperature, was concentrated by rotary evaporation to dryness, extracted with ethyl acetate, washed with brine, sulfuric acid 6 magnesium dried and concentrated by rotary evaporation to dryness, a mixed solvent of ethyl acetate and n-hexane was recrystallized to give N- [5- (3- chlorophenyl) -3-7-chloro-2-carbonyl] glycine methyl ester, white solid (19.7g), yield 76.4%, this reaction step is as follows:

C) Preparation of N_ [5- (3- chlorophenyl) -3-methoxy-pyridine-2-carbonyl] glycine:

N- [5- (3- chlorophenyl) -3-chloropyridine-2-carbonyl] glycine methyl ester (19 (^, 56111 111〇1) and sodium methoxide (7.6g, 0.14mol) was dissolved in methanol (150 mL), the reaction mixture was heated to 65 ° C, the reaction was stirred at reflux for 24 hours, TLC determined gussets completion of the reaction the reaction solution was cooled to room temperature, water (300mL) was stirred for 3h, cooled to 0 ° C, stirred for 2h, precipitated solid was filtered, the filter cake was dried to give N- [5- (3- chlorophenyl) -3-methoxy-pyridine-2-carbonyl] glycine, off-white solid (17.4 g of), a yield of 96.5%, of the reaction steps are as follows:

D) Preparation Vadadustat:

N- [5- (3- chlorophenyl) -3-methoxy-pyridine-2-carbonyl] glycine (16.68,51.7111111〇1) and 48% hydrobromic acid solution (52mL, 0.46mol) added to the reaction bottle, the reaction mixture was heated to 100 ° C, the reaction was stirred at reflux for 24 hours, TLC determined gussets completion of the reaction the reaction solution cooled square ~ 5 ° C, was slowly added 50% sodium hydroxide solution was adjusted to pH 2 at 0 -5 ° C under crystallization 3h, the filter cake washed with ethyl acetate and n-hexane mixed solvent of recrystallization, in finished Vadadustat, off-white solid (15.6g), a yield of 98.0%, this reaction step is as follow

PATENT

WO-2016153996

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016153996&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescriptiohttps://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016153996&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

PATENT

WO 2015073779

https://www.google.com/patents/WO2015073779A1?cl=en

Form A of Compound (I):

(I),

which has an X-ray powder diffraction pattern as shown in FIG. 1. In certain embodiments, Form A of Compound (I) has an X-ray powder diffraction pattern comprising one, two, three, four, or five peaks at approximately 18.1 , 20.3, 22.9, 24.0, and 26.3 °2Θ; and wherein the crystalline Compound (I) is substantially free of any other crystalline form of Compound (I).

PATENT

US 20120309977

  • FIG. 1 depicts an outline of one embodiment for preparing the disclosed prolyl hydroxylase inhibitors.
    FIG. 2 depicts an outline of one embodiment for preparing the disclosed prolyl hydroxylase inhibitor ester prodrugs.
    FIG. 3 depicts an outline of one embodiment for preparing the disclosed prolyl hydroxylase inhibitor amide prodrugs.

Example 1 describes a non-limiting example of the disclosed process for the preparation of a prolyl hydroxylase ester pro-drug

Figure US20120309977A1-20121206-C00044

Figure US20120309977A1-20121206-C00045

EXAMPLE 1Methyl {[5-(3-chlorophenyl)-3-hydroxypyridin-2-yl]amino}acetate (4)

Preparation of 5-(3-chlorophenyl)-3-chloro-2-cyanopyridine (1): To a 100 mL round bottom flask adapted for magnetic stirring and equipped with a nitrogen inlet was charged (3-chlorophenyl)boronic acid (5 g, 32 mmol), 3,5-dichloro-2-cyanopyridine (5.8 g, 34 mmol), K2CO3 (5.5 g, 40 mmol), [1,1′-bis(diphenyphosphino)ferrocene]dichloro-palladium(II) [PdCl2(dppf)] (0.1 g, 0.13 mmol), dimethylformamide (50 mL) and water (5 mL). The reaction solution was agitated and heated to 45° C. and held at that temperature for 18 hours after which the reaction was determined to be complete due to the disappearance of 3,5-dichloro-2-cyanopyridine as measured by TLC analysis using ethyl acetate/methanol (4:1) as the mobile phase and UV 435 nm to visualize the reaction components. The reaction solution was then cooled to room temperature and the contents partitioned between ethyl acetate (250 mL) and saturated aqueous NaCl (100 mL). The organic phase was isolated and washed a second time with saturated aqueous NaCl (100 mL). The organic phase was dried for 4 hours over MgSO4, the MgSO4 removed by filtration and the solvent removed under reduced pressure. The residue that remained was then slurried in methanol (50 mL) at room temperature for 20 hours. The resulting solid was collected by filtration and washed with cold methanol (50 mL) then hexanes (60 mL) and dried to afford 5.8 g (73% yield) of an admixture containing a 96:4 ratio of the desired regioisomer. 1H NMR (DMSO-d6) δ 9.12 (d, 1H), 8.70 (d, 1H), 8.03 (t, 1H) 7.88 (m, 1H), and 7.58 (m, 2H)

Preparation of 5-(3-chlorophenyl)-3-methoxy-2-cyanopyridine (2): To a 500 mL round bottom flask adapted for magnetic stirring and fitted with a reflux condenser and nitrogen inlet was charged with 5-(3-chlorophenyl)-3-chloro-2-cyanopyridine, 1, (10 g, 40 mmol), sodium methoxide (13.8 mL, 60 mmol) and methanol (200 mL). With stirring, the reaction solution was heated to reflux for 20 hours. The reaction was determined to be complete due to the disappearance of 5-(3-chlorophenyl)-3-chloro-2-cyanopyridine as measured by TLC analysis using hexane/ethyl acetate (6:3) as the mobile phase and UV 435 nm to visualize the reaction components. The reaction mixture was cooled to room temperature and combined with water (500 mL). A solid began to form. The mixture was cooled to 0° C. to 5° C. and stirred for 3 hours. The resulting solid was collected by filtration and washed with water, then hexane. The resulting cake was dried in vacuo at 40° C. to afford 9.4 g (96% yield) of the desired product as an off-white solid. 1H NMR (DMSO-d6) δ 8.68 (d, 1H), 8.05 (d, 1H), 8.01 (s, 1H) 7.86 (m, 1H), 7.59 (s, 1H), 7.57 (s, 1H) and 4.09 (s, 3H).

Preparation of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carboxylic acid (3): To a 50 mL round bottom flask adapted for magnetic stirring and fitted with a reflux condenser was charged 5-(3-chlorophenyl)-3-methoxy-2-cyanopyridine, 2, (1 g, 4 mmol) and a 48% aqueous solution of HBr (10 mL). While being stirred, the reaction solution was heated to reflux for 20 hours. The reaction was determined to be complete due to the disappearance of 5-(3-chlorophenyl)-3-methoxy-2-cyanopyridine as measured by TLC analysis using hexane/ethyl acetate (6:3) as the mobile phase and UV 435 nm to visualize the reaction components. The reaction contents was then cooled to 0° C. to 5° C. with stirring and the pH was adjusted to approximately 2 by the slow addition of 50% aqueous NaOH. Stirring was then continued at 0° C. to 5° C. for 3 hours. The resulting solid was collected by filtration and washed with water, then hexane. The resulting cake was dried in vacuo at 40° C. to afford 1.03 g (quantitative yield) of the desired product as an off-white solid. 1H NMR (DMSO-d6) δ 8.52 (d, 1H), 7.99 (d, 1H), 7.95 (s, 1H) 7.81 (t, 1H), 7.57 (s, 1H), and 7.55 (s, 1H).

Preparation of methyl {[5-(3-chlorophenyl)-3-hydroxypyridin-2-yl]amino}acetate (4): To a 50 mL round bottom flask adapted for magnetic stirring and fitted with a nitrogen inlet tube was charged 5-(3-chlorophenyl)-3-hydroxypyridine-2-carboxylic acid, 3, (1 gm, 4 mmol), N,N′-carbonyldiimidazole (CDI) (0.97 g, 6 mmol) and dimethyl sulfoxide (5 mL). The reaction mixture was stirred at 45° C. for about 1 hour then cooled to room temperature. Glycine methyl ester hydrochloride (1.15 g, 12 mmol) is added followed by the dropwise addition of diisopropylethylamine (3.2 mL, 19 mmol). The mixture was then stirred for 2.5 hours at room temperature after which water (70 mL) was added. The contents of the reaction flask was cooled to 0° C. to 5° C. and 1N HCl was added until the solution pH is approximately 2. The solution was extracted with dichloromethane (100 mL) and the organic layer was dried over MgSO4 for 16 hours. Silica gel (3 g) is added and the solution slurried for 2 hours after which the solids are removed by filtration. The filtrate is concentrated to dryness under reduced pressure and the resulting residue was slurried in methanol (10 mL) for two hours. The resulting solid was collected by filtration and washed with cold methanol (20 mL) then hexane and the resulting cake is dried to afford 0.85 g of the desired product as an off-white solid. The filtrate was treated to afford 0.026 g of the desired product as a second crop. The combined crops afford 0.88 g (68% yield) of the desired product. 1H NMR (DMSO-d6) δ 12.3 (s, 1H), 9.52 (t, 1H), 8.56 (d, 1H), 7.93 (s, 1H), 7.80 (q, 2H), 7.55 (t, 2H), 4.12 (d, 2H), and 3.69 (s, 3H).

The formulator can readily scale up the above disclosed synthesis. Disclosed herein below is a synthesis wherein the disclosed process is scaled up for commercial use

EXAMPLE 2Methyl {[5-(3-chlorophenyl)-3-hydroxypyridin-2-yl]amino}acetate (4)

Preparation of 5-(3-chlorophenyl)-3-chloro-2-cyanopyridine (1): A 20 L reactor equipped with a mechanical stirrer, dip tube, thermometer and nitrogen inlet was charged with (3-chlorophenyl)boronic acid (550 g, 3.52 mol), 3,5-dichloro-2-cyanopyridine (639 g, 3.69 mol), K2CO3 (5.5 g, 40 mmol), [1,1′-bis(diphenyphosphino)ferrocene]dichloro-palladium(II) [PdCl2(dppf)] (11.5 g, 140 mmol), and dimethylformamide (3894 g, 4.125 L). The reaction solution was agitated and purged with nitrogen through the dip-tube for 30 minutes. Degassed water (413 g) was then charged to the reaction mixture while maintaining a temperature of less than 50° C. 25 hours. The reaction was determined to be complete due to the disappearance of 3,5-dichloro-2-cyanopyridine as measured by TLC analysis using ethyl acetate/methanol (4:1) as the mobile phase and UV 435 nm to visualize the reaction components. The reaction solution was then cooled to 5° C. and charged with heptane (940 g, 1.375 L) and agitated for 30 minutes. Water (5.5 L) was charged and the mixture was further agitated for 1 hour as the temperature was allowed to rise to 15° C. The solid product was isolated by filtration and washed with water (5.5 L) followed by heptane (18881 g, 2750 ML). The resulting cake was air dried under vacuum for 18 hours and then triturated with a mixture of 2-propanol (6908 g, 8800 mL0 and heptane (1 g, 2200 mL0 at 50° C. for 4 hours, cooled to ambient temperature and then agitated at ambient temperature for 1 hour. The product was then isolated by filtration and washed with cold 2-propanol (3450 g, 4395 mL) followed by heptane (3010 g, 4400 mL). The resulting solid was dried under high vacuum at 40° C. for 64 hours to afford 565.9 g (65% yield) of the desired product as a beige solid. Purity by HPLC was 98.3. 1H NMR (DMSO-d6) δ 9.12 (d, 1H), 8.70 (d, 1H), 8.03 (t, 1H) 7.88 (m, 1H), and 7.58 (m, 2H).

Preparation of 5-(3-chlorophenyl)-3-methoxy-2-cyanopyridine (2): A 20 L reactor equipped with a mechanical stirred, condenser, thermometer and nitrogen inlet was charged with 5-(3-chlorophenyl)-3-chloro-2-cyanopyridine, 1, (558 g, 2.24 mol) and sodium methoxide (25% solution in methanol, 726.0 g, 3.36 mol). With agitation, the reaction solution was heated to reflux for 24 hours, resulting in a beige-colored suspension. The reaction was determined to be complete due to the disappearance of 5-(3-chlorophenyl)-3-chloro-2-cyanopyridine as measured by TLC analysis using hexane/ethyl acetate (6:3) as the mobile phase and UV 435 nm to visualize the reaction components. The reaction mixture was cooled to 5° C. and then charged with water (5580 mL). The resulting slurry was agitated for 3 hours at 5° C. The solid product was isolated by filtration and washed with water (5580 mL) until the filtrate had a pH of 7. The filter cake was air dried under vacuum for 16 hours. The filter cake was then charged back to the reactor and triturated in MeOH (2210 g, 2794 mL) for 1 hour at ambient temperature. The solid was collected by filtration and washed with MeOH (882 g, 1116 mL, 5° C.) followed by heptane (205 mL, 300 mL), and dried under high vacuum at 45° C. for 72 hours to afford 448 g (82% yield) of the desired product as an off-white solid. Purity by HPLC was 97.9%. 1H NMR (DMSO-d6) δ 8.68 (d, 1H), 8.05 (d, 1H), 8.01 (s, 1H) 7.86 (m, 1H), 7.59 (s, 1H), 7.57 (s, 1H) and 4.09 (s, 3H).

Preparation of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carboxylic acid (3): A 20 L reactor equipped with a mechanical stirrer, condenser, thermometer, nitrogen inlet and 25% aqueous NaOH trap was charged 5-(3-chlorophenyl)-3-methoxy-2-cyanopyridine, 2, (440.6 g, 1.8 mol) and 37% aqueous solution of HCl (5302 g). While being agitated, the reaction solution was heated to 102° C. for 24 hours. Additional 37% aqueous HCl (2653 g) was added followed by agitation for 18 hours at 104° C. The reaction contents was then cooled to 5° C., charged with water (4410 g) and then agitated at 0° C. for 16 hours. The resulting precipitated product was isolated by filtration and washed with water until the filtrate had a pH of 6 (about 8,000 L of water). The filter cake was pulled dry under reduced pressure for 2 hours. The cake was then transferred back into the reactor and triturated in THF (1958 g, 2201 mL) at ambient temperature for 2 hours. The solid product was then isolated by filtration and washed with THF (778 g, 875 mL) and dried under reduced pressure at 5° C. for 48 hours to afford 385 g (89% yield) of the desired product as an off-white solid. HPLC purity was 96.2%. 1H NMR (DMSO-d6) δ 8.52 (d, 1H), 7.99 (d, 1H), 7.95 (s, 1H) 7.81 (t, 1H), 7.57 (s, 1H), and 7.55 (s, 1H).

Preparation of methyl {[5-(3-chlorophenyl)-3-hydroxypyridin-2-yl]amino}acetate (4): A 20 L reactor equipped with a mechanical stirrer, condenser, thermometer and nitrogen inlet was charged with 5-(3-chlorophenyl)-3-hydroxypyridine-2-carboxylic acid, 3, (380 g, 1.52 mol) and diisopropylethylamine (DIPEA) (295 g, 2.28 mol). With agitation, the solution was cooled to 3° C. and charged with trimethylacetyl chloride (275.7 g, 2.29 mol) while maintaining a temperature of less than 11° C., The mixture was then agitated at ambient temperature for 2 hours. The mixture was then cooled to 10° C. and charged with a slurry of glycine methyl ester HCl (573.3 g, 4. 57 mol) and THF (1689 g, 1900 mL), then charged with DIPEA (590.2 g, 4.57 mol) and agitated at ambient temperature for 16 hours. The mixture was then charged with EtOH (1500 g, 1900 mL) and concentrated under reduced pressure to a reaction volume of about 5.8 L. The EtOH addition and concentration was repeated twice more. Water (3800 g) was then added and the mixture was agitated for 16 hours at ambient temperature. The resulting solid product was isolated by filtration and washed with a mixture of EtOH (300 g, 380 mL) and water (380 g), followed by water (3800 g), dried under reduced pressure for 18 hours at 50° C. to afforded 443 g (91% yield) of the desired product as an off-white solid. Purity by HPLC was 98.9%. 1H NMR (DMSO-d6) δ 12.3 (s, 1H), 9.52 (t, 1H), 8.56 (d, 1H), 7.93 (s, 1H), 7.80 (q, 2H), 7.55 (t, 2H), 4.12 (d, 2H), and 3.69 (s, 3H).

Scheme II herein below outlines and Example 2 describes a non-limiting example of the disclosed process for preparing a prolyl hydroxylase inhibitor from an ester prodrug.

Figure US20120309977A1-20121206-C00046

EXAMPLE 3{[5-(3-Chlorophenyl)-3-hydroxypyridin-2-yl]amino}acetic acid (5)

Preparation of {[5-(3 -chlorophenyl)-3-hydroxypyridin-2-yl]amino}acetic acid (5): To a 50 mL flask is charged methyl {[5-(3-chlorophenyl)-3-hydroxypyridin-2-yl]amino}-acetate, 4, (0.45 g, 1.4 mmol), tetrahydrofuran (4.5 mL) and 1 M NaOH (4.5 mL, 4.5 mmol). The mixture was stirred for 2 hours at room temperature after which it was determined by TLC analysis using hexane/ethyl acetate (6:3) as the mobile phase and UV 435 nm to visualize the reaction components that the reaction was complete. The reaction solution was adjusted to pH 1 with concentrated HCl and the solution was heated at 35° C. under vacuum until all of the tetrahydrofuran had been removed. A slurry forms as the solution is concentrated. With efficient stirring the pH is adjusted to ˜2 with the slow addition of 1 M NaOH. The solid which forms was collected by filtration, washed with water, followed by hexane, then dried under vacuum to afford 0.38 g (88% yield) of the desired product as a white solid. 1H NMR (DMSO-d6) δ 12.84 (s, 1H), 12.39 (s, 1H), 9.39 (t, 1H), 8.56 (d, 1H), 7.94 (s, 1H), 7.81 (m, 2H), 7.55 (q, 2H), and 4.02 (d, 2H).

The formulator can readily scale up the above disclosed synthesis. Disclosed herein below is a synthesis wherein the disclosed process is scaled up for commercial use.

EXAMPLE 4{[5-(3-Chlorophenyl)-3-hydroxypyridin-2-yl]amino}acetic acid (5)

Preparation of {[5-(3-chlorophenyl)-3-hydroxypyridin-2-yl]amino}acetic acid (5): To a 20 L reactor equipped with a mechanical stirrer, condenser, thermometer and nitrogen inlet was charged methyl {[5-(3-chlorophenyl)-3-hydroxypyridin-2-yl]amino}-acetate, 4, (440 g, 1.42 mol), tetrahydrofuran (3912 g, 4400 mL) and 1 M NaOH (4400 mL). The mixture was stirred for 2 hours at room temperature after which it was determined by TLC analysis using hexane/ethyl acetate (6:3) as the mobile phase and UV 435 nm to visualize the reaction components that the reaction was complete. The reaction solution was acidified to a pH of 2 with slow addition of 2M HCl (2359 g). The resulting mixture was concentrated under reduced pressure to a volume of about 7.5 L. Ware (2210 g) was added and the solution cooled to ambient temperature and agitated for 18 hours. The solid product was isolated by filtration and washed with water (6 L). the crude product was transferred back into the reactor and triturated with 2215 g o deionized water at 70° C. for 16 hours. The mixture was cooled to ambient temperature, The solid product was isolated by filtration and washed with water (500 mL) and dried under reduced pressure at 70° C. for 20 hours to afford 368 g (87% yield) of the desired product as an off-white solid. Purity by HPLC was 99.3%. 1H NMR (DMSO-d6) δ 12.84 (s, 1H), 12.39 (s, 1H), 9.39 (t, 1H), 8.56 (d, 1H), 7.94 (s, 1H), 7.81 (m, 2H), 7.55 (q, 2H), and 4.02 (d, 2H).

Scheme III herein below outlines and Example 3 describes a non-limiting example of the disclosed process for preparing a prolyl hydroxylase amide prodrug.

Figure US20120309977A1-20121206-C00047

EXAMPLE 55-(3-Chlorophenyl)-N-(2-amino-2-oxoethyl)-3-hydroxylpyridin-2-yl amide

Preparation of 5-(3-chlorophenyl)-N-(2-amino-2-oxoethyl)-3-hydroxylpyridin-2-yl amide (6): To a solution of 5-(3-chlorophenyl)-3-hydroxypyridine-2-carboxylic acid, 3, (749 mg, 3 mmol) in DMF (20 mL) at room temperature under N2 is added 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide (EDCI) (0.925 g, 5.97 mmol) and 1-hydroxybenzo-triazole (HOBt) (0.806 g, 5.97 mmol). The resulting solution is stirred for 15 minutes then 2-aminoacetamide hydrochloride (0.66 g, 5.97 mmol) and diisopropylethylamine (1.56 ml, 8.96 mmol) are added. The reaction is monitored by TLC and when the reaction is complete the reaction mixture is concentrated under reduced pressure and H2O added. The product can be isolated by normal work-up: The following data have been reported for compound (6). 1H NMR (250 MHz, DMSO-d6) δ ppm 12.46 (1H, s), 9.17 (1H, t, J=5.9 Hz), 8.55 (1H, d, J=2.0 Hz), 7.93 (1H, d, J=0.9 Hz), 7.75-7.84 (2H, m), 7.49-7.60 (3H, m), 7.18 (1H, s), 3.91 (2H, d, J=5.9 Hz). HPLC-MS: m/z 306 [M+H]+.

Scheme IV herein below depicts a non-limiting example the hydrolysis of an amide pro-drug to a prolyl hydroxylase inhibitor after removal of a R10 protecting group

Figure US20120309977A1-20121206-C00048

PATENT

US 20070299086

https://www.google.com/patents/US20070299086

REF

http://akebia.com/wp-content/themes/akebia/img/media-kit/abstracts-posters-presentations/Akebia_NKF%202016%20Poster_FINAL.pdf

Beuck S, Schänzer W, Thevis M. Hypoxia-inducible factor stabilizers and other
small-molecule erythropoiesis-stimulating agents in current and preventive doping
analysis. Drug Test Anal. 2012 Nov;4(11):830-45. doi: 10.1002/dta.390. Epub 2012
Feb 24. Review. PubMed PMID: 22362605.

Abstracts, posters, and presentations

The effect of altitude on erythropoiesis-stimulating agent dose, hemoglobin level, and mortality in hemodialysis patients

Vadadustat, a novel oral HIF stabilizer, provides effective anemia treatment in nondialysisdependent chronic kidney disease

2016 ERA-EDTA: Poster
A Drug-Drug Interaction Study to Evaluate the Effect of Vadadustat on the Pharmacokinetics of Celecoxib—a CYP2C9 Substrate—in Healthy Volunteers

2016 NKF: Poster
Vadadustat — a Novel, Oral Treatment for Anemia of CKD — Maintains Stable Hemoglobin Levels in Dialysis Patients Converting From Erythropoiesis-Stimulating Agent (ESA)

2015 ASN: Posters
Vadadustat Demonstrates Controlled Hemoglobin Response in a Phase 2b Study for the Treatment of Anemia in Patients with Non-Dialysis Dependent Chronic Kidney Disease

Dose Exposure Relationship of Vadadustat is Independent of the Level of Renal Function

Vadadustat, a Novel, Oral Treatment for Anemia of CKD, Maintains Stable Hemoglobin Levels in Dialysis Patients Converting from Erythropoiesis-Stimulating Agents

Hemoglobin Response in a Phase 2b Study of Vadadustat for the Treatment of Anemia in Patients with Non-Dialysis Dependent Chronic Kidney Disease

The Effect of Altitude on Erythropoiesis-Stimulating Agent Dose, Hemoglobin Level, and Mortality in Hemodialysis Patients

Erythropoiesis-Stimulating Agent Hyporesponse Is Associated with Persistently Elevated Mortality among Hemodialysis Patients

Variability in Hemoglobin Levels in Hemodialysis Patients in the Current Era

2014 ASN: Posters
Phase 2 Study of AKB-6548, a novel hypoxia-inducible factor prolyl-hydroxylase inhibitor (HIF-PHI) in patients with end stage renal disease (ESRD) undergoing hemodialysis (HD)

Hemodialysis has minimal impact on the pharmacokinetics of AKB-6548, a once-daily oral inhibitor of hypoxia-inducible factor prolyl-hydroxylases (HIF-PHs) for the treatment of anemia related to chronic kidney disease (CKD)

2014 ERA-EDTA: Oral presentation
Controlled Hemoglobin Response in a Double-Blind, Placebo-Controlled Trial of AKB-6548 in Subjects with Chronic Kidney Disease

2012 ASN: Oral presentation
AKB-6548, A New Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor, Increases Hemoglobin in Chronic Kidney Disease Patients Without Increasing Basal Erythropoietin Levels

2011 ASN: Oral presentation
AKB-6548, A Novel Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Reduces Hepcidin and Ferritin while It Increases Reticulocyte Production and Total Iron Binding Capacity In Healthy Adults

2011 ASN: Poster
AKB-6548, A New Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Increases Hemoglobin While Decreasing Ferritin in a 28-day, Phase 2a Dose Escalation Study in Stage 3 and 4 Chronic Kidney Disease Patients With Anemia

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WO2013013609A1 * Jul 23, 2012 Jan 31, 2013 Zhejiang Beta Pharma Incorporation Polymorphic forms of compounds as prolyl hydroxylase inhibitor, and uses thereof
US20070299086 * Jun 26, 2007 Dec 27, 2007 The Procter & Gamble Company Prolyl hydroxylase inhibitors and methods of use
US20100331303 * Aug 20, 2010 Dec 30, 2010 Richard Masaru Kawamoto Prolyl hydroxylase inhibitors and methods of use
US20130203816 * Nov 20, 2012 Aug 8, 2013 Akebia Therapeutics Inc. Prolyl hydroxylase inhibitors and methods of use
WO2016118858A1 * Jan 22, 2016 Jul 28, 2016 Akebia Therapeutics, Inc. Solid forms of 2-(5-(3-fluorophenyl)-3-hydroxypicolinamido)acetic acid, compositions, and uses thereof

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Akebia Therapeutics

Oct 6, 2015

Akebia Reaches Agreement with FDA and EMA on Vadadustat Global Phase 3 Program

Plans to Initiate Phase 3 PRO2TECT Clinical Program by Year-End

CAMBRIDGE, Mass.–(BUSINESS WIRE)– Akebia Therapeutics, Inc. (NASDAQ: AKBA), a biopharmaceutical company focused on delivering innovative therapies to patients with kidney disease through the biology of hypoxia inducible factor (HIF), today announced the successful completion of the End-of-Phase 2 Meeting process with the United States Food and Drug Administration (FDA) and the Scientific Advice Process with the European Medicines Agency (EMA) for its lead product, vadadustat (formerly AKB-6548), for patients with anemia related to non-dialysis dependent chronic kidney disease (NDD-CKD). The company has reached agreement with both the FDA and EMA regarding key elements of the Phase 3 program, known as the PRO2TECTprogram, and expects to launch the program later this year.

The PRO2TECT™ program includes two separate studies and will collectively enroll approximately 3,100 NDD-CKD patients across 500 sites globally. The correction study will address anemia patients not currently being treated with recombinant erythropoiesis stimulating agents (rESAs). The conversion study includes patients currently receiving rESA who will be converted to either vadadustat or the active control with the goal of maintaining their baseline hemoglobin levels. Both studies will include a 1:1 randomization and an open label, active-control, non-inferiority design. Primary endpoints include an efficacy assessment of the hemoglobin response and an assessment of cardiovascular safety measured by major adverse cardiovascular events.

“Akebia’s Phase 3 program is designed to provide the medical community and regulators with a clear understanding of vadadustat’s potential benefit and safety advantages over rESAs, the current standard of care worldwide and, with a positive outcome, to establish vadadustat as the best-in-class treatment option for patients with renal anemia,” stated John P. Butler, President and Chief Executive Officer of Akebia. “We are pleased that the regulators are in agreement regarding the importance of an active-control trial as this design is the most clinically relevant and commercially valuable, and will allow us the quickest path to full enrollment. We are now moving rapidly to launch these studies and advance our goal of bringing forward new treatment options for patients suffering from renal anemia.”

