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ORGANIC SPECTROSCOPY

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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 30 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, Dr T.V. Radhakrishnan and Dr B. K. Kulkarni, 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 Open superstar 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 30 year tenure till date Dec 2017, Around 35 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, 50 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 19 lakh plus views on New Drug Approvals Blog in 216 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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TROFINETIDE


ChemSpider 2D Image | Trofinetide | C13H21N3O6
Trofinetide structure.png

Trofinetide

  • Molecular FormulaC13H21N3O6
  • Average mass315.322 Da

Tofinetide , NNZ-256610076853400-76-7[RN]
glycyl-2-methyl-L-prolyl-L-glutamic acid
H-Gly-PMe-Glu-OHL-Glutamic acid, glycyl-2-methyl-L-prolyl-UNII-Z2ME8F52QLZ2ME8F52QLтрофинетид [Russian] [INN]تروفينيتيد [Arabic] [INN]曲非奈肽 [Chinese] [INN]

IUPAC CondensedH-Gly-aMePro-Glu-OH
SequenceGXE
HELMPEPTIDE1{G.[*C(=O)[C@@]1(CCCN1*)C |$_R2;;;;;;;;_R1;$|].E}$$$$
IUPACglycyl-alpha-methyl-L-prolyl-L-glutamic acid

An (1-3) IGF-1 analog with neuroprotective activity.

OPTICAL ROT; -52.4 °   Conc: 0.19 g/100mL;  water ;  589.3 nm; Temp: 20 °C; Len: 1.0 dm…Tetrahedron 2005, V61(42), P10018-10035 

EU Customs Code CN, 29339980

Harmonized Tariff Code, 293399

  • L-Glutamic acid, glycyl-2-methyl-L-prolyl-
  • glycyl-2-methyl-L-prolyl-L-glutamic acid
  • Glycyl-L-2-methylprolyl-L-glutamic acid
2D chemical structure of 853400-76-7

Trofinetide (NNZ-2566) is a drug developed by Neuren Pharmaceuticals that acts as an analogue of the neuropeptide (1-3) IGF-1, which is a simple tripeptide with sequence GlyProGlu formed by enzymatic cleavage of the growth factor IGF-1 within the brain. Trofinetide has anti-inflammatory properties and was originally developed as a potential treatment for stroke,[1][2] but has subsequently been developed for other applications and is now in Phase II clinical trials against Fragile X syndrome and Rett syndrome.[3][4][5]

Trofinetide (NNZ-2566), a neuroprotective analogue of glypromate, is a novel molecule that has a profile suitable for both intravenous infusion and chronic oral delivery. It is currently in development to treat traumatic brain injury.

In February 2021, Neuren is developing trofinetide (NNZ-2566, phase 2 clinical ), a small-molecule analog of the naturally occurring neuroprotectant and N-terminus IGF-1 tripeptide Glypromate (glycine-proline-glutamate), for intravenous infusion treatment of various neurological conditions, including moderate to severe traumatic brain injury (TBI), stroke, chronic neurodegenerative disorders and peripheral neuropathies. At the same time, Neuren is also investigating an oral formulation of trofinetide (phase 3 clinical) for similar neurological indications, including mild TBI.

Autism Spectrum Disorders and neurodevelopment disorders (NDDs) are becoming increasingly diagnosed. According to the fourth edition of the American Psychiatric Association’s (APA) Diagnostic and Statistical Manual oƒ Mental Disorders (DSM-4), Autism spectrum disorders (ASD) are a collection of linked developmental disorders, characterized by abnormalities in social interaction and communication, restricted interests and repetitive behaviours. Current classification of ASD according to the DSM-4 recognises five distinct forms: classical autism or Autistic Disorder, Asperger syndrome, Rett syndrome, childhood disintegrative disorder and pervasive developmental disorder not otherwise specified (PDD-NOS). A sixth syndrome, pathological demand avoidance (PDA), is a further specific pervasive developmental disorder.

More recently, the fifth edition of the American Psychiatric Association’s (APA) Diagnostic and Statistical Manual oƒ Mental Disorders (DSM-5) recognizes recognises Asperger syndrome, childhood disintegrative disorder, and pervasive developmental disorder not otherwise specified (PDD-NOS) as ASDs.

This invention applies to treatment of disorders, regardless of their classification as either DSM-4 or DSM-5.

Neurodevelopment Disorders (NDDs) include Fragile X Syndrome (FXS), Angelman Syndrome, Tuberous Sclerosis Complex, Phelan McDermid Syndrome, Rett Syndrome, CDKL5 mutations (which also are associated with Rett Syndrome and X-Linked Infantile Spasm Disorder) and others. Many but not all NDDs are caused by genetic mutations and, as such, are sometimes referred to as monogenic disorders. Some patients with NDDs exhibit behaviors and symptoms of autism.

As an example of a NDD, Fragile X Syndrome is an X-linked genetic disorder in which affected individuals are intellectually handicapped to varying degrees and display a variety of associated psychiatric symptoms. Clinically, Fragile X Syndrome is characterized by intellectual handicap, hyperactivity and attentional problems, autism spectrum symptoms, emotional lability and epilepsy (Hagerman, 1997a). The epilepsy seen in Fragile X Syndrome is most commonly present in childhood, but then gradually remits towards adulthood. Hyperactivity is present in approximately 80 percent of affected males (Hagerman, 1997b). Physical features such as prominent ears and jaw and hyper-extensibility of joints are frequently present but are not diagnostic. Intellectual handicap is the most common feature defining the phenotype. Generally, males are more severely affected than females. Early impressions that females are unaffected have been replaced by an understanding of the presence of specific learning difficulties and other neuropsychiatric features in females. The learning disability present in males becomes more defined with age, although this longitudinal effect is more likely a reflection of a flattening of developmental trajectories rather than an explicit neurodegenerative process.

The compromise of brain function seen in Fragile X Syndrome is paralleled by changes in brain structure in humans. MRI scanning studies reveal that Fragile X Syndrome is associated with larger brain volumes than would be expected in matched controls and that this change correlates with trinucleotide expansion in the FMRP promoter region (Jakala et al, 1997). At the microscopic level, humans with Fragile X Syndrome show abnormalities of neuronal dendritic structure, in particular, an abnormally high number of immature dendritic spines (Irwin et al, , 2000).

Currently available treatments for NDDs are symptomatic – focusing on the management of symptoms – and supportive, requiring a multidisciplinary approach. Educational and social skills training and therapies are implemented early to address core issues of learning delay and social impairments. Special academic, social, vocational, and support services are often required. Medication, psychotherapy or behavioral therapy may be used for management of co-occurring anxiety, ADHD, depression, maladaptive behaviors (such as aggression) and sleep issues, Antiepileptic drugs may be used to control seizures.

Patent

WO 2014085480,

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2014085480

str1-1

EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acids Gly-Pro-Glu) and its di-peptide derivatives Gly-Pro and Pro-Glu. EP 0 366 638 discloses that GPE is effective as a neuromodulator and is able to affect the electrical properties of neurons.

WO95/172904 discloses that GPE has neuroprotective properties and that administration of GPE can reduce damage to the central nervous system (CNS) by the prevention or inhibition of neuronal and glial cell death.

WO 98/14202 discloses that administration of GPE can increase the effective amount of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and nitric oxide synthase (NOS) in the central nervous system (CNS).

WO99/65509 discloses that increasing the effective amount of GPE in the CNS, such as by administration of GPE, can increase the effective amount of tyrosine hydroxylase (TH) in the CNS to increase TH-mediated dopamine production in the treatment of diseases such as Parkinson’s disease.

WO02/16408 discloses certain GPE analogs having amino acid substitutions and certain other modification that are capable of inducing a physiological effect equivalent to GPE within a patient. The applications of the GPE analogs include the treatment of acute brain injury and neurodegenerative diseases, including injury or disease in the CNS.

EXAMPLES

The following examples are intended to illustrate embodiments of this invention, and are not intended to limit the scope to these specific examples. Persons of ordinary skill in the art can apply the disclosures and teachings presented herein to develop other embodiments without undue experimentation and with a likelihood of success. All such embodiments are considered part of this invention.

Example 1: Synthesis of N,N-Dimethylglycyl-L-prolyl)-L-glutamic acid

The following non-limiting example illustrates the synthesis of a compound of the invention, N,N-Dimethylglycyl-L-prolyl-L-glutamic acid

All starting materials and other reagents were purchased from Aldrich; BOC=tert-butoxycarbonyl; Bn=benzyl.

BOC-L-proline-(P-benzyl)-L-glutamic acid benzyl ester

To a solution of BOC-proline [Anderson GW and McGregor AC: J. Amer. Chem. Soc: 79, 6810, 1994] (10 mmol) in dichloromethane (50 mi), cooled to 0°C, was added triethylamine (1 .39 ml, 10 mmol) and ethyl chloroformate (0.96 ml, 10 mmol). The resultant mixture was stirred at 0 °C for 30 minutes. A solution of dibenzyl-L-glutamate (10 mmol) was then added and the mixture stirred at 0° C for 2 hours then warmed to room temperature and stirred overnight. The reaction mixture was washed with aqueous sodium bicarbonate and citric acid (2 mol 1-1) then dried (MgSO4) and concentrated at reduced pressure to give BOC-L-proline-L-glutamic acid dibenzyl ester (5.0 g, 95%).

L-proline-L-glutamic acid dibenzyl ester

A solution of BOC-L-glutamyl-L-proline dibenzyl ester (3.4 g, 10 mmol), cooled to 0 °C, was treated with trifluoroacetic acid (25 ml) for 2 h. at room temperature. After removal of the volatiles at reduced pressure the residue was triturated with ether to give L-proline-L-glutamic acid dibenzyl ester.

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid

A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 ml) was added to a stirred and cooled (0 °C) solution of L-proline-L-glutamic acid dibenzyl ester (10 mmol), N,N-dimethylglycine (10 mmol) and triethylamine ( 10.3 mmol) in dichloromethane (30 ml). The mixture was stirred at 0°C overnight and then at room temperature for 3 h. After filtration, the filtrate was evaporated at reduced pressure. The resulting crude dibenzyl ester was dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml) containing 10% palladium on charcoal (0.5 g) then hydrogenated at room temperature and pressure until the uptake of hydrogen ceased. The filtered solution was evaporated and the residue recrystallised from ethyl acetate to yield the tripeptide derivative.

It can be appreciated that following the method of the Examples, and using alternative amino acids or their amides or esters, will yield other compounds of Formula 1.

Eample 2: Synthesis of Glycyl-L-2-Methyl-L-Prolyl-L-Glutamate

L-2-Methylproline and L-glutamic acid dibenzyl ester p-toluenesulphonate were purchased from Bachem, N-benzyloxycarbonyl-glycine from Acros Organics and bis(2-oxo-3-oxazolidinyl)phosphinic chloride (BoPCl, 97%) from Aldrich Chem. Co.

Methyl L-2-methylprolinate hydrochloride 2

Thionyl chloride (5.84 cm3, 80.1 mmol) was cautiously added dropwise to a stirred solution of (L)-2-methylproline 1 (0.43 g, 3.33 mmol) in anhydrous methanol (30 cm3) at -5 °C under an atmosphere of nitrogen. The reaction mixture was heated under reflux for 24 h, and the resultant pale yellow-coloured solution was. concentrated to dryness in vacuo. The residue was dissolved in a 1 : 1 mixture of methanol and toluene (30 cm3) then concentrated to dryness to remove residual thionyl chloride. This procedure was repeated twice more, yielding hydrochloride 2 (0.62 g, 104%) as an hygroscopic, spectroscopically pure, off-white solid: mp 127- 131 °C; [α]D -59.8 (c 0.24 in CH2Cl2); vmax (film)/cm-1 3579, 3398 br, 2885, 2717, 2681 , 2623, 2507, 1743, 1584, 1447, 1432, 1374, 1317, 1294, 1237, 1212, 1172, 1123, 981 , 894, 861 and 764; δH (300 MHz; CDCl3; Me4Si) 1.88 (3H, s, Proα-CH3), 1 .70-2.30 (3H, br m, Proβ-HAΗΒ and Proγ-H2), 2.30-2.60 (1H, br m, Proβ-HAΗΒ), 3.40-3.84 (2H, br m, Proδ-H2), 3.87 (3H, s, CO2CH3), 9.43 (1H, br s, NH) and 10.49 ( 1H, br s, HCl); δC (75 MHz; CDCl3) 21.1 (CH3, Proα-CH3), 22.4 (CH2, Proγ-C), 35.6 (CH2, Proβ-C), 45.2 (CH2, Proδ-C), 53.7 (CH3, CO2CH3), 68.4 (quat., Proα-C) and 170.7 (quat, CO); m/z (FAB+) 323.1745 [M2.H35Cl.H+: (C7H13NO2)2. H35Cl.H requires 323.1738] and 325.1718 [M2.H37Cl.H+: (C7H13NOz)2. H37Cl.H requires 325.1708],

N-Benxyloxycarbonyl-glycyl-L-2-methylproline 5

Anhydrous triethylamine (0.45 cm3, 3.23 mmol) was added dropwise to a mixture of methyl L-2-methylprolinate hydrochloride 2 (0.42 g, 2.34 mmol) and N-benzyloxycarbonyl-glycine (98.5%) 3 (0.52 g, 2.45 mmol) in methylene chloride (16 cm3), at 0 °C, under an atmosphere of nitrogen. The resultant solution was stirred for 20 min and a solution of 1 ,3-dicyclohexylcarbodiimide (0.56 g, 2.71 mmol) in methylene chloride (8 cm3) at 0 °C was added dropwise and the reaction mixture was warmed to room temperature and stirred for a further 20 h. The resultant white mixture was filtered through a Celite™ pad to partially remove 1 ,3-dicyclohexylurea, and the pad was washed with methylene chloride (50 cm3). The filtrate was washed successively with 10% aqueous hydrochloric acid (50 cm3) and saturated aqueous sodium hydrogen carbonate (50 cm3), dried (MgSO4), filtered, and concentrated to dryness in vacuo. Further purification of the residue by flash column chromatography (35 g SiO2; 30-70% ethyl acetate – hexane; gradient elution) afforded tentatively methyl N-benzyloxycarbonyl-glycyl-L-2-methylprolinate 4 (0.56 g), containing 1 ,3-dicyclohexylurea, as a white semi-solid: Rf 0.65 (EtOAc); m/z (ΕI+) 334.1534 (M+. C17H22N2O5 requires 334.1529) and 224 ( 1 ,3-dicyclohexylurea).

To a solution of impure prolinate 4 (0.56 g, ca. 1.67 mmol) in 1,4-dioxane (33 cm3) was added dropwise 1 M aqueous sodium hydroxide (10 cm3, 10 mmol) and the mixture was stirred for 19 h at room temperature. Methylene chloride ( 100 cm3) was then added and the organic layer extracted with saturated aqueous sodium hydrogen carbonate (2 x 100 cm3). The combined aqueous layers were carefully acidified with hydrochloric acid (32%), extracted with methylene chloride (2 x 100 cm3), and the combined organic layers dried (MgSO4), filtered, and

concentrated to dryness in vacuo. Purification of the ensuing residue (0.47 g) by flash column chromatography ( 17 g SiO2; 50% ethyl acetate – hexane to 30% methanol – dichloromethane; gradient elution) gave N-protected dipeptide 5 (0.45 g, 60%) as a white foam in two steps from hydrochloride 2. Dipeptide 5 was shown to be exclusively the frafw-orientated conformer by NMR analysis: Rf 0.50 (20% MeOH – CH2Cl2); [α]D -62.3 (c 0.20 in CH2Cl2); vmax (film)/cm-1 3583, 3324 br, 2980, 2942, 1722, 1649, 1529, 1454, 1432, 1373, 1337, 1251 , 1219, 1179, 1053, 1027, 965, 912, 735 and 698; δH (300 MHz; CDCl3; Me4Si) 1.59 (3H, s, Proα-CH3), 1 .89 (1H, 6 lines, J 18.8, 6.2 and 6.2, Proβ-HAHB), 2.01 (2H, dtt, J 18.7, 6.2 and 6.2, Proγ-H2), 2.25-2.40 (1H, m, Proβ-HAΗΒ), 3.54 (2H, t, J 6.6, Proδ-H2), 3.89 (1H, dd, J 17.1 and 3.9, Glyα-HAHB), 4.04 (1H, dd, J 17.2 and 5.3, Glyα-HAΗΒ), 5.11 (2H, s, OCH2Ph), 5.84 (I H, br t, J 4.2, N-H), 7.22-7.43 (5H, m, Ph) and 7.89 (1 H, br s, -COOH); δC (75 MHz; CDCl3) 21.3 (CH3, Proα-CH3), 23.8 (CH2, Proγ-C), 38.2 (CH2, Proβ-C), 43.6 (CH2, Glyα-C), 47.2 (CH2, Proδ-C), 66.7 (quat, Proα-C), 66.8 (CH2, OCH2Ph), 127.9 (CH, Ph), 127.9 (CH, Ph), 128.4, (CH, Ph), 136.4 (quat., Ph), 156.4 (quat., NCO2), 167.5 (quat., Gly-CON) and 176.7 (quat., CO); m/z (EI+) 320.1368 (M+. C16Η20Ν2Ο5 requires 320.1372).