“This Phase 3 program builds on the positive data from our Phase 2 program in NDD-CKD patients which demonstrated that once-daily vadadustat can control and maintain hemoglobin levels in a clinically relevant range while minimizing fluctuations in hemoglobin levels that are associated with increased cardiovascular safety risks,” stated Brad Maroni, M.D., Chief Medical Officer at Akebia. “These two Phase 3 event-driven studies are designed to establish the safety and efficacy of vadadustat in the setting of contemporary clinical practice patterns, and support regulatory approvals globally.”

In addition, Akebia discussed with the FDA and EMA a parallel Phase 3 program, known as the INNO2VATE™ program, for vadadustat in patients with anemia related to chronic kidney disease who are undergoing dialysis (DD-CKD). Akebia expects to formalize its Phase 3 program in DD-CKD patients after presenting the results from its recently completed Phase 2 study to both regulatory agencies.

About Vadadustat (Formerly AKB-6548)

Vadadustat is an oral therapy currently in development for the treatment of anemia related to chronic kidney disease (CKD). Vadadustat is designed to stabilize HIF, a transcription factor that regulates the expression of genes involved with red blood cell (RBC) production in response to changes in oxygen levels, by inhibiting the hypoxia-inducible factor prolyl hydroxylase (HIF-PH) enzyme. Vadadustat exploits the same mechanism of action used by the body to naturally adapt to lower oxygen availability associated with a moderate increase in altitude. At higher altitudes, the body responds to lower oxygen availability with increased production of HIF, which coordinates the interdependent processes of iron mobilization and erythropoietin (EPO) production to increase RBC production and, ultimately, improve oxygen delivery.

As a HIF stabilizer with best-in-class potential, vadadustat raises hemoglobin levels predictably and sustainably, with a dosing regimen that allows for a gradual and controlled titration. Vadadustat has been shown to improve iron mobilization, potentially eliminating the need for intravenous iron administration and reducing the overall need for iron supplementation.

About Anemia Related to CKD

Approximately 30 million people in the United States have CKD, with an estimated 1.8 million of these patients suffering from anemia. Anemia results from the body’s inability to coordinate RBC production in response to lower oxygen levels due to the progressive loss of kidney function, which occurs in patients with CKD. Left untreated, anemia significantly accelerates patients’ overall deterioration of health with increased morbidity and mortality. Renal anemia is currently treated with injectable rESAs, which are associated with inconsistent hemoglobin responses and well-documented safety risks.

About Akebia Therapeutics

Akebia Therapeutics, Inc. is a biopharmaceutical company headquartered in Cambridge, Massachusetts, focused on delivering innovative therapies to patients with kidney disease through HIF biology. The company has completed Phase 2 development of its lead product candidate, vadadustat, an oral therapy for the treatment of anemia related to CKD in both non-dialysis and dialysis patients.

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Akebia Announces Positive Top-Line Results from its Phase 2 Study of Vadadustat in Dialysis Patients with Anemia Related to Chronic Kidney Disease

-Treatment with Vadadustat Successfully Maintained Mean Hemoglobin Levels Following Conversion from rESA Therapy-

-Vadadustat Demonstrated a Favorable Safety Profile with Once Daily and Three Times per Week Dosing-

CAMBRIDGE, Mass.–(BUSINESS WIRE)–Akebia Therapeutics, Inc. (NASDAQ:AKBA), a biopharmaceutical company focused on delivering innovative therapies to patients with kidney disease through the biology of hypoxia inducible factor (HIF), today announced positive top-line results from its Phase 2 study of vadadustat (formerly AKB-6548) in dialysis patients with anemia related to chronic kidney disease (CKD). The study achieved its primary objective, indicating that vadadustat maintained stable hemoglobin (HGB) levels throughout the 16-week treatment period following conversion from recombinant erythropoiesis-stimulating agent (rESA) therapy. Vadadustat demonstrated a favorable safety profile with no drug-related serious adverse events and no deaths. The results highlight the potential of vadadustat, dosed either once daily or three times per week, to safely and predictably manage and sustain HGB levels in CKD patients undergoing dialysis.

“This study was a clear success, demonstrating the potential of vadadustat to effectively and safely treat anemia in dialysis patients switching from injectable rESA therapy”

The open-label, multi-center, 94 patient study was designed to evaluate the ability of vadadustat to maintain hemoglobin levels in patients undergoing hemodialysis who were previously being treated with rESAs. Patients were assigned to one of three dose cohorts: once daily vadadustat at a starting dose of 300mg, once daily vadadustat at a starting dose of 450mg, or vadadustat three times per week in conjunction with the patient’s hemodialysis schedule at a starting dose of 450mg. The study achieved its primary endpoints of maintaining stable hemoglobin levels over 16 weeks of treatment in all three cohorts of patients converting from rESAs to vadadustat.

Mean Hemoglobin Levels (g/dL)* Baseline Week 7/8 Week 15/16
300mg Daily Dose 10.4 10.4 10.3
450mg Daily Dose 10.6 10.3 10.5
450mg Three Times per Week Dose 10.5 10.2 10.4

* Modified intent-to-treat (MITT) population, n=94

Vadadustat was well tolerated among patients in all three dose cohorts. Treatment-emergent adverse events (TEAEs) with vadadustat were balanced across the cohorts. Serious adverse events (SAEs) were reported in 13 subjects (13.8%), well within the expected range for this patient population. There were no drug-related SAEs and no deaths reported in the study.

“This study was a clear success, demonstrating the potential of vadadustat to effectively and safely treat anemia in dialysis patients switching from injectable rESA therapy,” said Brad Maroni, M.D., Chief Medical Officer at Akebia. “We are impressed with the consistency in hemoglobin levels across the duration of the study, which highlights the ability of vadadustat to control and maintain hemoglobin levels in this patient population. Furthermore, the results indicate that daily and three times per week dosing regimens are both viable options for patients on dialysis.”

John P. Butler, President and Chief Executive Officer of Akebia, stated, “These results further confirm vadadustat as a potential best-in-class anemia treatment for CKD patients, and reinforce our confidence in this product candidate as we advance toward our Phase 3 program. Adding these results to the 12 other clinical studies we have completed, we are confident in the potential for vadadustat to treat anemia in a broad array of patients with CKD. We are pleased to have successfully completed this stage of our drug development and look forward to initiating Phase 3 studies.”

Complete efficacy and safety data from this Phase 2 study will be presented at an upcoming medical meeting.

About the Phase 2 Study Design of Vadadustat in Dialysis Patients with Anemia Related to CKD

The Phase 2 multi-center, open-label study evaluated 94 patients over 16 weeks of treatment, at 20 dialysis centers in the United States, including an assessment of HGB response to the starting dose of vadadustat during the first 8 weeks, followed by an assessment of HGB response to algorithm-guided dose adjustments of vadadustat during the subsequent 8 weeks of treatment. The study enrolled three cohorts, each consisting of approximately 30 CKD patients with anemia undergoing dialysis who were switched from injectable rESA therapy to vadadustat. Patients in the first two cohorts received once daily doses of vadadustat, while patients in the third cohort received vadadustat three times per week in conjunction with their hemodialysis schedule.

///////////VADADUSTAT, PHASE 3, AKB-6548, PG-1016548, B-506

c1cc(cc(c1)Cl)c2cc(c(nc2)C(=O)NCC(=O)O)O

Evofosfamide, эвофосфамид , إيفوفوسفاميد , 艾伏磷酰胺 ,


str1

TH-302.svg

Evofosfamide, HAP-302 , TH-302, TH 302

эвофосфамид ,  إيفوفوسفاميد ,  艾伏磷酰胺 ,

  • Molecular Formula C9H16Br2N5O4P
  • Average mass 449.036 Da

(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N’-bis(2-bromoethyl)phosphorodiamidate

(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl-N,N’-bis(2-bromethyl)phosphorodiamidat
918633-87-1

TH-302 is a nitroimidazole-linked prodrug of a brominated derivative of an isophosphoramide mustard previously used in cancer drugs

  • Originator Threshold Pharmaceuticals
  • Developer Merck KGaA; Threshold Pharmaceuticals
  • Class Antineoplastics; Nitroimidazoles; Phosphoramide mustards; Small molecules
  • Mechanism of Action Alkylating agents
  • Orphan Drug Status Yes – Soft tissue sarcoma; Pancreatic cancer
  • On Fast track Pancreatic cancer; Soft tissue sarcoma
  • Suspended Glioblastoma; Leukaemia; Malignant melanoma; Multiple myeloma; Non-small cell lung cancer; Solid tumours
  • Discontinued Pancreatic cancer; Soft tissue sarcoma

Most Recent Events

  • 01 Aug 2016 Threshold plans a clinical trial for Solid tumours
  • 01 Aug 2016 Threshold announces intention to submit NDA to the Pharmaceuticals and Medical Device Agency in Japan
  • 16 Jun 2016 Merck KGaA terminates a phase II trial in Soft tissue sarcoma (Combination therapy, Inoperable/Unresectable, Metastatic disease, Late-stage disease) in Japan (IV) due to negative results from the phase III SARC021 trial (NCT02255110)

Evofosfamide (first disclosed in WO2007002931), useful for treating cancer.

Image result for Evofosfamide

Threshold Pharmaceuticals and licensee Merck Serono are codeveloping evofosfamide, the lead in a series of topoisomerase II-inhibiting hypoxia-activated prodrugs and a 2-nitroimidazole-triggered bromo analog of ifosfamide, for treating cancer, primarily soft tissue sarcoma and pancreatic cancer (phase 3 clinical, as of April 2015).

In November 2014, the FDA granted Fast Track designation to the drug for the treatment of previously untreated patients with metastatic or locally advanced unresectable soft tissue sarcoma.

Evofosfamide (INN,[1] USAN;[2] formerly known as TH-302) is an investigational hypoxia-activated prodrug that is in clinical development for cancer treatment. The prodrug is activated only at very low levels of oxygen (hypoxia). Such levels are common in human solid tumors, a phenomenon known as tumor hypoxia.[3]

Evofosfamide is being evaluated in clinical trials for the treatment of multiple tumor types as a monotherapy and in combination with chemotherapeutic agents and other targeted cancer drugs.

Dec 2015 : two Phase 3 trials fail, Merck will not apply for a license

Collaboration

Evofosfamide was developed by Threshold Pharmaceuticals Inc. In 2012, Threshold signed a global license and co-development agreement for evofosfamide with Merck KGaA, Darmstadt, Germany (EMD Serono Inc. in the US and Canada), which includes an option for Threshold to co-commercialize evofosfamide in the United States. Threshold is responsible for the development of evofosfamide in the soft tissue sarcoma indication in the United States. In all other cancer indications, Threshold and Merck KGaA are developing evofosfamide together.[4] From 2012 to 2013, Merck KGaA paid 110 million US$ for upfront payment and milestone payments to Threshold. Additionally, Merck KGaA covers 70% of all evofosfamide development expenses.[5]

Mechanism of prodrug activation and Mechanism of action (MOA) of the released drug[edit]

Evofosfamide is a 2-nitroimidazole prodrug of the cytotoxin bromo-isophosphoramide mustard (Br-IPM). Evofosfamide is activated by a process that involves a 1-electron (1 e) reduction mediated by ubiquitous cellular reductases, such as the NADPH cytochrome P450, to generate a radical anion prodrug:

  • A) In the presence of oxygen (normoxia) the radical anion prodrug reacts rapidly with oxygen to generate the original prodrug and superoxide. Therefore, evofosfamide is relatively inert under normal oxygen conditions, remaining intact as a prodrug.
  • B) When exposed to severe hypoxic conditions (< 0.5% O2; hypoxic zones in many tumors), however, the radical anion undergoes irreversible fragmentation, releasing the active drug Br-IPM and an azole derivative. The released cytotoxin Br-IPM alkylates DNA, inducing intrastrand and interstrand crosslinks.[6]

Evofosfamide is essentially inactive under normal oxygen levels. In areas of hypoxia, evofosfamide becomes activated and converts to an alkylating cytotoxic agent resulting in DNA cross-linking. This renders cells unable to replicable their DNA and divide, leading to apoptosis. This investigational therapeutic approach of targeting the cytotoxin to hypoxic zones in tumors may cause less broad systemic toxicity that is seen with untargeted cytotoxic chemotherapies.[7]

The activation of evofosfamide to the active drug Br-IPM and the mechanism of action (MOA) via cross-linking of DNA is shown schematically below:

Activation of eofosfamide to the active drug Br-IPM, and mechanism of action via cross-linking of DNA

Drug development history

Phosphorodiamidate-based, DNA-crosslinking, bis-alkylator mustards have long been used successfully in cancer chemotherapy and include e.g. the prodrugs ifosfamide andcyclophosphamide. To demonstrate that known drugs of proven efficacy could serve as the basis of efficacious hypoxia-activated prodrugs, the 2-nitroimidizole HAP of the active phosphoramidate bis-alkylator derived from ifosfamide was synthesized. The resulting compound, TH-281, had a high HCR (hypoxia cytotoxicity ratio), a quantitative assessment of its hypoxia selectivity. Subsequent structure-activity relationship (SAR) studies showed that replacement of the chlorines in the alkylator portion of the prodrug with bromines improved potency about 10-fold. The resulting, final compound is evofosfamide (TH-302).[8]

Synthesis

Evofosfamide can be synthesized in 7 steps.[9][10]

  1. CPhI.cn: Synthetic routes to explore anti-pancreatic cancer drug Evofosfamide, 22 Jan 2015
  2.  Synthetic route Reference: International patent application WO2007002931A2

Formulation

The evofosfamide drug product formulation used until 2011 was a lyophilized powder. The current drug product formulation is a sterile liquid containing ethanol,dimethylacetamide and polysorbate 80. For intravenous infusion, the evofosfamide drug product is diluted in 5% dextrose in WFI.[11]

Diluted evofosfamide formulation (100 mg/ml evofosfamide, 70% ethanol, 25% dimethylacetamide and 5% polysorbate 80; diluted to 4% v/v in 5% dextrose or 0.9% NaCl) can cause leaching of DEHP from infusion bags containing PVC plastic.[12]

Clinical trials

Overview and results

Evofosfamide (TH-302) is currently being evaluated in clinical studies as a monotherapy and in combination with chemotherapy agents and other targeted cancer drugs. The indications are a broad spectrum of solid tumor types and blood cancers.

Evofosfamide clinical trials (as of 21 November 2014)[13] sorted by (Estimated) Primary Completion Date:[14]


Both, evofosfamide and ifosfamide have been investigated in combination with doxorubicin in patients with advanced soft tissue sarcoma. The study TH-CR-403 is a single arm trial investigating evofosfamide in combination with doxorubicin.[35] The study EORTC 62012 compares doxorubicin with doxorubicin plus ifosfamide.[36] Doxorubicin and ifosfamide are generic products sold by many manufacturers.Soft tissue sarcoma

The indirect comparison of both studies shows comparable hematologic toxicity and efficacy profiles of evofosfamide and ifosfamide in combination with doxorubicin. However, a longer overall survival of patients treated with evofosfamide/doxorubicin (TH-CR-403) trial was observed. The reason for this increase is probably the increased number of patients with certain sarcoma subtypes in the evofosfamide/doxorubicin TH-CR-403 trial, see table below.

However, in the Phase 3 TH-CR-406/SARC021 study (conducted in collaboration with the Sarcoma Alliance for Research through Collaboration (SARC)), patients with locally advanced unresectable or metastatic soft tissue sarcoma treated with evofosfamide in combination with doxorubicin did not demonstrate a statistically significant improvement in OS compared with doxorubicin alone (HR: 1.06; 95% CI: 0.88 – 1.29).

Metastatic pancreatic cancer

Both, evofosfamide and protein-bound paclitaxel (nab-paclitaxel) have been investigated in combination with gemcitabine in patients with metastatic pancreatic cancer. The study TH-CR-404 compares gemcitabine with gemcitabine plus evofosfamide.[39] The study CA046 compares gemcitabine with gemcitabine plus nab-paclitaxel.[40] Gemcitabine is a generic product sold by many manufacturers.

The indirect comparison of both studies shows comparable efficacy profiles of evofosfamide and nab-paclitaxel in combination with gemcitabine. However, the hematologic toxicity is increased in patients treated with evofosfamide/gemcitabine (TH-CR-404 trial), see table below.

In the Phase 3 MAESTRO study, patients with previously untreated, locally advanced unresectable or metastatic pancreatic adenocarcinoma treated with evofosfamide in combination with gemcitabine did not demonstrate a statistically significant improvement in overall survival (OS) compared with gemcitabine plus placebo (hazard ratio [HR]: 0.84; 95% confidence interval [CI]: 0.71 – 1.01; p=0.0589).

Drug development risks

Risks published in the quarterly/annual reports of Threshold and Merck KGaA that could affect the further development of evofosfamide (TH-302):

Risks related to the formulation

The evofosfamide formulation that Threshold and Merck KGaA are using in the clinical trials was changed in 2011[43] to address issues with storage and handling requirements that were not suitable for a commercial product. Additional testing is ongoing to verify if the new formulation is suitable for a commercial product. If this new formulation is also not suitable for a commercial product another formulation has to be developed and some or all respective clinical phase 3 trials may be required to be repeated which could delay the regulatory approvals.[44]

Risks related to reimbursement

Even if Threshold/Merck KGaA succeed in obtaining regulatory approvals and bringing evofosfamide to the market, the amount reimbursed for evofosfamide may be insufficient and could adversely affect the profitability of both companies. Obtaining reimbursement for evofosfamide from third-party and governmental payors depend upon a number of factors, e.g. effectiveness of the drug, suitable storage and handling requirements of the drug and advantages over alternative treatments.

There could be the case that the data generated in the clinical trials are sufficient to obtain regulatory approvals for evofosfamide but the use of evofosfamide has a limited benefit for the third-party and governmental payors. In this case Threshold/Merck KGaA could be forced to provide supporting scientific, clinical and cost effectiveness data for the use of evofosfamide to each payor. Threshold/Merck KGaA may not be able to provide data sufficient to obtain reimbursement.[45]

Risks related to competition

Each cancer indication has a number of established medical therapies with which evofosfamide will compete, for example:

  • If approved for commercial sale for pancreatic cancer, evofosfamide would compete with gemcitabine (Gemzar), marketed by Eli Lilly and Company; erlotinib (Tarceva), marketed by Genentech and Astellas Oncology; protein-bound paclitaxel (Abraxane), marketed by Celgene; and FOLFIRINOX, which is a combination of generic products that are sold individually by many manufacturers.
  • If approved for commercial sale for soft tissue sarcoma, evofosfamide could potentially compete with doxorubicin or the combination of doxorubicin and ifosfamide, generic products sold by many manufacturers.[46]

Risks related to manufacture and supply

Threshold relies on third-party contract manufacturers for the manufacture of evofosfamide to meet its and Merck KGaA’s clinical supply needs. Any inability of the third-party contract manufacturers to produce adequate quantities could adversely affect the clinical development and commercialization of evofosfamide. Furthermore, Threshold has no long-term supply agreements with any of these contract manufacturers and additional agreements for more supplies of evofosfamide will be needed to complete the clinical development and/or commercialize it. In this regard, Merck KGaA has to enter into agreements for additional supplies or develop such capability itself. The clinical programs and the potential commercialization of evofosfamide could be delayed if Merck KGaA is unable to secure the supply.[47]

History

Date Event
Jun 2005 Threshold files evofosfamide (TH-302) patent applications in the U.S.[48]
Jun 2006 Threshold files an evofosfamide (TH-302) patent application in the EU and in Japan[49]
Sep 2011 Threshold starts a Phase 3 trial (TH-CR-406) of evofosfamide in combination with doxorubicin in patients with soft tissue sarcoma
Feb 2012 Threshold signs an agreement with Merck KGaA to co-develop evofosfamide
Apr 2012 A Phase 2b trial (TH-CR-404) of evofosfamide in combination with gemcitabine in patients with pancreatic cancer meets primary endpoint
Jan 2013 Merck KGaA starts a global Phase 3 trial (MAESTRO) of evofosfamide in combination with gemcitabine in patients with pancreatic cancer
Dec 2015 two Phase 3 trials fail, Merck will not apply for a license

CLIP

CLIP

Efficient synthesis of 2-nitroimidazole derivatives and the bioreductive clinical candidate Evofosfamide (TH-302)

*Corresponding authors
aDepartment of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford, UK
E-mail: stuart.conway@chem.ox.ac.uk
bCancer Research UK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, UK
Org. Chem. Front., 2015,2, 1026-1029

DOI: 10.1039/C5QO00211G

http://pubs.rsc.org/en/content/articlelanding/2015/qo/c5qo00211g/unauth#!divAbstract

http://www.rsc.org/suppdata/c5/qo/c5qo00211g/c5qo00211g1.pdf

Hypoxia, regions of low oxygen, occurs in a range of biological environments, and is involved in human diseases, most notably solid tumours. Exploiting the physiological differences arising from low oxygen conditions provides an opportunity for development of targeted therapies, through the use of bioreductive prodrugs, which are selectively activated in hypoxia. Herein, we describe an improved method for synthesising the most widely used bioreductive group, 2-nitroimidazole. The improved method is applied to an efficient synthesis of the anti-cancer drug Evofosfamide (TH-302), which is currently in Phase III clinical trials for treatment of a range of cancers.

Graphical abstract: Efficient synthesis of 2-nitroimidazole derivatives and the bioreductive clinical candidate Evofosfamide (TH-302)

Image result for Evofosfamide

(1-Methyl-2-nitro-1H-imidazol-5-yl)-N,N–bis(2-bromoethyl) phosphordiamidate (TH- 302)

The residue was then purified by semi-preparative HPLC on a Phenomenex Luna (C18(2), 10 µm, 250 × 10 mm) column, eluting with H2O and methanol (50 – 70% methanol over 10 min, then 1 min wash with methanol, 5 mL/min flow rate) to afford TH-302 as a yellow gum: vmax (solid) cm-1 : 3212 (br), 1489 (m), 1350 (m), 1105 (m), 1004 (s); δH (DMSO-D6, 400 MHz) 7.25 (1H, s, CH), 5.10–4.90 (2H, m, NHCH2CH2Br), 4.98 (2H, d, J 7.8, CH2O), 3.94 (3H, s, CH3), 3.42 (4H, t, J 7.0, NHCH2CH2Br), 3.11 (4H, dt, J 9.8, 7.2, NHCH2CH2Br); δC (DMSO-D6, 126 MHz) 146.1, 134.2 (d, J 7.5, OCH2CN), 128.2, 55.6 (d, J 4.6, CH2O), 42.7, 34.2 (d, J 26.4, CH2Br), 34.1; δP (DMSO-D6, 202 MHz) 15.4; HRMS m/z (ESI− ) [found; (M-H)− 447.9216, C9H16 79Br81BrN5O4P requires (M-H)− 447.9213]; m/z (ESI+ ) 448.0 ([M-H]− , 60%, [C9H15 79Br81BrN5O4P] − ), 493.9 ([M+formate] − , 100%, [C10H17 79Br81BrN5O6P] − ). These data are in good agreement with the literature values.4

4 J.-X. Duan, H. Jiao, J. Kaizerman, T. Stanton, J. W. Evans, L. Lan, G. Lorente, M. Banica, D. Jung, J. Wang, H. Ma, X. Li, Z. Yang, R. M. Hoffman, W. S. Ammons, C. P. Hart and M. Matteucci, J. Med. Chem., 2008, 51, 2412–2420.

J. Med. Chem., 2008, 51, 2412–2420/……………….1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N-bis(2-bromoethyl)
phosphordiami-date (3b). Compound 3b was synthesized by a procedure similar to that described for 3a and obtained as an off-white solid in 47.6% yield.

1H NMR (DMSO-d6) δ: 7.22 (s, 1H), 5.10–5.00 (m, 2H), 4.97 (d, J ) 7.6 Hz, 2H), 3.94 (s, 3H), 3.42 (t, J ) 7.2 Hz, 4H), and 3.00–3.20 (m, 4H).

13C NMR (DMSOd6)δ: 146.04, 134.16 (d, J ) 32 Hz), 128.17, 55.64, 42.70, 34.33,and 34.11 (d, J ) 17.2 Hz).

31P NMR (DMSO-d6) δ: -11.25.
HRMS: Calcd for C9H16N5O4PBr2, 446.9307; found, 446.9294.

CLIP

Synthesis Route reference WO2007002931A2

Med J.. Chem. 2008, 51, 2412-2420

From compound S-1 starting aminoacyl protection is S-2 , a suspension of NaH grab α -proton, offensive, ethyl, acidification, introduction of an aldehyde group, S-3followed by condensation with the amino nitrile, off N- acyl ring closure, migration rearrangement amino imidazole compound S-. 8 , the amino and sodium nitrite into a diazonium salt, raising the temperature, nitrite anion nucleophilic attack diazonium salt obtained nitro compound S-9, under alkaline conditions ester hydrolysis gives acid S-10 , followed by NEt3 under the action of isobutyl chloroformate and the reaction mixed anhydride formed by of NaBH 4 reduction to give the alcohol S-. 11 , [use of NaBH 4 reduction of the carboxyl group is another way and the I 2 / of NaBH 4 ] , to give S-11 later, the DIAD / PPh3 3 under the action via Mitsunobu linking two fragments obtained reaction Evofosfamide

Image result for Evofosfamide.

PATENT

http://www.google.co.in/patents/WO2015051921A1?cl=en

EXAMPLE 1

1

N-Formylsarcosine ethyl ester 1 (1 ,85 kg) was dissolved in toluene (3,9 kg) and ethyl formate (3,28 kg) and cooled to 10 °C. A 20 wt-% solution of potassium tert-butoxide (1 ,84 kg) in tetrahydrofuran (7,4 kg) was added and stirring was continued for 3h. The reaction mixture was extracted 2x with a solution of sodium chloride in water (10 wt-%) and the combined water extracts were washed lx with toluene.

Aqueous hydrogen chloride (25% wt-%; 5,62 kg) was added to the aqueous solution, followed by ethylene glycol (2,36 kg). The reaction mixture was heated to 55-60 °C for lh before only the organic solvent residues were distilled off under vacuum.

Aqueous Cyanamide (50 wt-%, 2,16 kg) was then added at 20 °C, followed by sodium acetate (3,04 kg). The resulting reaction mixture was heated to 85-90 °C for 2h and cooled to 0-5 °C before a pH of ~ 8-9 was adjusted via addition of aqueous sodium hydroxide (32% wt-%; 4,1 kg). Compound 3 (1,66 kg; 75%) was isolated after filtration and washing with water.

Ή-NMR (400 MHz, d6-DMSO): δ= 1,24 (3H, t, J= 7,1 Hz); 3,53 (3H, s); 4,16 (2H, q, J= 7,0 Hz) ; 6,15 (s, 2 H); 7,28 (s, 1H).

HPLC (Rt = 7,7 min): 97,9% (a/a).

HPLC data was obtained using Agilent 1100 series HPLC from agilent technologies using an Column: YMC-Triart CI 8 3μ, 100 x 4,6 mm Solvent A: 950 ml of ammonium acetate/acetic acid buffer at pH = 6 + 50 ml acetonitril; Solvent B: 200 ml of ammonium acetate/acetic acid buffer at pH = 6 + 800 ml acetonitril; Flow: 1,5 ml/min; Gradient: 0 min: 5 % B, 2 min: 5 % B, 7 min: 20 % B, 17 min: 85% B, 17, 1 min: 5% B, 22 min: 5% B.

PATENT

WO2007002931

http://www.google.com/patents/WO2007002931A2?cl=en

Example 8

Synthesis of Compounds 25, 26 [0380] To a solution of 2-bromoethylammmonium bromide (19.4 g) in DCM (90 mL) at – 1O0C was added a solution OfPOCl3 (2.3 mL) in DCM (4 mL) followed by addition of a solution of TEA (14.1 mL) in DCM (25 mL). The reaction mixture was filtered, the filtrate concentrated to ca. 30% of the original volume and filtered. The residue was washed with DCM (3×25 mL) and the combined DCM portions concentrated to yield a solid to which a mixture of THF (6 mL) and water (8 mL) was added. THF was removed in a rotary evaporator, the resulting solution chilled overnight in a fridge. The precipitate obtained was filtered, washed with water (10 mL) and ether (30 mL), and dryed in vacuo to yield 2.1 g of:

Figure imgf000127_0001

Isophosphoramide mustard

Figure imgf000127_0002

can be synthesized employing the method provided in Example 8, substituting 2- bromoethylammmonium bromide with 2-chloroethylammmonium chloride. Synthesis of Isophosphoramide mustard has been described (see for example Wiessler et al., supra).

The phosphoramidate alkylator toxin:

Figure imgf000127_0003

was transformed into compounds 24 and 25, employing the method provided in Example 6 and the appropriate Trigger-OH.

Example 25

Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid

Figure imgf000143_0002

A suspension of the nitro ester (39.2 g, 196.9 rnmol) in IN NaOH (600 mL) and water (200 mL) was stirred at rt for about 20 h to give a clear light brown solution. The pH of the reaction mixture was adjusted to about 1 by addition of cone. HCl and the reaction mixture extracted with EA (5 x 150 mL). The combined ethyl acetate layers were dried over MgS O4 and concentrated to yield l-N-methyl-2-nitroimidazole-5-carboxylis acid (“nitro acid”) as a light brown solid (32.2 g, 95%). Example 26

Synthesis of l-N-methyl-2-nitroimidazole-5-carboxylis acid

Figure imgf000144_0001

A mixture of the nitro acid (30.82 g, 180.23 mmol) and triethylamine (140 niL, 285 mmol) in anhydrous THF (360 mL) was stirred while the reaction mixture was cooled in a dry ice-acetonitrile bath (temperature < -20 0C). Isobutyl chloroformate (37.8 mL, 288 mmol) was added drop wise to this cooled reaction mixture during a period of 10 min and stirred for 1 h followed by the addition of sodium borohydride (36 g, 947 mmol) and dropwise addition of water during a period of 1 h while maintaining a temperature around or less than O0C. The reaction mixture was warmed up to O0C. The solid was filtered off and washed with THF. The combined THF portions were evaporated to yield l-N-methyl-2- nitroimidazole-5-methanol as an orange solid (25 g) which was recrystallized from ethyl acetate.

PATENT

WO-2015051921

EXAMPLE 1

1

N-Formylsarcosine ethyl ester 1 (1 ,85 kg) was dissolved in toluene (3,9 kg) and ethyl formate (3,28 kg) and cooled to 10 °C. A 20 wt-% solution of potassium tert-butoxide (1 ,84 kg) in tetrahydrofuran (7,4 kg) was added and stirring was continued for 3h. The reaction mixture was extracted 2x with a solution of sodium chloride in water (10 wt-%) and the combined water extracts were washed lx with toluene.

Aqueous hydrogen chloride (25% wt-%; 5,62 kg) was added to the aqueous solution, followed by ethylene glycol (2,36 kg). The reaction mixture was heated to 55-60 °C for lh before only the organic solvent residues were distilled off under vacuum.

Aqueous Cyanamide (50 wt-%, 2,16 kg) was then added at 20 °C, followed by sodium acetate (3,04 kg). The resulting reaction mixture was heated to 85-90 °C for 2h and cooled to 0-5 °C before a pH of ~ 8-9 was adjusted via addition of aqueous sodium hydroxide (32% wt-%; 4,1 kg). Compound 3 (1,66 kg; 75%) was isolated after filtration and washing with water.

Ή-NMR (400 MHz, d6-DMSO): δ= 1,24 (3H, t, J= 7,1 Hz); 3,53 (3H, s); 4,16 (2H, q, J= 7,0 Hz) ; 6,15 (s, 2 H); 7,28 (s, 1H).

HPLC (Rt = 7,7 min): 97,9% (a/a).

PATENT

WO 2016011195

http://google.com/patents/WO2016011195A1?cl=en

Figure 1 provides the differential scanning calorimetry (DSC) data of crystalline solid form A of TH-302.

Figure 2 shows the 1H-NMR of crystalline solid form A of TH-302.

Figure 5 shows the Raman Spectra of TH-302 (Form A)

Scheme 1 illustrates a method of preparing TH-302.

Scheme 1: Process for the Preparation of TH-302

NaOH (RGT)

Step 1. Imidazole Purified water (SLV)

Carboxylic Acid IPC: NMT 1.0% SM by HPLC

HCI (RGT)

IPC: pH 1.0 ± 0.5

IPC: NMT 1.0% water by KF

TH-302

MW = 449.0

SM = Starting Material INT = Intermediate IPC = In-process Control RGT = Reagent SLV = Solvent MW = Molecular Weight LOD = Loss on drying NMT = Not more than NLT = Not less than

TH-302 can be prepared by hydro lyzing (l-methyl-2-nitro-lH-imidazol-5-yl) ethyl ester above for example under aqueous conditions with a suitable base catalyst (e.g. NaOH in water at room temperature). The imidazole carboxylic acid prepared by this method can be used without further purification. However, it has been found that treating the dried crude intermediate product with a solvent such as acetonitrile, ethyl acetate, n-heptane, acetone, dimethylacetamide, dimethylformamide, 1, 4-dioxane, ethylene glycol, 2-propanol, 1-propanol, tetrahydrofuran (1 : 10 w/v) or combinations thereof in a vessel with heating, followed by cooling and filtration through a filtration aid with acetone decreased the number and levels of impurities in the product. The number and levels of impurities could be further reduced by treating the dried crude product with water (1 :5.0 w/v) in a vessel with heating followed by cooling and filtration through a filtration aid with water.

The carboxylic acid of the imidazole can then be reduced using an excess of a suitable reducing agent (e.g. sodium borohydride in an appropriate solvent, typically aqueous. The reaction is exothermic (i.e. potentially explosive) releasing borane and hydrogen gases over several hours. It was determined that the oxygen balance of the product imidazole alcohol is about 106.9, which suggests a high propensity for rapid decomposition. It has been found that using NaOH, for example 0.01M NaOH followed by quenching the reaction with an acid. Non-limiting examples of acids include, but are not limited to water, acetic acid, hydrobromic acid, hydrochloric acid, sodium hydrogen phosphate, sulfuric acid, citric acid, carbonic acid, phosphoric acid, oxalic acid, boric acid and combinations thereof. In some embodiments, the acid may diluted with a solvent, such as water and/or tetrahydrofuran. In some embodiments, acetic acid or hydrochloric acid provide a better safety profile, presumably because it is easier to control the temperature during the addition of the reducing agent and the excess reducing agent is destroyed after the reaction is complete. This also results in improved yields and fewer impurities, presumably due to reduced impurities from the reducing agent and decomposition of the product. Using this process, greater than 98.5% purity could be achieved for this intermediate. The formation of ether linkage can be accomplished by treating the product imidazole alcohol with solution of N,N’-Bis(2-bromoethyl)phosphorodiamidic acid (Bromo IPM), a trisubstituted phosphine and diisopropyl azodicarboxylate in tetrahydrofuran at room temperature to afford TH-302. It has been found that by recrystallizing the product from a solvents listed in the examples, one could avoid further purfication by column chromatography, which allowed for both reduced solvent use especially on larger scales.

Scheme 2 illustrates an alternative method of preparing TH-302.

Scheme 2: Process for the Preparation of TH-302

(SM)

ethylamine mide (SM) 04.9 ) SLV) , RGT) ter by KF

NT)

MW = 449.0

Example 1: Synthesis of TH-302

Step 1 – Preparation intermediate imidazole carboxylic

I T)

Crude imidazole carboxylic acid ethyl ester (1 : 1.0 w/w) was taken in water (1 : 10.0 w/v) at 25± 5°C and cooled to 17± 3°C. A 2.5 N sodium hydroxide solution (10 V) was added slowly at 17±3°C. The reaction mass was warmed to 25±5°C and monitored by HPLC. After the completion of reaction, the reaction mass was cooled to 3±2°C and pH of the reaction mass adjusted to 1=1=0.5 using 6 M HC1 at 3±2°C. The reaction mass was then warmed to 25±5°C and extracted with ethyl acetate (3 x 10 V). The combined organic layers

were washed with water (1 x 10 V) followed by brine (1 x 10 V). The organic layer was dried over sodium sulfate (3 w/w), filtered over Celite and concentrated. n-Heptane (1.0 w/v) was added and the the reaction mixture was concentrated below 45°C to 2.0 w/v. The reaction mass was cooled to 0±5°C. The solid was filtered, and the bed was washed with n-heptane (1 x 0.5 w/v) and dried at 35±5°C. In a vessel, acetone (1 : 10 w/v) was added. Dry crude imidazole carboxylic acid (ICA) from 1.12 was added to the acetone. The mixture was warmed to 45±5°C and was stirred for 30 minutes. The mass was cooled to 28±3°C and filtered through a Celite bed. The filter bed was washed with 1 : 1.0 w/v of acetone. Water (1 :5.0 w/v) was added to the filtrate and the mixture was concentrated. The concentrated mass was cooled to 5±5°C and stirred for 30 minutes. The material was filtered and the solid was washed 2 x 1 : 1.0 w/v of water at 3±2°C. The product was dried for 2 hours at 25±5°C and then at 45±5°C. As can be seen below, the number and levels of impurities are decreased.

Table I: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Imidazole alcohol:

CI^Oi-Bu

T

o

Imidazole carboxylic acid (1.0 w/w) was taken in tetrahydrofuran (10 w/v) under nitrogen atmosphere at 25±5°C. The reaction mass was cooled to -15±5°C. Triethylamine (1 : 1.23 w/v) was added slowly over a period of 1 hour maintaining the temperature at – 15±5°C. The reaction mass was stirred at -15±5°C for 15-20 min. Isobutylchloroformate (1 : 1.14 w/v) was added slowly over a period of 1 hour maintaining the temperature at – 15±5°C. The reaction mass was stirred at -15±5°C for 30-40 min. A solution of sodium borohydride (1 : 1.15 w/w) in 0.01 M aqueous sodium hydroxide (2.2 w/v) was divided into 6 lots and added to the above reaction mass while maintaining the temperature of the reaction mass between 0±10°C for 40-60 min for each lot. The reaction mass was warmed to 25±5°C and stirred until imidazole carboxylic acid content < 5.0 % w/w. The reaction mass was filtered and the bed was washed with tetrahydrofuran (1 :2.5 w/v). The filtrate was quenched with 10 % acetic acid in water at 25±5°C. Reaction mass stirred for 50-60 minutes at 25±5°C. The filtrate was concentrated below 45°C until no distillate was observed. The mass was cooled to 5±5°C and stirred for 50-60 minutes. The reaction mass was filtered and the solid was taken in ethanol (1 :0.53 w/v). The reaction mass was cooled 0±5°C and stirred for 30-40 min. The solid was filtered and the bed was washed ethanol (1 :0.13 w/v). The solid was dried at 40±5 °C.

Step 3 – Synthesis of intermediate Br-IPM:

P

o

M
W = 286.7 MW = 204.9 Purified water (SLV, RGT)

Acetone (SLV)

IPC: NMT 1.0% water by KF

2-Bromoethylamine hydrobromide (1 : 1.0 w/w) and POBr^ (1 :0.7 w/w) were taken in DCM (1 :2 w/v) under nitrogen atmosphere. The reaction mixture was cooled to -70±5°C. Triethylamine (1 : 1.36 w/v) in DCM (1 :5 w/v) was added to the reaction mass at -70±5°C. The reaction mass was stirred for additional 30 min at -70±5°C. Reaction mass was warmed to 0±3°C and water (1 :1.72 w/v) was added. The reaction mixture was stirred at 0±3°C for 4 hrs. The solid obtained was filtered and filter cake was washed with ice cold water (2 x 1 :0.86 w/v) and then with chilled acetone (2 x 1 :0.86 w/v). The solid was dried in at 20±5°C.

Step 4 Synthesis ofTH-302

TH-302

MW = 449.0

Imidazole alcohol (IA) (1 : 1.0 w/w), Bromo-IPM (1 :2.26 w/w) and

triphenylphosphine (1 :2.0 w/w) were added to THF (1 : 13.5 w/v) at 25±5°C. The reaction

mass was cooled to 0±5°C and DIAD (1.5 w/v) was added. The reaction mixture warmed to 25±5°C and stirred for 2 hours. Progress of the reaction was monitored by HPLC. Solvent was removed below 50°C under vacuum. Solvent exchange with acetonitrile (1 :10.0 w/v) below 50°C was performed. The syrupy liquid was re-dissolved in acetonitrile (1 : 10.0 w/v) and the mixture was stirred at -20±5°C for 1 hour. The resulting solid was filtered and the filtrate bed was washed with chilled acetonitrile (1 : 1.0 w/v). The acetonitrile filtrate was concentrated below 50°C under vacuum. The concentrated mass was re-dissolved in ethyl acetate (1 : 10.0 w/v) and concentrated below 50°C under vacuum. The ethyl acetate strip off was repeated two more times. Ethyl acetate (1 : 10.0 w/v) and silica gel (230-400 mesh, 1 :5.3 w/w) were added to the concentrated reaction mass. The mixture was concentrated below 40°C under vacuum. n-Heptane (1 :5.0 w/v) was charged to the above mass and the mixture was evaporated below 40°C under vacuum. n-Heptane (1 :5.0 w/v) was again added to the above mass and the solid was filtered and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in a mixture oftoluene (1 :7.1 w/v) and n-heptane (1 :21.3 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane

(1 : 1.0 w/v). The solid was re-suspended in a mixture of toluene (1 : 10.6 w/v) and n-heptane (1 : 10.6 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in acetone (1 : 19.0 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with acetone (1 : 1.0 w/v). The acetone washes were repeated 3 more times. Filtrates from the above acetone washings were combined and concentrated below 40°C under vacuum. The residue dissolved in ethyl acetate (1 : 10.0 w/v) and concentrated below 40°C under vacuum. The ethyl acetate strip off was repeated one more time. The residue was re-dissolved in ethyl acetate (1 :5.5 w/v), cooled to 0±3°C and stirred at 0±3°C for 2 h and then at -20±5°C for 2 h. The solid was filtered and the solid was washed with ethyl acetate (1 :0.10 w/v). The solid was dissolved in ethyl acetate (1 : 10.0 w/v) at 50±5°C and the resulting solution was filtered through a cartridge filter. The filtrate was concentrated to ~4.0 w/w and stirred at 0±3°C for 4 hours. The solid was filtered and washed with ethyl acetate (1 :0.10 w/v). The crystallization from ethyl acetate was repeated and TH-302 was dried at 25±5°C. Table 2 shows how the process reduces solvent use.

Table 2: Solvent and Silica Gel Usage for 10 kg Column and 10 kg Column-free Purification

“Amounts are estimated from a 5 kg batch

b Amounts are estimated

Example 2: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA was prepared according to the method described in Example 1. In a vessel, water (1 :7.0 w/v) was added. Dry crude ICA was added to the water. The reaction mixture was heated to 85±5°C until a clear solution was obtained. The reaction mass was cooled to 20±5°C and filtered through a Celite bed. The filter bed was washed with 2 x 5.0 of n-heptane. The material was dried for 2 hours at 25±5°C and then 45±5°C. As can be seen below, the number and levels of impurities decreased.

Table 3: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Example 3: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA was prepared according to the method described in Example 1. In a vessel

ethanol (1 :30.0 w/v) and ICA (1 : 1.0 w/w) were mixed. The reaction mixture was stirred at

25±5°C for 30 minutes and filtered. Water (1 :50.0 w/v) was added and the mixture was

stirred at 50±5°C for 30 minutes. The reaction mass was cooled to 20±5°C and filtered. The isolated solid was dried at 25±5°C for 24 hours. As can be seen below, the number and levels

of impurities generally decreased.

Table 4: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Example 4: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA was prepared according to the method described in Example 1. In a vessel

acetonitrile (1 :20.0 w/v) and ICA (1 : 1.0 w/w) were mixed at 25±5°C for one hour. The

reaction mixture was filtered and the solution was concentrated to ~ 6 volumes. The mixture

was then cooled to 0±5°C, stirred at this temperature for one hour and filtered. The isolated

solid was dried at 25±5°C for 24 hours. As can be seen below the number of impurities

decreased and except for TH-2717, the amounts also decreased.

Table 5: Purity and Impurity Profile Comparison of Typical Crude ICA and Purified

ICA

Example 5: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by treatment with dimethylacetamide and water.

Example 6: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by treatment with dimethylforamide and water.

Example 7: Synthesis ofTH-302 using alternative procedure to purify ICA:

[0109] Crude ICA is prepared according to the method described in Example 1 and purified by crystallization from a 1,4-dioxane and water mixture.

Example 8: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by crystallization from a mixture of ethylene glycol and water.

Example 9: Synthesis ofTH-302 using alternative procedure to purify ICA:

Crude ICA is prepared according to the method described in Example 1 and purified by treatment with 2-propanol and water.

Example 10: Synthesis ofTH-302 using alternative procedure to purify ICA:

[0112] Crude ICA is prepared according to the method described in Example 1 and purified by treatment with 1-propanol and water.

Example 11: Synthesis ofTH-302 using alternative procedure to purify ICA:

[0113] Crude ICA is prepared according to the method described in Example 1 and purified by crystallization from a mixture of tetrahydrofuran and water.

Example 12: Synthesis ofTH-302 using alternative procedure to quench IA:

[0114] The reduction of ICA to IA was carried out according to Example 1 except that after reaction completion and filtration of the inorganics, the filtrate was quenched with 1.5 M hydrochloric acid.

Example 13: Synthesis ofTH-302 using alternative procedure to quench IA:

[0115] The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with 1.5 M

hydrobromic acid.

Example 14: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with

hydrobromic acid in acetic acid.

Example 15: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was treated with sodium

hydrogen phosphate.

Example 16: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with 10% acetic

acid in tetrahydrofuran.

Example 17: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA was carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate was quenched with water.

Example 18: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is quenched with sulfuric acid.

Example 19: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is quenched with citric acid.

Example 20: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is treated with carbonic acid.

Example 21: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is treated with phosphoric

acid.

Example 22: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after

reaction completion and filtration of the inorganics, the filtrate is quenched with oxalic acid.

Example 23: Synthesis ofTH-302 using alternative procedure to quench IA:

The reduction of ICA to IA is carried out according to Example 1 except that after reaction completion and filtration of the inorganics, the filtrate is quenched with boric acid.

Example 24: Synthesis ofTH-302 using alternative procedure to purify TH-302:

[0126] Coupling of bromo-IPM and IA was performed according to Example 1 except that after concentration of the reaction mixture, ethyl acetate (1 : 10 w/v) was added to the concentrated mass. The mixture was stirred at -55±5°C for 2 hours. The resulting solid was filtered and washed with chilled EtOAc (1 :2.0 w/v). The solid was reslurried in ethyl acetate (1 : 10 w/v) at -55±5°C for 2 hours, filtered and the solid was washed with chilled ethyl acetate (1 : 1.0 w/v). The filtrates from both filtrations were combined and treated with silica gel (1 :5.3 w/w) of silica gel (230-400 mesh). The mixture was concentrated below 40°C under vacuum. n-Heptane (1 :5.0 w/v) was again added to the above mass and the solid was filtered and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in a mixture of toluene (1 :7.1 w/v) and n-heptane (1 :21.3 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was re-suspended in a mixture of toluene (1 : 10.6 w/v) and n-heptane (1 :10.6 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with n-heptane (1 : 1.0 w/v). The solid was suspended in acetone (1 : 19.0 w/v), stirred at 35±5°C for 15-20 minutes, filtered off and the bed was washed with acetone (1 : 1.0 w/v). The acetone washes were repeated 3 more times. Filtrates from the above acetone washings were combined and concentrated below 40°C under vacuum. The residue dissolved in ethyl acetate (1 :5.5 w/v), cooled to 0±3°C and stirred at 0±3°C for 2 h and then at -20±5°C for 2 h. The solid was filtered and the solid was washed with ethyl acetate (1 :0.10 w/v). The solid was dissolved in ethyl acetate (1 :27 w/v), stirred at 50±5°C and filtered through Celite. The filtrate was concentrated to ~4.0 w/w and stirred at 0±5°C for 4 hours. The recrystallization from ethyl acetate was repeated and TH- 302 was dried at 25±5°C. Table 4 shows how the process reduced solvent use.

Table 4: Estimated Solvent and Silica Gel Usage for Column and 10 kg Column-free

(EtOAc) Purification

References

  1.  WHO Drug Information; Recommended INN: List 73
  2.  Adopted Names of the United States Adopted Names Council
  3.  Duan J; Jiao, H; Kaizerman, J; Stanton, T; Evans, JW; Lan, L; Lorente, G; Banica, M; et al. (2008). “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs”. J. Med. Chem. 51 (8): 2412–20. doi:10.1021/jm701028q.PMID 18257544.
  4. Jump up^ Threshold Pharmaceuticals and Merck KGaA Announce Global Agreement to Co-Develop and Commercialize Phase 3 Hypoxia-Targeted Drug TH-302 – Press release from 3 February 2012
  5. Jump up^ Threshold Pharmaceuticals Form 8-K from 3 Nov 2014
  6. Jump up^ Weiss, G.J., Infante, J.R., Chiorean, E.G., Borad, M.J., Bendell, J.C., Molina, J.R., Tibes, R., Ramanathan, R.K., Lewandowski, K., Jones, S.F., Lacouture, M.E., Langmuir, V.K., Lee, H., Kroll, S., Burris, H.A. (2011) Phase 1 Study of the Safety, Tolerability, and Pharmacokinetics of TH-302, a Hypoxia-Activated Prodrug, in Patients with Advanced Solid Malignancies. Clinical Cancer Research 17, 2997–3004.doi:10.1158/1078-0432.CCR-10-3425
  7.  J. Thomas Pento (2011). “TH-302”. Drugs of the Future. 36 (9): 663–667.doi:10.1358/dof.2011.036.09.1678337.
  8. Jump up^ Duan J; Jiao, H; Kaizerman, J; Stanton, T; Evans, JW; Lan, L; Lorente, G; Banica, M; et al. (2008). “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs”. J. Med. Chem. 51 (8): 2412–20. doi:10.1021/jm701028q.PMID 18257544.
  9. Jump up^ CPhI.cn: Synthetic routes to explore anti-pancreatic cancer drug Evofosfamide, 22 Jan 2015
  10.  Synthetic route Reference: International patent application WO2007002931A2
  11. Jump up^ FDA Advisory Committee Briefing Materials Available for Public Release, TH-302: Pediatric oncology subcommittee of the oncologic drugs advisory committee (ODAC) meeting, December 4, 2012
  12. Jump up^ AAPS 2014 – Measurement of Diethylhexyl Phthalate (DEHP) Leached from Polyvinyl Chloride (PVC) Containing Plastics by Infusion Solutions Containing an Organic Parenteral Formulation – Poster W4210, Nov 5, 2014
  13. Jump up^ ClinicalTrials.gov
  14.  The Primary Completion Date is defined as the date when the final subject was examined or received an intervention for the purposes of final collection of data for the primary outcome.
  15. Jump up^ Detailed Results From Positive Phase 2b Trial of TH-302 in Pancreatic Cancer at AACR Annual Meeting – Press release from 30 March 2012
  16. Jump up^ TH-302 Plus Gemcitabine vs. Gemcitabine in Patients with Untreated Advanced Pancreatic Adenocarcinoma. Borad et al. Presentation at the European Society for Medical Oncology (ESMO) 2012 Congress, September 2012. (Abstract 6660)
  17. Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  18.  Updated Phase 2 Results Including Analyses of Maintenance Therapy With TH-302 Following Induction Therapy With TH-302 Plus Doxorubicin in Soft Tissue Sarcoma – Press release from 15 November 2012
  19.  TH-302 Maintenance Following TH-302 Plus Doxorubicin Induction: The Results pf a Phase 2 Study of TH-302 in Combination with Doxorubicin in Soft Tissue Sarcoma. Ganjoo et al. Connective Tissue Oncology Society (CTOS) 2012 Meeting, November 2012
  20. Jump up^ Chawla, S.P., Cranmer, L.D., Van Tine, B.A., Reed, D.R., Okuno, S.H., Butrynski, J.E., Adkins, D.R., Hendifar, A.E., Kroll, S., Ganjoo, K.N., 2014. Phase II Study of the Safety and Antitumor Activity of the Hypoxia-Activated Prodrug TH-302 in Combination With Doxorubicin in Patients With Advanced Soft Tissue Sarcoma. Journal of Clinical Oncology 32, 3299–3306.doi:10.1200/JCO.2013.54.3660
  21. Jump up^ Follow-Up Data From a Phase 1/2 Clinical Trial of TH-302 in Solid Tumors – Press release from 12 October 2010
  22.  TH-302 Continues to Demonstrate Promising Activity in Pancreatic Cancer Phase 1/2 Clinical Trial – Press release from 24 January 2011
  23. Jump up^ TH-302, a tumor selective hypoxia-activated prodrug, complements the clinical benefits of gemcitabine in first line pancreatic cancer. Borad et al. ASCO Gastrointestinal Cancers Symposium, January 2011
  24. Jump up^ Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  25. Jump up^ Borad et al., ESMO Annual Meeting, October 2010
  26. Jump up^ Video interview of Stefan Oschmann, CEO Pharma at Merck – Merck Serono Investor & Analyst Day 2014 – 18 Sept 2014 – 2:46 min – Youtube
  27. Jump up^ The Phase 3 Trial of TH-302 in Patients With Advanced Soft Tissue Sarcoma Will Continue as Planned Following Protocol-Specified Interim Analysis – Press release from 22 September 2014
  28. Jump up^ Threshold Pharmaceuticals’ Partner Merck KGaA, Darmstadt, Germany, Completes Target Enrollment in the TH-302 Phase 3 MAESTRO Study in Patients With Locally Advanced or Metastatic Pancreatic Adenocarcinoma – Press release from 3 November 2014
  29.  Data From Ongoing Phase 1/2 Trial of TH-302 Plus Bevacizumab (Avastin(R)) in Patients With Recurrent Glioblastoma – Press release from 30 May 2014
  30. Jump up^ Phase 1/2 Study of Investigational Hypoxia-Targeted Drug, TH-302, and Bevacizumab in Recurrent Glioblastoma Following Bevacizumab Failure. Brenner, et al. 2014 ASCO, 7 – 30 May 2014
  31. Jump up^ Phase 1/2 Interim Data Signaling Activity of TH-302 Plus Bevacizumab (Avastin(R)) in Patients With Glioblastoma – Press release from 17 November 2014
  32. Jump up^ Threshold Pharmaceuticals’ Partner Merck KGaA, Darmstadt, Germany, Completes Target Enrollment in the TH-302 Phase 3 MAESTRO Study in Patients With Locally Advanced or Metastatic Pancreatic Adenocarcinoma – Press release from 3 November 2014
  33. Jump up^ Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  34. Jump up^ Stifel 2014 Healthcare Conference; Speaker: Harold Selick – 18 November 2014
  35. Jump up^ Chawala SP, et al. J Clin Oncol. 2014 (54) 3660 doi:10.1200/JCO.2013.54.3660
  36. Jump up^ Judson I, et al. Lancet Oncol. 2014 Apr;15(4):415-23doi: 10.1016/S1470-2045(14)70063-4
  37. Jump up^ Judson I, et al. Lancet Oncol. 2014 Apr;15(4):415-23doi: 10.1016/S1470-2045(14)70063-4
  38. Jump up^ Chawala SP, et al. J Clin Oncol. 2014 (54) 3660 doi:10.1200/JCO.2013.54.3660
  39. Jump up^ Borad, M. J. et al. Randomized Phase II Trial of Gemcitabine Plus TH-302 Versus Gemcitabine in Patients With Advanced Pancreatic Cancer. Journal of Clinical Oncology (2014). doi: 10.1200/JCO.2014.55.7504
  40. Jump up^ Von Hoff, D. D. et al. Increased Survival in Pancreatic Cancer with nab-Paclitaxel plus Gemcitabine. New England Journal of Medicine 369, 1691–1703 (2013). doi:10.1056/NEJMoa1304369
  41. Jump up^ Von Hoff, D. D. et al. Increased Survival in Pancreatic Cancer with nab-Paclitaxel plus Gemcitabine. New England Journal of Medicine 369, 1691–1703 (2013). doi:10.1056/NEJMoa1304369
  42. Jump up^ Borad, M. J. et al. Randomized Phase II Trial of Gemcitabine Plus TH-302 Versus Gemcitabine in Patients With Advanced Pancreatic Cancer. Journal of Clinical Oncology (2014). doi: 10.1200/JCO.2014.55.7504
  43. Jump up^ Threshold Pharmaceuticals 10-K Annual report 2011 from 15 Mar 2012
  44. Jump up^ Threshold Pharmaceuticals 10-Q Quarterly report Q3/2014 from 3 Nov 14
  45. Jump up^ Threshold Pharmaceuticals Form 8-K from 9 Oct 14
  46. Jump up^ Threshold Pharmaceuticals Form 8-K from 9 Oct 14
  47.  Threshold Pharmaceuticals Form 8-K from 9 Oct 14
  48.  Phosphoramidate alkylator prodrugs US8003625B2,US8507464B2, US8664204B2
  49.  Phosphoramidate alkylator prodrugs EP1896040B1and JP5180824B2
WO2007002931A2 * Jun 29, 2006 Jan 4, 2007 Threshold Pharmaceuticals, Inc. Phosphoramidate alkylator prodrugs
WO2008083101A1 * Dec 21, 2007 Jul 10, 2008 Threshold Pharmaceuticals, Inc. Phosphoramidate alkylator prodrugs for the treatment of cancer
WO2010048330A1 * Oct 21, 2009 Apr 29, 2010 Threshold Pharmaceuticals, Inc. Treatment of cancer using hypoxia activated prodrugs
WO2015051921A1 * Oct 10, 2014 Apr 16, 2015 Merck Patent Gmbh Synthesis of 1-alkyl-2-amino-imidazol-5-carboxylic acid ester via calpha-substituted n-alkyl-glycine ester derivatives
Reference
1 * DUAN, J.-X. ET AL.: “Potent and Highly Selective Hypoxia-Activated Achiral Phosphoramidate Mustards as Anticancer Drugs“, JOURNAL OF MEDICINAL CHEMISTRY, vol. 51, 2008, pages 2412 – 2420, XP008139620, DOI: doi:10.1021/jm701028q
Evofosfamide
TH-302.svg
Names
IUPAC name