Dibenzyl N-benzyloxycarbonyl-glycyl-L-2-methylprolyl-L-glutamate 7

Triethylamine (0.50 cm3, 3.59 mmol) was added dropwise to a solution of dipeptide 5 (0.36 g, 1.12 mmol) and L-glutamic acid dibenzyl ester /Moluenesulphonate 6 (0.73 g, 1.46 mmol) in methylene chloride (60 cm3) under nitrogen at room temperature, and the reaction mixture stirred for 10 min. Bis(2-oxo-3-oxazoIidinyl)phosphinic chloride (BoPCl, 97%) (0.37 g, 1.41 mmol) was added and the colourless solution stirred for 17 h. The methylene chloride solution was washed successively with 10% aqueous hydrochloric acid (50 cm3) and saturated aqueous sodium hydrogen carbonate (50 cm3), dried (MgSO4), filtered, and evaporated to dryness in vacuo. Purification of the resultant residue by repeated (2x) flash column chromatography (24 g SiO2; 30-70% ethyl acetate – hexane; gradient elution) yielded ƒully protected tripeptide 7 (0.63 g, 89%) as a colourless oil. Tripeptide 7 was shown to be exclusively the trans-orientated conformer by NMR analysis: Rf 0.55 (EtOAc); [α]D -41.9 (c 0.29 in CH2Cl2); vmax (film)/cm-1 3583, 3353 br, 2950, 1734, 1660, 1521, 1499, 1454, 1429, 1257, 1214, 1188, 1166, 1051, 911, 737 and 697; δH (400 MHz; CDCl3; Me4Si) 1.64 (3H, s, Proot-CH3), 1.72 (1H, dt, J 12.8, 7.6 and 7.6, Proβ-HAHB), 1.92 (2H, 5 lines, J 6.7, Proγ-H2), 2.04 (1H, 6 lines, J 7.3 Gluβ-HAHB), 2.17-2.27 (1H, m, Gluβ-HAΗΒ), 2.35-2.51 (3H, m, Proβ-HAΗΒ and Gluγ-H2), 3.37-3.57 (2H, m, Proδ-H2), 3.90 (1 H, dd, J 17.0 and 3.6, Glyα-HAHB), 4.00 (1H, dd, J 17.1 and 5.1, Glyα-HAΗΒ), 4.56 (1H, td, J 7.7 and 4.9, Glyα-H), 5.05-5.20 (6H, m, 3 x OCH2Ph), 5.66-5.72 (1H, br m, Gly-NH), 7.26-7.37 (15H, m, 3 x Ph) and 7.44 (1H, d, J 7.2, Glu-NH); δC (100 MHz; CDCl3) 21.9 (CH3, Proα-CH3), 23.4 (CH2, Proγ-C), 26.6 (CH2, Gluβ-C), 30.1 (CH2, Gluγ-C), 38.3 (CH2, Proβ-C),

43.9 (CH2, Glyα-C), 47.6 (CH2, Proδ-C), 52.2 (CH, Glua-C), 66.4 (CH2, OCH2Ph), 66.8 (CH2, OCH2Ph), 67.1 (CH2, OCH2Ph), 68.2 (quat, Proα-C), 127.9 (CH, Ph), 128.0 (CH, Ph), 128.1, (CH, Ph), 128.2, (CH, Ph), 128.2, (CH, Ph), 128.3, (CH, Ph), 128.4, (CH, Ph), 128.5, (CH, Ph), 128.5, (CH, Ph), 135.2 (quat., Ph), 135.7 (quat., Ph), 136.4 (quat, Ph), 156.1 (quat, NCO2), 167.3 (quat., Gly-CO), 171.4 (quat., CO), 172.9 (quat., CO) and 173.4 (quat., CO); m/z (FAB+) 630.2809 (MH+. C35H40N3O8 requires 630.2815).

Glycyl-L-2-methylprolyl-L-glutamic acid (G-2-MePE)

A mixture of the protected tripeptide 7 (0.63 g, 1.00 mmol) and 10 wt % palladium on activated carbon (0.32 g, 0.30 mmol) in 91 :9 methanol – water (22 cm3) was stirred under an atmosphere of hydrogen at room temperature, protected from light, for 23 h. The reaction mixture was filtered through a Celite™ pad and the pad washed with 75 :25 methanol – water (200 cm3). The filtrate was concentrated to dryness under reduced pressure and the residue triturated with anhydrous diethyl ether to afford a 38: 1 mixture of G-2-MePE and tentatively methylamine 8 (0.27 g, 86%) as an extremely hygroscopic white solid. Analytical reverse-phase HPLC studies on the mixture [Altech Econosphere C 18 Si column, 150 x 4.6 mm, 5 ☐m; 5 min flush with H2O (0.05% TFA) then steady gradient over 25 min to MeCN as eluent at flow rate of 1 ml/min; detection using diode array] indicated it was a 38: 1 mixture of two eluting peaks with retention times of 13.64 and 14.44 min at 207 and 197 nm, respectively. G-2-MePE was shown to be a 73 :27 trans:cis mixture of conformers by 1H NMR analysis (the ratio was estimated from the relative intensities of the double doublet and triplet at δ 4.18 and 3.71 , assigned to the Gluα-H protons of the major and minor conformers, respectively):

mp 144 °Cɸ;

[ α]D -52.4 (c 0.19 in H2O);

δα (300 MHz; D2O; internal MeOH) 1.52 (3H, s, Proα-CH3), 1.81-2.21 (6H, m, Proβ-H2, Proγ-H, and Gluβ-H2), 2.34 (1.46H, t, J 7.2, Gluy-H2), 2.42* (0.54H, t, 77.3, Gluγ-H2), 3.50-3.66 (2H, m, Pro6-H2), 3.71 * (0.27H, t, J 6.2, Gluoc-H), 3.85 (1H, d, J 16.6, Glyα-HAHB), 3.92 (1H, d, J 16.6, Glyα-HAΗΒ) and 4.18 (0.73H, dd, J 8.4 and 4.7, Glua-H);

δC (75 MHz; D2O; internal MeOH) 21.8 (CH3, Proα-CH3), 25.0 (CH2, Proγ-C), 27.8* (CH2: Gluβ-C), 28.8 (CH2, Gluβ-C), 32.9 (CH2, Gluγ-C), 40.8 (CH2, Proβ-C), 42.7 (CH2, Glyα-C), 49.5 (CH2, Proδ-C), 56.0* (CH, Gluα-C), 56.4 (CH, Gluα-C), 69.8 (quat, Proα-C), 166.5 (quat., Gly-CO), 177.3 (quat., Pro-CON), 179.2 (quat., Gluα-CO), 180.2* (quat., Gluγ-CO) and 180.6 (quat., Gluγ-CO);

m/z (FAB+) 3 16.1508 (MH+. C13H22N3O6 requires 316.1509).

PATENT

WO02094856

Example

The following non-limiting example illustrates the synthesis of a compound of the invention, NN-dimethylglycyl-L-prolyl-L-glutamic acid.

All starting materials and other reagents were purchased from Aldrich;
BOC = tert-butoxycarbonyl; Bn = benzyl.

BOC-(γ-benzyl)-L-prolyl-L-glutamic acid benzyl ester
To a solution of BOC-proline [Anderson GW and McGregor AC: J. Amer. Chem.

Soc: 79, 6180, 1957] (10 mmol) in dichloromethane (50 ml), cooled to 0 °C, was added triethylamine (1.39 ml, 10 mmol) and ethyl chloroformate (0.96 ml, 10 mmol). The resultant mixture was stirred at 0 °C for 30 minutes. A solution of dibenzyl L-glutamate (10 mmol) was then added and the mixture stirred at 0 °C for 2 hours then warmed to room temperature and stirred overnight. The reaction mixture was washed with aqueous sodium bicarbonate and citric acid (2 mol l“1) then dried (MgS04) and concentrated at reduced pressure to give BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (5.0 g, 95%).

(7-Benzyl)-L-prolyl-L-glutamic acid dibenzyl ester
A solution of BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (3.4 g, 10 mmol), cooled to 0 °C, was treated with trifluoroacetic acid (25 ml) for 2 hr at room temperature. After removal of the volatiles at reduced pressure the residue was triturated with ether to give (γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (I).

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid
A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 ml) was added to a stirred and cooled (0 °C) solution of (7-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (10 mmol), TVN-dimethylglycine (10 mmol) and triethylamine
(10.3 mmol) in dichloromethane (30 ml). The mixture was stirred at 0 °C overnight and then at room temperature for 3 h. After filtration, the filtrate was evaporated at reduced pressure. The resulting crude dibenzyl ester was dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml) containing 10% palladium on charcoal (0.5 g) then hydrogenated at room temperature and pressure until the uptake of hydrogen ceased. The filtered solution was evaporated and the residue recrystallized from ethyl acetate to yield the tri-peptide derivative.

It will be evident that following the method of the Example, and using alternative amino acids or their amides or esters, will yield other compounds of Formula 1.

Testing; Material and Methods
The following experimental protocol followed guidelines approved by the

University of Auckland animal ethics committee.
Preparation of cortical astrocyte cultures for harvest of metabolised cell culture supernatant

One cortical hemisphere from a postnatal day 1 rat was used and collected into

4ml of DMEM. Trituration was done with a 5ml glass pipette and subsequently through an 18 gauge needle. Afterwards, the cell suspension was sieved through a lOOμm cell strainer and washed in 50ml DMEM (centrifugation for 5min at 250g). The sediment was resuspended into 20ml DMEM+10% fetal calf serum. 10 Milliliters of suspension was added into each of two 25cm3 flasks and cultivated at 37°C in the presence of 10% C02, with a medium change twice weekly. After cells reached confluence, they were washed three times with PBS and adjusted to Neurobasal/B27 and incubated for another 3 days. This supernatant was frozen for transient storage until usage at -80°C.

Preparation of striatal and cortical tissue from rat E18/E19 embryos
A dam was sacrificed by C02-treatment in a chamber for up to 4 minutes and was prepared then for cesarean section. After surgery, the embryos were removed from their amniotic sacs, decapitated and the heads put on ice in DMEM/F12 medium for striatum and PBS + 0.65% D(+)-glucose for cortex.

Striatal tissue extraction procedure and preparation of cells
Whole brain was removed from the skull with the ventral side facing upside in DMEM/F12 medium. The striatum was dissected out from both hemispheres under a stereomicroscope and the striatal tissue was placed into the Falcon tube on ice.

The collected striatal tissue was triturated by using a PI 000 pipettor in 1ml of volume. The tissue was triturated by gently pipetting the solution up and down into the pipette tip about 15 times, using shearing force on alternate outflows. The tissue pieces settled to the bottom of the Falcon tube within 30 seconds, subsequently the supernatant was transferred to a new sterile Falcon tube on ice. The supernatant contained a suspension of dissociated single cells. The tissue pieces underwent a second trituration to avoid excessively damaging cells already dissociated by over triturating them. 1 Milliliter of ice-cold DMEM/F12 medium was added to the tissue pieces in the first tube and triturated as before. The tissue pieces were allowed to settle and the supernatant was removed to a new sterile Falcon tube on ice. The cells were centrifuged at 250g for 5 minutes at 4°C. The resuspended cell pellet was ready for cell counting.

Plating and cultivation of striatal cells
Striatal cells were plated into Poly-L-Lysine (O.lmg/ml) coated 96-well plates (the inner 60 wells only) at a density of 200,000 cells /cm2 in Neurobasal/B27 medium (Invitrogen). The cells were cultivated in the presence of 5% C02 at 37°C under 100% humidity. Complete medium was changed on days 1, 3 and 6.

Cortical tissue extraction procedure and preparation of cells
The two cortical hemispheres were carefully removed by a spatula from the whole brain with the ventral side facing upside into a PBS +0.65% D(+)-glucose containing petri dish. Forcips were put into the rostral part (near B. olfactorius) of the cortex for fixing the tissue and two lateral – sagittal oriented cuttings were done to remove the paraform and entorhinal cortices. The next cut involved a frontal oriented cut at the posterior end to remove the hippocampal formation. A final frontal cut was done a few millimeters away from the last cut in order to get hold of area 17/18 of the visual cortex.

The collected cortices on ice in PBS+0.65% D(+)-glucose were centrifuged at 350g for 5min. The supernatant was removed and trypsin/EDTA (0.05%/0.53mM) was added for 8min at 37°C. The reaction was stopped by adding an equal amount of DMEM+10%) fetal calf serum. The supernatant was removed by centrifugation followed by two subsequent washes in Neurobasal/B27 medium.

The cells were triturated once with a glass Pasteur pipette in 1 ml of
Neurobasal/B27 medium and subsequently twice by using a 1ml insulin syringe with a 22 gauge needle. The cell suspension was passed through a lOOμm cell strainer and subsequently rinsed by 1ml of Neurobasal B27 medium. Cells were counted and adjusted to 50,000 cells per 60μl.

Plating and cultivation of cortical cells

96-well plates were coated with 0.2mg/ml Poly-L-Lysine and subsequently coated with 2μg/ml laminin in PBS, after which 60μl of cortical astrocyte-conditioned medium was added to each well. Subsequently, 60μl of cortical cell suspension was added. The cells were cultivated in the presence of 10% C02 at 37°C under 100%) humidity. At day 1, there was a complete medium change (1:1- Neurobasal/B27 and astrocyte-conditioned medium) with addition of lμM cytosine-β-D-arabino-furanoside (mitosis inhibitor). On the second day, 2/3 of medium was changed. On day 5, 2/3 of the medium was changed again.

Cerebellar microexplants from P8 animals: preparation, cultivation and fixation

The laminated cerebellar cortices of the two hemispheres were explanted from a P8 rat, cut into small pieces in PBS + 0.65% D(+)glucose solution and triturated by a 23gauge needle and subsequently pressed through a 125 μm pore size sieve. The microexplants that were obtained were centrifuged (60 g) twice (media exchange) into serum-free BSA-supplemented START V-medium (Biochrom). Finally, the
microexplants were reconstituted in 1500 μl STARTV-medium (Biochrom). For cultivation, 40μl of cell suspension was adhered for 3 hours on a Poly-D-Lysine
(O.lmg/ml) coated cover slip placed in 35mm sized 6-well plates in the presence of 5% C02 under 100% humidity at 34°C. Subsequently, 1ml of STARTV-medium was added together with the toxins and drugs. The cultures were monitored (evaluated) after 2-3 days of cultivation in the presence of 5% C02 under 100% humidity. For cell counting analysis, the cultures were fixed in rising concentrations of paraformaldehyde (0.4%, 1.2%, 3% and 4% for 3min each) followed by a wash in PBS.
Toxin and drug administration for cerebellar, cortical and striatal cells: analysis

All toxin and drug administration experiments were designed that 1/100 parts of okadaic acid (30nM and lOOnM concentration and 0.5mM 3-nitropropionic acid for cerebellar microexplants only), GPE (InM -ImM) and G-2Methyl-PE (InM-lmM) were used respectively at 8DIV for cortical cultures and 9DIV for striatal cultures. The incubation time was 24hrs. The survival rate was determined by a colorimetric end-point MTT-assay at 595nm in a multi-well plate reader. For the cerebellar microexplants four windows (field of 0.65 mm2) with highest cell density were chosen and cells displaying neurite outgrowth were counted.

Results
The GPE analogue G-2Methyl-PE exhibited comparable neuroprotective capabilities within all three tested in vitro systems (Figures 12-15).

The cortical cultures responded to higher concentrations of GPE (Figure 12) /or

G-2Methyl-PE (lOμM, Figure 13) with 64% and 59% neuroprotection, respectively.

Whereas the other 2 types of cultures demonstrated neuroprotection at lower doses of G-2Methyl-PE (Figures 14 and 15). The striatal cells demonstrated
neuroprotection within the range of InM to ImM of G-2Methyl-PE (Figure 15) while the postnatal cerebellar microexplants demonstrated neuroprotection with G-2Methyl-PE in the dose range between InM and lOOnM (Figure 14).

While this invention has been described in terms of certain preferred embodiments, it will be apparent to a person of ordinary skill in the art having regard to that knowledge and this disclosure that equivalents of the compounds of this invention may be prepared and administered for the conditions described in this application, and all such equivalents are intended to be included within the claims of this application.

PATENT

WO-2021026066

Composition and kits comprising trofinetide and other related substances. Also claims a process for preparing trofinetide and the dosage form comprising the same. Disclosed to be useful in treating neurodegenerative conditions, autism spectrum disorders and neurodevelopmental disorders.

Trofinetide is a synthetic compound, having a similar core structure to Glycyl-Prolyl-Glutamic acid (or “GPE”). Trofinetide has been found to be useful in treating neurodegenerative conditions and recently has been found to be effective in treating Autism Spectrum disorders and Neurodevelopmental disorders.

Formula (Ila),

Example 1: Trofinetide Manufacturing Process

In general, trofinetide and related compounds can be manufactured from a precursor peptide or amino acid reacted with a silylating or persilylating agent at one or more steps. In the present invention, one can use silylating agents, such as N-trialkylsilyl amines or N-trialkylsilyl amides, not containing a cyano group.

Examples of such silylating reagents include N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, 3-(trimethylsilyl)-2-oxazolidone.

Step 1: Preparation of Z-Gly-OSu

Several alternative procedures can be used for this step.

Procedure 1A

One (1) eq of Z-Gly-OH and 1.1 eq of Suc-OH were solubilized in 27 eq of iPrOH and 4 eq of CH2Cl2 at 21 °C. The mixture was cooled and when the temperature reached -4 °C, 1.1 eq of EDC.HCl was added gradually, keeping the temperature below 10 °C. During the reaction a dense solid appeared. After addition of EDC.HCl, the mixture was allowed to warm to 20 °C. The suspension was cooled to 11 °C and filtered. The cake was washed with 4.9 eq of cold iPrOH and 11 eq of IPE before drying at 34 °C (Z-Gly-OSu dried product -Purity: 99.5%; NMR assay: 96%; Yield: 84%).

Procedure 1B

This Procedure is for a variant of Procedure 1A, and differs by replacing iPrOH with ACN. One (1) eq of Z-Gly-OH and 1.1 eq of Suc-OH were solubilized in 22 eq of ACN at 35 °C. The mixture was cooled in an ice bath. When the temperature reached 1 °C, 0.9 eq of DCC in 5.5 eq of ACN was added gradually to keep the temperature below 5 °C. The coupling reaction took about 20 hrs. During the reaction, DCU precipitated and was removed by filtration at the end of the coupling. After filtration, DCU was washed with ACN to recover the product. The mixture of Z-Gly-OSu was then concentrated to reach 60% by weight. iPrOH (17 eq) was added to initiate the crystallization. Quickly after iPrOH addition a dense solid appeared. An additional 17 eq of iPrOH was needed to liquify the suspension. The suspension was cooled in an ice bath and filtered. The solid was washed with 9 eq of iPrOH before drying at 45 °C (Z-Gly-OSu dried product – Purity: 99.2%; HPLC assay: 99.6%; Yield: 71%).

Step 2: Preparation of Z-Gly-MePro-OH

Several alternative procedures can be used for this step.

Procedure 2A

 One (1) eq of MePro.HCl was partially solubilized in 29 eq of CH2Cl2 at 35 °C with 1.04 eq of TEA and 1.6 eq of TMA. The mixture was heated at 35 °C for 2 hrs to perform the silylation. Then 1.02 eq of Z-Gly-OSu was added to the mixture. The mixture was kept at 35 °C for 3 hrs and then 0.075 eq of butylamine was added to quench the reaction. The mixture was allowed to return to room temperature and mixed for at least 15 min. The Z-Gly-MePro-OH was extracted once with 5% w/w NaHCO3 in 186 eq of water, then three times successively with 5% w/w NaHCO3 in 62 eq of water. The aqueous layers were pooled and the pH was brought to 2.2 by addition of 34 eq of HCl as 12N HCl at room temperature. At this pH, Z-Gly-MePro-OH formed a sticky solid that was solubilized at 45 °C with approximately 33 eq of EtOAc and 2.3 eq of iButOH. Z-Gly-MePro-OH was extracted into the organic layer and washed with 62 eq of demineralized water. The organic layer was then dried by azeotropic distillation with 11.5 eq of EtOAc until the peptide began to precipitate. Cyclohexane (12 eq) was added to the mixture to complete the precipitation. The suspension was cooled at 5 °C for 2 hrs and filtered. The solid was washed with 10 eq of cyclohexane before drying at 45 °C (Z-Gly-MePro-OH dried product – Purity: 100%; HPLC assay: 100%; Yield 79%).

Procedure 2B

This Procedure is for a variant of Procedure 2A. One (1) eq of MePro.HCl was partially solubilized in 36.6 eq of CH2Cl2 at 34 °C with 1.01 eq of TEA and 0.1 eq of TMA. Then 1.05 eq of Z-Gly-OSu was added to the mixture, followed by 1.0 eq of TEA. The mixture was maintained at 35 °C for approximately 1 hr, cooled to 25 to 30 °C and 0.075 eq of DMAPA was added to stop the reaction. One hundred (100) eq of water, 8.6 eq of HCl as 12N HCl and 0.3 eq of KHSO4 were added to the mixture (no precipitation was observed, pH=1.7). Z-Gly-MePro-OH was extracted into the organic layer and washed twice with 97 eq of demineralized water with 0.3 eq of KHSO4, then 100 eq of demineralized water, respectively. EtOAc (23 eq) was added to the mixture and CH2Cl2 was removed by distillation until the peptide began to precipitate. Cyclohexane (25 eq) was added to the mixture to complete the precipitation. The suspension was cooled at -2 °C overnight and filtered. The solid was washed with 21 eq of cyclohexane before drying at 39 °C (Z-Gly-MePro-OH dried product – Purity: 98.7%; NMR assay: 98%; Yield 86%).

Procedure 2C


In reactor 1, MePro.HCl (1 eq) was suspended in EtOAc (about 7 eq). DIPEA (1 eq) and TMA (2 eq) were added, and the mixture heated to dissolve solids. After dissolution, the solution was cooled to 0 °C. In reactor 2, Z-Gly-OH (1 eq) was suspended in EtOAc (about 15 eq). DIPEA (1 eq), and pyridine (1 eq) were added. After mixing, a solution was obtained, and cooled to -5 °C. Piv-Cl (1 eq) was added to reactor 2, and the contents of reactor 1 added to reactor 2. Upon completed addition, the contents of reactor 2 were taken to room temperature. The conversion from Z-Gly-OH to Z-Gly-MePro-OH was monitored by HPLC. When the reaction was complete, the reaction mixture was quenched with DMAPA (0.1 eq), and washed with an aqueous solution comprised of KHSO4, (about 2.5 wt%), NaCl (about 4 wt%), and conc. HCl (about 6 wt%) in 100 eq H2O. The aqueous layer was re-extracted with EtOAc, and the combined organic layers washed with an aqueous solution comprised of KHSO4 (about 2.5 wt%) and NaCl (about 2.5 wt%) in 100 eq H2O, and then with water (100 eq). Residual water was removed from the organic solution of Z-Gly-MePro-OH by vacuum distillation with EtOAc. The resulting suspension was diluted with heptane (about 15 eq) and cooled to 0 °C. The product was isolated by filtration, washed with cold heptane (about 7 eq), and dried under vacuum at 45 °C. Z-Gly-MePro-OH (85% yield) was obtained.

Step 3: Preparation of Z-Gly-MePro-Glu-OH

Several alternative procedures can be used in this step.

Procedure 3A

 H-Glu-OH (1.05 eq) was silylated in 2 eq of CH2Cl2 with 3.5 eq of TMA at 65 °C. Silylation was completed after 2 hrs. While the silylation was ongoing, 1.0 eq of Z-Gly-MePro-OH and 1.0 eq of Oxyma Pure were solubilized in 24 eq of CH2Cl2 and 1.0 eq of DMA at room temperature in another reactor. EDC.HCl (1.0 eq.) was added. The activation rate reached 97% after 15 min. The activated Oxyma Pure solution, was then added to silylated H-Glu-OH at 40 °C and cooled at room temperature. Coupling duration was approximately 15 min, with a coupling rate of 97%. Addition of 8.2% w/w NaHCO3 in 156 eq of water to the mixture at room temperature (with the emission of CO2) was performed to reach pH 8. Z-Gly-MePro-Glu-OH was extracted in water. The aqueous layer was washed twice with 29 eq of CH2Cl2. Residual CH2Cl2 was removed by concentration. The pH was brought to 2.5 with 2.5N HCl, followed by 1.4 eq of solid KHSO4 to precipitate Z-Gly-MePro-Glu-OH. The mixture was filtered and the solid was washed with 3 x 52 eq of water. The filtered solid was added to 311 eq of demineralized water and heated to 55-60 °C. iPrOH (29 eq) was added gradually until total solubilization of the product. The mixture was slowly cooled to 10 °C under moderate mixing during 40 min to initiate the crystallization. The peptide was filtered and washed with 2 x 52 eq of water before drying at 45 °C (Z-Gly-MePro-Glu-OH dried product – Purity: 99.5%; NMR assay: 96%; Yield 74%).

Procedure 3B

One (1) eq of Z-Gly-MePro-OH and 1.05 eq of Suc-OH were solubilized in 40 eq of ACN and 30 eq of CH2Cl2 at room temperature. The mixture was cooled in an ice bath, and when the temperature was near 0 °C, 1.05 eq of DCC dissolved in 8 eq of ACN was added gradually, keeping the temperature below 5 °C. After addition of DCC, the mixture was progressively heated from 0 °C to 5 °C over 1 hr, then to 20 °C between 1 to 2 hrs and then to 45 °C between 2 to 5 hrs. After 5 hrs, the mixture was cooled to 5 °C and maintained overnight. The activation rate reached 98% after approximately 24 hrs. DCU was removed by filtration and washed with 13.5 eq of ACN. During the activation step, 1.1 eq of H-Glu-OH was silylated in 30 eq of ACN with 2.64 eq of TMA at 65 °C. Silylation was completed after 2 hrs. Z-Gly-MePro-OSu was then added gradually to the silylated H-Glu-OH at room temperature, with 0.4 eq of TMA added to maintain the solubility of the H-Glu-OH. The mixture was heated to 45 °C and 0.7 eq of TMA was added if precipitation occurred. The coupling duration was about 24 hrs to achieve a coupling rate of approximately 91%. The reaction was quenched by addition of 0.15 eq of butylamine and 2.0 eq of TEA. Water (233 eq) was added and the mixture concentrated until gelation occurred. Z-Gly-MePro-Glu-OH was extracted in water by addition of 5% w/w NaHCO3 in 233 eq of water and 132 eq of CH2Cl2. The aqueous layer was washed twice with 44 eq of CH2Cl2. Residual CH2Cl2 was removed by distillation. The pH was brought to 2.0 with 24 eq of HCl as 12N HCl followed by 75 eq of HCl as 4N HCl. At this pH, Z-Gly-MePro-Glu-OH precipitated. The mixture was cooled in an ice bath over 1 hr and filtered. The solid was washed with 186 eq of cold water before drying at 45 °C (Z-Gly-MePro-Glu-OH dried product – HPLC Purity: 98.4%; NMR assay: 100%; Yield 55%).

Procedure 3C

This Procedure is for a variant of Procedure 3A. H-Glu-OH (1.05 eq) was silylated in 3.7 eq of CH2Cl2 with 3.5 eq of TMA at 62 °C. Silylation was completed after approximately 1.5 to 2 hrs, as evidenced by solubilization. During the silylation step, 1.0 eq of Z-Gly-MePro-OH and 1.0 eq of Oxyma Pure were solubilized in 31.5 eq of CH2Cl2 at 22 °C. One (1.06) eq of EDC.HCl was added to complete the activation. The silylated H-Glu-OH was then added to the activated Oxyma Pure solution. The temperature was controlled during the addition to stay below 45 °C. Desilylation was performed by addition of a mixture of 2.5% w/w KHSO4 in 153 eq of water and 9 eq of iPrOH to reach a pH of 1.65. Residual CH2Cl2 was removed by concentration. The mixture was cooled to 12 °C to precipitate the Z-Gly-MePro-Glu-OH. The mixture was filtered and the solid was washed with 90 eq of water before drying at 36 °C.

Procedure 3D

This Procedure is for a variant of Procedure 3A. H-Glu-OH (1.05 eq.) was silylated in 3.9 eq of CH2Cl2 with 3.5 eq of TMA at 62 °C. Silylation was completed after 2 hrs, as evidenced by Solubilization. During the silylation step, 1 eq of Z-Gly-MePro-OH and 1 eq of Oxyma Pure were solubilized in 25 eq of CH2Cl2 at 23 °C. One (1) eq of EDC.HCl was added. To complete the activation, an additional 0.07 eq of EDC. HCl was added. Silylated H-Glu-OH was then added to the activated Oxyma Pure solution. Temperature was controlled during the addition to stay below 45 °C. Desilylation was performed by addition of a mixture of 2.5% w/w KHSO4 in 160 eq of water and 9.6 eq of iPrOH to reach pH 1.63.

Residual CH2Cl2 was removed by concentration. The mixture was cooled to 20 °C to precipitate the Z-Gly-MePro-Glu-OH. The mixture was filtered and the solid was washed with 192 eq of water before drying at about 25 °C for 2.5 days. The solid was then solubilized at 64 °C by addition of 55 eq of water and 31 eq of iPrOH. After solubilization, the mixture was diluted with 275 eq of water and cooled to 10 °C for crystallization. The mixture was filtered and the solid was washed with 60 eq of water before drying at 27 °C (Z-Gly-MePro-Glu-OH dried product – Purity: 99.6%; NMR assay: 98%; Yield 74%).

Procedure 3E

 In reactor 1, H-Glu-OH (1.05 eq) was suspended in ACN (about 2.2 eq). TMA (about 3.5 eq) added, and the mixture was heated to dissolve solids. After dissolution, the solution was cooled to room temperature. In reactor 2, Z-Gly-MePro-OH (1 eq) was suspended in ACN (14 eq). Oxyma Pure (1 eq) and EDC.HCl (1 eq) were added. The mixture was stirred at room temperature until the solids dissolved. The contents of reactor 2 were added to reactor 1. The conversion from Z-Gly-MePro-OH to Z-Gly-MePro-Glu-OH was monitored by HPLC. Upon completion the reaction mixture was added to an aqueous solution comprised of KHSO4 (about 2.5 wt%) dissolved in about 100 eq H2O. ACN was removed from the aqueous suspension of Z-Gly-MePro-Glu-OH by vacuum distillation with H2O. After stirring at room temperature, the product in the resulting suspension was isolated by filtration and washed with water. The solid obtained was dissolved in an aqueous solution comprised of NaHCO3 (about 5 wt%) in 110 eq H2O, and recrystallized by addition of an aqueous solution comprised of KHSO4 (about 10 wt%) in 90 eq H2O. The product was isolated by filtration, washed with water, and dried under vacuum at 45 °C. Z-Gly-MePro-Glu-OH (75% yield) was obtained.

Step 4: Deprotection and Isolation of Trofinetide

Several alternative procedures can be used in this step.

Procedure 4A

 Z-Gly-MePro-Glu-OH (1 eq) was suspended in water (about 25 eq) and EtOAc (about 15 eq). Pd/C (0.025 eq by weight and containing 10% Pd by weight) was added, and the reaction mixture hydrogenated by bubbling hydrogen through the reaction mixture at room temperature. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC, and upon reaction completion the catalyst was removed by filtration, and the layers separated. Residual EtOAc was removed from the aqueous solution containing trofinetide by sparging with nitrogen or washing with heptane. The aqueous solution was spray-dried to isolate the product. Trofinetide (90% yield) was obtained. Alternatively, deprotection can be accomplished using MeOH only, or a combination of iPrOH and MeOH, or by use of ethyl acetate in water.

Procedure 4B

This Procedure is for a variant of Procedure 4A, excluding EtOAc. Z-Gly-MePro-Glu-OH (1 eq) was suspended in water (about 50 eq). Pd/C (0.05 eq, 5% Pd by weight) was added, and the reaction mixture hydrogenated at room temperature with a pressure of 5 bar. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC. Upon

reaction completion the catalyst was removed by filtration, and the aqueous layer washed with EtOAc (about 5 eq). Residual EtOAc was removed from the aqueous solution containing trofinetide by sparging with nitrogen or washing with heptane. The aqueous solution was spray-dried to isolate the product. Trofinetide (90% yield) was obtained.

Procedure 4C

This Procedure is for a variant of Procedure 4A, replacing EtOAc with MeOH. Z-Gly-MePro-Glu-OH (1 eq) was suspended in MeOH (100 eq) and water (12 eq). Pd/Si (0.02 eq by weight) was added and the mixture was heated at 23 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC, and upon reaction completion the catalyst was removed by filtration and the layers were washed with MeOH and iPrOH. The solvents were concentrated under vacuum at 45 °C, and trofinetide precipitated. The precipitate was filtered and dried at 45 °C to provide trofinetide.

Procedure 4D

This Procedure is for a variant of Procedure 4A, replacing Pd/C with Pd/Si. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 105 eq of MeOH and 12 eq of water. Pd/Si (0.02 eq by weight) was added and the mixture was heated at 23 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (conversion rate approximately 99% after 1 hr), the catalyst was filtered off and washed with 20-30 eq of MeOH. iPrOH (93 eq) was added and MeOH was replaced by iPrOH by concentration at 45 °C under vacuum. The peptide was concentrated until it began to precipitate. The peptide was filtered and dried at 45 °C (H-Gly-MePro-Glu-OH dried product: Purity: 98.1%; NMR assay: 90%; Yield 81%).

Procedure 4E

This Procedure is for a variant of Procedure 4A, removing H2O and replacing Pd/C with Pd/Si. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 44 eq of MeOH. Pd/Si type 340 (0.02 eq by weight) was added and the mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (conversion rate about 99.9%, after 3-3.5 hrs), the catalyst was filtered off and washed with 8 eq of MeOH. Deprotected peptide was then precipitated in 56 eq of iPrOH. After 30 min at 5 °C, the peptide was filtered and washed with three times with 11 eq of iPrOH before drying at 25 °C (H-Gly-MePro-Glu-OH dried product: Purity: 99.4%; HPLC assay: ~98%; Yield: 81%).