(1-Methyl-2-nitro-1H-imidazol-5-yl)methyl N,N’-bis(2-bromoethyl)phosphorodiamidate
Other names

TH-302; HAP-302
Identifiers
918633-87-1 Yes
ChemSpider 10157061 Yes
Jmol-3D images Image
PubChem 11984561
Properties
C9H16Br2N5O4P
Molar mass 449.04 g·mol−1
6 to 7 g/l

///////////Orphan Drug Status, soft tissue sarcoma,  Pancreatic cancer, Fast track,  TH-302, TH 302, эвофосфамид ,  إيفوفوسفاميد ,  艾伏磷酰胺 , Evofosfamide, 918633-87-1, PHASE 3

O=[N+]([O-])c1ncc(COP(=O)(NCCBr)NCCBr)n1C

DAROLUTAMIDE


STR1

ODM-201.svg

ChemSpider 2D Image | ODM-201 | C19H19ClN6O2

Darolutamide

N-((S)-1-(3-(3-Chloro-4-cyanophenyl)-1H-pyrazol-1-yl)-propan-2-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide

N-((S)- 1 -(3-(3-chloro-4-cyanophenyl)- lH-pyrazol- 1 -yl)-propan-2-yl)-5-(l-hydroxyethyl)-lH-pyrazole-3-carboxamide

  • MF C19H19ClN6O2
  • MW 398.846

BAY 1841788; ODM-201

1H-Pyrazole-3-carboxamide, N-[(1S)-2-[3-(3-chloro-4-cyanophenyl)-1H-pyrazol-1-yl]-1-methylethyl]-5-(1-hydroxyethyl)-
BAY-1841788
N-{(2S)-1-[3-(3-Chlor-4-cyanphenyl)-1H-pyrazol-1-yl]-2-propanyl}-5-(1-hydroxyethyl)-1H-pyrazol-3-carboxamid
N-{(2S)-1-[3-(3-Chloro-4-cyanophenyl)-1H-pyrazol-1-yl]-2-propanyl}-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide
N-{(2S)-1-[3-(3-Chloro-4-cyanophényl)-1H-pyrazol-1-yl]-2-propanyl}-5-(1-hydroxyéthyl)-1H-pyrazole-3-carboxamide
ODM-201
1297538-32-9  CAS
UNII:X05U0N2RCO
  • Originator Orion
  • Developer Bayer HealthCare; Orion
  • Class Antineoplastics
  • Mechanism of Action Androgen receptor antagonists
  • Phase III Prostate cancer
  • Most Recent Events

    • 03 Jun 2016 Bayer and Orion plan the phase III ARASENS trial for Prostate cancer
    • 03 Jun 2016 Bayer and Orion expand the licensing agreement to include joint development of ODM 201 for Metastatic hormone-sensitive prostate cancer (mHSPC)
    • 06 May 2016 Long-term combined adverse events data from the the ARADES (phase I/II) and the ARAFOR (phase I) trials in Prostate cancer presented at the 111th Annual Meeting of the American Urological Association (AUA -2016)

Darolutamide (INN) (developmental code names ODM-201, BAY-1841788) is a non-steroidal antiandrogen, specifically, a full and high-affinity antagonist of the androgen receptor (AR), that is under development by Orion and Bayer HealthCare[1] for the treatment of advanced, castration-resistant prostate cancer (CRPC).[2][3]

Orion and licensee Bayer are co-developing darolutamide, an androgen receptor antagonist, for treating castration-resistant prostate cancer and metastatic hormone-sensitive prostate cancer. In August 2016, darolutamide was reported to be in phase 3 clinical development. The drug appears to be first disclosed in WO2011051540, claiming novel heterocyclic derivatives as tissue-selective androgen receptor modulators, useful for the treatment of prostate cancer.

Mode of action

Relative to enzalutamide (MDV3100 or Xtandi) and apalutamide (ARN-509), two other recent non-steroidal antiandrogens, darolutamide shows some advantages.[3] Darolutamide appears to negligibly cross the blood-brain-barrier.[3] This is beneficial due to the reduced risk of seizures and other central side effects from off-target GABAA receptor inhibition that tends to occur in non-steroidal antiandrogens that are structurally similar to enzalutamide.[3] Moreover, in accordance with its lack of central penetration, darolutamide does not seem to increase testosterone levels in mice or humans, unlike other non-steroidal antiandrogens.[3] Another advantage is that darolutamide has been found to block the activity of all tested/well-known mutant ARs in prostate cancer, including the recently-identified clinically-relevant F876L mutation that produces resistance to enzalutamide and apalutamide.[3] Finally, darolutamide shows higher affinity and inhibitory efficacy at the AR (Ki = 11 nM relative to 86 nM for enzalutamide and 93 nM for apalutamide; IC50 = 26 nM relative to 219 nM for enzalutamide and 200 nM for apalutamide) and greater potency/efficaciousness in non-clinical models of prostate cancer.[3]

ORM-15341 is the main active metabolite of darolutamide.[3] It, similarly, is a full antagonist of the AR, with an affinity (Ki) of 8 nM and an IC50 of 38 nM.[3]

Clinical trials

Darolutamide has been studied in phase I and phase II clinical trials and has thus far been found to be effective and well-tolerated,[4] with the most commonly reported side effects including fatigue, nausea, and diarrhea.[5][6] No seizures have been observed.[6][7] As of July 2015, darolutamide is in phase III trials for CRPC.[3]

Representative binding affinities of ODM-201, ORM-15341, enzalutamide, and ARN-509 measured in competition with [3H]mibolerone using wtAR isolated from rat ventral prostates (C). All data points are means of quadruplicates ±SEM. Ki values are presented in parentheses. D. Antagonism to wtAR was determined using AR-HEK293 cells treated with ODM-201, ORM-15341, enzalutamide, or ARN-509 together with 0.45 nM testosterone in steroid-depleted medium for 24 hours before luciferase activity measurements. All data points are means of triplicates ±SEM. IC50 values are presented in parentheses.

WHIPPANY, N.J., Sept. 16, 2014 /PRNewswire/ — Bayer HealthCare and Orion Corporation, a pharmaceutical company based in Espoo, Finland, have begun to enroll patients in a Phase III trial with ODM-201, an investigational oral androgen receptor inhibitor in clinical development. The study, called ARAMIS, evaluates ODM-201 in men with castration-resistant prostate cancer who have rising Prostate Specific Antigen (PSA) levels and no detectable metastases. The trial is designed to determine the effects of the treatment on metastasis-free survival (MFS).

“The field of treatment options for prostate cancer patients is evolving rapidly.  However, once prostate cancer becomes resistant to conventional anti-hormonal therapy, many patients will eventually develop metastatic disease,” said Dr. Joerg Moeller, Member of the Bayer HealthCare Executive Committee and Head of Global Development. “The initiation of a Phase III clinical trial for ODM-201 marks the starting point for a potential new treatment option for patients whose cancer has not yet spread.  This is an important milestone for Bayer in our ongoing effort to meet the unmet needs of men affected by prostate cancer.”

Earlier this year, Bayer and Orion entered into a global agreement under which the companies will jointly develop ODM-201, with Bayer contributing a major share of the costs of future development. Bayer will commercialize ODM-201 globally, and Orion has the option to co-promote ODM-201 in Europe. Orion will be responsible for the manufacturing of the product.

About the ARAMIS Study
The ARAMIS trial is a randomized, Phase III, multicenter, double-blind, placebo-controlled trial evaluating the safety and efficacy of oral ODM-201 in patients with non-metastatic CRPC who are at high risk for developing metastatic disease. About 1,500 patients are planned to be randomized in a 2:1 ratio to receive 600 mg of ODM-201 twice a day or matching placebo. Randomisation will be stratified by PSA doubling time (PSADT less than or equal to 6 months vs. > 6 months) and use of osteoclast-targeted therapy (yes vs. no).

The primary endpoint of this study is metastasis-free survival (MFS), defined as time between randomization and evidence of metastasis or death from any cause. The secondary objectives of this study are overall survival (OS), time to first symptomatic skeletal event (SSE), time to initiation of first cytotoxic chemotherapy, time to pain progression, and characterization of the safety and tolerability of ODM-201.

About ODM-201
ODM-201 is an investigational androgen receptor (AR) inhibitor that is thought to block the growth of prostate cancer cells. ODM-201 binds to the AR and inhibits receptor function by blocking its cellular function.

About Oncology at Bayer
Bayer is committed to science for a better life by advancing a portfolio of innovative treatments. The oncology franchise at Bayer now includes three oncology products and several other compounds in various stages of clinical development. Together, these products reflect the company’s approach to research, which prioritizes targets and pathways with the potential to impact the way that cancer is treated.

About Bayer HealthCare Pharmaceuticals Inc.
Bayer HealthCare Pharmaceuticals Inc. is the U.S.-based pharmaceuticals business of Bayer HealthCare LLC, a subsidiary of Bayer AG. Bayer HealthCare is one of the world’s leading, innovative companies in the healthcare and medical products industry, and combines the activities of the Animal Health, Consumer Care, Medical Care, and Pharmaceuticals divisions. As a specialty pharmaceutical company, Bayer HealthCare provides products for General Medicine, Hematology, Neurology, Oncology and Women’s Healthcare. The company’s aim is to discover and manufacture products that will improve human health worldwide by diagnosing, preventing and treating diseases.

Bayer® and the Bayer Cross® are registered trademarks of Bayer.

SYNTHESIS

STR1

str1

PATENT

US 2015203479

http://www.google.com/patents/WO2011051540A1?cl=en

PATENT

WO 2012143599

http://www.google.com/patents/US20140094474?cl=de

PATENTS

WO2011051540

https://www.google.com/patents/WO2011051540A1?cl=en

PATENT

IN 2011KO00570

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016120530&recNum=1&maxRec=&office=&prevFilter=&sortOption=&queryString=&tab=PCTDescription

WO-2016120530

Compound of (I) (5 g) was dissolved in an acetonitrile and distilled water. The reaction mixture was heated at 75 °C and then slowly cooled down at RT and stirred at RT for 3 days. The solid obtained was filtered, washed twice with the acetonitrile: water and dried under vacuum at 40 °C and 60 °C to yield crystalline form of (I) (4.42 g) with 88% of yield (example 1, page 10).

Compound (I) can be synthetized using the procedures described in WO

201 1/051540.

Pure diastereomers (la) and (lb) can be suitably synthetized, for example, using ketoreductase enzymes (KREDs) for both S- and R-selective reduction of compound 1 to compound 2 as shown in Scheme 1, wherein R is H or Ci_6 alkyl.

Scheme 1.

For example, Codexis KRED-130 and KRED -NADH-110 enzymes are useful for obtaining excellent stereoselectivity, even stereospecificity. In Scheme 1 the starting material 1 is preferably an ester (R= Ci_6 alkyl), for example ethyl ester (R=ethyl), such as to facilitate extraction of the product into the organic phase as the compound where R=H has a tendency to remain in the water phase. Intermediate 2 can be protected, preferably with silyl derivatives such as tert-butyldiphenylsilyl, in order to avoid esterification in amidation step. In the case of R=Ci_6 alkyl, ester hydrolysis is typically performed before amidation step, preferably in the presence of LiOH, NaOH or KOH. Amidation from compound 3 to compound 5_is suitably carried out using EDCI HBTU, DIPEA system but using other typical amidation methods is also possible. Deprotection of 5 give pure diastereomers (la) and (lb).

Pyrazole ring without NH substitution is known tautomerizable functionality and is described here only as single tautomer but every intermediate and end product here can exist in both tautomeric forms at the same time.

The stereochemistry of the compounds can be confirmed by using optically pure starting materials with known absolute configuration as demonstrated in Scheme 2, wherein R=H or Ci_6 alkyl, preferably alkyl, for example ethyl. The end products of Scheme 2 are typically obtained as a mixture of tautomers at +300K 1H-NMR analyses in DMSO.

Scheme 2. Synthesis pathway to stereoisomers by using starting materials with known absolute configuration

The crystalline forms I, Γ and Γ ‘ of compounds (I), (la) and (lb), respectively, can be prepared, for example, by dissolving the compound in question in an

acetonitrile: water mixture having volume ratio from about 85: 15 to about 99: 1, such as from about 90: 10 to about 98:2, for example about 95:5, under heating and slowly cooling the solution until the crystalline form precipitates from the solution. The concentration of the compound in the acetonitrile: water solvent mixture is suitably about 1 kg of the compound in 5-25 liters of acetonitrile: water solvent mixture, for example 1 kg of the compound in 10-20 liters of acetonitrile: water solvent mixture. The compound is suitably dissolved in the acetonitrile: water solvent mixture by heating the solution, for example near to the reflux temperature, for example to about 60-80 °C, for example to about 75 °C, under stirring and filtering if necessary. The solution is suitably then cooled to about 0-50 °C, for example to about 5-35 °C, for example to about RT, over about 5 to about 24 hours, for example over about 6 to 12 hours, and stirred at this temperature for about 3 to 72 hours, for example for about 5 to 12 hours. The obtained crystalline product can then be filtered, washed, and dried. The drying is suitably carried out in vacuum at about 40 to 60 °C, for example at 55 °C, for about 1 to 24 hours, such as for about 2 to 12 hours, for example 2 to 6 hours.

The crystalline forms I, Γ and I” of compounds (I), (la) and (lb), respectively, are useful as medicaments and can be formulated into pharmaceutical dosage forms, such as tablets and capsules for oral administration, by mixing with pharmaceutical excipients known in the art.

The disclosure is further illustrated by the following examples.

Example 1. Crystallization of N-((S)- 1 -(3 -(3 -chloro-4-cyanophenyl)- 1 H-pyrazol- 1 -yl)-propan-2-yl)-5 -( 1 -hydroxyethyl)- 1 H-pyrazole-3 -carboxamide (I)

N-((iS)- 1 -(3 -(3 -chloro-4-cyanophenyl)- 1 H-pyrazol- 1 -yl)-propan-2-yl)-5 -( 1 -hydroxyethyl)-! H-pyrazole-3 -carboxamide (I) (5 g), 71.25 ml of acetonitrile, and 3.75 ml of distilled water were charged to a flask, and the mixture was heated up to 75 °C. The mixture was slowly cooled down to RT and stirred at RT for 3 days. The solid obtained was filtered and washed twice with acetonitrile: water (9.5 ml:0.5 ml). The product was dried under vacuum at 40 °C and finally at 60°C to obtain 4.42 g of crystalline title compound (yield of 88 %) which was used in X-ray diffraction study.

Example 3. Synthesis of N-((S)- 1 -(3-(3-chloro-4-cyanophenyl)- lH-pyrazol- 1 -yl)-propan-2-yl)-5-((S)- 1 -hy droxy ethyl)- lH-pyrazole-3-carboxamide (la)

a) Ethyl-5 -((S) 1 -hydroxy ethyl)- 1 H-pyrazole-3 -carboxylate

HO

MgS04 x7H20 (341 mg), NADP monosodium salt (596 mg), D(+)-glucose (9.26 g) and optimized enzyme CDX-901 lyophilized powder (142 mg) were added to 0.2 mM of KH2P04 buffer (pH 7.0, 709 ml) to prepare solution I. To this solution I was added solution II which contained ethyl-5 -acetyl- 1 H-pyrazole-3 -carboxylate (8.509 g; 46.70 mmol), EtOH (28 ml) and K ED-130 (NADPH ketoreductase, 474 mg). The mixture was agitated at 30-32°C for 5.5 h (monitoring by HPLC) and allowed to cool to RT. The mixture was evaporated to smaller volume and the residue was agitated with diatomaceous earth and filtered. The mother liquid was extracted with 3×210 ml of EtOAc and dried. The solution was filtered through silica (83 g) and evaporated to dryness to give 7.40 g of the title compound. The optical purity was 100 % ee.

b) Ethyl 5-((S)-l -((tert-butyldiphenylsilyl)oxy)ethyl)- 1 H-pyrazole-3 -carboxylate

Diphenyl-tert-butyl chlorosilane (7.48 g, 27.21 mmol) was added in 26 ml of DMF to a mixture of compound of Example 3(a) (5.00 g, 27.15 mmol) and imidazole (2.81 g, 41.27 mmol) in DMF (50 ml) at RT. The mixture was stirred at RT for 24 h.

Saturated aqueous NaHC03 (56 ml) and water (56 ml) were added and the mixture was stirred at RT for 20 min. The mixture was extracted with 2×100 ml of EtOAc. Combined organic phases were washed with water (1×100 ml, 1×50 ml), dried (Na2S04), filtered and concentrated to give 10.92 g of crude title compound.

c) 5-((S)-l -((tert-Butyldiphenylsilyl)oxy)ethyl)- 1 H-pyrazole-3 -carboxylic acid

2 M NaOH (aq) (38.8 ml; 77.5 mmol) was added to a solution of the compound of Example 3(b) (10.9 g, 25.8 mmol) in 66 ml of THF. The mixture was heated up to reflux temperature. Heating was continued for 2.5 h and THF was removed in vacuum. Water (40 ml) and EtOAc (110 ml) were added. Clear solution was obtained after addition of more water (10 ml). Layers were separated and aqueous phase was extracted with 100 ml of EtOAc. Combined organic phases were dried (Na2S04), filtered and concentrated to give 9.8 g of the title compound.

d) 5-((S)- 1 -((tert-Butyldiphenylsilyl)oxy)ethyl)-N-((S)- 1 -(3-(3-chloro-4-cyano-phenyl)- 1 H-pyrazol- 1 -yl)propan-2-yl)- 1 H-pyrazole-3 -carboxamide

Under nitrogen atmosphere HBTU (0.84 g; 2.22 mmol), EDCIxHCl (3.26 g; 17.02 mmol) and (S)-4-(l-(2-aminopropyl)-lH-pyrazol-3-yl)-2-chlorobenzonitrile (3.86 g; 14.80 mmol) were added to a mixture of crude compound of Example 3(c) (8.68g; purity 77.4 area-%) and DIPEA (2.20 g; 17.02 mmol) in DCM (50 ml). The mixture was stirred at RT for 46 h (6 ml of DCM was added after 20 h). The mixture was washed with 3×20 ml of water, dried (Na2S04), filtered and concentrated to give 13.7 g of crude title compound.

e) N-((S)- 1 -(3-(3-chloro-4-cyanophenyl)- lH-pyrazol- 1 -yl)propan-2-yl)-5-((S)- 1 -hydroxy ethyl)- 1 H-pyrazole-3 -carboxamide (la)

TBAF hydrate (Bu4NF x 3H20; 2.34 g; 7.40 mmol) in 10 ml of THF was added to the solution of the compound of Example 3(d) (9.43 g; 14.79 mmol) in THF (94 ml) at 0 °C under nitrogen atmosphere. Stirring was continued at RT for 21.5 h and the mixture was concentrated. DCM (94 ml) was added to the residue and the solution was washed with 3×50 ml of water, dried (Na2S04), filtered and concentrated. Crude product was purified by flash chromatography (EtOAc/n-heptane) to give 2.1 g of the title compound. 1H-NMR (400MHz; d6-DMSO; 300K): Major tautomer (-85 %): δ 1.11 (d, 3H), 1.39 (d, 3H), 4.24-4.40 (m, 2H), 4.40-4.50 (m, 1H), 6.41(s, 1H), 6.93 (d, 1H), 7.77-7.82 (m, 1H), 7.88-8.01 (m, 2H), 8.08 (s, 1H), 8.19 (d, 1H), 13.02 (broad s, 1H). Minor tautomer (-15 %) δ 1.07-1.19 (m, 3H), 1.32-1.41 (m, 3H), 4.24-4.40 (m, 2H), 4.40-4.50 (m, 1H), 6.80 (broad s, 1H), 6.91-6-94 (m, 1H), 7.77-7.82 (m, 1H), 7.88-8.01 (m, 2H), 8.05-8.09 (m, 1H), 8.31 (d, 1H), 13.10 (broad s, 1H).

Example 4. Crystallization of N-((S)- 1 -(3-(3-chloro-4-cyanophenyl)- lH-pyrazol- 1 -yl)propan-2-yl)-5-((S)- 1 -hy droxy ethyl)- lH-pyrazole-3-carboxamide (la)

N-((S)- 1 -(3-(3-chloro-4-cyanophenyl)- lH-pyrazol- 1 -yl)propan-2-yl)-5-((S)- 1 -hydroxyethyl)-lH-pyrazole-3-carboxamide (la) (5.00 g; 12.54 mmol) was mixed with 47.5 ml of ACN and 2.5 ml of water. The mixture was heated until compound (la) was fully dissolved. The solution was allowed to cool slowly to RT to form a precipitate. The mixture was then further cooled to 0 °C and kept in this temperature for 30 min. The mixture was filtered and the precipitate was dried under vacuum to obtain 4.50 g of crystalline title compound which was used in the X-ray diffraction study.

Example 6. Synthesis of N-((S)- 1 -(3-(3-chloro-4-cyanophenyl)- lH-pyrazol- 1 -yl)-propan-2-yl)-5-((R)- 1 -hy droxy ethyl)- lH-pyrazole-3-carboxamide (lb)

a) Ethyl-5 -((R)- 1 -hydroxy ethyl)- 1 H-pyrazole-3 -carboxylate

Potassium dihydrogen phosphate buffer (Solution I) was prepared by dissolving potassium dihydrogen phosphate (11.350 g, 54.89 mmol) to water (333 ml) and adjusting pH of the solution to 7.0 by addition of 5 M solution of NaOH. MgS04 x 7 H20 (1.650 g), NAD monosodium salt (0.500 g), D(+)-glucose (10.880 g) and optimised enzyme CDX-901 lyophilised powder (0.200 g) were added to Solution I. To this solution (Solution II) were added KRED-NADH- 110 (0.467 g), ethyl-5-acetyl-1 H-pyrazole-3 -carboxylate (10.00 g; 54.89 mmol) and 2-methyltetrahydro-furan (16 ml). The mixture was agitated at 30° C for 11 h and allowed to cool to RT overnight. The pH of the mixture was kept at 7 by addition of 5 M solution of NaOH. The mixture was evaporated to a smaller volume. The evaporation residue was agitated for 10 min with diatomaceous earth (40 g) and activated charcoal (0.54 g), and filtered. Material on the filter was washed with water (40 ml) and the washings were combined with the filtrate. Layers were separated and aqueous phase was extracted with EtOAc (450 ml and 2×270 ml). Combined organic phases were dried over Na2S04, filtered and evaporated to dryness to give 9.85 g of the title compound (100 % ee).

b) Ethyl-5 -((R)- 1 -((tert-butyldiphenylsilyl)oxy)ethyl)- 1 H-pyrazole-3 -carboxylate

Imidazole (5.32 g; 78.08 mmol) was added to a DCM (67 ml) solution of the compound of Example 6(a) (9.85 g; 53.48). The mixture was stirred until all reagent was dissolved and tert-butyldiphenyl chlorosilane (13.21 ml; 50.80 mmol) was added to the mixture. The mixture was stirred for 1.5 h, 70 ml of water was added and stirring was continued for 15 min. Layers were separated and organic phase was washed with 2×70 ml of water and dried over Na2S04, filtered and concentrated to give 22.07 g of crude title compound.

c) 5 -((R)- 1 -((tert-Butyldiphenylsilyl)oxy)ethyl)- 1 H-pyrazole-3 -carboxylic acid

Compound of Example 6(b) (11.3 g; 26.74 mmol; theoretical yield from the previous step) was dissolved in 34 ml of THF and 50 ml of 2 M NaOH (aq.) was added. The mixture was heated under reflux temperature for 70 min. The mixture was extracted with 2×55 ml of EtOAc and combined organic phases were washed with brine, dried over Na2S04, filtered and concentrated. Evaporation residue was triturated in 250 ml of n-heptane, filtered and dried to give 17.58 g of crude title compound.

d) 5-((R)- 1 -((tert-Butyldiphenylsilyl)oxy)ethyl)-N-((S)- 1 -(3-(3-chloro-4-cyano-phenyl)- 1 H-pyrazol- 1 -yl)propan-2-yl)- 1 H-pyrazole-3 -carboxamide

A mixture of the compound of Example 6(c) (11.14 g; 26.75 mmol; theoretical yield from the previous step), 91 ml of DCM, HBTU (1.52 g; 4.01 mmol), EDCIxHCl

(5.90 g; 30.76 mmol), (S)-4-(l-(2-aminopropyl)-lH-pyrazol-3-yl)-2-chlorobenzo-nitrile (6.97 g; 26.75 mmol) and DIPEA (3.98 g; 30.76 mmol) was stirred at RT for 3 h and at 30° C for 22 h. The mixture was washed with 2×90 ml of 0.5 M HC1 and 4×90 ml of water, dried over Na2S04, filtered and concentrated. Crude product was purified by flash column chromatography (n-heptane-EtOAc) to give 16.97 g of title compound.

e) N-((S)- 1 -(3-(3-chloro-4-cyanophenyl)- lH-pyrazol- 1 -yl)propan-2-yl)-5-((R)- 1 -hydroxy ethyl)- 1 H-pyrazole-3 -carboxamide (lb)

A mixture of the compound of Example 6(d) (6.09 g; 9.56 mmol), 61 ml of THF and TBAF was stirred at 40 °C for 6.5 h. The mixture was concentrated and 61 ml of EtOAc was added to the evaporation residue. Solution was washed with 2×50 ml of 0.5 M HC1 and 4×50 ml of water, dried over Na2S04, filtered and concentrated. Crude product was purified by flash column chromatography (n-heptane-EtOAc) to give 1.71 g of the title compound. 1H-NMR (400MHz; d6-DMSO; 300K): Major tautomer (~85%): 5 1.10 (d, 3H), 1.38 (d, 3H), 4.14-4.57 (m, 2H), 5.42 (d, 1H),

6.39(s, 1H), 6.86-6.98 (m, 1H), 7.74-7.84 (m, 1H), 7.86-8.02 (m, 2H), 8.08 (s, 1H), 8.21 (d, 1H), 13.04 (broad s, 1H). Minor tautomer (-15%) δ 0.95-1.24 (m, 3H), 1.25-1.50 (m, 3H), 4.14-4.57 (m, 2H), 4.60-4.90 (m, 1H), 5.08 (d, 1H), 6.78 (broad s, 1H), 6.86-6.98 (m, 1H), 7.77-7.84 (m, 1H), 7.86-8.02 (m, 2H), 8.02-8.12 (m, 1H), 8.32 (d, 1H), 13.1 1 (broad s, 1H).