Procedure 4F

This Procedure is for a variant of Procedure 4A. One (1) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 14 eq of EtOAc and 25 eq of water. Pd/C (0.01 eq by weight) was added and the mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (conversion rate about 100%, after about 3.5 hrs), the catalyst was filtered off and washed with a mixture of 3.5 eq of EtOAc and 6 eq of water. The aqueous layer was then ready for spray-drying (Aqueous H-Gly-MePro-Glu-OH peptide solution: Purity: 98.6%; Yield: ~95%).

Procedure 4G

This Procedure is for a variant of Procedure 4A, replacing Pd/C with Pd/Si, EtOAc with MeOH, and removing H2O. Pd/Si type 340 (0.02 eq by weight) was added to 2.9 vols of MeOH for pre-reduction during 30 min. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 34 eq of MeOH. The reduced palladium was then transferred to the peptide mixture. The mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. Pd/C type 39 (0.007 eq by weight) was added to the mixture to increase reaction kinetics. At the end of the deprotection, the catalyst was filtered off and washed with 13.6 eq of MeOH. The deprotected peptide was then precipitated in 71 eq of iPrOH. After about 40 min, the peptide was filtered and washed with 35 eq of iPrOH. The peptide was dried below 20 °C and was then ready for solubilization in water and spray-drying.

Procedure 4H

This Procedure is for a variant of Procedure 4A. One (1.0) eq of Z-Gly-MePro-Glu-OH was partially solubilized in 24.8 eq of water and 13.6 eq of EtOAc. Pd/C type 39 (0.025 eq by weight) was added to the peptide mixture. The mixture was kept at 20 °C for the hydrogenolysis. Solubilization of the peptide occurred during the deprotection. At the end of the deprotection (19 hrs), the catalyst was removed by filtration and washed with 5.3 eq of water and 2.9 eq of EtOAc. The biphasic mixture was then decanted to remove the upper organic layer. The aqueous layer was diluted with water to reach an H-Gly-MePro-Glu-OH concentration suitable for spray-drying the solution.

Example 2: Alternative Trofinetide Manufacturing Process

An alternative method for synthesis of Trofinetide is based on U.S. Patent No.

8,546,530 adapted for a tripeptide as follows.

The persilylated compounds used to synthesis Formula (Ia) (trofinetide) are obtained by silylating a corresponding peptide or amino acid by reaction with a silylating agent, optionally in an organic solvent. The persilylated peptide or amino acid can be isolated and purified if desired. One can use the persilylated peptide or amino acid in situ, e.g. by combining a solution containing persilylated peptide or amino acid with a solution containing, optionally activated, peptide or amino acid.

In step 2, the persilylated compound of an amino acid is obtained by silylating a corresponding amino acid (for example, H-MePro-OH) by reaction with a silylating agent, optionally in an organic solvent. The persilylated amino acid can be isolated and purified if desired. One can use the persilylated amino acid in situ, e.g. by combining a solution containing the persilylated amino acid with a solution containing, optionally activated, amino acid (for example, Z-Gly-OH).

In step 3, the persilylated compound of an amino acid is obtained by silylating a corresponding amino acid (for example, H-Glu-OH) by reaction with a silylating agent, optionally in an organic solvent. The persilylated amino acid or peptide can be isolated and purified if desired. It is however useful to use the persilylated amino acid or peptide in situ, e.g. by combining a solution containing the persilylated amino acid with a solution containing, optionally activated (for example, by using EDC.HCl and Oxyma Pure), peptide (for example, Z-Gly-MePro-OH).

In the present invention, it is useful to use silylating agents, such as N-trialkylsilyl amines or N-trialkylsilyl amides, not containing a cyano group. Examples of such silylating reagents include N,O-bis(trimethylsilyl)acetamide (BSA), N,O-bis(trimethylsilyl)trifluoroacetamide, hexamethyldisilazane, N-methyl-N-(trimethylsilyl)acetamide (TMA), N-methyl-N-(trimethylsilyl)trifluoroacetamide, N-(trimethylsilyl)acetamide, N-(trimethylsilyl)diethylamine, N-(trimethylsilyl)dimethylamine, 1-(trimethylsilyl)imidazole, 3-(trimethylsilyl)-2-oxazolidone.

The reaction of step 2 is generally carried out at a temperature from 0 °C to 100 °C, optionally from 10 °C to 40 °C, and optionally from 15 °C to 30 °C.

The reaction of step 3 is generally carried out at a temperature from 0 °C to 100 °C, optionally from 10 °C to 60 °C, optionally from 15 °C to 50 °C.

In the reaction of step 2, generally 0.5 to 5 equivalents, optionally 1 to 3 equivalents, optionally about 1.5 to 2.5 equivalents of silylating agent are used relative to the molar amount of functional groups to be silylated. Use of 2 to 4 equivalents of silylating agent relative to the molar amount of functional groups to be silylated is also possible. “Functional groups to be silylated” means particular groups having an active hydrogen atom that can react with the silylating agent such as amino, hydroxyl, mercapto or carboxyl groups.

In the reaction of step 3, generally 0.5 to 5 equivalents, optionally 2 to 4.5 equivalents, optionally about 3 to 4 equivalents of silylating agent are used relative to the molar amount of functional groups to be silylated. Use of 2.5 to 4.5 equivalents of silylating agent relative to the molar amount of functional groups to be silylated is also possible.

It is understood that “persilylated” means an amino acid or peptide or amino acid analogue or peptide analogue in which the groups having an active hydrogen atom that can react with the silylating agent are sufficiently silylated to ensure that a homogeneous reaction medium for a coupling step is obtained.

In the process according to the invention, the reaction between the amino acid or peptide and the persilylated amino acid or peptide is often carried out in the presence of a carboxyl group activating agent. In that case the carboxylic activating reagent is suitably selected from carbodiimides, acyl halides, phosphonium salts and uronium or guanidinium salts. More optionally, the carboxylic activating agent is an acyl halide, such as isobutyl chloroformate or pivaloyl chloride or a carbodiimide, such as EDC.HC1 or DCC.

Good results are often obtained when using additional carboxylic activating reagents which reduce side reactions and/or increase reaction efficiency. For example, phosphonium and uronium salts can, in the presence of a tertiary base, for example, N,N-diisopropylethylamine (DIPEA) and triethylamine (TEA), convert protected amino acids into activated species. Other reagents help prevent racemization by providing a protecting reagent. These reagents include carbodiimides (for example, DCC) with an added auxiliary nucleophile (for example, 1-hydroxy-benzo triazole (HOBt), 1-hydroxy-azabenzotriazole (HOAt), or Suc-OH) or derivatives thereof. Another reagent that can be utilized is TBTU. The mixed anhydride method, using isobutyl chloroformate, with or without an added auxiliary nucleophile, is also used, as is the azide method, due to the low racemization associated with it. These types of compounds can also increase the rate of carbodiimide-mediated couplings. Typical additional reagents include also bases such as N,N-diisopropylethylamine (DIPEA), triethylamine (TEA) or N-methylmorpholine (NMM).

When the silylation is carried out in the presence of a solvent, said solvent is optionally a polar organic solvent, more optionally a polar aprotic organic solvent. An amide type solvent such as N,N-dimethylformamide (DMF) or N,N-dimethylacetamide (DMAC)

can be used. In the present invention for step 2, one can use an alkyl acetate solvent, in particular ethyl acetate is more particularly optional.

In the present invention for step 3, one can use a chlorinated hydrocarbon solvent or alkyl cyanide solvent, in particular dichloromethane or acetonitrile are more particularly optional.

In another embodiment, silylation is carried out in a liquid silylation medium consisting essentially of silylating agent and amino acid or peptide.

In the present invention, amino acid or peptide is understood to denote in particular an amino acid or peptide or amino acid analogue or peptide analogue which is bonded at its N-terminus or optionally another position, to a carboxylic group of an amino protected amino acid or peptide.

Example 3: Specifications for Compositions Containing Compounds of Formula (I)

1 ICH guideline Q3C on impurities: guideline for residual solvents

Example 4: Alternative Manufacturing of Trofinetide Example 1, Step 4, Procedure 4B

This Procedure is for a variant of Step 4, Procedure 4B. Z-Gly-MePro-Glu-OH (1 eq) was added in portions to Pd/C (0.027 eq by weight and containing 5% Pd by weight) in about 50 eq of water. The reaction mixture was hydrogenated at 20 °C at a pressure of 5 bar for at least 4 cycles of 4 hrs each. Pd/C (0.0027 eq by weight) was charged between cycles, as needed, to speed up the reaction. The conversion from Z-Gly-MePro-Glu-OH to trofinetide was monitored by HPLC. Upon reaction completion the catalyst was removed by filtration, washed with water (12.5 eq) and the aqueous layer washed with EtOAc (about 14 eq). After phase separation, residual EtOAc was removed from the aqueous solution containing

trofinetide by sparging with nitrogen under vacuum at 20 °C for about 3 hrs. The aqueous solution was filtered. The final concentration of trofinetide was about 25 wt% and the solution was then ready for spray-drying to isolate the product.

Example 5: Alternative Composition of Trofinetide

A composition comprising a compound of Formula (I)

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (II):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, and/or a compound of Formula (III):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 independently are selected from the group consisting of hydrogen and C1-4 alkyl, provided that least one of R1, R2, R3 and R4 is C1-4 alkyl, and wherein the composition comprises at least 90 wt%, such as 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, or 97 wt% of the compound of Formula (I) on an anhydrous basis.

Example 6: Alternative Composition of Trofinetide

A composition comprising a compound of Formula (Ia)

or a hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (II):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, and/or a compound of Formula (III):

or a stereoisomer, hydrate, or pharmaceutically acceptable salt thereof, wherein R1, R2, R3 and R4 independently are selected from the group consisting of hydrogen and C1-4 alkyl, provided that least one of R1, R2, R3 and R4 is C1-4 alkyl, and wherein the composition comprises at least 90 wt%, such as 91 wt%, 92 wt%, 93 wt%, 94 wt%, 95 wt%, 96 wt%, or 97 wt% of the compound of Formula (Ia) on an anhydrous basis.

Example 7: A Product of Trofinetide

A product, including a kit containing a dosage form with instructions for use, comprising a compound of Formula (Ia)

or a hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (IIa)


or a hydrate, or pharmaceutically acceptable salt thereof, wherein the product comprises between 95 wt% and 105 wt%, such as 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101

wt%, 102 wt%, 103 wt%, or 104 wt% of the specified amount of the compound of Formula (Ia) in the product.

Example 8: A Product of Trofinetide

A product, including a kit containing a dosage form with instructions for use, comprising a compound of Formula (Ia)

or a hydrate, or pharmaceutically acceptable salt thereof, and a compound of Formula (IIa)

 or a hydrate, or pharmaceutically acceptable salt thereof, and additionally comprising one or more compounds selected from the group consisting of Formula (III), Formula (IIIa), Formula (IV), Formula (V), Formula (VI), Formula (VII), Formula (VIII), and Formula (IX), wherein the composition comprises between 95 wt% and 105 wt%, such as 96 wt%, 97 wt%, 98 wt%, 99 wt%, 100 wt%, 101 wt%, 102 wt%, 103 wt%, or 104 wt% of the specified amount of the compound of Formula (Ia) in the product.

Example 9: Analysis of Products and Compositions

The products and compositions disclosed herein may be analyzed by liquid chromatography, a suitable chromatographic method using UPLC, e.g. using materials and conditions such as Waters Acquity CSH C18, 1.7 µm, 150 x 2.1 mm column, water with 0.1 % TFA (mobile phase A), and water/ACN 70/30 + 0.1 % TFA (mobile phase B), ranging from (4% phase A/6% phase B to 100% phase B and flushed with 4% phase A/6% phase B).

Flow rate: 0.35 ml/min, Column temperature: 40 °C, autosampler temperature: 4 °C, injection volume: 4 ml (e.g. prepared by weighing about 10 mg of powder in a 10 ml volumetric flask and diluted to volume with water). Examples of detectors are UV (ultraviolet, UV 220 nm) and MS (mass spectrometry).

INDUSTRIAL APPLICABILITY

This invention finds use in the pharmaceutical, medical, and other health care fields.

PATENT

WO2014085480 ,

claiming use of trofinetide for treating autism spectrum disorders including autism, Fragile X Syndrome or Rett Syndrome.

EP 0 366 638 discloses GPE (a tri-peptide consisting of the amino acids Gly-Pro- Glu) and its di-peptide derivatives Gly-Pro and Pro-Glu. EP 0 366 638 discloses that GPE is effective as a neuromodulator and is able to affect the electrical properties of neurons.

W095/172904 discloses that GPE has neuroprotective properties and that administration of GPE can reduce damage to the central nervous system (CNS) by the prevention or inhibition of neuronal and glial cell death.

WO 98/14202 discloses that administration of GPE can increase the effective amount of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and nitric oxide synthase (NOS) in the central nervous system (CNS).

WO99/65509 discloses that increasing the effective amount of GPE in the CNS, such as by administration of GPE, can increase the effective amount of tyrosine hydroxylase (TH) in the CNS for increasing TH-mediated dopamine production in the treatment of diseases such as Parkinson’s disease.

WO02/16408 discloses GPE analogs capable of inducing a physiological effect equivalent to GPE within a patient. The applications of the GPE analogs include the treatment of acute brain injury and neurodegenerative diseases, including but not limited to, injury or disease in the CNS.

Example

The following non-limiting example illustrates the synthesis of a compound of the invention, NN-dimethylglycyl-L-prolyl-L-glutamic acid.

All starting materials and other reagents were purchased from Aldrich;
BOC = tert-butoxycarbonyl; Bn = benzyl.

BOC-(γ-benzyl)-L-prolyl-L-glutamic acid benzyl ester
To a solution of BOC-proline [Anderson GW and McGregor AC: J. Amer. Chem.

Soc: 79, 6180, 1957] (10 mmol) in dichloromethane (50 ml), cooled to 0 °C, was added triethylamine (1.39 ml, 10 mmol) and ethyl chloroformate (0.96 ml, 10 mmol). The resultant mixture was stirred at 0 °C for 30 minutes. A solution of dibenzyl L-glutamate (10 mmol) was then added and the mixture stirred at 0 °C for 2 hours then warmed to room temperature and stirred overnight. The reaction mixture was washed with aqueous sodium bicarbonate and citric acid (2 mol l“1) then dried (MgS04) and concentrated at reduced pressure to give BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (5.0 g, 95%).

(7-Benzyl)-L-prolyl-L-glutamic acid dibenzyl ester
A solution of BOC-(γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (3.4 g, 10 mmol), cooled to 0 °C, was treated with trifluoroacetic acid (25 ml) for 2 hr at room temperature. After removal of the volatiles at reduced pressure the residue was triturated with ether to give (γ-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (I).

N,N-Dimethylglycyl-L-prolyl-L-glutamic acid
A solution of dicyclohexylcarbodiimide (10.3 mmol) in dichloromethane (10 ml) was added to a stirred and cooled (0 °C) solution of (7-benzyl)-L-prolyl-L-glutamic acid dibenzyl ester (10 mmol), TVN-dimethylglycine (10 mmol) and triethylamine
(10.3 mmol) in dichloromethane (30 ml). The mixture was stirred at 0 °C overnight and then at room temperature for 3 h. After filtration, the filtrate was evaporated at reduced pressure. The resulting crude dibenzyl ester was dissolved in a mixture of ethyl acetate (30 ml) and methanol (30 ml) containing 10% palladium on charcoal (0.5 g) then hydrogenated at room temperature and pressure until the uptake of hydrogen ceased. The filtered solution was evaporated and the residue recrystallized from ethyl acetate to yield the tri-peptide derivative.

It will be evident that following the method of the Example, and using alternative amino acids or their amides or esters, will yield other compounds of Formula 1.

PAPER

Tetrahedron (2005), 61(42), 10018-10035.  (CLICK HERE)

The synthesis of ten proline-modified analogues of the neuroprotective tripeptide GPE is described. Five of the analogues incorporate a proline residue with a hydrophobic group at C-2 and two further analogues have this side chain locked into a spirolactam ring system. The pyrrolidine ring was also modified by replacing the γ-CH2 group with sulfur and/or incorporation of two methyl groups at C-5.

Graphical Abstract

PAPER

Bioorganic & Medicinal Chemistry Letters (2005), 15(9), 2279-2283

A series of GPE analogues, including modifications at the Pro and/or Glu residues, was prepared and evaluated for their NMDA binding and neuroprotective effects. Main results suggest that the pyrrolidine ring puckering of the Pro residue plays a key role in the biological responses, while the preference for cis or trans rotamers around the Gly-Pro peptide bond is not important.

Graphical abstract

A series of Pro and/or Glu modified GPE analogues is described. Compounds incorporating PMe and dmP showed higher affinity for glutamate receptors than GPE and neuroprotective effects similar to those of this endogenous tripeptide in culture hippocampal neurons exposed to NMDA.

PATENT

US 20060251649

WO 2006127702

US 20070004641

US 20080145335

WO 2012102832

WO 2014085480

US 20140147491

References

  1. ^ Bickerdike MJ, Thomas GB, Batchelor DC, Sirimanne ES, Leong W, Lin H, et al. (March 2009). “NNZ-2566: a Gly-Pro-Glu analogue with neuroprotective efficacy in a rat model of acute focal stroke”. Journal of the Neurological Sciences278 (1–2): 85–90. doi:10.1016/j.jns.2008.12.003PMID 19157421S2CID 7789415.
  2. ^ Cartagena CM, Phillips KL, Williams GL, Konopko M, Tortella FC, Dave JR, Schmid KE (September 2013). “Mechanism of action for NNZ-2566 anti-inflammatory effects following PBBI involves upregulation of immunomodulator ATF3”Neuromolecular Medicine15 (3): 504–14. doi:10.1007/s12017-013-8236-zPMID 23765588S2CID 12522580.
  3. ^ Deacon RM, Glass L, Snape M, Hurley MJ, Altimiras FJ, Biekofsky RR, Cogram P (March 2015). “NNZ-2566, a novel analog of (1-3) IGF-1, as a potential therapeutic agent for fragile X syndrome”. Neuromolecular Medicine17 (1): 71–82. doi:10.1007/s12017-015-8341-2PMID 25613838S2CID 11964380.
  4. ^ Study Details – Rett Syndrome Study
  5. ^ Neuren’s trofinetide successful in Phase 2 clinical trial in Fragile X
PHASESTATUSPURPOSECONDITIONSCOUNT
3Enrolling by InvitationTreatmentRett’s Syndrome1
3RecruitingTreatmentRett’s Syndrome1
2CompletedSupportive CareInjuries, Brain1
2CompletedTreatmentFragile X Syndrome (FXS)1
2CompletedTreatmentInjuries, Brain1
2CompletedTreatmentRett’s Syndrome2
2TerminatedTreatmentConcussions1
1CompletedTreatmentBrain Injuries,Traumatic2
Legal status
Legal statusUS: Investigational New Drug
Identifiers
IUPAC name[show]
CAS Number853400-76-7 
PubChem CID11318905
ChemSpider9493869
UNIIZ2ME8F52QL
Chemical and physical data
FormulaC13H21N3O6
Molar mass315.322 g·mol−1
3D model (JSmol)Interactive image
SMILES[hide]C[C@]1(CCCN1C(=O)CN)C(=O)N[C@@H](CCC(=O)O)C(=O)O
InChI[hide]InChI=1S/C13H21N3O6/c1-13(5-2-6-16(13)9(17)7-14)12(22)15-8(11(20)21)3-4-10(18)19/h8H,2-7,14H2,1H3,(H,15,22)(H,18,19)(H,20,21)/t8-,13-/m0/s1Key:BUSXWGRAOZQTEY-SDBXPKJASA-N

////////////Tofinetide , NNZ 2566, PHASE 2, PHASE 3. NEUREN, Amino Acids, Peptides, Proteins,

CC1(CCCN1C(=O)CN)C(=O)NC(CCC(=O)O)C(=O)O

Telacebec


ChemSpider 2D Image | Telacebec | C29H28ClF3N4O2
Image result for Telacebec
Image result for Telacebec

Telacebec

  • Molecular FormulaC29H28ClF3N4O2
  • Average mass557.006 Da

Telacebec, IAP6, CAS No. 1334719-95-7телацебек [Russian] [INN]تيلاسيبيك [Arabic] [INN]特雷贝克105731334719-95-7[RN]55G92WGH3X
6-Chloro-2-ethyl-N-(4-{4-[4-(trifluoromethoxy)phenyl]-1-piperidinyl}benzyl)imidazo[1,2-a]pyridine-3-carboxamide
Imidazo[1,2-a]pyridine-3-carboxamide, 6-chloro-2-ethyl-N-[[4-[4-[4-(trifluoromethoxy)phenyl]-1-piperidinyl]phenyl]methyl]-Q203Q-203T56 AN DNJ C2 HG BVM1R D- AT6NTJ DR DOXFFF

Qurient Therapeutics and Russia licensee Infectex are developing telacebec, an oral formulation which targets QcrB subunit of the cytochrome bc1 complex, for treating multi drug resistant or extensively drug resistant Mycobacterium tuberculosis infection. Qurient is also investigating telacebec for treating buruli ulcer (an infection caused by Mycobacterium ulcerans ). In January 2021, a global phase II trial was expected to begin by December 2021 for the treatment of buruli ulcer.

syn

Angewandte Chemie, International Edition, 57(4), 1108-1111; 2018

PATENT

WO-2021018387

Novel crystalline forms of telacebec , processes for their preparation and compositions comprising them are claimed. Also claimed is their use for treating bacterial infection.

Different forms of 6-chloro-2-ethyl-AT-(4-(4-(4- (trifluoromethoxy)phenvDpiperidine-i-vDbenzvDimidazolT.2-alpyridine- 3-carboxamide

The present invention relates to different forms of the compound 6-chloro-2-ethyl-lV-(4-(4-(4-(trifhioromethoxy)phenyl)piperidine-i-yl)benzyl)imidazo[i,2-a]pyridine-3-carboxamide and to methods of making such forms/compounds. The present invention furthermore relates to mono-acid addition salts thereof, to methods of making such mono-acid addition salts and to pharmaceutical compositions comprising any of the aforementioned compounds. Furthermore, the present invention relates to uses of any of these compounds.

Tuberculosis as a disease continues to result in millions of deaths each year. Inadequate use of chemotherapy has led to an increasing number of drug resistant cases. This situation is likely to worsen with the emergence of extremely resistant strains to all currently known drugs. Current chemotherapy consists of compounds that directly target Mycobacterium tuberculosis, either by neutralizing general information pathways and critical processes such as RNA polymerization and protein synthesis inhibition or by interfering with mycobacterial specific cell envelop synthesis. The most widely used dedicated anti-tubercular drugs isoniazid, ethionamide, and pyriazin amide are pro-drugs that first require activation. They are administered to a patient for a course of several months. Patients infected with multi-drug resistant strains of M. tuberculosis may have to undergo combination therapies for extended periods of time.

WO 2011/113606 describes various anti-tubercular compounds and their use in the treatment of bacterial infections, including compound“Q203” which chemically is 6-chloro-2-ethyl-!V-(4-(4-(4-(trifluoromethoxy)phenyl)piperidine-i-yl)benzyl)imidazo[i,2-a]pyridine-3-carboxamide. In a publication by Pethe et al. (Nature Medicine, 19, 1157-1160 (2013), this compound is reported to be active against tuberculosis by interfering with the bacterial energy metabolism, inhibiting cytochrome bci activity which is an essential component of the electron transport chain required for synthesis of ATP.

Whilst the compound shows promise for future therapy of tuberculosis and related infections, there continues to be a need for forms thereof that are particularly suitable for pharmaceutical administration. In particular there is a need to provide forms that are showing an improved solubility in comparison to the free base of this compound. Furthermore, there is a need in the art to provide for forms that show an improved stability.

In a first aspect the present invention relates to a compound 6-chloro-2-ethyl-N-(4-(4-(4-(trifluoromethoxy)phenyl)piperidine-i-yl)benzyl)imidazo[i,2-a]pyridine-3-carboxamide ditosylate having the structure

PATENT

WO2011113606 .

WO 2017049321

WO 2012143796

PAPER

Scientific reports (2019), 9(1), 8608.

Angewandte Chemie, International Edition (2018), 57(4), 1108-1111.

European journal of medicinal chemistry (2017), 136, 420-427.

European Journal of Medicinal Chemistry (2017), 136, 420-427.

 European journal of medicinal chemistry (2017), 125, 807-815.

Nature communications (2016), 7, 12393.

Nature medicine (2013), 19(9), 1157-60

PAPER

Journal of Medicinal Chemistry (2014), 57(12), 5293-5305.

https://pubs.acs.org/doi/10.1021/jm5003606J. Med. Chem. 2014, 57, 12, 5293–5305

Publication Date:May 28, 2014
https://doi.org/10.1021/jm5003606

Abstract Image

A critical unmet clinical need to combat the global tuberculosis epidemic is the development of potent agents capable of reducing the time of multi-drug-resistant (MDR) and extensively-drug-resistant (XDR) tuberculosis therapy. In this paper, we report on the optimization of imidazo[1,2-a]pyridine amide (IPA) lead compound 1, which led to the design and synthesis of Q203 (50). We found that the amide linker with IPA core is very important for activity against Mycobacterium tuberculosis H37Rv. Linearity and lipophilicity of the amine part in the IPA series play a critical role in improving in vitro and in vivo efficacy and pharmacokinetic profile. The optimized IPAs 49 and 50 showed not only excellent oral bioavailability (80.2% and 90.7%, respectively) with high exposure of the area under curve (AUC) but also displayed significant colony-forming unit (CFU) reduction (1.52 and 3.13 log10 reduction at 10 mg/kg dosing level, respectively) in mouse lung.

6-Chloro-2-ethyl-N-(4-{4-[4-(trifluoromethoxy)phenyl]piperidin-1-yl}benzyl)imidazo[1,2-a]pyridine-3-carboxamide (50)

Mp = 164.0 °C; 1H NMR (400 MHz, CDCl3) δ 1.37 (t, J = 7.6 Hz, 3H), 1.82–1.97 (m, 4H), 2.64–2.70 (m, 1H), 2.80–2.87 (m, 2H), 2.93 (q, J = 7.6 Hz, 2H), 3.80–3.83 (m, 2H), 4.61 (d, J = 5.2 Hz, 2H), 6.00 (br t, J = 5.2 Hz, 1H), 6.96–6.99 (m, 2H), 7.15 (d, J = 8.0 Hz, 2H), 7.24–7.30 (m, 5H), 7.52 (dd, J = 9.6, 0.8 Hz, 1H), 9.53 (dd, J = 2.0, 0.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 13.3, 23.6, 33.4, 42.0, 43.3, 50.4, 115.4, 117.0, 121.2, 121.6, 121.9, 126.3, 128.2, 128.3, 128.7, 128.9, 144.5, 144.7, 147.7, 151.4, 151.5, 161.2; 19F NMR (376 MHz, CDCl3) δ 58.31 (s, 3F); LC/MS (ESI) m/z 557 [M + H]+; HRESIMS calcd for C29H29ClF3N4O2 [M + H]+ 557.1926, found 557.1918.

str1
str2
str3

19F NMR (376 MHz, CDCl3) δ 58.31 (s, 3F); 

13C NMR (100 MHz, CDCl3) δ 13.3, 23.6, 33.4, 42.0, 43.3, 50.4, 115.4, 117.0, 121.2, 121.6, 121.9, 126.3, 128.2, 128.3, 128.7, 128.9, 144.5, 144.7, 147.7, 151.4, 151.5, 161.2; 

1H NMR (400 MHz, CDCl3) δ 1.37 (t, J = 7.6 Hz, 3H), 1.82–1.97 (m, 4H), 2.64–2.70 (m, 1H), 2.80–2.87 (m, 2H), 2.93 (q, J = 7.6 Hz, 2H), 3.80–3.83 (m, 2H), 4.61 (d, J = 5.2 Hz, 2H), 6.00 (br t, J = 5.2 Hz, 1H), 6.96–6.99 (m, 2H), 7.15 (d, J = 8.0 Hz, 2H), 7.24–7.30 (m, 5H), 7.52 (dd, J = 9.6, 0.8 Hz, 1H), 9.53 (dd, J = 2.0, 0.8 Hz, 1H);

CLIP

June 3, 2019.  Qurient press release:

SEONGNAM-SI, South Korea–(BUSINESS WIRE)– Qurient Co. Ltd. today announced positive results from the Phase 2a EBA (early bactericidal activity) clinical trial for telacebec (Q203), a first-in-class, orally-available antibiotic for the treatment of tuberculosis (TB). Telacebec is a selective inhibitor with high specificity for the cytochrome bc1 complex of Mycobacterium tuberculosis. This complex is a critical component of the electron transport chain, and inhibition disrupts the bacterium’s ability to generate energy.

The EBA trial assessed the pharmacokinetics, safety, and activity of telacebec in three dose strength (100 mg, 200 mg and 300 mg) in the treatment of adult patients with pulmonary TB. Telacebec met the primary objective of rate of change in the time to positivity (TTP) in sputum over days 0 to 14. Telacebec was safe and well tolerated throughout the different dose strengths. Full results from EBA trial are expected to be presented at future scientific meetings.


Phase 2. EBA began July 2018 in South Africa.  As of March 2019, study is active, not enrolling.


June 2018. Q203 has a non-proprietary name assigned: telacebec. USAN: -cebec Cytochrome bc1 complex inhibitors in Mycobacterium tuberculosis.


Phase 1. Description from clinicaltrials.gov:  Randomized, double-blind, placebo-controlled, dose-escalation study in healthy male and female volunteers. Subjects randomly assigned to 1 of 7 treatment cohorts (Cohorts 1 – 7) of 8 subjects each, receiving either Q203 or placebo (6 active treatment : 2 placebo) in a fasting state. Dose escalation to the next cohort may be considered when at least 6 out of 8 subjects, in a cohort, completes all procedures and none of the subjects has a clinically significant adverse event (AE) that is being followed, or at the discretion of the PI if no drug-related serious adverse events (SAEs) have occurred. A food effect cohort will be enrolled to test administration of Q203 in a fed state, at 100 mg dose level (this dose level may change based on PK analysis results). Subjects who received 100mg dose in a fasting state will return and receive the second dose, with food. Subjects will be followed up for AEs, SAE or pregnancy for 30 days postdrug administration.

Related Links


Qurient Press Release. June 2019.Kalia NP et al. 2017. Exploiting the synthetic lethality between terminal respiratory oxidases to kill M. tuberculosis and clear host infection.. PNAS.114.7426

Related Links


//////////////Telacebec,  IAP6, 1334719-95-7, PHASE 2, QURIENT, TUBERCULOSIS, телацебек , تيلاسيبيك , 特雷贝克 , Q 203

BMS 262084


2-Azetidinecarboxylic acid, 3-(3-((aminoiminomethyl)amino)propyl)-1-((4-(((1,1-dimethylethyl)amino)carbonyl)-1-piperazinyl)carbonyl)-4-oxo-, (2S,3R)-.png
ChemSpider 2D Image | BMS-262084 | C18H31N7O5

BMS-262084

CAS 253174-92-4

  • Molecular FormulaC18H31N7O5
  • Average mass425.483 Da

NII-I0IR71971G

I0IR71971G

(2S,3R)-1-[4-(tert-butylcarbamoyl)piperazine-1-carbonyl]-3-[3-(diaminomethylideneamino)propyl]-4-oxoazetidine-2-carboxylic acid(2S,3R)-1-{[4-(tert-butylcarbamoyl)piperazin-1-yl]carbonyl}-3-{3-[(diaminomethylidene)amino]propyl}-4-oxoazetidine-2-carboxylic acid
(2S,3R)-3-{3-[(Diaminomethylene)amino]propyl}-1-({4-[(2-methyl-2-propanyl)carbamoyl]-1-piperazinyl}carbonyl)-4-oxo-2-azetidinecarboxylic acid253174-92-4[RN]2-Azetidinecarboxylic acid, 3-[3-[(diaminomethylene)amino]propyl]-1-[[4-[[(1,1-dimethylethyl)amino]carbonyl]-1-piperazinyl]carbonyl]-4-oxo-, (2S,3R)-

Factor XIa inhibitors (thrombosis), BMS; Factor XIa inhibitors (thrombosis), Bristol-Myers Squibb; BMS-654457; Factor XIa inhibitors (cardiovascular diseases), BMS; BMS-724296

Novel crystalline forms of BMS-262084  as Factor XIa antagonist useful for treating cardiovascular diseases.

PHASE 2

PAPER

Bioorganic & Medicinal Chemistry Letters (2002), 12(21), 3229-3233.

https://www.sciencedirect.com/science/article/pii/S0960894X02006881

Abstract

A series of N1-activated C4-carboxy azetidinones was prepared and tested as inhibitors of human tryptase. The key stereochemical and functional features required for potency, serine protease specificity and aqueous stability were determined. From these studies compound 2, BMS-262084, was identified as a potent and selective tryptase inhibitor which, when dosed intratracheally in ovalbumin-sensitized guinea pigs, reduced allergen-induced bronchoconstriction and inflammatory cell infiltration into the lung.

BMS-262084 was identified as a potent and selective tryptase inhibitor that, when dosed intratracheally in ovalbumin-sensitized guinea pigs, reduced allergen-induced bronchoconstriction and inflammatory cell infiltration into the lung.

PAPER

https://pubs.acs.org/doi/10.1021/jo010757o

Journal of Organic Chemistry (2002), 67(11), 3595-3600.

A highly stereoselective synthesis of the novel tryptase inhibitor BMS-262084 was developed. Key to this synthesis was the discovery and development of a highly diastereoselective demethoxycarbonylation of diester 12 to form the trans-azetidinone 13. BMS-262084 was prepared in 10 steps from d-ornithine in 30% overall yield.

1 as a white powder (3.18 g, 99% yield). Mp:  213-215 °C dec. [α]25D = −65.9 (c 0.99, MeOH). 1H NMR (CD3OD):  δ 4.17 (d, J = 3.29 Hz, 1H), 3.61−3.11 (m, 11H), 1.94−1.75 (m, 4H), 1.32 (s, 9H). 13C NMR (CD3OD):  δ 176.6, 168.7, 159.4, 158.7, 152.3, 58.7, 53.2, 51.8, 46.5, 45.0, 41.8, 29.6, 27.4, 26.3. HRMS:  calcd for C18H32N7O5(M+ + H) 426.2465, found 426.2470. IR (KBr):  3385, 3184, 1775, 1657, 1535, 1395, 1259, 1207, 996, 763 cm1. Anal. Calcd for C18H31N7O5:  C, 50.81, H, 7.34, N, 23.04. Found:  C, 50.65, H, 7.42, N, 22.72. Chiral HPLC:  ee 99.6%; Chiralpak OD column, 250 × 4.6 mm, 10 μm; mobile phase hexane/EtOH (85:15, v/v); isocratic at ambient temperature, 1.0 mL/min, 220 nm; concentration 0.25 mg/mL, 10 μL injection; RT = 18.6 min (enantiomer, RT = 15.7 min).