Example 7. Crystallization of N-((S)- 1 -(3-(3-chloro-4-cyanophenyl)- lH-pyrazol- 1 -yl)propan-2-yl)-5-((R)- 1 -hy droxy ethyl)- 1 H-pyrazole-3 -carboxamide (lb)

N-((S)- 1 -(3-(3-chloro-4-cyanophenyl)- lH-pyrazol- 1 -yl)propan-2-yl)-5-((R)- 1 -hydroxyethyl)-l H-pyrazole-3 -carboxamide (lb) (3.7 g; 9.28 mmol) was mixed with 70 ml of ACN and 3.5 ml of water. The mixture was heated to reflux temperature until compound (lb) was fully dissolved. The solution was allowed to cool slowly. The mixture was filtered at 50 °C to obtain 6.3 mg of the precipitate. Mother liquid was cooled to 41 °C and filtered again to obtain 20.7 mg of the precipitate. Obtained mother liquid was then cooled to 36 °C and filtered to obtain 173 mg of the precipitate. The final mother liquid was cooled to RT, stirred overnight, cooled to 0 °C, filtered, washed with cold ACN: water (1 : 1) and dried to obtain 2.71 g of the precipitate. The precipitates were checked for optical purity and the last precipitate of crystalline title compound (optical purity 100 %) was used in the X-ray diffraction study.

Example 9. Synthesis of Ethyl-5 -((S) 1 -hydroxy ethyl)- 1 H-pyrazole-3 -carboxylate

HO

Zinc trifluoromethanesulfonate (0.259 g; 0.713 mmol) and (S)-(-)-3-butyn-2-ol (0.25 g; 3.57 mmol) were added to 0.75 ml (5.35 mmol) of Et3N under nitrogen

atmosphere. Ethyldiazoacetate (0.45 ml; 4.28 mmol) was added slowly and the

mixture was heated at 100 °C for 2 h. The mixture was cooled to RT and 5 ml of water was added. The mixture was washed with 15 ml of DCM, 5 ml of water was added and phases were separated. Water phase was washed twice with DCM, all organic layers were combined, dried with phase separator filtration and evaporated to dryness to give 0.523 g of crude material. The product was purified by normal phase column chromatography (0-5 % MeOH:DCM) to give 0.165 mg of the title compound. 1H-NMR (400MHz; d6-DMSO; temp +300 K): Tautomer 1 (major 77%): δ 1.28 (t, 3H), 1.39 (d, 3H), 4.20-4.28 (m, 2H), (d, 1H), 4.75-4.85 (m, 1H) 5.43 (broad d, 1H), 6.54 (broad s, 1H), 13.28 (broad s, 1H). Tautomer 2 (minor 23%): δ 1.28 (t, 3H), 1.39 (d, 3H), 4.20-4.28 (m, 2H), 4.66-4.85 (m, 1H), 5.04-5.15 (broad s, 1H), 6.71 (broad s, 1H), 13.60 (broad s, 1H).

Exam le 10. Ethyl-5 -((R)- 1 -hydroxy ethyl)- 1 H-pyrazole-3 -carboxylate

Zinc trifluoromethanesulfonate (1.037 g; 2.85 mmol) and (R)-(+)-3-butyn-2-ol (1.00 g; 14.27 mmol) were added to 2.98 ml (21.40 mmol) of Et3N under nitrogen atmosphere. Ethyldiazoacetate (1.80 ml; 21.40 mmol) was added slowly and then refluxed for 3 h. The mixture was cooled to RT and 45 ml of water was added. The mixture was extracted with 3×50 ml of DCM, organic layers were combined, dried with phase separator filtration and evaporated to dryness to give 2.503 g of crude material which was purified by normal phase column chromatography (0-10 % MeOH:DCM) to give 0.67 lmg of the title compound. 1H-NMR (400MHz; d6-DMSO; temp +300 K): Tautomer 1 (major 78%): δ 1.28 (t, 3H), 1.39 (d, 3H), 4.18-4.35 (m, 2H), (d, 1H), 4.75-4.85 (m, 1H) 5.42 (broad d, 1H), 6.54 (s, 1H), 13.29 (broad s, 1H). Tautomer 2 (minor 22%): δ 1.28 (t, 3H), 1.39 (d, 3H), 4.18-4.35 (m, 2H), 4.66-4.85 (m, 1H), 5.09 (broad s, 1H), 6.71 (broad s, 1H), 13.61 (broad s, 1H).

References

  1.  “Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies.”. Sci Rep. 5: 12007. 2015. doi:10.1038/srep12007. PMC 4490394free to read. PMID 26137992.
  2.  Fizazi K, Albiges L, Loriot Y, Massard C (2015). “ODM-201: a new-generation androgen receptor inhibitor in castration-resistant prostate cancer”. Expert Rev Anticancer Ther. 15(9): 1007–17. doi:10.1586/14737140.2015.1081566. PMID 26313416.
  3.  Moilanen AM, Riikonen R, Oksala R, Ravanti L, Aho E, Wohlfahrt G, Nykänen PS, Törmäkangas OP, Palvimo JJ, Kallio PJ (2015). “Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies”. Sci Rep. 5: 12007.doi:10.1038/srep12007. PMC 4490394free to read. PMID 26137992.
  4.  “ODM-201 is safe and active in metastatic castration-resistant prostate cancer”. Cancer Discov. 4 (9): OF10. 2014. doi:10.1158/2159-8290.CD-RW2014-150. PMID 25185192.
  5. Pinto Á (2014). “Beyond abiraterone: new hormonal therapies for metastatic castration-resistant prostate cancer”. Cancer Biol. Ther. 15 (2): 149–55. doi:10.4161/cbt.26724.PMC 3928129free to read. PMID 24100689.
  6. Fizazi K, Massard C, Bono P, Jones R, Kataja V, James N, Garcia JA, Protheroe A, Tammela TL, Elliott T, Mattila L, Aspegren J, Vuorela A, Langmuir P, Mustonen M (2014). “Activity and safety of ODM-201 in patients with progressive metastatic castration-resistant prostate cancer (ARADES): an open-label phase 1 dose-escalation and randomised phase 2 dose expansion trial”. Lancet Oncol. 15 (9): 975–85. doi:10.1016/S1470-2045(14)70240-2. PMID 24974051.
  7.  Agarwal N, Di Lorenzo G, Sonpavde G, Bellmunt J (2014). “New agents for prostate cancer”. Ann. Oncol. 25 (9): 1700–9. doi:10.1093/annonc/mdu038. PMID 24658665.

External links

Fenner A. Prostate cancer: ODM-201 tablets complete phase I. Nat Rev Urol. 2015 Dec;12(12):654. doi: 10.1038/nrurol.2015.268. Epub 2015 Nov 3. PubMed PMID: 26526759.

2: Massard C, Penttinen HM, Vjaters E, Bono P, Lietuvietis V, Tammela TL, Vuorela A, Nykänen P, Pohjanjousi P, Snapir A, Fizazi K. Pharmacokinetics, Antitumor Activity, and Safety of ODM-201 in Patients with Chemotherapy-naive Metastatic Castration-resistant Prostate Cancer: An Open-label Phase 1 Study. Eur Urol. 2015 Oct 10. pii: S0302-2838(15)00964-1. doi: 10.1016/j.eururo.2015.09.046. [Epub ahead of print] PubMed PMID: 26463318.

3: Fizazi K, Albiges L, Loriot Y, Massard C. ODM-201: a new-generation androgen receptor inhibitor in castration-resistant prostate cancer. Expert Rev Anticancer Ther. 2015;15(9):1007-17. doi: 10.1586/14737140.2015.1081566. PubMed PMID: 26313416; PubMed Central PMCID: PMC4673554.

4: Bambury RM, Rathkopf DE. Novel and next-generation androgen receptor-directed therapies for prostate cancer: Beyond abiraterone and enzalutamide. Urol Oncol. 2015 Jul 7. pii: S1078-1439(15)00269-0. doi: 10.1016/j.urolonc.2015.05.025. [Epub ahead of print] Review. PubMed PMID: 26162486.

5: Moilanen AM, Riikonen R, Oksala R, Ravanti L, Aho E, Wohlfahrt G, Nykänen PS, Törmäkangas OP, Palvimo JJ, Kallio PJ. Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms to androgen signaling-directed prostate cancer therapies. Sci Rep. 2015 Jul 3;5:12007. doi: 10.1038/srep12007. PubMed PMID: 26137992; PubMed Central PMCID: PMC4490394.

6: Thibault C, Massard C. [New therapies in metastatic castration resistant prostate cancer]. Bull Cancer. 2015 Jun;102(6):501-8. doi: 10.1016/j.bulcan.2015.04.016. Epub 2015 May 26. Review. French. PubMed PMID: 26022286.

7: Bjartell A. Re: activity and safety of ODM-201 in patients with progressive metastatic castration-resistant prostate cancer (ARADES): an open-label phase 1 dose-escalation and randomised phase 2 dose expansion trial. Eur Urol. 2015 Feb;67(2):348-9. doi: 10.1016/j.eururo.2014.11.019. PubMed PMID: 25760250.

8: De Maeseneer DJ, Van Praet C, Lumen N, Rottey S. Battling resistance mechanisms in antihormonal prostate cancer treatment: Novel agents and combinations. Urol Oncol. 2015 Jul;33(7):310-21. doi: 10.1016/j.urolonc.2015.01.008. Epub 2015 Feb 21. Review. PubMed PMID: 25708954.

9: Boegemann M, Schrader AJ, Krabbe LM, Herrmann E. Present, Emerging and Possible Future Biomarkers in Castration Resistant Prostate Cancer (CRPC). Curr Cancer Drug Targets. 2015;15(3):243-55. PubMed PMID: 25654638.

10: ODM-201 is safe and active in metastatic castration-resistant prostate cancer. Cancer Discov. 2014 Sep;4(9):OF10. doi: 10.1158/2159-8290.CD-RW2014-150. Epub 2014 Jul 9. PubMed PMID: 25185192.

11: Fizazi K, Massard C, Bono P, Jones R, Kataja V, James N, Garcia JA, Protheroe A, Tammela TL, Elliott T, Mattila L, Aspegren J, Vuorela A, Langmuir P, Mustonen M; ARADES study group. Activity and safety of ODM-201 in patients with progressive metastatic castration-resistant prostate cancer (ARADES): an open-label phase 1 dose-escalation and randomised phase 2 dose expansion trial. Lancet Oncol. 2014 Aug;15(9):975-85. doi: 10.1016/S1470-2045(14)70240-2. Epub 2014 Jun 25. PubMed PMID: 24974051.

12: Agarwal N, Di Lorenzo G, Sonpavde G, Bellmunt J. New agents for prostate cancer. Ann Oncol. 2014 Sep;25(9):1700-9. doi: 10.1093/annonc/mdu038. Epub 2014 Mar 20. Review. PubMed PMID: 24658665.

13: Pinto Á. Beyond abiraterone: new hormonal therapies for metastatic castration-resistant prostate cancer. Cancer Biol Ther. 2014 Feb;15(2):149-55. doi: 10.4161/cbt.26724. Epub 2013 Nov 1. Review. PubMed PMID: 24100689; PubMed Central PMCID: PMC3928129.

14: Yin L, Hu Q, Hartmann RW. Recent progress in pharmaceutical therapies for castration-resistant prostate cancer. Int J Mol Sci. 2013 Jul 4;14(7):13958-78. doi: 10.3390/ijms140713958. Review. PubMed PMID: 23880851; PubMed Central PMCID: PMC3742227.

15: Leibowitz-Amit R, Joshua AM. Targeting the androgen receptor in the management of castration-resistant prostate cancer: rationale, progress, and future directions. Curr Oncol. 2012 Dec;19(Suppl 3):S22-31. doi: 10.3747/co.19.1281. PubMed PMID: 23355790; PubMed Central PMCID: PMC3553559.

Darolutamide
ODM-201.svg
Systematic (IUPAC) name
N-((S)-1-(3-(3-chloro-4-cyanophenyl)-1H-pyrazol-1-yl)propan-2-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide[1]
Identifiers
ChemSpider 38772320
UNII X05U0N2RCO Yes
Chemical data
Formula C19H19ClN6O2
Molar mass 398.85 g·mol−1

//////////// Bayer HealthCare,  Orion,  Antineoplastics,  Androgen receptor antagonists, Phase III, Prostate cancer, BAY 1841788,  ODM-201

O=C(N[C@@H](C)Cn1ccc(n1)c2ccc(C#N)c(Cl)c2)c3cc(nn3)C(O)C

Day 8 of the 2016 Doodle Fruit Games! Find out more at g.co/fruit

Doravirine, MK-1439


Doravirine.svg

Image for unlabelled figure

Doravirine.png

Doravirine, MK-1439……….. AN ANTIVIRAL

3-Chloro-5-({1-[(4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl]-2-oxo-4-(trifluoromethyl)-1,2-dihydro-3-pyridinyl}oxy)benzonitrile

Benzonitrile, 3-chloro-5-[[1-[(4,5-dihydro-4-methyl-5-oxo-1H-1,2,4-triazol-3-yl)methyl]-1,2-dihydro-2-oxo-4-(trifluoromethyl)-3-pyridinyl]oxy]-

3-chloro-5-({1-[(4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl]-2-oxo-4-(trifluoromethyl)-1,2-dihydropyridin-3-yl}oxy)benzonitrile

(3-Chloro-5-((1-((4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl)-2-oxo-4-(trifluoromethyl)-1,2-dihydropyridin-3-yl)oxy)benzonitrile)

1338225-97-0 CAS

MF  C17H11ClF3N5O3
MW 425.7  Merck Sharp & Dohme Corp

Merck Frosst Canada Ltd. INNOVATOR

Jason Burch, Bernard Cote, Natalie Nguyen,Chun Sing Li, Miguel St-Onge, Danny Gauvreau,

Reverse transcriptase inhibitor

UNII:913P6LK81M

  • Originator Merck & Co
  • Class Antiretrovirals; Nitriles; Pyridones; Small molecules; Triazoles
  • Mechanism of Action Non-nucleoside reverse transcriptase inhibitors
  • Phase III HIV-1 infections

Most Recent Events

  • 16 Jul 2016 No recent reports of development identified for phase-I development in HIV-1-infections(Monotherapy, Treatment-naive) in Germany (PO, Tablet)
  • 01 Jun 2016 Merck Sharp & Dohme completes a phase I pharmacokinetics trial in subjects requiring methadone maintenance therapy in USA (PO, Tablet) (NCT02715700)
  • 01 May 2016 Merck completes a phase I trial in severe renal impairment in USA (NCT02641067)

SYNTHESIS COMING………

WO  2015084763

STR1

CONTD………………………

STR1

img_pgene01.jpg

SPECTRAL DATA

19F DMSOD6
STR1

13C NMR DMSOD6

STR1

1H NMR DMSOD6

STR1

3-chloro-5-((2-oxo-1-((5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl)-4-(trifluoromethyl)-1,2-dihydropyridin-3-yl)oxy)benzonitrile.

1H NMR (400 MHz, DMSO-d6) δ 11.47 (br. s., 1H), 11.40 (s, 1H), 7.93 (d, J = 7.3 Hz, 1H), 7.75 (t, J =1.5 Hz, 1H), 7.58 (dd, J = 1.2, 2.3 Hz, 1H), 7.51 (t, J = 2.1 Hz, 1H), 6.66 (d, J = 7.3 Hz, 1H), 5.02 (s, 2H)

13C NMR (101 MHz, DMSO-d6) δ 157.25, 156.20, 155.97, 142.52, 140.09 (q, JC-F = 2.0 Hz), 137.74,134.97, 130.17 (q, JC-F = 31.2 Hz), 126.53, 121.70 (q, JC-F = 274.7 Hz), 121.16, 118.37, 116.96, 113.70,99.96 (q, JC-F = 4.0 Hz), 44.90

19F NMR (376 MHz, DMSO-d6) δ -62.24 (s, 1F)
HRMS [M + H]+ for C16H10ClF3N5O3 calcd, 412.0419; found, 412.0415.
mp 148.46-156.11 °C

REF Org. Process Res. Dev., Article ASAP, DOI: 10.1021/acs.oprd.6b00163

http://pubs.acs.org/doi/suppl/10.1021/acs.oprd.6b00163

STR1

str2

Doravirine (MK-1439) is a non-nucleoside reverse transcriptase inhibitor under development by Merck & Co. for use in the treatment of HIV/AIDS. Doravirine demonstrated robust antiviral activity and good tolerability in a small clinical study of 7-day monotherapy reported at the 20th Conference on Retroviruses and Opportunistic Infections in March 2013. Doravirine appeared safe and generally well-tolerated with most adverse events being mild-to-moderate.[2][3]

Highly active antiretroviral therapy (HAART) is the standard of care for the treatment of HIV infection. Typically, this protocol recommends the combination of two nucleoside reverse-transcriptase inhibitors (NRTIs) with either a non-nucleoside reverse-transcriptase inhibitor (NNRTI), a ritonavir-boosted protease inhibitor or an integrase inhibitor. 

NNRTI-based combinations have become first-line therapy mainly because of their demonstrated efficacies, convenient dosing regimen and relatively low toxicities. These inhibitors block the polymerase activity of the HIV reverse transcriptase by binding to an allosteric hydrophobic pocket adjacent to the active site. Efavirenz (1, ) is a first generation NNRTI that has been conveniently co-formulated with NRTIs tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) as a once-a-day fixed-dose combination (Atripla®). Although recommended for the therapy of treatment-naïve patients, efavirenz suffers from neurocognitive side effects, teratogenicity and exacerbation of hyperlipidemia. Moreover, the low barrier to genetic resistance of first generation NNRTIs led to the emergence of resistant viruses bearing mutations K103N and Y181C in patients failing therapy.

Structures of marketed and lead NNRTIs.

Figure .

Structures of marketed and lead NNRTIs.

Second generation NNRTIs etravirine (2) and rilpivirine (3) efficiently suppress the replication of the K103N resistant mutants as shown by an improved activity in cell culture assays . Etravirine (200 mg, bid) is approved for use in treatment-experienced adult patients with multi-drug resistance. With an improved pharmacokinetic profile, the close analog rilpivirine (25 mg, qd) was recently approved for use in treatment-naïve patients. Phase III data reveal that at the 96-week point, a rilpivirine/truvada®  combination was better tolerated than efavirenz/truvada®. However, the virologic failure rate was twice as high for rilpivirine (14%) than it was for efavirenz (8%). For patients with viral load greater than 500,000 copies/mL, the response rate is 62% (rilpivirine) versus 81% (efavirenz). As a result, rilpivirine is not recommended for treating HIV patients with viral load >500,000 copies/mL. This difference in treatment durability could be explained by the much higher ratio of trough concentration over the antiviral activity for efavirenz versus rilpivirine.

Investigational next-generation, non-nucleoside reverse transcriptase inhibitor (NNRTI), at the 21st Conference on Retroviruses and Opportunistic Infections (CROI). Interim data demonstrating potent antiretroviral (ARV) activity for four doses (25, 50, 100 and 200 mg) of once-daily, oral doravirine in combination with tenofovir/emtricitabine in treatment-naïve, HIV-1 infected adults after 24 weeks of treatment were presented during a late-breaker oral session. Based on these findings as well as other data from the doravirine clinical program, Merck plans to initiate a Phase 3 clinical trial program for doravirine in combination with ARV therapy in the second half of 2014.

“Building on our long-standing commitment to the HIV community, Merck continues to evaluate new candidates we believe have the potential to make a meaningful difference in the lives of HIV patients,” said Daria Hazuda, Ph.D., vice president, Infectious Diseases, Merck Research Laboratories. “We look forward to advancing doravirine into Phase 3 clinical trials in the second half of 2014.”

Doravirine Clinical Data

This randomized, double-blind clinical trial examined the safety, tolerability and efficacy of once-daily doravirine (25, 50, 100 and 200 mg) in combination with once-daily tenofovir/emtricitabine versus efavirenz (600 mg), in treatment-naïve, HIV-1 infected patients. The primary efficacy analysis was percentage of patients achieving virologic response (< 40 copies/mL).

At 24 weeks, doravirine doses of 25, 50, 100, and 200 mg showed virologic response rates consistent with those observed for efavirenz at a dose of 600 mg. All treatment groups showed increased CD4 cell counts.

Proportion of Patients with Virologic
Response at 24 weeks (95% CI)

Mean CD4 Change
from Baseline (95% CI)

Treatment* Dose (mg) n/N

% <40
copies/mL

cells/μL

Doravirine 25 32/40 80.0 (64.6, 90.9) 158 (119, 197)
50 32/42 76.2 (60.5, 87.9) 116 (77, 155)
100 30/42 71.4 (55.4, 84.3) 134 (100, 167)
200 32/41 78.0 (62.4, 89.4) 141 (96, 186)
Efavirenz 600 27/42 64.3 (48.0, 78.4) 121 (73, 169)
Missing data approach: Non-completer = Failure Observed Failure

*In combination with tenofovir/emtricitabine

The incidence of drug-related adverse events was comparable among the doravirine-treated groups. The overall incidence of drug-related adverse events was lower in the doravirine-treated groups (n=166) than the efavirenz-treated group (n=42), 35 percent and 57 percent, respectively. The most common central nervous system (CNS) adverse events at week 8, the primary time point for evaluation of CNS adverse experiences, were dizziness [3.0% doravirine (overall) and 23.8% efavirenz], nightmare [1.2% doravirine (overall) and 9.5% efavirenz], abnormal dreams [9.0% doravirine (overall) and 7.1% efavirenz], and insomnia [5.4% doravirine (overall) and 7.1% efavirenz].

Based on the 24-week data from this dose-finding study, a single dose of 100 mg doravirine was chosen to be studied for the remainder of this study, up to 96 weeks.

About Doravirine

DORAVIRINE

Doravirine, also known as MK-1439, is an investigational next-generation, NNRTI being evaluated by Merck for the treatment of HIV-1 infection. In preclinical studies, doravirine demonstrated potent antiviral activity against HIV-1 with a characteristic profile of resistance mutations selected in vitro compared with currently available NNRTIs. In early clinical studies, doravirine demonstrated a pharmacokinetic profile supportive of once-daily dosing and did not show a significant food effect.

Merck’s Commitment to HIV

For more than 25 years, Merck has been at the forefront of the response to the HIV epidemic, and has helped to make a difference through our proud legacy of commitment to innovation, collaborating with the community, and expanding global access to medicines. Merck is dedicated to applying our scientific expertise, resources and global reach to deliver healthcare solutions that support people living with HIV worldwide.

About Merck

Today’s Merck is a global healthcare leader working to help the world be well. Merck is known as MSD outside the United States and Canada. Through our prescription medicines, vaccines, biologic therapies, and consumer care and animal health products, we work with customers and operate in more than 140 countries to deliver innovative health solutions. We also demonstrate our commitment to increasing access to healthcare through far-reaching policies, programs and partnerships. For more information, visit www.merck.com and connect with us on TwitterFacebook and YouTube.

PATENT

WO 2014089140

The compound 3 -chloro-5-( { 1 – [(4-methyl-5 -oxo-4,5 -dihydro- 1 H- 1 ,2,4-triazol-3 – yl)methyl]-2-oxo-4-(trifluoromethyl)-l,2-dihydropyridin-3-yl}oxy)benzonitrile has the following chemical structure.

Figure imgf000017_0001

Anhydrous 3 -chloro-5-( { 1 – [(4-methyl-5 -oxo-4,5 -dihydro- 1 H- 1 ,2,4-triazol-3 -yl)methyl] -2-oxo-4- (trifluoromethyl)-l,2-dihydropyridin-3-yl}oxy)benzonitrile is known to exist in three crystalline forms – Form I, Form II and Form III. The differential scanning calorimetry (DSC) curve for crystalline anhydrous Form II shows an endotherm with an onset at 230.8° C, a peak maximum at 245.2°C, and an enthalpy change of 3.7 J/g, which is due to polymorphic conversion of anhydrous Form II to anhydrous Form I, and a second melting endotherm with an onset at 283.1°C, a peak maximum at 284.8°C, and an enthalpy change of 135.9 J/g, due to melting of Anhydrous Form I. Alternative production and the ability of this compound to inhibit HIV reverse transcriptase is illustrated in WO 201 1/120133 Al, published on October 6, 201 1, and US 201 1/0245296 Al, published on October 6, 201 1, both of which are hereby incorporated by reference in their entirety.

The process of the present invention offers greater efficiency, reduced waste, and lower cost of goods relative to the methods for making the subject compounds existing at the time of the invention. Particularly, the late stage cyanation and methylation steps are not required.

The following examples illustrate the invention. Unless specifically indicated otherwise, all reactants were either commercially available or can be made following procedures known in the art. The following abbreviations are used:

EXAMPLE 1

Figure imgf000018_0001
Figure imgf000018_0002

Step 1

Figure imgf000018_0003

1 2

3-(Chloromethyl)-l-(2-methoxypropan-2-yl)-4-methyl-lH-l,2,4-triazol-5(4H)-one (2): A

100 ml round bottom flask equipped with stir bar and a nitrogen inlet was charged with 1 (5 g, 33.9 mmol) and (lS)-(+)-10-camphorsulfonic acid (0.39 g, 1.694 mmol) at ambient temperature. After 2,2-dimethoxy propane (36.0 g, 339 mmol) was charged at ambient temperature, the resulting mixture was heated to 45°C. The resulting mixture was stirred under nitrogen at 45°C for 18 hours and monitored by HPLC for conversion of the starting material (< 5% by HPLC). After the reaction was completed, the batch was taken on to the next step without further workup or isolation. ‘H NMR (CDCI3, 500 MHz): 4.45 (s, 2H), 3.35 (s, 3H), 3.21 (s, 3H), 1.83 (s, 6H).

Step 2

Figure imgf000019_0001

3-Fluoro-l-((l-(2-methoxypropan-2-yl)-4-methyl-5-oxo-4,5-dihydro-lH-l,2,4-triazol-3- yl)methyl)-4-(trifluoromethyl)pyridin-2(lH)-one (3): A mixture of 2 (100 mg, 93.1% purity, 0.49 mmol), pyridone (1 17 mg, 97.6% purity, 0.49 mmol) and K2CO3 (82 mg, 0.59 mmol) in DMF (0.5 ml) was aged with stirring at ambient temperature for 3h. After the reaction was completed, the batch was taken on to the next step without further work up or isolation.

Step 3

Figure imgf000019_0002

3-Chloro-5-((l-((l-(2-methoxypropan-2-yl)-4-methyl-5-oxo-4,5-dihydro-lH-l,2,4-triazol-3- yl)methyl)-2-oxo-4-(trifluoromethyl)-l,2-dihydropyridin-3-yl)oxy)benzonitrile (4): To a mixture of compound 3 in DMF (reaction mixture from the previous step) was added 3-chloro-5- hydroxybenzonitrile (1.77 g, 1 1.5 mmol) at ambient temperature. The resulting mixture was then heated to 95-100°C and held for 20 hours.

Upon completion (typically 18-20 hours), the reaction was cooled to room temperature, diluted with ethyl acetate and washed with water. The aqueous cut was back extracted with ethyl acetate. The organic layers were combined and then concentrated to an oil. MeOH (80 ml) was added and the resulting slurry was taken on to the next step. XH NMR (CDC13, 500 MHz): 7.60 (d, IH), 7.42 (s, IH), 7.23 (s, IH), 7.12 (s, IH), 6.56 (d, IH), 5.14 (s, 2H), 3.30 (s, 3H), 3.22 (s, 3H), 1.82 (s, 6H).

Step 4

Figure imgf000020_0001

4 5

3-Chloro-5-((l-((4-methyl-5-oxo-4,5-dihydro-lH-l,2,4-triazol-3-yl)methyl)-2-oxo-4- (trifluoromethyl)-l,2-dihydropyridin-3-yl)oxy)benzonitrile (5): To a solution of 4 (5.74 g., 1 1.53 mmol) in MeOH (from previous step) was added concentrated hydrochloric acid (lml, 12.18 mmol) at ambient temperature. The resulting mixture was agitated for 1 hour at room temperature.

The resulting solids were collected by filtration and dried under a nitrogen sweep, providing 5 as a white solid (2.63 g, 46% yield): XH NMR (DMSO, 400 MHz): 1 1.74 (S, IH), 7.92 (d, IH), 7.76 (s, IH), 7.61 (s, IH), 7.54 (s, IH), 6.69 (d, IH), 5.15 (s, 2H), 3.10 (s, 3H)

EXAMPLE 2

Figure imgf000021_0001

Step 1

Figure imgf000021_0002

Phenyl methylcarbamate: 40% Aqueous methylamine (500 g, 6.44 mol) was charged to a 2 L vessel equipped with heat/cool jacket, overhead stirrer, temperature probe and nitrogen inlet. The solution was cooled to -5 °C. Phenyl chloroformate (500.0 g, 3.16 mol) was added over 2.5 h maintaining the reaction temperature between -5 and 0 °C. On complete addition the white slurry was stirred for lh at ~0 °C.