PATENT

WO2018133793

claiming macrocyclic compounds.

PATENT

WO-2020259366

Novel crystalline and solid forms of BMS-262084 (designates as monohydrate or 1.5 hydrate), processes for their preparation and compositions comprising them are claimed. BMS-262084 is disclosed to be Factor XIa antagonist, useful for treating cardiovascular diseases.MS-262084 (CAS number: 253174-92-4), the chemical name is (2S,3R)-1-[4-(tert-butylcarbamoyl)piperazine-1-carbonoyl]-3-[3- (Diaminomethylamino)propyl]-4-cyclopropanamide-2-carboxylic acid, also called compound (1) in the present invention, is developed by BMS (Bristol-Myers-Squibb) to treat cardiovascular diseases The drug, as an oral coagulation factor XIa inhibitor for thrombus, has the advantage of significantly reducing the risk of bleeding, and its structure is shown in formula (1): 

Patent application WO 9967215A1 discloses the BMS-262084 compound, but the specific molecular formula of the solid substance obtained by the disclosed preparation process is C 18 H 31 N 7 O 5 ·1.56H 2 O, which is similar to the crystal of BMS-262084 described in this application. Type and amorphous water have different molecular weights.

“A stereoselective synthesis of BMS-262084 an azetidinone-based tryptase inhibitor” (Source: Journal of Organic Chemistry, 2002,67(11):3595-3600; Journal of Organic Chemistry,2002,67(11):3595-3600) It is mentioned that the preparation method of BMS-262084 is that hydrogenolysis under neutral conditions eliminates the benzene and Cbz protection groups, and obtains BMS-262084 (melting point 213-215℃). The inventors conducted experiments based on part of the contents disclosed in the document, and the test results obtained crystal form A and crystal form B. The X-ray powder diffraction patterns are shown in Figure 1 and Figure 2 respectively.Example 1 
“A stereoselective synthesis of BMS-262084 an azetidinone-based tryptase inhibitor” (Source: Journal of Organic Chemistry, 2002,67(11):3595-3600; Journal of Organic Chemistry,2002,67(11):3595-3600) Only ethanol solvents are mentioned in the literature. Since no specific crystal refining process was provided, only part of the experiment was performed using ethanol solvent. 
1) Ethanol solvent volatilization at room temperature: 50mg of BMS-262084 (amorphous) was added to 1.0 mL of ethanol solvent and completely dissolved at room temperature (about 25°C). After volatilizing at room temperature for two days, the solid product was obtained and its crystal form was tested. It is crystal form A, as shown in Figure 1. It is considered that it contains a small amount of amorphous form; but it is unstable and will undergo crystal transformation at room temperature. After standing for one day, the XRPD was tested, and it was found that it was converted to a mixture containing crystal form A, other crystal forms and amorphous forms. 
2) Ethanol solvent high-temperature volatilization: 50mg BMS-262084 is added to 1.0mL ethanol solvent, completely dissolved at high temperature (about 60℃), and high-temperature volatilization is carried out in the open to obtain a solid product. The crystal form of the solid product is detected, and the crystal form is B (contains a lot of amorphous), see Figure 2.

SYN1

WO 9967215

The condensation of N-(tert-butyldimethylsilyl)-4-oxoazetidine-2(S)-carboxylic acid (I) with 1-chloro-3-iodopropane (II) by means of BuLi and triisopropylamine (TIA) in THF, followed by treatment with HCl, gives the 3(R)-(3-chloropropyl) derivative (III), which is treated with tetrabutylammonium azide and tetrabutylammonium iodide in DMF to yield the 3-azidopropyl derivative (IV). The reduction of (IV) with H2 over Pd/C in DMF affords the 3-aminopropyl compound (V), which is treated with 1-[N,N’-bis(benzyloxycarbonyl)-1H-pyrazole] (VI) in the same solvent to provide the protected 3-guanidinopropyl compound (VII). The esterification of (VII) with NaHCO3, tetrabutylammonium iodide and Bn-Br in DMF gives the benzyl ester (VIII), which is condensed with N-tert-butylpiperazine-1-carboxamide (IX) and phosgene by means of TEA in toluene to yield the protected precursor (X). Finally, this compound is debenzylated by hydrogenation with H2 over Pd/C in dioxane to give the target azetidine-carboxylic acid.

SYN 2

Ethyl nipecotate (I) was protected as the N-Boc derivative (II) and subsequently reduced to alcohol (III) by means of LiAlH4. Conversion of alcohol (III) into iodide (IV) was achieved by treatment with iodine and triphenylphosphine. The dianion of the chiral azetidinecarboxylic acid (V) was alkylated with iodide (IV) to furnish adduct (VI) as a diastereomeric mixture that was desilylated to (VII) using tetrabutylammonium fluoride. Benzyl ester (VIII) was then obtained by reaction of carboxylic acid (VII) with benzyl bromide and NaHCO3.

SYN 3

Coupling of 6-phenylhexanoic acid (X) with N-Boc-piperazine (IX) to give (XI), followed by acid deprotection of the Boc group of (XI), provided (6-phenylhexanoyl)piperazine (XII). This was converted to the carbamoyl chloride (XIII) upon treatment with phosgene. The condensation of carbamoyl chloride (XIII) with azetidinone (VIII) gave rise to the urea derivative (XIV). After acid cleavage of the Boc protecting group of (XIV), the resulting piperidine (XV) was condensed with N,N’-dicarbobenzoxy-S-methylisothiourea (XVI) in the presence of HgCl2, yielding the protected guanidine (XVII). This was finally deprotected by catalytic hydrogenolysis over Pd/C.

////////////////////////BMS-262084, BMS 262084,  BMS 724296, Factor XIa inhibitors, thrombosis, Bristol-Myers Squibb,  BMS 654457, PHASE 2

CC(C)(C)NC(=O)N1CCN(CC1)C(=O)N2C(C(C2=O)CCCN=C(N)N)C(=O)O

Catequentinib, Anlotinib


Anlotinib.png
ChemSpider 2D Image | ANLOTINIB | C23H22FN3O3

Catequentinib

C23H22FN3O3  407.4 g/mol

1-[[4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6-methoxyquinolin-7-yl]oxymethyl]cyclopropan-1-amine

1058156-90-3

CAS No. 1360460-82-7 DI HCL

Molecular Weight480.36
FormulaC23H22FN3O3 • 2HCl

Anlotinib

AL3818

UNII-GKF8S4C432

Chia Tai Tianqing Pharmaceutical Group Co Ltd

Launched (Metastatic non small cell lung cancer – China – May-2018)

Orphan Drug; Priority Review

MOA:VEGFR inhibitor

Indication:advanced gastric adenocarcinoma; Advanced renal cell carcinoma (RCC); Medullary thyroid cancer (MTC); Metastatic colorectal cancer (CRC); Non small cell lung cancer (NSCLC); Soft tissue sarcoma; Ovarian cancerStatus:Phase III (Active)

AL-3818 ; AL-3818, Jiangsu Chia-tai Tianqing Pharmaceutical ; FOCUS-V ; FuKeWei ; VEGFR2/VEGFR3 inhibitor (capsule, cancer), Jiangsu Chia Tai Tianqing Pharmaceutical ; anlotinib ; anlotinib dihydrochloride ; catequentinib ; catequentinib ; catequentinib dihydrochloride

NMR  https://file.selleckchem.com/downloads/nmr/S872601-Anlotinib-AL3818-hnmr-selleck.pdf

Anlotinib (AL3818) is a highly potent and selective VEGFR2 inhibitor with IC50 less than 1 nM. It has broad-spectrum antitumor potential in clinical trials.

Anlotinib dihydrochloride is in phase II/III clinical trials for the treatment of metastatic colorectal cancer and advanced gastric adenocarcinoma. The compound was co-developed by CTTQ Pharmaceutical (正大天晴) and Advenchen Laboratory.

It is also in phase II clinical trials for the treatment of ovarian cancer, endometrial cancer, non small cell lung cancer (NSCLC), medullary thyroid cancer (MTC), soft tissue sarcoma and advanced renal cell carcinoma (RCC).

In 2015, orphan drug designation was received in the U.S. for the treatment of ovarian cancer.

PATENT

WO 2016179123

https://patents.google.com/patent/WO2016179123A1/en

new process to synthesize l-((4-(4-Fluoro-2-methyl- lH- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropanamine (AL3818) by condensing intermediate (XI) with (Yl) in a solvent at the presence of KI or Nal, or intermediate (X2) with (Y2) in a solvent to form intermediate (Z) which is deprotected to give the final compound (AL3818) in Scheme I. A stable crystalline form of l-((4-(4-Fluoro-2 -methyl- lH-indol-5-yloxy)-6- methoxyquinolin-7-yloxy)-methyl)cyclopropanamine and its salts as well as crystalline forms of salts have also been prepared.

Figure imgf000004_0001

Wherein, R is selected from H and Ci-Cealkoxy.

Process A

Figure imgf000007_0001

R is selected from H and C1 -C6 alkoxy

The final compound (AL3818) was prepared according to Process Al when R is H by deprotecting intermediate (Z-l) with HCOONH4 (ammonium formate) and Pd/C in an alcoholic solvent, such as MeOH, at 25°C-80°C for 0.1-4 hours. (Z-l) was prepared by reacting intermediate (XI) with (Yl-1) at the presence of KI or Nal with K2CO3 in a solvent, such as acetone or DMF, at a temperature of 60°C-160°C for 2-24 hours.

Process Al (R=H)

Figure imgf000008_0001

The final compound (AL3818) was prepared according to Process A2 when R is 4-OMe by deprotecting intermediate (Z-2) with TFA in DCM at 0°C-30°C for 1-24 hours. (Z-2) was prepared by reacting intermediate (XI) with (Y 1-2) at the presence of KI or Nal with K2C03 in a solvent, such as acetone or DMF, at a temperature of 60°C -160°C for 2-24 hours.

Process A2 (R=4-OMe)

Figure imgf000008_0002

The present invention relates a new process to synthesize l-((4-(4-Fluoro-2 -methyl- 1H- indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropanamine (AL3818) by reacting intermediate (X2) with (Y2) in a solvent to form intermediate (Z) which is deprotected to give the final compound (AL3818) according to Process B. Proce B

Figure imgf000009_0001

R is selected from H and C1-C6 alkoxy

The final compound (AL3818) was prepared according to Process Bl when R is H by deprotecting intermediate (Z-1) with HCOONH4 (ammonium formate) and Pd/C in an alcoholic solvent, such as MeOH, at 25°C-80°C for 0.1-4 hours. (Z-1) was prepared by reacting intermediate (X2-1) with (Y2) in a solvent, such as pyridine or lutidine, at a temperature of 60°C – 160°C for 1-12 hours.

Process Bl R=H)

Figure imgf000009_0002

The final compound (AL3818) was prepared according to Process B2 when R is 4-OMe by deprotecting intermediate (Z-2) with TFA in DCM at 0°C-30°C for 1-24 hours. (Z-2) was prepared by reacting intermediate (X2-2) with (Y2) in a solvent, such as pyridine or lutidine, at a temperature of 60°C -160°C for 1-12 hours.

Process B2 (R=4-OMe)

Figure imgf000009_0003

The following examples further illustrate the present invention, but should not be construed as in any way to limit its scope.

Example 1

Representation of Process A, Process Al

Process for preparation of l-((4-(4-Fluoro-2 -methyl- lH-indol-5-yloxy)-6-methoxy- quinolin-7-yloxy)methyl)cyclopropanamine (AL3818)

To a stirred mixture of benzyl l-(hydroxymethyl)cyclopropylcarbamate (50 g) and DCM (200 ml) was added DIPEA (39g). The result solution was cooled to 0-5 °C with ice/water and further stirred under this temperature for 15 min. MsCl (30g) was added via an addition funnel dropwise keeping temperature below 5°C for about 1.5 hours. After completion of addition, the reaction mixture was allowed stirring at 0-5°C for 30 min and quenched with saturated NaHC03 (150 ml). The solution was extracted with 150 ml DCM twice. The combined DCM layer was washed with 0.1 N HCl (400 ml) followed by brine. It was dried over Na2S04 and concentrated to obtain an off-white solid 60 gram as (l-(benzyloxycarbonylamino)cyclopropyl)methyl methanesulfonate (Yl-1), MS: (M+l) 300.

To a stirred mixture of (Yl-1) (16 g), XI [(4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6- methoxy-7-hydroxyquinoline, 12 g] , K2CO3 (21 g) and KI (21 g) was added DMF (100 ml), the reaction suspension was heated at 80°C for 10 hours and (Yl-l) (10 g) was added to continuously heated 80°C for 10 hours. The reaction then was quenched with water (150 ml) and extracted with 150 ml DCM twice. The combined DCM layer was washed with 2 N NaOH (100 ml) followed by water and brine. It was dried over Na2SC>4 and concentrated, further recrystallized from EtOH to obtain a yellow solid as benzyl l-((4-(4-fluoro-2-methyl-lH-indol-5-yloxy)-6-methoxyquinolin- 7-yloxy)methyl)cyclopropylcarbamate (Z-l) 9.5 g. MS: (M+l) 542.

To a stirred mixture of (Z-l) (9.5 g), HCOONH4 (4.7 g) and Pd/C (10%, wet 50%, 4.7g) was added MeOH, the reaction mixture was heated at 45°C for 1.5 hours. It was then cooled and filtered through Celite, further evaporated. 2N HCl (200 ml) was added and extracted with DCM/MeOH (10/1, 100 ml) twice. The aqueous layer was basified with 3N NaOH to adjust pH 11-12 to generate a solid precipitation. The solid was filtered and washed with water to neutral, further suction dry. The solid was dissolved into a mixture of DCM/MeOH (250 ml, 10/1) and further washed with water and brine. It was dried with MgS04 and filtered, further evaporated to give a light yellow solid 5.5 g crude product. Further purification was conducted by dissolving the crude product into DCM/MeOH (40 ml, 10/1) to triturate with petroleum ether (40 ml) for 2 hours slow stirring. The precipitate was filtered and dried in an oven to give the final crystalline product 4.4 g (MP: 203-208 C) and it can be further purified by recrystallizing from EtOH to give purer final product as a same crystalline form. MS: (M+l) 408; ¾ NMR(DMSO-dg) δ 0.60- 0.63(d, 4H), 2.41(s, 1H), 2.42-2.5 l(t, 2H), 3.3 l(s, 2H), 3.96(s, 3H), 4.04(s, 2H), 6.27(s, 1H), 6.31-6.32(m, 1H), 6.97-7.02(t, 1H), 7.20-7.22(d, 1H), 7.36(s, 1H), 7.60(s, 1H), 8.40-8.42(d, 1H), 1 1.41(s, 1H). MP: 208-210°C; DSC Melting Range (Endo): 207-220°C with Peak Temp=216°CPATENTWO 2019154273https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=11C1DF5485B11ADA40E45C9488AB5679.wapp1nB?docId=WO2019154273&tab=FULLTEXT
Tyrosine kinases are a group of enzymes that catalyze the phosphorylation of protein tyrosine residues. They play an important role in intracellular signal transduction. They are involved in the regulation, signal transmission and development of normal cells, and are also related to tumor cells. Proliferation, differentiation, migration and apoptosis are closely related. Many receptor tyrosine kinases are related to the formation of tumors, and can be divided into epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), and vascular endothelial cell growth factor receptor according to the structure of their extracellular region. Body (VEGFR), Fibroblast Growth Factor Receptor (FGFR), etc.[0003]WO2008112407 discloses the compound 1-((4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy in Example 24 )Methyl)cyclopropylamine and its preparation method, its structural formula is shown in formula I:[0004]

[0005]It is a multi-target receptor tyrosine kinase inhibitor that can inhibit the activity of vascular endothelial cell growth factor receptors (VEGFR1, VEGFR2/KDR and VEGFR3), stem cell factor receptors, platelet-derived growth factor receptors and other kinase activities. Inhibit the downstream signal transduction mediated by VEGFR2, thereby inhibiting tumor angiogenesis.[0006]Solid drugs generally have multiple crystal forms, such as polymorphs, solvates (hydrates), salts, and co-crystals. The change in the crystal form of the same drug usually results in different melting points, solubility, stability, biological activity, etc., which are important factors that affect the difficulty of drug preparation, storage stability, preparation difficulty, and bioavailability. . When the compound has multiple crystal forms, due to the specific thermodynamic properties and stability of the specific crystal form of the drug, it is important to understand the crystal form of the compound used in each dosage form during the preparation process to ensure the production process Use the same form of medicine. Therefore, it is necessary to ensure that the compound is a single crystal form or a known mixture of some crystal forms.[0007]WO2016179123 discloses the crystalline form 1 of the free base anhydrate of the compound of formula I and a preparation method thereof. CN201010245688.1 discloses the anhydrate and dihydrate crystals of quinoline derivative dihydrochloride and the preparation method thereof.[0008]The discovery of a variety of new crystal forms of medicinal compounds provides an opportunity to improve the physical properties of the drug, that is, to expand all the properties of the substance, which can better guide the research of the compound and its preparation. Therefore, the quinoline derivative provided in this application The crystals and pharmaceutical compositions containing the crystals have commercial value in the manufacture of medicines and other applications.Example 1 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropylamine (Formula I compound) preparation[0081]

[0082]Put intermediate 1 (its chemical name is (1-((4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxy Quinolin-7-yl)oxy)methyl)cyclopropyl)benzyl carbamate) 100g, 10% palladium on carbon 30g, ammonium formate 50g and methanol 800ml. Incubate the reaction at 45-55°C, TLC tracking showed that the reaction was complete, filtered, the filter cake was washed with a small amount of methanol, the filtrate was concentrated to dryness under reduced pressure, ethyl acetate and 2mol/L hydrochloric acid were added, stirred for 10 minutes, and then stood for 10 minutes. Separate the aqueous phase, adjust the pH to above 12 with 4N sodium hydroxide, and a large amount of solids will precipitate out. After washing with water until neutral, the aqueous phase is filtered to obtain the crude product of the title compound.[0083]Example 2 Preparation of amorphous compound of formula I[0084]According to the preparation method disclosed in Example 24 of WO2008112407, 1-((4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yl (Oxy)methyl)cyclopropylamine is composed of (1-(((4-(4-fluoro-2-methyl-1H-indol-5-yl)oxy-6-methoxyquinolin-7-yl )Oxy)methyl)cyclopropyl)benzyl carbamate (Intermediate 1) was prepared according to the following methods 2.1 and 2.2.[0085]2.1 Take 100 mg of Intermediate 1 and Pd/C (10%, 40 mg) into ethanol (20 ml), and hydrogenate at 50 psi for 12 hours. The reaction solution was filtered with diatomaceous earth, and evaporated to obtain an amorphous compound of formula I, and its X-ray powder diffraction (XRD) pattern was obtained as shown in FIG. 11.[0086] 
2.2 Take 100 mg of Intermediate 1, acetic acid (1ml) and 33% hydrobromic acid/acetic acid (1ml) and mix. The reaction was stirred for 1 hour at room temperature, diluted with ethyl acetate/water, and then basified with sodium carbonate. The organic layer is dried, concentrated, and purified by silica gel column to obtain the amorphous compound of formula I.PATENTUS 20160326138https://patents.google.com/patent/US20160326138A1/enNew process has been outlined in Scheme I.