The slurry was filtered, washed with water (500 mL) and dried under 2 sweep overnight to afford 465g (96%> yield) of the desired product as a white crystalline solid; 1H NMR (CDCI3, 500 MHz): δ 7.35 (t, J = 8.0 Hz, 2H), 7.19 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 8.0 Hz, 2H), 4.95 (br s, 1H), 2.90 (d, J = 5 Hz, 3H).

Step 2

Figure imgf000022_0001

2-(2-Hydroxyacetyl)-N-methylhydrazinecarboxamide: Part A: Phenyl methylcarbamate (300 g, 1.95 mol) was charged to a 2 L vessel with cooling jacket, overhead stirrer, temperature probe, reflux condenser and nitrogen inlet. IPA (390 mL) was added at 23 °C. Hydrazine hydrate (119 g, 2.33 mol) was added and the slurry heated to 75 °C for 6 h.

Part B: On complete reaction (>99% conversion by HPLC), IPA (810 mL) and glycolic acid (222 g, 2.92 mol) were added and the mixture stirred at 83-85 °C for 10-12 h. The reaction mixture is initially a clear colorless solution. The mixture is seeded with product (0.5 g) after 4h at 83-85 °C. The slurry was slowly cooled to 20 °C over 2h and aged for lh.

The slurry was filtered and washed with IPA (600 mL). The cake was dried under 2 sweep to afford 241.8g (81% yield) of the desired product as a white crystalline solid: XH NMR (D20, 500 MHz): δ 4.11 (s, 2H), 2.60 (s, 3H).

Step 3

Figure imgf000022_0002

3-(Hydroxymethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one: 2-(2-Hydroxyacetyl)-N- methylhydrazinecarboxamide (130 g @ ~95wt%, 0.84 mol), w-propanol (130 mL) and water (130 mL) were charged to a 1 L vessel with jacket, overhead stirrer, temperature probe, reflux condenser and nitrogen inlet. Sodium hydroxide (pellets, 16.8 g, 0.42 mol) was added and the slurry warmed to reflux for 3h. The reaction mixture was cooled to 20 °C and the pH adjusted to 6.5 (+/- 0.5) using cone hydrochloric acid (28.3 mL, 0.34 mol). Water was azeotropically removed under vacuum at 40-50 °C by reducing the volume to -400 mL and maintaining that volume by the slow addition of n-propanol (780 mL). The final water content should be <3000 ug/mL. The resultant slurry (~ 400 mL) was cooled to 23 °C and heptane (390 ml) was added. The slurry was aged lh at 23 °C, cooled to 0 °C and aged 2h. The slurry was filtered, the cake washed with 1 :2 n-PrOH/heptane (100 mL) and dried to provide 125g (85% yield) of an off- white crystalline solid. The solid is ~73 wt% due to residual inorganics (NaCl): ‘H NMR (CD3OD, 500 MHz): δ 3.30 (s, 3H), 4.46 (s, 2H).

Step 4

Figure imgf000023_0001

3-(Chloromethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one (1): A mixture of 3- (Hydroxymethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one (54 g, at 73wt%, 307 mmol) in ethyl acetate (540 mL) was stirred at 45 °C. SOCI2 (26.9 mL, 369 mmol) was added over 30-45 min and aged at 50 °C for 2h. Monitor reaction progress by HPLC. On complete reaction (>99.5% by area at 210nm.), the warm suspension was filtered and the filter cake (mainly NaCl) was washed with ethyl acetate (108 mL). The combined filtrate and wash were concentrated at 50-60 °C under reduced pressure to approximately 150 mL. The resulting slurry was cooled to -10 °C and aged lh. The slurry was filtered and the filter cake washed with ethyl acetate (50 mL). The cake was dried under 2 sweep to afford 40. lg (86% yield) of the desired product as a bright yellow solid: ‘H NMR (CD3OD, 500 MHz): δ 3.30 (s, 3H), 4.58 (s, 2H).

EXAMPLE 3

Figure imgf000023_0002

3-fluoro-4-(trifluoromethyl)pyridin-2(lH)-one (2): To a 250 ml round bottom flask equipped with overhead stirring and a nitrogen inlet was added a mixture of sulfuric acid (24.31 ml, 437 mmol) and water (20.00 ml). To this was added 2,3-difluoro-4-(trifluoromethyl)pyridine (6.83 ml, 54.6 mmol) and the mixture was heated to 65 °C and stirred for 4 h. By this time the reaction was complete, and the mixture was cooled to room temperature. To the flask was slowly added 5M sodium hydroxide (43.7 ml, 218 mmol), maintaining room temperature with an ice bath. The title compound precipitates as a white solid during addition. Stirring was maintained for an additional lh after addition. At this time, the mixture was filtered, the filter cake washed with 20 mL water, and the resulting white solids dried under nitrogen. 3-fluoro-4- (trifluoromethyl)pyridin-2(lH)-one (2) was obtained as a white crystalline solid (9.4g, 51.9 mmol, 95 % yield): ¾ NMR (CDC13, 400 MHz): 12.97 (br s, 1H), 7.36 (d, 1H), 6.44 (m, 1H).

EXAMPLE 4

Step 1 – Ethyl Ester Synthesis Experimental Procedure;

Figure imgf000024_0001

Ethyl 2-(3-chloro-5-cyanophenoxy)acetate (A): A 1L round bottom flask equipped with overhead stirring was charged with 3-chloro-5-hydroxybenzonitrile (50.0 g, 98 wt% purity, 319 mmol) and 15% aqueous DMF (200 mL DMF + 35.5 mL FLO). To the resulting solution was added diisopropylethylamine (61.3 mL, 99.0% purity, 1.1 equiv) and ethyl 2-bromoacetate (35.7 g, 98% purity, 1.15 equiv) at ambient temperature. The resulting solution was warmed to 50°C under nitrogen and aged for 12 h. Upon completion of the reaction the batch was cooled to 0- 5°C. To the clear to slightly cloudy solution was added 5% seed (3.8g, 16.0 mmol). H20 (64.5mL) was added to the thin suspension via syringe pump over 3h while maintaining the temp at 0-5 °C. Additional FLO (200mL) was added over lh while maintaining the temp at 0-5 °C. The final DMF/FLO ratio is 1 : 1.5 (10 vol). The resulting slurry was typically aged lh at 0-5 °C. The batch was filtered and the cake slurry washed with 2: 1 DMF/water (150 mL, 3 vol), followed by water (200 mL, 4 vol). The wet cake was dried on the frit with suction under a nitrogen stream at 20-25 °C; note: heat must not be applied during drying as product mp is 42 °C. The cake is considered dry when H20 is <0.2%. Obtained 73.4 g ethyl ester as a light tan solid, 96% yield (corrected), 99.5 LCAP: XH NMR (CDC13, 400 MHz) δ = 7.29 (s, 1H), 7.15 (s, 1H), 7.06 (s, 1H), 4.67 (s, 2H), 4.32 (q, 2H), 1.35 (t, 3H) ppm. Step 2 – Pyridone Synthesis

Synthetic Scheme; batch

TEA, TFAA, 10 °C;

then MeOH, rt

Figure imgf000025_0001

[isolated solid, A] [PhMe exit stream, B]

Figure imgf000025_0002

[PhMe/MeOH solution, C] [PhMe/MeOH/NH3 solution, D] [isolated solid, E]

Experimental Procedures;

Aldol Condensation, Ester A to Diene C

(2E/Z,4E)-Ethyl 2-(3-chloro-5-cyanophenoxy)-5-ethoxy-3-(trifluoromethyl)penta-2,4- dienoate (C): Ester A (25.01 g, 104.4 mmol, 1.00 equiv) was charged to toluene (113.43 g, 131 mL, 5.24 vol) and 4-ethoxy-l, l, l-trifluoro-3-buten-2-one (26.43 g, 157.2 mmol, 1.51 equiv) was added.

The flow reactor consisted of two feed solution inlets and an outlet to a receiving vessel. The flow reactor schematic is shown in Figure 1.

The ester solution was pumped to one flow reactor inlet. Potassium tert-pentoxide solution was pumped to the second reactor inlet. Trifluoroacetic anhydride was added continuously to the receiver vessel. Triethylamine was added continuously to the receiver vessel. The flow rates were: 13 mL/min ester solution, 7.8 mL/min potassium tert-pentoxide solution, 3.3 mL/min trifluoroacetic anhydride and 4.35 mL/min triethylamine.

Charged toluene (50 mL, 2 vol) and potassium trifluoroacetate (0.64 g, 4.21 mmol, 0.04 equiv) to the receiver vessel. The flow reactor was submerged in a -10 °C bath and the pumps were turned on. The batch temperature in the receiver vessel was maintained at 5 to 10 °C throughout the run using a dry ice/acetone bath. After 13.5 min the ester solution was consumed, the reactor was flushed with toluene (10 mL) and the pumps were turned off.

The resulting yellow slurry was warmed to room temperature and aged for 4.5 h. Charged methanol (160 mL) to afford a homogeneous solution which contained 81.20 area percent diene C by HPLC analysis.

The solution of diene C (573 mL) was used without purification in the subsequent reaction. Cyclization, Diene C to E

3-Chloro-5-((2-oxo-4-(trifluoromethyl)-l,2-dihydropyridin-3-yl)oxy)benzonitrile (E): To a solution of diene C in PhMe/MeOH (573 mL; 40.69 g, 104.4 mmol theoretical C) was charged methanol (25 mL, 0.61 vol). Ammonia (32 g, 1.88 mol, 18 equiv based on theoretical C) was added and the solution was warmed to 60 °C. The reaction was aged at 60 °C for 18 h. The temperature was adjusted to 35-45 °C and the pressure was decreased maintain a productive distillation rate. The batch volume was reduced to -300 mL and methanol (325 mL, 8 vol) was charged in portions to maintain a batch volume between 250 and 350 mL. The heating was stopped and the system vented. The resulting slurry was cooled to room temperature and aged overnight.

The batch was filtered and the cake washed with methanol (3x, 45 mL). The wet cake was dried on the frit with suction under a nitrogen stream to afford 18.54 g of a white solid: XH NMR (DMSO-i/6, 500 MHz): δ 12.7 (br s, 1H), 7.73 (t, 1H, J= 1.5 Hz), 7.61-7.59 (m, 2H), 7.53 (t, 1H, J= 2.0 Hz), 6.48 (d, 1H, J= 7.0 Hz) ppm.

Step 3 – Chlorination, Alkylation and Isolation of 3-Chloro-5-({l-[(4-methyl-5-oxo-4,5-dihydro- lH-l,2,4-triazol-3-yl)methyl]-2-oxo-4-(trifluoromethyl)-l,2-dihydropyridin-3-yl}oxy)benzonitrile

Figure imgf000027_0001

3-(Chloromethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one: 3-(Hydroxymethyl)-4-methyl-lH- l,2,4-triazol-5(4H)-one (1.638 kg of 68wt%, 8.625 mol) and N-methylpyrrolidinone (8.9 L) was charged into a 30 L vessel. The suspension was aged for lOh at ambient temperature. The slurry was filtered through a 4L sintered glass funnel under 2 and the filter cake (mainly NaCl) was washed with NMP (2.23 L). The combined filtrate and wash had a water content of 5750 μg/mL. The solution was charged to a 75L flask equipped with a 2N NaOH scrubber to capture off-gasing vapors. Thionyl chloride (0.795 L, 10.89 mol) was added over lh and the temperature rose to 35 °C. HPLC analysis indicated that the reaction required an additional thionyl chloride charge (0.064 L, 0.878 mol) to bring to full conversion. The solution was warmed to 50 °C, placed under vacuum at 60 Torr (vented to a 2N NaOH scrubber), and gently sparged with subsurface N2 (4 L/min). The degassing continued for lOh until the sulfur dioxide content in the solution was <5 mg/mL as determined by quantitative GC/MS. The tan solution of 3-(chloromethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one in NMP weighed 13.0 kg and was assayed at 9.63 wt% providing 1.256 kg (97% yield).

3-chloro-5-((l-((4-methyl-5-oxo-4,5-dihydro-lH-l,2,4-triazol-3-yl)methyl)-2-oxo-4- (trifluoromethyl)-l,2-dihydropyridin-3-yl)oxy)benzonitrile: To a 75L flask was charged a 9.63wt% solution of 3-(chloromethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one in NMP (1 1.6 kg, 7.55 mol), 3 -chloro-5 -((2-oxo-4-(trifluoromethyl)- 1 ,2-dihydropyridin-3 -yl)oxy)benzonitrile (2.00 kg, 6.29 mol), NMP (3.8 L) and 2-methyl-2-butanol (6.0 L). To the resulting suspension was slowly added N,N-diisopropylethylamine (4.38 L, 25.2 mol) over 4h. The reaction was aged 18h at ambient temperature. The reaction is considered complete when HPLC indicates <1% 3 -chloro-5 -((2-oxo-4-(trifluoromethyl)-l,2-dihydropyridin-3-yl)oxy)benzonitrile remaining. The tan solution was quenched with acetic acid (1.26 L, 22.0 mol) and aged at ambient temperature overnight. The tan solution was warmed to 70 °C. Water (2.52 L) was added and the batch was seed with anhydrate Form II (134 g). The thin suspension was aged lh at 70 °C. Additional water (14.3 L) was added evenly over 7 h. The slurry was aged 2h at 70 °C and then slowly cooled to 20 °C over 5 h. The slurry was filtered and washed with 2 : 1 NMP/water (6 L), followed by water washes (6 L x 2). The filter cake was dried over a 2 sweep to give 2.53 kg (85% yield – corrected) of a white solid that was confirmed to be crystalline Form II by X-ray powder detraction analysis.

PATENT

WO 2015084763

The following scheme is an example of Step 3A.

EXAMPLE 1

1

Step 1

c| 0. h CH3NH3 Me.NA0.Ph

H

Phenyl methylcarbamate: 40% Aqueous methylamine (500 g, 6.44 mol) was charged to a 2 L vessel equipped with heat/cool jacket, overhead stirrer, temperature probe and nitrogen inlet. The solution was cooled to -5 °C. Phenyl chloroformate (500.0 g, 3.16 mol) was added over 2.5 h maintaining the reaction temperature between -5 and 0 °C. On complete addition the white slurry was stirred for lh at ~0 °C.

The slurry was filtered, washed with water (500 mL) and dried under a nitrogen sweep overnight to afford 465g (96% yield) of the desired product as a white crystalline solid; XH NMR (CDCI3, 500 MHz): δ 7.35 (t, J = 8.0 Hz, 2H), 7.19 (t, J = 8.0 Hz, 1H), 7.12 (d, J = 8.0 Hz, 2H), 4.95 (br s, 1H), 2.90 (d, J = 5 Hz, 3H).

Step 2

2-(2-Hydroxyacetyl)-N-methylhydrazinecarboxamide: Part A: Phenyl methylcarbamate (300 g, 1.95 mol) was charged to a 2 L vessel with cooling jacket, overhead stirrer, temperature probe, reflux condenser and nitrogen inlet. IPA (390 mL) was added at 23 °C. Hydrazine hydrate (119 g, 2.33 mol) was added and the slurry heated to 75 °C for 6 h.

Part B: On complete reaction (>99% conversion by HPLC), IPA (810 mL) and glycolic acid (222 g, 2.92 mol) were added and the mixture stirred at 83-85 °C for 10-12 h. The reaction mixture was initially a clear colorless solution. The mixture was seeded with product (0.5 g) after 4h at 83-85 °C. The slurry was slowly cooled to 20 °C over 2h and aged for lh. Seed was used to advance the crystallization, but the crystalline product can be precipitated and isolated without seed by allowing the solution to age at 83-85 °C for 4 hours.

The slurry was filtered and washed with IPA (600 mL). The cake was dried under a nitrogen sweep to afford 241.8g (81% yield) of the desired product as a white crystalline solid: XH NMR (D20, 500 MHz): δ 4.11 (s, 2H), 2.60 (s, 3H).

Step 3

3-(Hydroxymethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one: 2-(2-Hydroxyacetyl)-N-methylhydrazinecarboxamide (130 g @ ~95wt%, 0.84 mol), w-propanol (130 mL) and water (130 mL) were charged to a 1 L vessel with jacket, overhead stirrer, temperature probe, reflux condenser and nitrogen inlet. Sodium hydroxide (pellets, 16.8 g, 0.42 mol) was added and the slurry warmed to reflux for 3h. The reaction mixture was cooled to 20 °C and the pH adjusted to 6.5 (+/- 0.5) using concentrated hydrochloric acid (28.3 mL, 0.34 mol). Water was

azeotropically removed under vacuum at 40-50 °C by reducing the volume to -400 mL and maintaining that volume by the slow addition of n-propanol (780 mL). The final water content was <3000 ug/mL. The resultant slurry (~ 400 mL) was cooled to 23 °C and heptane (390 ml) was added. The slurry was aged lh at 23 °C, cooled to 0 °C and aged 2h. The slurry was filtered, the cake washed with 1 :2 n-PrOH/heptane (100 mL) and the filter cake was dried under a nitrogen sweep to provide 125g (85% yield) of an off-white crystalline solid. The solid was -73 wt% due to residual inorganics (NaCl): ¾ NMR (CD3OD, 500 MHz): δ 3.30 (s, 3H), 4.46 (s, 2H).

Step 4

3-(Chloromethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one (1): A mixture of 3-(Hydroxymethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one (54 g, at 73wt%, 307 mmol) in ethyl acetate (540 mL) was stirred at 45 °C. SOCl2 (26.9 mL, 369 mmol) was added over 30-45 min and aged at 50 °C for 2h. The reaction progress was monitored by HPLC. On complete reaction (>99.5% by area at 210nm), the warm suspension was filtered and the filter cake (mainly NaCl) was washed with ethyl acetate (108 mL). The combined filtrate and wash were concentrated at 50-60 °C under reduced pressure to approximately 150 mL. The resulting slurry was cooled to – 10 °C and aged lh. The slurry was filtered and the filter cake washed with ethyl acetate (50 mL). The cake was dried under a nitrogen sweep to afford 40. lg (86% yield) of the desired product as a bright yellow solid: XH NMR (CD3OD, 500 MHz): δ 3.30 (s, 3H), 4.58 (s, 2H).

EXAMPLE 2

Step 1 – Ethyl Ester Synthesis

Experimental Procedure;

A

Ethyl 2-(3-chloro-5-cyanophenoxy)acetate (A): A 1L round bottom flask equipped with overhead stirring was charged with 3-chloro-5-hydroxybenzonitrile (50.0 g, 98 wt% purity, 319 mmol) and 15% aqueous DMF (200 mL DMF + 35.5 mL Η20). To the resulting solution was added diisopropylethylamine (61.3 mL, 99.0% purity, 1.1 equiv) and ethyl 2-bromoacetate (35.7 g, 98% purity, 1.15 equiv) at ambient temperature. The resulting solution was warmed to 50°C under nitrogen and aged for 12 h. Upon completion of the reaction the batch was cooled to 0-5°C. To the clear to slightly cloudy solution was added 5% seed (3.8g, 16.0 mmol). H20 (64.5mL) was added to the thin suspension via syringe pump over 3h while maintaining the temperature at 0-5 °C. Additional H20 (200mL) was added over lh while maintaining the temp at 0-5 °C. The final DMF/H20 ratio is 1 : 1.5. The resulting slurry was aged lh at 0-5 °C. The batch was filtered and the cake slurry washed with 2: 1 DMF/water (150 mL), followed by water (200 mL). The wet cake was dried on the frit with suction under a nitrogen stream at 20-25 °C. The cake is considered dry when H20 is <0.2%. Obtained 73.4 g ethyl ester as a light tan solid, 96% yield: XH NMR (CDC13, 400 MHz) δ = 7.29 (s, 1H), 7.15 (s, 1H), 7.06 (s, 1H), 4.67 (s, 2H), 4.32 (q, 2H), 1.35 (t, 3H) ppm. Seed was used to advance the crystallization, but the crystalline product can be precipitated and isolated without seed by allowing the solution to age at 0-5 °C for at least about 2 hours.

Step 2 – Pyridone Synthesis

Synthetic Scheme;

Experimental Procedures;

Aldol Condensation

(2E/Z,4E)-Ethyl 2-(3-chloro-5-cyanophenoxy)-5-ethoxy-3-(trifluoromethyl)penta-2,4-dienoate (C): Ethyl 2-(3-chloro-5-cyanophenoxy)acetate (25.01 g, 104.4 mmol, 1.00 equiv) was charged to toluene (113.43 g, 131 mL) and 4-ethoxy-l, l,l-trifluoro-3-buten-2-one (26.43 g, 157.2 mmol, 1.51 equiv) was added.

The flow reactor consisted of two feed solution inlets and an outlet to a receiving vessel. The flow reactor schematic is shown in Figure 1.

The ester solution was pumped to one flow reactor inlet. Potassium tert-amylate solution was pumped to the second reactor inlet. Trifluoroacetic anhydride was added continuously to the receiver vessel. Triethylamine was added continuously to the receiver vessel.

The flow rates were: 13 mL/min ester solution, 7.8 mL/min potassium tert-amylate solution, 3.3 mL/min trifluoroacetic anhydride and 4.35 mL/min triethylamine.

Charged toluene (50 mL) and potassium trifluoroacetate (0.64 g, 4.21 mmol, 0.04 equiv) to the receiver vessel. The flow reactor was submerged in a -10 °C bath and the pumps were turned on. The batch temperature in the receiver vessel was maintained at 5 to 10 °C throughout the run using a dry ice/acetone bath. After 13.5 min the ester solution was consumed, the reactor was flushed with toluene (10 mL) and the pumps were turned off.

The resulting yellow slurry was warmed to room temperature and aged for 4.5 h. Charged methanol (160 mL) to afford a homogeneous solution which contained 81.20 LCAP diene .

The solution of diene (573 mL) was used without purification in the subsequent reaction.

Cyclization

3-Chloro-5-((2-oxo-4-(trifluoromethyl)-l,2-dihydropyridin-3-yl)oxy)benzonitrile (E): To a solution of diene in PhMe/MeOH (573 mL; 40.69 g, 104.4 mmol theoretical) was charged methanol (25 mL). Ammonia (32 g, 1.88 mol, 18 equiv based on theoretical) was added and the solution was warmed to 60 °C. The reaction was aged at 60 °C for 18 h. The temperature was adjusted to 35-45 °C and the pressure was decreased to maintain a productive distillation rate. The batch volume was reduced to -300 mL and methanol (325 mL) was charged in portions to maintain a batch volume between 250 and 350 mL. The heating was stopped and the system vented. The resulting slurry was cooled to room temperature and aged overnight.

The batch was filtered and the cake washed with methanol (3x, 45 mL). The wet cake was dried on the frit with suction under a nitrogen stream to afford 18.54 g of a white solid: XH NMR (DMSO-ifc, 500 MHz): δ 12.7 (br s, 1H), 7.73 (t, 1H, J= 1.5 Hz), 7.61-7.59 (m, 2H), 7.53 (t, 1H, J= 2.0 Hz), 6.48 (d, 1H, J= 7.0 Hz) ppm.

Step 3 – Chlorination, Alkylation and Isolation of 3-Chloro-5-({l-[(4-methyl-5-oxo-‘ dihydro-lH-l,2,4-triazol-3-yl)methyl]-2-oxo-4-(trifluoromethyl)-l,2-dihydropyridin-3-yl}oxy)benzonitrile

3-(Chloromethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one: 3-(Hydroxymethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one (1.638 kg of 68wt%, 8.625 mol) and N-methylpyrrolidinone (8.9 L) was charged into a 30 L vessel. The suspension was aged for lOh at ambient temperature. The slurry was filtered through a 4L sintered glass funnel under 2 and the filter cake (mainly NaCl) was washed with NMP (2.23 L). The combined filtrate and wash had a water content of 5750 μg/mL. The solution was charged to a 75L flask equipped with a 2N NaOH scrubber to capture off-gasing vapors. Thionyl chloride (0.795 L, 10.89 mol) was added over lh and the temperature rose to 35 °C. HPLC analysis indicated that the reaction required an additional thionyl chloride charge (0.064 L, 0.878 mol) to bring to full conversion. The solution was warmed to 50 °C, placed under vacuum at 60 Torr (vented to a 2N NaOH scrubber), and gently sparged with subsurface nitrogen (4 L/min). The degassing continued for lOh until the sulfur dioxide content in the solution was <5 mg/mL as determined by quantitative GC/MS. The tan solution of 3-(chloromethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one in NMP weighed 13.0 kg and was assayed at 9.63 wt% providing 1.256 kg (97% yield).

3-chloro-5-((l-((4-methyl-5-oxo-4,5-dihydro-lH-l,2,4-triazol-3-yl)methyl)-2-oxo-4-(trifluoromethyl)-l,2-dihydropyridin-3-yl)oxy)benzonitrile: To a 75L flask was charged a 9.63wt% solution of 3-(chloromethyl)-4-methyl-lH-l,2,4-triazol-5(4H)-one in NMP (1 1.6 kg, 7.55 mol), 3-chloro-5-((2-oxo-4-(trifluoromethyl)-l,2-dihydropyridin-3-yl)oxy)benzonitrile (2.00 kg, 6.29 mol), NMP (3.8 L) and 2-methyl-2-butanol (6.0 L). To the resulting suspension was slowly added N,N-diisopropylethylamine (4.38 L, 25.2 mol) over 4h. The reaction was aged 18h at ambient temperature. The reaction is considered complete when HPLC indicated <1% 3-chloro-5-((2-oxo-4-(trifluoromethyl)-l,2-dihydropyridin-3-yl)oxy)benzonitrile remaining. The tan solution was quenched with acetic acid (1.26 L, 22.0 mol) and aged at ambient temperature overnight. The tan solution was warmed to 70 °C. Water (2.52 L) was added and the batch was seeded with anhydrate Form II (134 g)(procedures for making anhydrate Form II are described in WO2014/052171). The thin suspension was aged lh at 70 °C. Additional water (14.3 L) was added evenly over 7 h. The slurry was aged 2h at 70 °C and then slowly cooled to 20 °C over 5 h. The slurry was filtered and washed with 2 : 1 NMP/water (6 L), followed by water washes (6 L x 2). The filter cake was dried under N2 to give 2.53 kg (85% yield) of a white solid that was confirmed to be crystalline Form II of the title compound by X-ray powder detraction analysis.

EXAMPLE 3

Ethyl 2-(3-chloro-5-cyanophenoxy)acetate (A):

70%

Step 3

Three step one pot sequence

Steps 1 and 2:

To an oven dried 250mL round bottom flask was added sodium 2-methylpropan-2-olate (12.85 g, 134 mmol) and BHT (0.641 g, 2.91 mmol) then added DMF (30mL). After lOmin, a light yellow solution resulted. 2-Phenylethanol (7.66 ml, 63.9 mmol) was added and the solution exothermed to 35 °C. The light yellow solution was warmed to 55 °C and then a solution of 3,5-dichlorobenzonitrile (10 g, 58.1 mmol) in DMF (15mL) was added over 2h via syringe pump. The resulting red-orange suspension was aged at 55-60 °C. After 2h, HPLC showed >98% conversion to the sodium phenolate.

Step 3:

The suspension was cooled to 10 °C, then ethyl 2-bromoacetate (8.70 ml, 78 mmol) was added over lh while maintaining the temperature <20 °C. The resulting mixture was aged at ambient temperature. After lh, HPLC showed >99% conversion to the title compound.

Work-up and isolation:

To the suspension was added MTBE (50mL) and H20 (50mL) and the layers were separated. The organic layer was washed with 20% aq brine (25mL). The organic layer was assayed at 12.5g (90% yield). The organic layer was concentrated to -38 mL, diluted with hexanes (12.5mL) and then cooled to 5 °C. The solution was seeded with 0.28g (2 wt%) of crystalline ethyl 2-(3-chloro-5-cyanophenoxy)acetate and aged 0.5h at 5 °C to give a free flowing slurry. Hexane (175mL) was added to the slurry over lh at 0-5 °C. The slurry was filtered at 0-5 °C, washed with hexane (50 mL) and dried under a nitrogen sweep to give 9.8g (70% yield) of the title compound as a white crystalline solid. Seed was used to advance the crystallization, but the crystalline product can be precipitated and isolated without seed by allowing the solution to age at 0-5 °C for at least about 2 hours.

Paper

Discovery of MK-1439, an orally bioavailable non-nucleoside reverse transcriptase inhibitor potent against a wide range of resistant mutant HIV viruses
Bioorg Med Chem Lett 2014, 24(3): 917

http://www.sciencedirect.com/science/article/pii/S0960894X13014546

The optimization of a novel series of non-nucleoside reverse transcriptase inhibitors (NNRTI) led to the identification of pyridone 36. In cell cultures, this new NNRTI shows a superior potency profile against a range of wild type and clinically relevant, resistant mutant HIV viruses. The overall favorable preclinical pharmacokinetic profile of 36 led to the prediction of a once daily low dose regimen in human. NNRTI 36, now known as MK-1439, is currently in clinical development for the treatment of HIV infection.

Full-size image (16 K)

Full-size image (10 K)

Scheme 1. 

Reagents and conditions: (a) K2CO3, NMP, 120 °C; (b) KOH, tert-BuOH, 75 °C; (c) Zn(CN)2, Pd(PPh3)4, DMF, 100 °C.

Full-size image (12 K)

Scheme 3.

Reagents and conditions: (a) K2CO3, DMF, −10 °C; (b) MeI or EtI, K2CO3, DMF.

36 IS DORAVIRINE

PATENT

WO 2011120133

http://www.google.com/patents/WO2011120133A1?cl=en

Scheme I depicts a method for preparing compounds of Formula I in which hydroxypyridine 1-1 is alkylated with chlorotriazolinone 1-2 to provide 1-3 which can be selectively alkylated with an alkyl halide (e.g., methyl iodide, ethyl iodide, etc.) to afford the desired 1-4. Scheme I

Figure imgf000039_0001

Scheme II depicts an alternative route to compounds of the present invention, wherein fluorohydroxypyridine II-l can be alkylated with chlorotriazolinone II-2 to provide the alkylated product II-3 which can be converted to the desired II-5 via nucleophilic aromatic substitution (S] fAr) using a suitable hydroxyarene II-4.

Scheme II

Figure imgf000039_0002

Hydroxypyridines of formula I-l (Scheme 1) can be prepared in accordance with Scheme III, wherein a SNAr reaction between pyridine III-l (such as commercially available 2- chloro-3-fluoro-4-(trifluoromethyl)pyridine) and hydroxyarene H-4 can provide chloropyridine III-2, which can be hydrolyzed under basic conditions to the hydroxypyridine I-l. Scheme III

Figure imgf000040_0001

Another method for preparing hydroxypyridines of formula I-l is exemplified in Scheme IV, wherein S Ar coupling of commercially available 2-chloro-3-fluoro-4- nitropyridone-N-oxide IV-1 with a suitable hydroxyarene II-4 provides N-oxide IV-2, which can first be converted to dihalides IV-3 and then hydro lyzed to hydroxypyridine IV-4. Further derivatization of hydroxypyridine IV-4 is possible through transition metal-catalyzed coupling processes, such as Stille or boronic acid couplings using a PdLn catalyst (wherein L is a ligand such as triphenylphosphine, tri-tert-butylphosphine or xantphos) to form hydroxypyridines IV-5, or amination chemistry to form hydroxypyridines IV-6 in which R2 is N(RA)RB.

Scheme IV

Figure imgf000040_0002

IV-1

Figure imgf000040_0003

– – Scheme V depicts the introduction of substitution at the five-position of the hydroxypyridines via bromination, and subsequent transition metal-catalyzed chemistries, such as Stille or boronic acid couplings using PdLn in which L is as defined in Scheme IV to form hydroxypyridines V-3, or amination chemistry to form hydroxypyridines V-4 in which R3 is N(RA)RB.

Scheme V

Figure imgf000041_0001

As shown in Scheme IV, fiuorohydroxypyridines II-l (Scheme II) are available from the commercially available 3-fluoroypridines VI- 1 through N-oxide formation and rearrangement as described in Konno et al., Heterocycles 1986, vol. 24, p. 2169.

Scheme VI

Figure imgf000041_0002

The following examples serve only to illustrate the invention and its practice. The examples are not to be construed as limitations on the scope or spirit of the invention.

The term “room temperature” in the examples refers to the ambient temperature which was typically in the range of about 20°C to about 26°C.

EXAMPLE 1

3-Chloro-5-({ l-[(4-methyl-5-oxo-4,5-dihydro-lH-l ,2,4-triazol-3-yl)methyl]-2-oxo-4- (trifluoromethyl)-l ,2-dihydropyridin-3-yl}oxy)benzonitrile (1-1)

Figure imgf000042_0001

Step 1(a):

Figure imgf000042_0002

A mixture of the 3-bromo-5-chlorophenol (3.74 g; 18.0 mmol), 2-chloro-3-fluoro- 4-(trifluoromethyl)pyridine (3.00 g; 15.0 mmol) and 2CO3 (2.49 g; 18.0 mmol) in NMP (15 mL) was heated to 120°C for one hour, then cooled to room temperature. The mixture was then diluted with 250 mL EtOAc and washed with 3 x 250 mL 1 :1 H20:brine. The organic extracts were dried (Na2S04) and concentrated in vacuo. Purification by ISCO CombiFlash (120 g column; load with toluene; 100:0 to 0:100 hexanes:CH2Cl2 over 40 minutes) provided title compound (1-2) as a white solid. Repurification of the mixed fractions provided additional title compound. lH NMR (400 MHz, CDCI3): δ 8.55 (d, J = 5.0 Hz, 1 H); 7.64 (d, J = 5.0 Hz, 1 H);

7.30 (s, 1 H); 6.88 (s, 1 H); 6.77 (s, 1 H).

3-(3-bromo-5-chlorophenoxy)-4-(trifluoromethyl)pyridin-2-ol (1-3)

Figure imgf000042_0003

To a suspension of 3-(3-bromo-5-chlorophenoxy)-2-chloro-4- (trifluoromethyl)pyridine (1-2; 3.48 g; 8.99 mmol) in lBuOH (36 mL) was added KOH (1.51 g; 27.0 mmol) and the mixture was heated to 75°C overnight, at which point a yellow oily solid had precipitated from solution, and LCMS analysis indicated complete conversion. The mixture was cooled to room temperature, and neutralized by the addition of -50 mL saturated aqueous NH4CI. The mixture was diluted with 50 mL H2O, then extracted with 2 x 100 mL EtOAc. The combined organic extracts were dried (Na2S04) and concentrated in vacuo. Purification by ISCO CombiFlash (120 g column; dry load; 100:0 to 90: 10 CH2Cl2:MeOH over 40 minutes) provided the title compound (1-3) as a fluffy white solid. lH NMR (400 MHz, DMSO): δ 12.69 (s, 1 H); 7.59 (d, J = 6.9 Hz, 1 H); 7.43 (t, J = 1.7 Hz, 1 H); 7.20 (t, J = 1.9 Hz, 1 H); 7.13 (t, J = 2.0 Hz, 1 H); 6.48 (d, J = 6.9 Hz, 1 H).

3-chloro-5-{[2-hydroxy-4-(trifluoromethyl)pyridin-3-yl]oxy}benzonitrile (1-4)

Figure imgf000043_0001

To a suspension of 3-(3-bromo-5-chlorophenoxy)-4-(trifluoromethyl)pyridin-2-ol (1-3; 3.25 g; 8.82 mmol) in NMP (29 mL) was added CuCN (7.90 g; 88 mmol) and the mixture was heated to 175°C for 5 hours, then cooled to room temperature slowly. With increased fumehood ventilation, 100 mL glacial AcOH was added, then 100 mL EtOAc and the mixture was filtered through Celite (EtOAc rinse). The filtrate was washed with 3 x 200 mL 1 : 1 H20:brine, then the organic extracts were dried (Na2S04) and concentrated in vacuo.

Purification by ISCO CombiFlash (120 g column; dry load; 100:0 to 90:10 CH2Cl2:MeOH over 40 minutes), then trituration of the derived solid with Et20 (to remove residual NMP which had co-eluted with the product) provided the title compound (1-4). lH NMR (400 MHz, DMSO): δ 12.71 (s, 1 H); 7.75 (s, 1 H); 7.63-7.57 (m, 2 H); 7.54 (s, 1 H); 6.49 (d, J = 6.9 Hz, 1 H).

Step 1(d): 5-(chloromethyl)-2,4-dihydro-3H-l,2,4-triazol-3-one (1-5)

Figure imgf000043_0002

The title compound was prepared as described in the literature: Cowden, C. J.; Wilson, R. D.; Bishop, B. C; Cottrell, I. F.; Davies, A. J.; Dolling, U.-H. Tetrahedron Lett. 2000, 47, 8661.

3 -chloro-5 -( { 2-oxo- 1 – [(5 -oxo-4,5 -dihydro- 1 H- 1 ,2,4-triazol-3 -yl)methyl] – 4- (trifiuoromethyl)- 1 ,2-dihydropyridin-3 -yl } oxy)benzonitrile (1-6)

Figure imgf000044_0001

A suspension of the 3-chloro-5-{[2-hydroxy-4-(trifluoromethyl)pyridin-3- yl]oxy}benzonitrile (1-4; 2.00 g; 6.36 mmol), 5-(chloromethyl)-2,4-dihydro-3H-l,2,4-triazol-3- one (1-5; 0.849 g; 6.36 mmol) and K2CO3 (0.878 g; 6.36 mmol) in DMF (32 mL) was stirred for 2 hours at room temperature, at which point LCMS analysis indicated complete conversion. The mixture was diluted with 200 mL Me-THF and washed with 150 mL 1 : 1 : 1 H20:brine:saturated aqueous NH4CI, then further washed with 2 x 150 mL 1 : 1 H20:brine. The aqueous fractions were further extracted with 150 mL Me-THF, then the combined organic extracts were dried (Na2S04) and concentrated in vacuo. Purification by ISCO CombiFlash (80 g column; dry load; 100:0 to 90:10 EtOAc:EtOH over 25 minutes) provided the title compound (1-6) as a white solid. lH NMR (400 MHz, DMSO): δ 1 1.46 (s, 1 H); 1 1.39 (s, 1 H); 7.93 (d, J = 7.3 Hz, 1 H); 7.76 (s, 1 H); 7.58 (s, 1 H); 7.51 (s, 1 H); 6.67 (d, J = 7.3 Hz, 1 H); 5.02 (s, 2 H).

Step 1(f): 3 -chloro-5 -( { 1 – [(4-methyl-5-oxo-4,5 -dihydro- 1 H- 1 ,2,4-triazol-3 -yl)methyl] -2- oxo-4-(trifluoromethyl)- 1 ,2-dihydropyridin-3 -yl } oxy)benzonitrile (1 -1 )

A solution of 3-chloro-5-({2-oxo-l -[(5-oxo-4,5-dihydro-lH-l,2,4-triazol-3- yl)methyl]- 4-(trifluoromethyl)-l ,2-dihydropyridin-3-yl}oxy)benzonitrile (1-6; 2.37 g; 5.76 mmol) and K2CO3 (0.796 g; 5.76 mmol) in DMF (58 mL) was cooled to 0°C, then methyl iodide (0.360 mL; 5.76 mmol) was added. The mixture was allowed to warm to room

temperature, and stirred for 90 minutes, at which point LCMS analysis indicated >95%

conversion, and the desired product of -75% LCAP purity, with the remainder being unreacted starting material and 6/s-methylation products. The mixture was diluted with 200 mL Me-THF, and washed with 3 x 200 mL 1 : 1 H20:brine. The aqueous fractions were further extracted with 200 mL Me-THF, then the combined organic extracts were dried (Na2S04) and concentrated in vacuo. The resulting white solid was first triturated with 100 mL EtOAc, then with 50 mL THF, which provided (after drying) the title compound (1-1) of >95% LCAP. Purification to >99% LCAP is possible using Prep LCMS (Max-RP, 100 x 30 mm column; 30-60% CH3CN in 0.6% aqueous HCOOH over 8.3 min; 25 mL/min). lH NMR (400 MHz, DMSO): δ 1 1.69 (s, 1 H); 7.88 (d, J = 7.3 Hz, 1 H); 7.75 (s, 1 H); 7.62 (s, 1 H); 7.54 (s, 1 H); 6.67 (d, J = 7.3 Hz, 1 H); 5.17 (s, 2 H); 3.1 1 (s, 3 H). EXAMPLE 1A

3-Chloro-5-({ l-[(4-methyl-5-oxo-4,5-dihydro-lH-l ,2,4-triazol-3-yl)methyl]-2- (trifluoromethyl)-l ,2-dihydropyridin-3-yl}oxy)benzonitrile (1-1)

Figure imgf000045_0001

Step lA(a): 2-chloro-3-(3-chloro-5-iodophenoxy)-4-(trifluoromethyl)pyridine (1A-2)

Figure imgf000045_0002

A mixture of the 3-chloro-l-iodophenol (208 g; 816.0 mmol), 2-chloro-3-fluoro-

4-(trifluoromethyl)pyridine (155 g; 777.0 mmol) and K2CO3 (161 g; 1 165.0 mmol) in NMP (1.5 L) was held at 60°C for 2.5 hours, and then left at room temperature for 2 days. The mixture was then re-heated to 60°C for 3 hours, then cooled to room temperature. The mixture was then diluted with 4 L EtOAc and washed with 2 L water + 1 L brine. The combined organics were then washed 2x with 500 mL half brine then 500 mL brine, dried over MgS04 and concentrated to afford crude 1A-2. lH NMR (500 MHz, DMSO) δ 8.67 (d, J = 5.0 Hz, 1 H), 7.98 (d, J = 5.0 Hz, 1 H), 7.63-7.62 (m, 1 H), 7.42-7.40 (m, 1 H), 7.22 (t, J = 2.1 Hz, 1 H).

Step lA(b): 2-chloro-3-(3-chloro-5-iodophenoxy)-4-(trifluoromethyl)pyridine (1A-3)

Figure imgf000045_0003

To a suspension of 3-(3-chloro-5-iodophenoxy)-2-chloro-4- (trifluoromethyl)pyridine (1A-2; 421 g, 970 mmol) in t-BuOH (1 L) was added KOH (272 g, 4850 mmol) and the mixture was heated to 75°C for 1 hour, at which point HPLC analysis indicated >95% conversion. The t-BuOH was evaporated and the mixture diluted with water (7mL/g, 2.4L) and then cooled to 0°C, after which 12N HC1 (~240mL) was added until pH 5. This mixture was then extracted with EtOAc (20mL/g, 6.5L), back extracted with EtOAc 1 x 5mL/g (1.5L), washed 1 x water:brine 1 : 1 (l OmL/g, 3.2L), 1 x brine (lOmL/g, 3.2L), dried over MgS04, filtered and concentrated to afford a crude proudct. The crude product was suspended in MTBE (2.25 L, 7mL/g), after which hexanes (1 L, 3 mL/g) was added to the suspension over ten minutes, and the mixturen was aged 30minutes at room temperature. The product was filtered on a Buchner, rinsed with MTBE hexanes 1 :2 (2 mL/g = 640 mL), then hexanes

(640mL), and dried on frit to afford 1A-3. lH NMR (400 MHz, acetone-d6): δ 11.52 (s, 1 H); 7.63 (d, J = 7.01 Hz, 1 H); 7.50-7.48 (m, 1 H); 7.34-7.32 (m, 1 H); 7.09-7.07 (m, 1 H); 6.48 (d, J = 7.01 Hz, 1 H).

Step lA(c): 3-chloro-5-{[2-hydroxy-4-(trifluoromethyl)pyridin-3-yl]oxy}benzonitrile (1-4)

Figure imgf000046_0001

A solution of 3-(3-chloro-5-iodophenoxy)-4-(trifluoromethyl)pyridin-2-ol (1A-3; 190 g; 457 mmol) in DMF (914 mL) was degassed for 20 minutes by bubbling N2, after which CuCN (73.7 g; 823 mmol) was added, and then the mixture was degassed an additional 5 minutes. The mixture was then heated to 120°C for 17 hours, then cooled to room temperature and partitioned between 6 L MeTHF and 2 L ammonium buffer (4:3: 1 = NH4CI

sat/water/NH-iOH 30%). The organic layer washed with 2 L buffer, 1 L buffer and 1 L brine then, dried over MgS04 and concentrated. The crude solid was then stirred in 2.2 L of refluxing

MeCN for 45 minutes, then cooled in a bath to room temperature over 1 hour, aged 30 minutes, then filtered and rinsed with cold MeCN (2 x 400mL). The solid was dried on frit under N2 atm for 60 hours to afford title compound 1-4. lH NMR (400 MHz, DMSO): δ 12.71 (s, 1 H); 7.75 (s, 1 H); 7.63-7.57 (m, 2 H); 7.54 (s, 1 H); 6.49 (d, J = 6.9 Hz, 1 H).

Steps lA(d) and lA(e)

The title compound 1-1 was then prepared from compound 1-4 using procedures similar to those described in Steps 1(d) and 1(e) set forth above in Example 1.

Patent

WO-2014052171

Crystalline anhydrous Form II of doravirine, useful for the treatment of HIV-1 and HIV-2 infections. The compound was originally claimed in WO2008076223. Also see WO2011120133. Merck & Co is developing doravirine (MK-1439), for the oral tablet treatment of HIV-1 infection. As of April 2014, the drug is in Phase 2 trials.

CLIPS

The next-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) doravirine (formerly MK-1439) showed potent antiretroviral activity and good tolerability in combination with tenofovir/FTC (the drugs in Truvada) in a dose-finding study presented at the 21st Conference on Retroviruses and Opportunistic Infections (CROI) last week in Boston.

NNRTIs are generally well tolerated and well suited for first-line HIV treatment, but as a class they are susceptible to resistance. Pre-clinical studies showed that Merck’s doravirine has a distinct resistance profile and remains active against HIV with common NNRTI resistance mutations including K103N and Y181C.

As reported at last year’s CROI, doravirine reduced HIV viral load by about 1.3 log in a seven-day monotherapy study. Doravirine is processed by the CYP3A4 enzyme, but it is neither a CYP3A4 inducer nor inhibitor, so it is not expected to have major drug interaction concerns.

Javier Morales-Ramirez from Clinical Research Puerto Rico reported late-breaking findings from a phase 2b study evaluating the safety and efficacy of various doses of doravirine versus efavirenz (Sustiva) for initial antiretroviral therapy.

This study included 208 treatment-naive people living with HIV from North America, Europe and Asia. More than 90% were men, 74% were white, 20% were black and the median age was 35 years. At baseline, the median CD4 cell count was approximately 375 cells/mm3 and 13% had received an AIDS diagnosis. Study participants were stratified by whether their viral load was above (about 30%) or below 100,000 copies/ml; median HIV RNA was approximately 4.5 log10.

Morales-Ramirez reported 24-week results from part 1 of the study, which will continue for a total of 96 weeks. In this part, participants were randomly allocated into five equal-sized arms receiving doravirine at doses of 25, 50, 100 or 200mg once daily, or else efavirenz once daily, all in combination with tenofovir/FTC.

At 24 weeks, 76.4% of participants taking doravirine had viral load below 40 copies/ml compared with 64.3% of people taking efavirenz. Response rates were similar across doravirine doses (25mg: 80.0%; 50mg: 76.2%; 100mg: 71.4%; 200mg: 78.0%). More than 80% of participants in all treatment arms reached the less stringent virological response threshold of <200 copies/ml.

Both doravirine and efavirenz worked better for people with lower pre-treatment viral load in an ad hoc analysis. For people with <100,000 copies/ml at baseline, response rates (<40 copies/ml) ranged from 83 to 89% with doravirine compared with 74% with efavirenz. For those with >100,000 copies/ml, response rates ranged from 50 to 91% with doravirine vs 54% with efavirenz.

Median CD4 cell gains were 137 cells/mm3 for all doravirine arms combined and 121 cells/mmfor the efavirenz arm.

Doravirine was generally safe and well tolerated. People taking doravirine were less than half as likely as people taking efavirenz to experience serious adverse events (3.0% across all doravirine arms vs 7.1% with efavirenz) or to stop treatment for this reason (2.4 vs 4.8%). Four people taking doravirine and two people taking efavirenz discontinued due to adverse events considered to be drug-related.

The most common side-effects were dizziness (3.6% with doravirine vs 23.8% with efavirenz), abnormal dreams (9.0 vs 7.1%), diarrhoea (4.8 vs 9.5%), nausea (7.8 vs 2.4%) and fatigue (6.6 vs 4.8%). Other central nervous system (CNS) adverse events of interest included insomnia (5.4 vs 7.1%), nightmares (1.2 vs 9.5%) and hallucinations (0.6 vs 2.4%). Overall, 20.5% of people taking doravirine reported at least one CNS side-effect, compared with 33.3% of people taking efavirenz.

People taking doravirine had more favourable lipid profiles and less frequent liver enzyme (ALT and AST) elevations compared with people taking efavirenz.

The researchers concluded that doravirine demonstrated potent antiretroviral activity in treatment-naive patients, a favourable safety and tolerability profile, and fewer drug-related adverse events compared with efavirenz.

Based on these findings, the 100mg once-daily dose was selected for future development and will be used in part 2 of this study, a dose-confirmation analysis that will enrol an additional 120 participants.

In the discussion following the presentation, Daniel Kuritzkes from Harvard Medical School noted that sometimes it takes longer for viral load to go down in people who start with a high level, so with further follow-up past 24 weeks doravirine may no longer look less effective in such individuals.

Reference

Morales-Ramirez J et al. Safety and antiviral effect of MK-1439, a novel NNRTI (+FTC/TDF) in ART-naive HIV-infected patients. 21st Conference on Retroviruses and Opportunistic Infections, Boston, abstract 92LB, 2014.

Merck Moves Doravirine Into Phase 3 Clinical Trials

Wednesday Mar 19 | Posted by: roboblogger | Full story: EDGE

Earlier this month, at the 21st Conference on Retroviruses and Opportunistic Infections , Merck indicated plans to initiate a Phase 3 clinical trial program for doravirine in combination with ARV therapy in the second half of 2014.

PAPER

A Robust Kilo-Scale Synthesis of Doravirine

Process Research and Development, Merck Research Laboratories, 126 E. Lincoln Ave., Rahway, New Jersey 07065,United States
Process Research and Development, Merck Frosst Center for Therapeutic Research, 16711 Trans Canada Highway, Kirkland, Quebec H9H 3L1, Canada
WuXi AppTec Co., Ltd., No. 1 Building, No. 288 FuTe ZhongLu, WaiGaoQiao Free Trade Zone, Shanghai 200131, China
Org. Process Res. Dev., Article ASAP
Abstract Image

Doravirine is non-nucleoside reverse transcriptase inhibitor (NNRTI) currently in phase III clinical trials for the treatment of HIV infection. Herein we describe a robust kilo-scale synthesis for its manufacture. The structure and origin of major impurities were determined and their downstream fate-and-purge studied. This resulted in a redesign of the route to introduce the key nitrile functionality via a copper mediated cyanation which allowed all impurities to be controlled to an acceptable level. The improved synthesis was scaled to prepare ∼100 kg batches of doravirine to supply all preclinical and clinical studies up to phase III. The synthesis affords high-quality material in a longest linear sequence of six steps and 37% overall yield.

PAPER

Highly Efficient Synthesis of HIV NNRTI Doravirine

Department of Process Chemistry, Merck & Co., Inc., P.O. Box 2000, Rahway, New Jersey 07065, United States
Org. Lett., 2015, 17 (6), pp 1353–1356
DOI: 10.1021/ol503625z
Publication Date (Web): March 09, 2015
Copyright © 2015 American Chemical Society

Gauthier, D. R., Jr.; Sherry, B. D.; Cao, Y.; Journet, M.; Humphrey, G.; Itoh, T.; Mangion, I.; Tschaen, D. M.Org. Lett. 2015, 17, 1353, DOI: 10.1021/ol503625z………..http://pubs.acs.org/doi/full/10.1021/ol503625z

STR1

US20100034813 * 8 Nov 2007 11 Feb 2010 Yi Xia Substituted pyrazole and triazole compounds as ksp inhibitors
US20100256181 * 14 Nov 2008 7 Oct 2010 Tucker Thomas J Non-nucleoside reverse transcriptase inhibitors
US20110245296 * 6 Oct 2011 Jason Burch Non-nucleoside reverse transcriptase inhibitors
Reference
1 * COWDEN ET AL.: “A new synthesis of 1,2,4-triazolin-5-ones: application to the convergent synthesis of an NK1 antagonist.“, TETRAHEDRON LETTERS, vol. 41, no. 44, 2000, pages 8661 – 8664, XP004236142
Patent ID Date Patent Title
US2015329521 2015-11-19 PROCESS FOR MAKING REVERSE TRANSCRIPTASE INHIBITORS
US9150539 2015-10-06 Crystalline form of a reverse transcriptase inhibitor
US2015232447 2015-08-20 CRYSTALLINE FORM OF A REVERSE TRANSCRIPTASE INHIBITOR
US2013296382 2013-11-07 NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS
US2011245296 2011-10-06 NON-NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS

References

  1.  Collins, Simon; Horn, Tim. “The Antiretroviral Pipeline.” (PDF). Pipeline Report. p. 10. Retrieved 6 December 2015.
  2. Safety and Antiviral Activity of MK-1439, a Novel NNRTI, in Treatment-naïve HIV+ Patients. Gathe, Joseph et al. 20th Conference on Retroviruses and Opportunistic Infections. 3–6 March 2013. Abstract 100.
  3.  CROI 2013: MK-1439, a Novel HIV NNRTI, Shows Promise in Early Clinical Trials. Highleyman, Liz. HIVandHepatitis.com. 6 March 2013.
Doravirine
Doravirine structure.svg
Systematic (IUPAC) name
3-Chloro-5-({1-[(4-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)methyl]-2-oxo-4-(trifluoromethyl)-1,2-dihydro-3-pyridinyl}oxy)benzonitrile
Clinical data
Routes of
administration
Oral[1]
Legal status
Legal status
  • Investigational New Drug
Identifiers
CAS Number 1338225-97-0
ATC code none
PubChem CID 58460047
ChemSpider 28424197
UNII 913P6LK81M Yes
KEGG D10624
ChEMBL CHEMBL2364608
Synonyms MK-1439
PDB ligand ID 2KW (PDBe, RCSB PDB)
Chemical data
Formula C17H11ClF3N5O3
Molar mass 425.75 g/mol

//////////Doravirine, MK-1439, 1338225-97-0 , Merck Sharp & Dohme Corp, Reverse transcriptase inhibitor, ANTIVIRAL, Non-nucleoside reverse transcriptase, HIV, Triazolinone, Pyridone, Inhibitor,

Supporting Info

AND

Supporting Info

Cn1c(n[nH]c1=O)Cn2ccc(c(c2=O)Oc3cc(cc(c3)Cl)C#N)C(F)(F)F

OSILODROSTAT for Treatment of Cushing’s Syndrome


ChemSpider 2D Image | osilodrostat | C13H10FN3

OSILODROSTAT

LCI 699, LCI 699NX

Novartis Ag INNOVATOR

UNII-5YL4IQ1078, CAS 928134-65-0

Benzonitrile, 4-[(5R)-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluoro-
4-[(5R)-6,7-Dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl]-3-fluorobenzonitrile
(R)-4-(6,7-Dihydro-5H-pyrrolo[l,2-c]imidazol-5-yl)-3-fluoro- benzonitrile
  • Molecular FormulaC13H10FN3
  • Average mass227.237 Da
  • Originator Novartis
  • Class Antihypertensives; Fluorobenzenes; Imidazoles; Nitriles; Pyridines; Small molecules
  • Mechanism of Action Aldosterone synthase inhibitors
  • Phase III Cushing syndrome
  • Phase I Liver disorders
  • Discontinued Heart failure; Hypertension; Solid tumours

Most Recent Events

  • 27 Feb 2016 Novartis plans the phase III LINC-4 trial for Cushing’s syndrome in Greece, Thailand, Poland, Turkey, Russia, Brazil, Belgium, Spain, Denmark, Switzerland and USA (PO) (NCT02697734)
  • 12 Jun 2015 Novartis plans a phase II trial for Cushing syndrome in Japan (NCT02468193)
  • 01 Apr 2015 Phase-I clinical trials in Liver disorders in USA (PO)

Osilodrostat phosphate
CAS: 1315449-72-9

MF, C13-H10-F-N3.H3-O4-P

MW, 325.2347

  • LCI 699AZA

An orally active aldosterone-synthase inhibitor.

for Treatment of Cushing’s Syndrome

4-((5R)-6,7-Dihydro-5H-pyrrolo(1,2-c)imidazol-5-yl)-3-fluorobenzonitrile dihydrogen phosphate

Aromatase inhibitor; Cytochrome P450 11B1 inhibitor

MORE SYNTHESIS COMING, WATCH THIS SPACE…………………..