Figure US20160326138A1-20161110-C00001
  • The present invention relates a new process to synthesize 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropanamine (AL3818) by condensing intermediate (X1) with (Y1) in a solvent at the presence of KI or NaI to form intermediate (Z) which is deprotected to give the final compound (AL3818) according to Process A.
  • [0040]
    The final compound (AL3818) was prepared according to Process A1 when R is H by deprotecting intermediate (Z-1) with HCOONH(ammonium formate) and Pd/C in an alcoholic solvent, such as MeOH, at 25° C.-80° C. for 0.1-4 hours. (Z-1) was prepared by reacting intermediate (X1) with (Y1-1) at the presence of KI or NaI with K2COin a solvent, such as acetone or DMF, at a temperature of 60° C.-160° C. for 2-24 hours.
  • [0041]
    The final compound (AL3818) was prepared according to Process A2 when R is 4-OMe by deprotecting intermediate (Z-2) with TFA in DCM at 0° C.-30° C. for 1-24 hours. (Z-2) was prepared by reacting intermediate (X1) with (Y1-2) at the presence of KI or NaI with K2COin a solvent, such as acetone or DMF, at a temperature of 60° C.-160° C. for 2-24 hours.
  • [0042]
    The present invention relates a new process to synthesize 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropanamine (AL3818) by reacting intermediate (X2) with (Y2) in a solvent to form intermediate (Z) which is deprotected to give the final compound (AL3818) according to Process B.
  • [0043]
    The final compound (AL3818) was prepared according to Process B1 when R is H by deprotecting intermediate (Z-1) with HCOONH(ammonium formate) and Pd/C in an alcoholic solvent, such as MeOH, at 25° C.-80° C. for 0.1-4 hours. (Z-1) was prepared by reacting intermediate (X2-1) with (Y2) in a solvent, such as pyridine or lutidine, at a temperature of 60° C.-160° C. for 1-12 hours.
  • [0044]
    The final compound (AL3818) was prepared according to Process B2 when R is 4-OMe by deprotecting intermediate (Z-2) with TFA in DCM at 0° C.-30° C. for 1-24 hours. (Z-2) was prepared by reacting intermediate (X2-2) with (Y2) in a solvent, such as pyridine or lutidine, at a temperature of 60° C.-160° C. for 1-12 hours.
  • [0045]
    The following examples further illustrate the present invention, but should not be construed as in any way to limit its scope.

Example 1Representation of Process A, Process A1Process for preparation of 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-quinolin-7-yloxy)methyl)cyclopropanamine (AL3818)

  • [0046]
    To a stirred mixture of benzyl 1-(hydroxymethyl)cyclopropylcarbamate (50 g) and DCM (200 ml) was added DIPEA (39 g). The result solution was cooled to 0-5° C. with ice/water and further stirred under this temperature for 15 min. MsCl (30 g) was added via an addition funnel dropwise keeping temperature below 5° C. for about 1.5 hours. After completion of addition, the reaction mixture was allowed stirring at 0-5° C. for 30 min and quenched with saturated NaHCO(150 ml). The solution was extracted with 150 ml DCM twice. The combined DCM layer was washed with 0.1 N HCl (400 ml) followed by brine. It was dried over Na2SOand concentrated to obtain an off-white solid 60 gram as (1-(benzyloxycarbonylamino)cyclopropyl)methyl methanesulfonate (Y1-1), MS: (M+1) 300.
  • [0047]
    To a stirred mixture of (Y1-1) (16 g), X1 [(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-7-hydroxyquinoline, 12 g], K2CO(21 g) and KI (21 g) was added DMF (100 ml), the reaction suspension was heated at 80° C. for 10 hours and (Y1-1) (10 g) was added to continuously heated 80° C. for 10 hours. The reaction then was quenched with water (150 ml) and extracted with 150 ml DCM twice. The combined DCM layer was washed with 2 N NaOH (100 ml) followed by water and brine. It was dried over Na2SOand concentrated, further recrystallized from EtOH to obtain a yellow solid as benzyl 1-((4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)methyl)cyclopropylcarbamate (Z-1) 9.5 g. MS: (M+1) 542.
  • [0048]
    To a stirred mixture of (Z-1) (9.5 g), HCOONH(4.7 g) and Pd/C (10%, wet 50%, 4.7 g) was added MeOH, the reaction mixture was heated at 45° C. for 1.5 hours. It was then cooled and filtered through Celite, further evaporated. 2N HCl (200 ml) was added and extracted with DCM/MeOH (10/1, 100 ml) twice. The aqueous layer was basified with 3N NaOH to adjust pH 11-12 to generate a solid precipitation. The solid was filtered and washed with water to neutral, further suction dry. The solid was dissolved into a mixture of DCM/MeOH (250 ml, 10/1) and further washed with water and brine. It was dried with MgSOand filtered, further evaporated to give a light yellow solid 5.5 g crude product. Further purification was conducted by dissolving the crude product into DCM/MeOH (40 ml, 10/1) to triturate with petroleum ether (40 ml) for 2 hours slow stirring. The precipitate was filtered and dried in an oven to give the final crystalline product 4.4 g (MP: 203-208° C.) and it can be further purified by recrystallizing from EtOH to give purer final product as a same crystalline form. MS: (M+1) 408; 1H NMR (DMSO-d6) δ 0.60-0.63 (d, 4H), 2.41 (s, 1H), 2.42-2.51 (t, 2H), 3.31 (s, 2H), 3.96 (s, 3H), 4.04 (s, 2H), 6.27 (s, 1H), 6.31-6.32 (m, 1H), 6.97-7.02 (t, 1H), 7.20-7.22 (d, 1H), 7.36 (s, 1H), 7.60 (s, 1H), 8.40-8.42 (d, 1H), 11.41 (s, 1H). MP: 208-210° C.; DSC Melting Range (Endo): 207-220° C. with Peak Temp=216° C. TGA demonstrating as an unsolvated material with weight loss at about 210° C. (between 205-215° C.). XRPD having pattern comprising characteristic 10 peaks with intensity % greater than 10% expressed in d values and angles as follows:
  • Angle d value 13.344 6.62986 15.858 5.58405 16.799 5.27326 17.640 5.02377 18.770 4.72373 20.650 4.29771 21.633 4.10463 23.087 3.84934 25.128 3.54112 26.607 3.34755
  • [0049]
    It was similar prepared according to the preparation procedures of (Z-1) described in Example 1 by using 4-methoxybenzyl 1-(hydroxymethyl)cyclopropylcarbamate to first generate (1-((4-methoxybenzyloxy)carbonylamino)cyclopropyl)methyl methanesulfonate (Y1-2) then to give 4-methoxybenzyl 1-((4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxyquinolin-7-yloxy)-methyl)cyclopropylcarbamate (Z-2), MS: (M+1) 572
  • [0050]
    To a stirred mixture of (Z-2) (1.5 g) in DCM (15 ml) at 0° C. was added TFA (1.5 ml) for about 30 min and warmed up to RT. The reaction was stirred at RT for 2 hours and added into water (30 ml). The aqueous layer was extracted with DCM twice (100 ml×2) and basified with 2N NaOH to adjust pH 11-12. The mixture was extracted with DCM (100 ml×3) and further washed with brine (100 ml). It was dried with MgSOand filtered. The solution was evaporated to give 1.05 g crude final product. Further purification was conducted to dissolve the crude product into DCM/MeOH and triturated with petroleum ether and dried in an oven to give the final pure product 0.8 g AL3818 with the same crystalline form.

Example 3Representation of Process A, Process B1Process for preparation of 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-quinolin-7-yloxy)methyl)cyclopropanamine (AL3818)

  • [0051]
    To a mixture of benzyl 1-((4-chloro-6-methoxyquinolin-7-yloxy)methyl)cyclopropyl-carbamate (X2-1) (5 g), 4-fluoro-2-methyl-1H-indol-5-ol (Y2) (5 g) and DMAP (4 g) was added 1,6-lutidine (15 ml). The reaction was stirred and heated at 135° C. for 5 hours and was cooled followed by adding IPA with slow stirring for 2 hours at RT. The solid was filtered and further washed with IPA, dried to give (Z-1) 5.2 g as a solid. It was then similarly prepared according to deprotection procedures described of (Z-1) in Example 1 to give the final compound AL3818 with the same crystalline form.

Example 4Representation of Process A, Process B2Process for preparation of 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-quinolin-7-yloxy)methyl)cyclopropanamine (AL3818)

  • [0052]
    (Z-2) was similarly prepared according to the procedures described in Example 3 by using 4-methoxybenzyl 1-((4-chloro-6-methoxyquinolin-7-yloxy)methyl)cyclopropylcarbamate (X2-2) and (Y2). It was then similarly prepared according to deprotection procedures of (Z-2) described in Example 2 to give the final compound AL3818 with the same crystalline form.

Example 5

  • [0053]
    Preparation of 1-((4-(4-Fluoro-2-methyl-1H-indol-5-yloxy)-6-methoxy-quinolin-7-yloxy)-methyl)cyclopropanamine bishydrochloride acid salt and its crystalline
  • [0054]
    To a 25 ml flask was added 250 mg free base (AL3818), 4N HCl in dioxane 0.625 mL (2.5 mmol, 4 eq.) in 10 ml EtOH, the reaction was heated at 75° C. for 30 minutes, cooled to RT and stirred for O.N. The solid was filtered and rinsed with acetone twice. It was dried in oven at 50° C. for 4 hours to give 126 mg white solid as the bishydrochloride salt as a crystalline and further recrystallized from EtOH to give a purer product as a same crystalline form. 1H NMR (DMSO-d6) δ 1.09-1.24 (m, 4H), 2.43 (s, 3H), 4.08 (s, 3H), 4.40 (s, 2H), 6.32 (s, 1H), 6.76 (s, 1H), 7.05-7.11 (t, 1H), 7.27-7.30 (d, 1H), 7.65 (s, 1H), 7.82 (s, 1H), 8.64 (s, 2H), 8.70-8.73 (m, 1H), 11.51 (s, 1H). Chloride ion chromatography showed 2 molecular ratio ions (16.1%). DSC Melting Range (Exo): 249-280 with Peak Temp=268° C.
  • [0055]
    To a 10 mL flask, charged 140 mg of 3818-2HCl salt from above Example 4 and 0.7 mL (×5 with salt volume) of 80% MeOH in H2O. The result suspension was heated to 70° C. to form a solution and cooled to RT and further stirred for O.N. The solid was filtered and rinsed with acetone twice. It was dried in oven at 50° C. for 4 hours to obtain off-white solid 110 mg as the crystalline bishydrochloride hydrate salt. 1H NMR (DMSO-d6) δ 1.09 (s, 2H), 1.22 (s, 2H), 2.44 (s, 1H), 2.52 (s, 2H), 4.09 (s, 3H), 4.44 (s, 2H), 6.32 (s, 1H), 6.81-6.82 (d, 1H), 7.08-7.14 (t, 1H), 7.29-7.32 (d, 1H), 7.79 (s, 1H), 7.85 (s, 1H), 8.75-8.78 (d, 1H), 8.85 (s, 2H), 11.66 (s. 1H). Chloride ion chromatography showed 2 molecular ratio ions (17.8%). DSC Melting Range (Exo): 207-260° C. with Peak Temp=226° C. TGA demonstrating 2.68% (˜3%, 1 water) weight loss till 120° C. (between 115-125° C.) and further weight loss at about 170° C. (between 165-175° C.).

PATENT

US8148532B2.

https://patents.google.com/patent/US8148532B2/en

Patent

2. US20080227811A1.

/////////////catequentinib, ANLOTINIB, AL3818, AL 3818, PHASE 2, CHINA 2018

NC1(CC1)COc1cc2nccc(Oc3ccc4[NH]c(C)cc4c3F)c2cc1OC

ETRUMADENANT


str1

ETRUMADENANT

CAS 2239273-34-6

C23 H22 N8 O, 426.47

Benzonitrile, 3-[2-amino-6-[1-[[6-(1-hydroxy-1-methylethyl)-2-pyridinyl]methyl]-1H-1,2,3-triazol-4-yl]-4-pyrimidinyl]-2-methyl-

  • 3-[2-Amino-6-[1-[[6-(1-hydroxy-1-methylethyl)-2-pyridinyl]methyl]-1H-1,2,3-triazol-4-yl]-4-pyrimidinyl]-2-methylbenzonitrile
  • AB 928

Arcus Biosciences is developing etrumadenant, the lead from the small molecule adenosine (A2a/A2b) dual receptor antagonist program, for treating cancer. In November 2020, preliminary data from ARC-7 in metastatic NSCLC were expected to report in the first half of 2021.

  • OriginatorArcus Biosciences
  • ClassAmines; Antineoplastics; Nitriles; Pyridines; Pyrimidines; Small molecules; Triazoles
  • Mechanism of ActionAdenosine A2A receptor antagonists; Adenosine A2B receptor antagonists
  • Phase IINon-small cell lung cancer
  • Phase I/IIProstate cancer
  • Phase IBladder cancer; Breast cancer; Cancer; Colorectal cancer; Endometrial cancer; Gastrointestinal cancer; Head and neck cancer; Malignant melanoma; Merkel cell carcinoma; Oesophageal cancer; Ovarian cancer; Renal cancer
  • 19 Sep 2020Updated efficacy and adverse events data from a phase I/Ib trial in Non-small cell lung cancer presented at the 45th European Society for Medical Oncology Congress (ESMO-2020)
  • 06 Aug 2020Efficacy data from a phase I trial in Colorectal cancer presented at the American Association for Cancer Research Meeting (AACR-2020)
  • 13 Jul 2020Arcus Biosciences and Gilead Sciences complete closing of partnership agreement to co-develop and co-promote AB 928 in USA

PAPER

Organic Process Research & Development (2020), 24(7), 1254-1261.

https://pubs.acs.org/doi/10.1021/acs.oprd.0c00124

AB928 is a potent and selective dual antagonist of the A2a and A2b receptors, which is currently in clinical trials. Here, we report the development of two scalable and practical syntheses of AB928. The first-generation synthesis was used to successfully obtain AB928 in excellent yield and purity to support our preclinical and initial clinical studies. Recently, we have developed a second-generation synthesis of AB928 featuring a palladium-free protocol to access 3-(2-amino-6-chloropyrimidin-4-yl)-2-methylbenzonitrile, a key intermediate in the AB928 synthesis. The new method is scalable, practical, and significantly more cost-effective.

Abstract Image

PAPER

Tetrahedron Letters (2020), 61(20), 151855.