 

SYNTHESIS

STR1

ACS Medicinal Chemistry Letters, 4(12), 1203-1207; 2013

REMIND ME,  amcrasto@gmail.com, +919323115463

Osilodrostat, as modulators of 11-β-hydroxylase, useful for treating a disorder ameliorated 11-β-hydroxylase inhibition eg Cushing’s disease, hypertension, congestive heart failure, metabolic syndrome, liver diseases, cerebrovascular diseases, migraine headaches, osteoporosis or prostate cancer.

Novartis is developing osilodrostat, an inhibitor of aldosterone synthase and aromatase, for treating Cushing’s disease. In July 2016, osilodrostat was reported to be in phase 3 clinical development.

The somatostatin analog pasireotide and the 11β-hydroxylase inhibitor osilodrostat (LCI699) reduce cortisol levels by distinct mechanisms of action. There exists a scientific rationale to investigate the clinical efficacy of these two agents in combination. This manuscript reports the results of a toxicology study in rats, evaluating different doses of osilodrostat and pasireotide alone and in combination. Sixty male and 60 female rats were randomized into single-sex groups to receive daily doses of pasireotide (0.3mg/kg/day, subcutaneously), osilodrostat (20mg/kg/day, orally), osilodrostat/pasireotide in combination (low dose, 1.5/0.03mg/kg/day; mid-dose, 5/0.1mg/kg/day; or high dose, 20/0.3mg/kg/day), or vehicle for 13weeks. Mean body-weight gains from baseline to Week 13 were significantly lower in the pasireotide-alone and combined-treatment groups compared to controls, and were significantly higher in female rats receiving osilodrostat monotherapy. Osilodrostat and pasireotide monotherapies were associated with significant changes in the histology and mean weights of the pituitary and adrenal glands, liver, and ovary/oviduct. Osilodrostat alone was associated with adrenocortical hypertrophy and hepatocellular hypertrophy. In combination, osilodrostat/pasireotide did not exacerbate any target organ changes and ameliorated the liver and adrenal gland changes observed with monotherapy. Cmax and AUC0-24h of osilodrostat and pasireotide increased in an approximately dose-proportional manner. In conclusion, the pasireotide and osilodrostat combination did not exacerbate changes in target organ weight or toxicity compared with either monotherapy, and had an acceptable safety profile; addition of pasireotide to the osilodrostat regimen may attenuate potential adrenal gland hyperactivation and hepatocellular hypertrophy, which are potential side effects of osilodrostat monotherapy.

The somatostatin analog pasireotide and the 11β-hydroxylase inhibitor osilodrostat (LCI699) reduce cortisol levels by distinct mechanisms of action. There exists a scientific rationale to investigate the clinical efficacy of these two agents in combination. This manuscript reports the results of a toxicology study in rats, evaluating different doses of osilodrostat and pasireotide alone and in combination. Sixty male and 60 female rats were randomized into single-sex groups to receive daily doses of pasireotide (0.3 mg/kg/day, subcutaneously), osilodrostat (20 mg/kg/day, orally), osilodrostat/pasireotide in combination (low dose, 1.5/0.03 mg/kg/day; mid-dose, 5/0.1 mg/kg/day; or high dose, 20/0.3 mg/kg/day), or vehicle for 13 weeks. Mean body-weight gains from baseline to Week 13 were significantly lower in the pasireotide-alone and combined-treatment groups compared to controls, and were significantly higher in female rats receiving osilodrostat monotherapy. Osilodrostat and pasireotide monotherapies were associated with significant changes in the histology and mean weights of the pituitary and adrenal glands, liver, and ovary/oviduct. Osilodrostat alone was associated with adrenocortical hypertrophy and hepatocellular hypertrophy. In combination, osilodrostat/pasireotide did not exacerbate any target organ changes and ameliorated the liver and adrenal gland changes observed with monotherapy. Cmax and AUC0–24h of osilodrostat and pasireotide increased in an approximately dose-proportional manner.

In conclusion, the pasireotide and osilodrostat combination did not exacerbate changes in target organ weight or toxicity compared with either monotherapy, and had an acceptable safety profile; addition of pasireotide to the osilodrostat regimen may attenuate potential adrenal gland hyperactivation and hepatocellular hypertrophy, which are potential side effects of osilodrostat monotherapy.

The somatostatin class is a known class of small peptides comprising the naturally occurring somatostatin- 14 and analogues having somatostatin related activity, e.g. as disclosed by A.S. Dutta in Small Peptides, Vol.19, Elsevier (1993). By “somatostatin analogue” as used herein is meant any straight-chain or cyclic polypeptide having a structure based on that of the naturally occurring somatostatin- 14 wherein one or more amino acid units have been omitted and/or replaced by one or more other amino radical(s) and/or wherein one or more functional groups have been replaced by one or more other functional groups and/or one or more groups have been replaced by one or several other isosteric groups. In general, the term covers all modified derivatives of the native somatostatin- 14 which exhibit a somatostatin related activity, e.g. they bind to at least one of the five somatostatin receptor (SSTR), preferably in the nMolar range. Commonly known somatostatin analogs are octreotide, vapreotide, lanreotide, pasireotide.

Pasireotide, having the chemical structure as follow:

Figure imgf000002_0001

Pasireotide is called cyclo[{4-(NH2-C2H4-NH-CO-0-)Pro}-Phg-DTrp-Lys-Tyr(4-Bzl)- Phe], wherein Phg means -HN-CH(C6H5)-CO- and Bzl means benzyl, in free form, in salt or complex form or in protected form.

Cushing’s syndrome is a hormone disorder caused by high levels of Cortisol in the blood. This can be caused by taking glucocorticoid drugs, or by tumors that produce Cortisol or adrenocorticotropic hormone (ACTH) or CRH. Cushing’s disease refers to one specific cause of the syndrome: a tumor (adenoma) in the pituitary gland that produces large amounts of ACTH, which elevates Cortisol. It is the most common cause of Cushing’s syndrome, responsible for 70% of cases excluding glucocorticoid related cases. The significant decrease of Cortisol levels in Cushing’s disease patients on pasireotide support its potential use as a targeted treatment for Cushing’s disease (Colao et al. N Engl J Med 2012;366:32^12).

Compound A is potent inhibitor of the rate-limiting enzyme 1 1-beta-hydroxylase, the last step in the synthesis of Cortisol. WO 201 1/088188 suggests the potential use of compound A in treating a disease or disorder characterised by increased stress hormone levels and/or decreased androgen hormone levels, including the potential use of compound A in treating heart failure, cachexia, acute coronary syndrome, chronic stress syndrome, Cushing’s syndrome or metabolic syndrome.

Compound A, also called (R)-4-(6,7-Dihydro-5H-pyrrolo[l,2-c]imidazol-5-yl)-3-fluoro- benzonitrile, has formula (II).

Figure imgf000003_0001

Compound A can be synthesized or produced and characterized by methods as described in WO2007/024945.

PRODUCT PATENT

WO2007024945, hold protection in the EU states until August 2026, and expire in the US in March 2029 with US154 extension

PAPER

ACS Medicinal Chemistry Letters (2013), 4(12), 1203-1207.

http://pubs.acs.org/doi/abs/10.1021/ml400324c?source=chemport&journalCode=amclct

Discovery and in Vivo Evaluation of Potent Dual CYP11B2 (Aldosterone Synthase) and CYP11B1 Inhibitors

Novartis Institutes for BioMedical Research, 100 Technology Square, Cambridge, Massachusetts 02139, United States
Novartis Pharmaceuticals Corporation, East Hanover, New Jersey 07936, United States
ACS Med. Chem. Lett., 2013, 4 (12), pp 1203–1207
DOI: 10.1021/ml400324c
*(E.L.M.) Tel: 617-871-7586. Fax: 617-871-7045. E-mail: erik.meredith@novartis.com.
Abstract Image

Aldosterone is a key signaling component of the renin-angiotensin-aldosterone system and as such has been shown to contribute to cardiovascular pathology such as hypertension and heart failure. Aldosterone synthase (CYP11B2) is responsible for the final three steps of aldosterone synthesis and thus is a viable therapeutic target. A series of imidazole derived inhibitors, including clinical candidate 7n, have been identified through design and structure–activity relationship studies both in vitro and in vivo. Compound 7n was also found to be a potent inhibitor of 11β-hydroxylase (CYP11B1), which is responsible for cortisol production. Inhibition of CYP11B1 is being evaluated in the clinic for potential treatment of hypercortisol diseases such as Cushing’s syndrome.

PATENT

WO-2016109361

silodrostat (LCI699; 4-[(5R)-6,7-dihydro-5H-pyrrolo[l,2-c]imidazol-5-yl]-3-fluoro-benzonitrile; CAS# 928134-65-0). Osilodrostat is a Ι Ι-β-hydroxylase inhibitor.

Osilodrostat is currently under investigation for the treatment of Cushing’s disease, primary aldosteronism, and hypertension. Osilodrostat has also shown promise in treating drug-resistant hypertension, essential hypertension, hypokalemia, hypertension, congestive heart failure, acute heart failure, heart failure, cachexia, acute coronary syndrome, chronic stress syndrome, Cushing’s syndrome, metabolic syndrome, hypercortisolemia, atrial fibrillation, renal failure, chronic renal failure, restenosis, sleep apnea, atherosclerosis, syndrome X, obesity, nephropathy, post-myocardial infarction, coronary heary disease, increased formation of collagen, cardiac or myocardiac fibrosis and/or remodeling following hypertension and endothelial dysfunction, Conn’s disease, cardiovascular diseases, renal dysfunction, liver diseases, cerebrovascular diseases, vascular diseases, retinopathy, neuropathy, insulinopathy, edema, endothelial dysfunction, baroreceptor dysfunction, migraine headaches, arrythmia, diastolic dysfunction, diastolic heart failure, impaired diastolic filling, systolic dysfunction, ischemia, hypertrophic cardiomyopathy, sudden cardia death, impaired arterial compliance, myocardial necrotic lesions, vascular damage, myocardial infarction, left ventricular hypertrophy, decreased ej ection fraction, cardiac lesions, vascular wall hypertrophy, endothelial thickening, fibrinoid, necrosis of coronary arteries, ectopic ACTH syndrome, change in adrenocortical mass, primary pigmented nodular adrenocortical disease (PPNAD), Carney complex (CNC), anorexia nervosa, chronic alcoholic poisoning, nicotine withdrawal syndrome, cocaine withdrawal syndrome, posttraumatic stress syndrome, cognitive impairment after a stroke or cortisol-induced mineral corticoid excess, ventricular arrythmia, estrogen-dependent disorders, gynecomastia, osteoporosis, prostate cancer, endometriosis, uterine fibroids, dysfunctional uterine bleeding, endometrial hyperplasia, polycyctic ovarian disease, infertility, fibrocystic breast disease, breast cancer, and fibrocystic mastopathy. WO 2013109514; WO 2007024945; and WO 2011064376.

Osilodrostat

Osilodrostat is likely subject to extensive CYP45o-mediated oxidative metabolism. These, as well as other metabolic transformations, occur in part through polymorphically-expressed enzymes, exacerbating interpatient variability. Additionally, some metabolites of osilodrostat derivatives may have undesirable side effects. In order to overcome its short half-life, the drug likely must be taken several times per day, which increases the probability of patient incompliance and discontinuance. Adverse effects associated with osilodrostat include fatigue, nausea, diarrhea, headache, hypokalemia, muscle spasms, vomiting, abdominal discomfort, abdominal pain, arthralgia, arthropod bite, dizziness, increased lipase, and pruritis.

Scheme I

EXAMPLE 1

(R)-4-(6,7-dihvdro-5H-pyrrolo[l,2-elimidazol-5-yl)-3-fluorobenzonitrile

(osilodrostat)

[00144] 4-(bromomethyl)-3-fluorobenzonitrile: 3-Fluoro-4-methylbenzonitrile (40 g, 296 mmol), NBS (63.2 g, 356 mmol) and benzoyl peroxide (3.6 g, 14.8 mmol) were taken up in carbon tetrachloride (490 mL) and refiuxed for 16 h. The mixture was allowed to cool to room temperature and filtered. The filtrate was concentrated and purified via flash column chromatography (0-5% EtOAc/hexanes) to give 4-(bromomethyl)-3-fluorobenzonitrile (35.4 g, 56%).

[00145] 2-(l-trityl-lH-imidazol-4-yl)acetic acid: Trityl chloride (40 g, 143.88 mmol, 1.2 equiv) was added to a suspension of (lH-imidazol-4-yl) acetic acid hydrochloride (20 g, 123.02 mmol, 1.0 equiv) in pyridine (200 mL). This was stirred at 50 °C for 16 h. Then the mixture was cooled and concentrated under vacuum and the crude product was purified by recrystallization from ethyl acetate (1000 ml) to afford 42 g (90%) of 2-[l-(triphenylmethyl)-lH-imidazol-4-yl] acetic acid as an off-white solid. LCMS (ESI): m/z = 369.2 [M+H]+

Step 2

2 step 2

2-( 1 -trityl- lH-imidazol-4-yl)ethanol : 2-(l-Trityl-lH-imidazol-4-yl) acetic acid (42 g, 114.00 mmol, 1.0 equiv) was suspended in THF (420 mL) and cooled to 0 °C. To this was added BH3 (1M in THF, 228.28 mL, 2.0 equiv). The clear solution obtained was stirred at 0 °C for 60 min, then warmed to room temperature until LCMS indicated completion of the reaction. The solution was cooled again to 0 °C and quenched carefully with water (300 mL). The resulting solution was extracted with ethyl acetate (3 x 100 mL) and the organic layers combined and dried over anhydrous Na2S04 and evaporated to give a sticky residue which was taken up in ethanolamine (800 mL) and heated to 90 °C for 2 h. The reaction was transferred to a separatory funnel, diluted with EtOAc (1 L) and washed with water (3 x 600 mL). The organic phase was dried over anhydrous Na2S04 and evaporated afford 35 g (87%) of 2-[l-(triphenylmethyl)-lH-imidazol-4-yl]ethanol as a white solid, which was used in the next step without further purification. LCMS (ESI) : m/z = 355.1 [M+H]+.

Step 3

3 step 3 4

4-(2-(tert-butyldimethylsilyloxy)ethyl)-l-trityl-lH-imidazole: 2-(l-Trityl-lH-imidazol-4-yl) ethanol (35 g, 98.75 mmol, 1.00 equiv) was dissolved in DCM (210 mL). To this was added imidazole (19.95 g, 293.05 mmol, 3.00 equiv) and tert-butyldimethylsilylchloride (22.40 g, 149.27 mmol, 1.50 equiv). The mixture was stirred at room temperature until LCMS indicated completion of the reaction. Then the resulting solution was diluted with 500 mL of DCM. The resulting mixture was washed with water (3 x 300 mL). The residue was purified by a silica gel column, eluted with ethyl

acetate/petroleum ether (1 :4) to afford 40 g (77%) of 4-[2-[(tert-butyldimethylsilyl)oxy]ethyl]-l-(triphenylmethyl)-lH-imidazole as a white solid. LCMS (ESI) : m/z = 469.1 [M+H]+.

Step 4

4-((5-(2-(tert-butyldimethylsilyloxy )ethylVlH-iniidazol-l -vnmethylV3-fluorobenzonitrile: 4-(2-((tert-Butyldimethylsilanyl)oxy)ethyl)-l rityl-lH-irnidazole (40 g, 85.34 mmol, 1.00 equiv) and 4-(Bromomethyl)-3-fluorobenzonitrile (27.38 g, 127.92 mmol, 1.50 equiv) obtained as a product of step 0, were dissolved in MeCN (480 mL) and DCM (80 mL), and stirred at room temperature for 48 h. Et2NH (80 mL) and MeOH (480 mL) were then added and the solution was warmed 80 °C for 3 h. The solution was evaporated to dryness and the residue was purified via flash column chromatography (EtOAc/hexanes 1 :5 to EtOAc) to afford 4-((5-(2-((tert-Butyldimethylsilanyl)oxy)ethyl)-lH-imidazol-l -yl)methyl)-3-fluorobenzonitrile (15 g, 50%). ¾ NMR (400 MHz, CDCh) δ: 7.67 (s, 1H), 7.43 (m, 2H), 6.98 (s, 1H), 6.88-6.79 (m, 1H), 5.34 (s, 2H), 3.79 (t, J= 8.0 Hz, 2H), 2.67 (t, J = 8.0 Hz, 2H), 0.88 (s, 9H), 0.02 (s, 6H). LCMS (ESI) : m/z = 360.1 [M+H]+.

Step 5

5 6

Methyl 2-(5-(2-(tert-butyldimethylsilyloxy)ethyl)-lH-imidazol-l -yl)-2-(4-cvano-2-fluorophenvDacetate: 4-((5-(2-((tert-Butyldimethylsilanyl)oxy)ethyl)-lH-imidazol-l -yl)methyl)-3-fluorobenzonitrile (15 g, 41.72 mmol, 1.00 equiv) was dissolved in anhydrous THF (150 mL) and stirred at -78 °C, then a THF solution of LiHMDS (75 mL, 1.80 equiv, 1.0 M) was added dropwise over 15 min. After 30 min, methyl cyanoformate (4.3 g, 45.50 mmol, 1.10 equiv) was added dropwise over 10 min and the solution was stirred at -78 °C for 2 h. The excess LiHMDS was quenched with aqueous saturated NH4CI and the mixture was allowed to warm to room temperature. The mixture was then diluted with EtOAc and washed

with aqueous saturated NH4CI (200 mL). The organic layers was dried over anhydrous Na2S04 and evaporated. The crude residue was purified via flash column chromatography (EtOAc/PE 3: 10 to EtOAc) to give methyl 2-(5-(2-((tert-butyldimethylsilanyl)oxy)ethyl)-lH-imidazol-l-yl)-2-(4-cyano-2-fluorophenyl) acetate (15 g, 86%) as a light yellow solid.

¾ NMR (400 MHz, CDCL3) δ: 7.66 (s, 1H), 7.54-7.43 (m, 2H), 7.15 (t, J= 8.0 Hz 1H), 6.93 (s, 1H), 6.47 (s, 1H), 3.88-3.74 (m, 5H), 2.81-2.62 (m, 2H), 0.89 (s, 9H), 0.05 (s, 6H) . LCMS (ESI) : m/z = 418.2 [M+H]+.

Step 6

Methyl 2-(4-cvano-2-fluorophenyl)-2-(5-(2-hvdroxyethyl)-lH-imidazol-l-yl) acetate: Methyl 2-(5-(2-((tert-butyldimethylsilanyl)oxy)ethyl)-lH-imidazol-l-yl)-2-(4-cyano-2-fiuorophenyl)acetate (15 g, 35.92 mmol, 1.00 equiv) was added to a solution of HCl in 1,4-dioxane (89 mL, 4.0 M, 359.2 mmol) at 0 °C and the mixture was allowed to warm to room temperature and stirred for 2 h. The solution was concentrated to dryness to give the crude alcohol, methyl 2-(4-cyano-2-fluorophenyl )-2-(5-(2 -hydroxy ethyl)-lH-imidazol-l-yl)acetate (10 g, 92%), which was used without further purification. LCMS: m/z = 304.0 [M+H]+.

Step 7

7 8

Methyl 2-(4-cvano-2-fluorophenyl)-2-(5-(2-(methylsulfonyloxy)ethyl)-lH-imidazol-l-yl) acetate: The crude methyl 2-(4-cyano-2-fluorophenyl )-2-(5-(2-hydroxyethyl)-lH-imidazol-l-yl)acetate (10 g, 32.97 mmol, 1.00 equiv) was dissolved in DCM (200 mL) and stirred at 0 °C, then Et3N (20 g, 197.65 mmol, 6.00 equiv) and

methanesulfonyl chloride (4.52 g, 39.67 mmol, 1.20 equiv) were added. After completion of the reaction, the solution was diluted with DCM and washed with aqueous saturated

NaHCC . The organic layer was dried over anhydrous Na2S04, filtered and evaporated to give the crude methyl 2-(4-cyano-2-fluorophenyl)-2-(5-(2-((methylsulfonyl)oxy)ethyl)-lH-imidazol-l-yl)acetate (11.43 g, 91%), which was used in the next step without further purification. LCMS (ESI) : m/z = 382.0 [M+H]+.

Step 8

Methyl 5-(4-cvano-2-fluorophenyl)-6.7-dihvdro-5H-pyrrolo[1.2-elimidazole-5-carboxylate: The crude methyl 2-(4-cyano-2 -fluorophenyl )-2-(5-(2- ((methylsulfonyl)oxy)ethyl)-lH-imidazol-l-yl)acetate (11.43 g, 29.97 mmol, 1.00 equiv) was dissolved in MeCN (550 mL) and then K2CO3 (12.44 g, 90.01 mmol, 3.00 equiv), Nal (13.50 g, 90.00 mmol, 3.00 equiv) and Et3N (9.09 g, 89.83 mmol, 3.00 equiv) were added. The reaction was stirred at 80 °C for 42 h. The mixture was filtered. The solids were washed with DCM. The filtrate was concentrated and purified by flash column chromatography (EtOAc) to give methyl 5-(4-cyano-2-fluorophenyl)-6,7-dihydro-5H-pyrrolo[l,2-c]imidazole-5-carboxylate (4.2 g, 49% in 3 steps).

[00153] ¾ NMR (400 MHz, CDCb) δ: 7.61 (s, 1H), 7.47-7.47 (m, 2H), 6.88 (s, 1H), 6.79-6.75 (m, 1H), 4.17-4.12 (m, 1H), 3.87 (s, 3H), 3.78-3.70 (m, 1H), 3.08-3.02 (m, 1H), 2.84-2.71 (m, 2H). LCMS (ESI) : m/z = 286.0 [M+H]+.

Step 9

10

4-(6.7-dihvdro-5H-pyrrolo[1.2-elimidazol-5-yl)-3-fluorobenzonitrile: To a 40-mL sealed tube, was placed methyl 5-(4-cyano-2-fluorophenyl)-5H,6H,7H-pyrrolo[l,2-c]imidazole-5-carboxylate (1 g, 3.51 mmol, 1.00 equiv), DMSO (10 mL), water (5 mL). The final reaction mixture was irradiated with microwave radiation for 40 min at 140 °C. The resulting solution was diluted with 100 mL of EtOAc. The resulting mixture was washed with (3 x 20 mL) brine, dried over anhydrous Na2S04, filtered and concentrated. The residue was purified by a silica gel column, eluted with ethyl acetate/petroleum ether (4: 1) to afford 420 mg (44%) of 5-(4-cyano-2-fluorophenyl)-5H,6H,7H-pyrrolo[l,2-c]irnidazole-5-carboxylic acid as a light yellow solid.

¾ NMR (400 MHz, CDCL3) δ: 7.55-7.28 (m, 3H), 6.90-6.85 (m, 2H), 5.74-5.71 (m, 1H), 3.25-3.15 (m, 1H), 3.02-2.92 (m, 2H), 2.58-2.50 (m, 1H). LCMS (ESI) : m/z = 228.2 [M+H]+.

Step 10

10

(R)-4-(6 -dihvdro-5H-pyrrolo[1.2-elirnidazol-5-yl)-3-fluorobenzonitrile:

Resolution of the enantiomers of the title compound (300 mg) was performed by chiral HPLC: Column, Chiralpak IA2, 2*25cm, 20um; mobile phase, Phase A: Hex (50%, 0.1% DEA), Phase B: EtOH (50%) ; Detector, UV 254/220 nm to afford the (S)-enantiomer (RT = 17 min) and the (R)-enantiomer (97.6 mg, desired compound) (RT = 21 min).

 ¾ NMR (400 MHz, DMSO-<4) δ: 7.98-7.95 (m, 1H), 7.70-7.69 (m, 1H), 7.50 (s, 1H), 6.87 (t, J= 8.0 Hz, 1H), 6.70 (s, 1H), 5.79-5.76 (m, 1H), 3.15-3.06 (m, 1H), 2.92-2.74 (m, 2H), 2.48-2.43 (m, 1H). LCMS (ESI) : m/z = 228.1 [M+H]+.

PATENT

WO2013/153129

https://www.google.com/patents/WO2013153129A1?cl=en

PATENT

WO2007/024945

http://www.google.co.in/patents/WO2007024945A1?cl=en

PATENT

 EP 2815749

Aspect (iii) of the present invention relates to phosphate salt or nitrate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile according to Formula (III)

Figure imgb0004

abbreviated as ‘{drug3}’. In particular, the present invention relates to crystalline form of phosphate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile, abbreviated as ‘{drug3a}’; to crystalline Form A of phosphate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile, abbreviated as ‘{drug3b}’; to crystalline Form B of phosphate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile, abbreviated as ‘{drug3c}’; to crystalline Form C of phosphate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile, abbreviated as ‘{drug3d}’; to crystalline Form D of phosphate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile, abbreviated as ‘{drug3e}’; to crystalline Form E of phosphate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile, abbreviated as ‘{drug3f}’; to crystalline Form F of phosphate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile, abbreviated as ‘{drug3g}’; to crystalline Form G of phosphate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile, abbreviated as ‘{drug3h}’; to crystalline Form H of phosphate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile, abbreviated as ‘{drug3i}’; and to crystalline form of nitrate salt of 4-(R)-(6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-5-yl)-3-fluoro-benzonitrile, abbreviated as ‘{drug3j}’. {drug3a}, {drug3b}, {drug3c}, {drug3d}, {drug3e}, {drug3f}, {drug3g}, {drug3h}, {drug3i}, and {drug3j} are specific forms falling within the definition of {drug3}. Aspect (iii) of the invention is separate from aspects (i), (ii), (iv), (v), (vi), (vii), and (viii) of the invention. Thus, all embodiments of {drug3a}, {drug3b}, {drug3c}, {drug3d}, {drug3e}, {drug3f}, {drug3g}, {drug3h}, {drug3i}, and {drug3j}, respectively, are only related to {drug3}, but neither to {drug1}, nor to {drug2}, nor to {drug4}, nor to {drug5}, nor to {drug6}, nor to {drug7}, nor to {drug8}.

PAPER

Osilodrostat (LCI699), a potent 11β-hydroxylase inhibitor, administered in combination with the multireceptor-targeted somatostatin analog pasireotide: A 13-week study in rats

  • a Preclinical Safety, Novartis Institutes for BioMedical Research, East Hanover, NJ, USA
  • b Drug Metabolism and Pharmacokinetics, Novartis Institutes for BioMedical Research, East Hanover, NJ, USA
  • c Novartis Oncology Development, Basel, Switzerland

doi:10.1016/j.taap.2015.05.004http://www.sciencedirect.com/science/article/pii/S0041008X15001684

CLIPS

STR1

STR1

WO2011088188A1 * Jan 13, 2011 Jul 21, 2011 Novartis Ag Use of an adrenal hormone-modifying agent
Reference
1 * BOSCARO M ET AL: “Treatment of Pituitary-Dependent Cushing’s Disease with the Multireceptor Ligand Somatostatin Analog Pasireotide (SOM230): A Multicenter, Phase II Trial“, JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM, vol. 94, no. 1, January 2009 (2009-01), pages 115-122, XP002698507, ISSN: 0021-972X

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///////OSILODROSTAT, Novartis ,  osilodrostat, an inhibitor of aldosterone synthase and aromatase, treating Cushing’s disease,  July 2016, phase 3 clinical development, LCI 699, 928134-65-0, 1315449-72-9, PHASE 3, LCI 699NX, LCI 699AZA, CYP11B1 CYP11B2

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TEZACAFTOR, VX 661 for treatment of cystic fibrosis disease.


ChemSpider 2D Image | Tezacaftor | C26H27F3N2O6