PAPENT

WO 2020018680

Example 1: Synthesis of 3-[2-amino-6-(l-{[6-(2-hydroxypropan-2-yl)pyridin-2-yl]methyl}-lH-l,2,3-triazol-4-yl)pyrimidin-4-yl]-2-methylbenzonitrile (Compound I)

[0208] Step 1 : In a 250mL round bottom flask equipped with a magnetic stir bar was successively charged the boronic ester (3.89 g, 16 mmol) and the 2-amino-4,6-dichloropyrimidine (3.67 g, 22,4 mmol). Absolute ethanol (100 mL) was added followed by a solution of KHCO3 (4.81 g, 48 mmol) in deionized water (19 mL). The resulting suspension was degassed with nitrogen for 5 minutes. PdChiPPluk (112 mg, 1 mol%) was then added and the mixture was heated to 78 °C for 3 hours under a nitrogen atmosphere. Ethanol was evaporated under reduced pressure and deionized water (150 mL) was added. The suspension was filtered and the solid was washed with additional water (100 mL). The solid was then dissolved in acetone (220 mL) and collected in a 500 mL round bottom flask. A mixture of silica and celite (1 : 1, 150 g) was added and the solvent was removed under reduced pressure. The resulting crude material was purified by flash chromatography over silica gel (dichloromethane/ethyl acetate gradient 0% to 15%). The desired product was obtained as a white solid (1.91 g, 49%). LCMS: Method A, retention time = 2.93 min, ESI MS [M+H]+ for C12H9CIN4, calcd 245.7, found 245.2

[0209] Step 2 : In a round-bottom flask 5.1 g (20.8 mmol) of chloro-pyrimidine was suspended in 42 mL of degassed THF. To this suspension was added 8.68 mL (62.4 mmol) of Et3N and 5.95 mL (25.0 mmol) of TIPS-acetylene. The reaction mixture was stirred for 5 min, followed by addition of 219 mg (0.312 mmol) of PdCl2(PPh3)2 and 119 mg (0.624 mmol) of Cul. The reaction mixture was stirred at 50 °C for 5h under N2. After cooling the reaction to room temp., solvent was removed and the crude material was resuspended in 100 mL EtOAc from which insoluble solid was filtered off. The filtrate was washed with (1 : 1) NH4CI/NH4OH (2 x 100 mL) and 10% Na2S204 (1 x 100 mL). The organic layer was dried using Na2S04, concentrated and taken to next step without further purification.

[0210] Step 3 : In a round-bottom flask the crude TIPS product from previous step was dissolved in 42 mL dry THF and cooled to 0 °C. To this was added 25 mL (25.0 mmol) of TBAF (1.0 M in THF). The reaction was stirred at 0 °C for 15 min. Saturated NH4CI (100 mL) was added to quench the reaction. The organics were extracted from the aqueous layer with EtOAc (2 x 100 mL). The combined organic layer was washed with (1 : 1) NH4CI/NH4OH (2 x 100 mL) and 10% Na2S204 (1 x 100 mL). The organic layer was dried using Na2S04, concentrated and the pure product 5 was obtained by triturating with 40% CH2Cl2/Hexane as a light brown solid. Yield: 3.71 g (76%, 2-steps).

[0211] Step 4 : To a solution of methylmagnesium bromide (3 M in Et20, 40 mL, 120 mmol, 4.0 equiv) at 0 °C under N2 was added a solution of methyl 2-(hydroxymethyl)pyridine-2-carboxylate (5.0 g, 29.9 mmol) in THF (70 mL, 0.4 M) over the course of 30 minutes. The resulting mixture was allowed to warm to room temperature and stirred for 3 h. The reaction mixture was quenched with NH4CI aq (55 mL) and EtOAc (50 mL) was added. The organic phase was separated, and the aqueous phase was extracted with EtOAc (3 x 40 mL). The combined organic extracts were washed with saturated aqueous sodium bisulfite (7 x 20 mL), then dried (Ni^SCh), filtered and concentrated in vacuo to give the title compound (3.45 g, 69% yield; 96% purity as judged by LCMS) as a pale yellow liquid. LCMS: Method A, retention time = 0.722 and 1.06 min, ESI MS [M+H]+ for C9H13NO2, calcd 167.09, found 167.2

[0212] Step 5 : To a solution of 2-hydroxymethyl-6-(l -hydroxy- 1 -methyl ethyljpyri dine (5 g,

29.9 mmol, 1.0 equiv) in PhMe (33 mL, 0.9 M) at 0 °C under N2 was added diphenylphosphoryl azide (7.73 mL, 35.9 mmol, 1.2 equiv.), followed by l,8-diazabicyclo[5.4.0]undec-7-ene (5.37 mL, 35.9 mmol, 1.2 equiv.). The resulting mixture was to warm to room temperature and stirred for 14 h. Upon completion, diluted with ethyl acetate and washed with water, the organic layer was dried (Na2S04), filtered and concentrated. The residue was dissolved in 1N aq HC1 (2 eq, 60 mmol) and extracted with MTBE in hexanes (3:7, 100 mL), the organic layer was washed with water (50 mL) and the combined aqueous layer was neutralized with 2N aqueous NaOH and extracted with ethyl acetate (3X75 mL), dried the organic layer (Na2S04), filtered through a plug of cotton and concentrated the filtrate to afford the pure compound as pale yellow color liquid (3.75 g, 75%). LCMS: Method A, retention time = 2.67 min, ESI MS [M+H]+ for C9H12N4O, calcd 193.1, found 193.2

[0213] Step 6: A mixture of azide (3.34 g, 17.4 mmol), alkyne (3.71 g, 15.8 mmol), copper(II) sulfate (39 mg; 0.158 mmol), and sodium ascorbate (156 mg, 0.790 mmol) in 2: 1 /-BuOH/EbO (158 mL) was heated at 60 °C for 13 h. The solvent was removed in vacuo, the residue dry loaded onto silica gel, and purified by silica gel chromatography (0-100% EtOAc in hexanes) to afford the desired product as an off-white solid (6.08 g, 90%). ‘H NMR (400 MHz, DMSO-cfc) d 8.69 (s, 1H), 7.90 (d, J= 7.8 Hz, 1H), 7.80 (t, J= 7.8 Hz, 1H), 7.76 (d, J= 7.8 Hz, 1H), 7.61 (d, J= 8.0 Hz, 1H), 7.51 (t, /= 7.8 Hz, 1H), 7.28 (s, 1H), 7.10 (d, J= 7.6 Hz, 2H), 6.90 (s, 2H), 5.81 (s, 2H), 5.23 (s, 1H), 2.55 (s, 3H), 1.38 (s, 6H). ESI MS [M+H]+ for C23H23N8O, calcd 427.2, found 427.3.

Example 2: Preparation of Crystalline Solid Form of 3-[2-amino-6-(l-{[6-(2-hydroxypropan-2-yl)pyridin-2-yl]methyl}-lH-l,2,3-triazol-4-yl)pyrimidin-4-yl]-2-methylbenzonitrile

[0214] The product from Example 1, Step 6 (7.53 g) was dissolved in acetone (109 mL) by heating to reflux at which point water (218 mL) was added at a rate of 10 mL/min to initiate crystallization. The mixture was cooled and the solids were collected by filtration, washed with 1 :2 acetone/water (109 mL), and dried under vacuum to afford Form I of Compound I as a white solid (7.08 g; 94%).

PATENT

WO 2019161054

PATENT

WO2020185859 , claiming method for treating a subject identified as having an oncogene driven cancer comprising an agent (eg AB-928) targeting the extracellular production of adenosine and/or antagonizing the activation by adenosine of one of its receptors.

PATENT

WO-2020247789

Processes for preparing aminopyrimidine compounds, particularly etrumadenant (AB-928).

Example 1: Trifluoroethanol Assisted Condensation of B-Ketoesters to Provide a

Hydroxypyrimidine (and Chloropyrimidine).

bromo-2-methylaniline (18.6 g, 100 mmol) dropwise so that a fine white suspension forms. The mixture was cooled to 0 °C and a solution of sodium nitrite (7.31 g, 106 mmol) in water (15.1 mL) was added dropwise. The mixture was stirred at 0 °C for 30 minutes. To the resultant homogeneous mixture at 0 °C was added sodium bicarbonate (17.8 g, 212 mmol) at such a rate to avoid excessive gas evolution. The aqueous phase of the resultant brown suspension was found to have pH ~7. This suspension was maintained at 0 °C.

[0070] In a separate flask, copper cyanide (9.85 g, 110 mmol), potassium cyanide (13.0 g, 200 mmol), and water (31 mL) were heated to 60 °C to form a homogeneous solution. To this solution at 60 °C with stirring was added the above suspension dropwise to avoid excessive gas evolution. After addition, the mixture was stirred at 100 °C for 30 minutes. The mixture was cooled, MTBE (200 mL) was added, the mixture agitated, and filtered to remove any solids, washing with MTBE. The organic phase was dried over Na2SO4 and concentrated. The resultant crude product was purified by vacuum distillation to afford the desired product as a light orange solid (13.6 g, 69%).

[0071] Step 2: In a two liter two-necked flask, aryl bromide (101.9 g, 520 mmol, 1.0 equiv.) was dissolved in THF (520 mL) under an atmosphere of N2, and the mixture was cooled in an

ice-water bath. iPrMgClLiCl (400 mL, 1.3 M in THF, 520 mmol, 1.0 equiv.) was added by cannula. Upon completion of the addition, the ice bath was removed. After four hours, the flask was cooled in an ice-water bath and dry ice (~ 230 g, 5.2 mol, 10 equiv.) was added portionwise to prevent overheating or bubbling over (note: CO2 gas can be bubbled through the solution in place of solid dry ice). When bubbling from the addition was complete, the mixture was diluted with MTBE (500 mL) and 2M HC1 (250 mL). The layers were separated, and the aqueous layer was washed with additional MTBE (500 mL). The organic layer was extracted with 10% NaOH (190 mL x 2), and the combined aqueous layers were cooled in an ice-water bath and acidified with concentrated HC1 until a white precipitate formed. The precipitate was isolated by filtration and washed with water before being dried overnight in a vacuum oven at 80° C to afford the benzoic acid as a white solid (64.1 g, 76% yield).

[0072] Step 3: The benzoic acid (50 g, 311 mmol, 1.0 equiv.) was suspended in CH2CI2, and oxalyl chloride (40 mL, 466 mmol, 1.5 equiv.) was added, followed by DMF (~ 30 drops). Off gassing was observed immediately, and the reaction flask was open to the atmosphere under positive pressure of N2. Upon complete consumption of the starting acid as determined by LCMS and visual inspection (complete dissolution of starting material), the reaction mixture was concentrated. Excess oxalyl chloride was removed by azeotropic distillation with toluene to afford the corresponding acid chloride as a tannish-brown solid.

[0073] In a separate two-necked flask equipped with an overhead stirrer, potassium ethyl malonate (66.1 g, 388 mmol, 1.25 equiv.), triethylamine (108 mL, 777 mmol, 2.5 equiv.) and MeCN (777 mL) were cooled in a salt/ice-brine bath. Solid MgCl2 (74 g, 777 mmol, 2.5 equiv.) was added, and the resulting suspension was vigorously stirred at ~ -10° C. After one hour, the solid acid chloride was added at a rate to ensure dissolution into the thick suspension. The suspension rapidly became homogenous, and the stirring rate was reduced to avoid splashing.

The ice bath was removed. Upon complete consumption of the starting material as determined by TLC analysis, the reaction mixture was cooled in an ice-water bath, and 2M HC1 (971 mL, 1.9 mol, 6.25 equiv.) was added, and the ice bath was removed. After 30 minutes, the layers were separated, and the aqueous layer was extracted with MTBE. The combined organic layers were washed with saturated NaHCO3 and brine, dried over sodium sulfate, filtered, and concentrated to afford the keto-ester as a tannish-brown solid (67 g, 93% yield).

[0074] Step 4: A round-bottom flask was charged with 42.0 g (181.8 mmol) of the b-keto-ester, 32.7 g (181.8 mmol) of guanidinium carbonate and 227 mL of trifluoroethanol. The suspension was then heated to reflux under N2 for 16 h.

[0075] Work-up: The reaction was cooled to room temperature and solvent was evaporated under reduced pressure to obtain a viscus red oil. The oil was re-dissolved in 250 mL H2O and the aqueous solution was extracted with dichloromethane (2 x 250 mL). The aqueous phase is then acidified to pH ~2-3 using 1.0 M HCl(aq ). The precipitated product was collected by filtration, washed thoroughly with H2O and dried in a vacuum oven at 70 °C. Yield 30.81 g (75%), Purity >99%.

[0076] Step 5: A round-bottom flask was charged with 50.0 g (221.2 mmol) pyrimidone from step 4 and 100.8 g (442.2 mmol) of benzyltriethylammonium chloride. The mixture was suspended in 442.2 mL of dry acetonitrile and 31.0 mL (331.8 mmol) of POCI3 was added. The suspension thus obtained was then heated to reflux under N2 for 4 h.

[0077] Work-up: The reaction was cooled to room temperature and ~200 g crushed ice was added. The mixture was then stirred for 30 min flowed by dropwise addition of ice-cold 15% aqueous NH4OH to ~ pH 10 -11. {Note: Slow addition of cold NH4OH is recommended to avoid sudden exotherm due to quenching of excess POCI3). The suspension was then stirred at room temperature for an additional 1.5 h. The precipitated product was collected by filtration, washed thoroughly with H2O and dried in a vacuum oven at 70 °C. Yield 48.2 g (89%), Purity >99%.

HPLC conditions

HPLC: Agilent 1 100

Column: YMC-HPLC Column; 250 x 4.6; S-5 pm, 20 nm; AQ20S05-2546WT; No.0425058945

Solvent: H2O / MeCN with 0.1% HCO2H

Flow Rate: 0.8 mL/min

Column Temperature: 30 °C

Method:

Example 2: Comparative Pyrimidine Coupling

[0078] The synthetic route for preparing 3-[2-amino-6-(l- {[6-(2-hydroxypropan-2-yl)pyridin-2-yl]methyl}-1H-1 ,2,3-triazol-4-yl)pyrimidin-4-yl]-2-methylbenzonitrile utilizing boronic ester benzonitrile to linked the phenyl and pyrimidine rings is shown below and is also provided in WO2018/136700.

[0079] The scheme below displays the synthetic route used to prepare the boronic ester benzonitrile used in the process above and subsequent reaction with pyrimidine to form a compound of Formula (I). Notably, the desired linkage between the pyrimidine and the phenyl provides a yield of less than 50%.

[0080] The below scheme displays the synthetic route used to prepare a compound of Formula (I) that utilized a conversion of a b-diketoester to a pyrimidine using guanidine. The route provides a 75% yield.

PATENT

WO 2018136700

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2018136700

Example 1: Synthesis of 3-[2-amino-6-(1-{[6-(2-hydroxypropan-2-yl)pyridin-2-yl]methyl}-1H-1,2,3-triazol-4-yl)pyrimidin-4-yl]-2-methylbenzonitrile

[0269] Step 1: In a 250mL round bottom flask equipped with a magnetic stir bar was successively charged the boronic ester (3.89 g, 16 mmol) and the 2-amino-4,6- dichloropyrimidine (3.67 g, 22,4 mmol). Absolute ethanol (100 mL) was added followed by a solution of KHCO3 (4.81 g, 48 mmol) in deionized water (19 mL). The resulting suspension was degassed with nitrogen for 5 minutes. PdCl2(PPh3)2 (112 mg, 1 mol%) was then added and the mixture was heated to 78 °C for 3 hours under a nitrogen atmosphere. Ethanol was evaporated under reduced pressure and deionized water (150 mL) was added. The suspension was filtered and the solid was washed with additional water (100 mL). The solid was then dissolved in acetone (220 mL) and collected in a 500 mL round bottom flask. A mixture of silica and celite (1:1, 150 g) was added and the solvent was removed under reduced pressure. The resulting crude material was purified by flash chromatography over silica gel (dichloromethane/ethyl acetate gradient 0% to 15%). The desired product was obtained as a white solid (1.91 g, 49%). LCMS: Method A, retention time = 2.93 mm, ESI MS [M+H]+ for C12H9ClN4, calcd 245.7, found 245.2

[0270] Step 2: In a round-bottom flask 5.1 g (20.8 mmol) of chloro-pyrimidine was suspended in 42 mL of degassed THF. To this suspension was added 8.68 mL (62.4 mmol) of Et3Ν and 5.95 mL (25.0 mmol) of TIPS -acetylene. The reaction mixture was stirred for 5 min, followed by addition of 219 mg (0.312 mmol) of PdCl2(PPh3)2 and 119 mg (0.624 mmol) of Cul. The reaction mixture was stirred at 50 °C for 5h under N2. After cooling the reaction to room temp., solvent was removed and the crude material was resuspended in 100 mL EtOAc from which insoluble solid was filtered off. The filtrate was washed with (1:1) NH4C1/NH4OH (2 × 100 mL) and 10% Na2S2O4 (1 × 100 mL). The organic layer was dried using Na2SO4, concentrated and taken to next step without further purification.

[0271] Step 3: In a round-bottom flask the crude TIPS product from previous step was dissolved in 42 mL dry THF and cooled to 0 °C. To this was added 25 mL (25.0 mmol) of TBAF (1.0 M in THF). The reaction was stirred at 0 °C for 15 mm. Saturated NH4Cl (100 mL) was added to quench the reaction. The organics were extracted from the aqueous layer with EtOAc (2 x 100 mL). The combined organic layer was washed with (1:1) NH4Cl/NH4OH (2 x 100 mL) and 10% Na2S2O4 (1 x 100 mL). The organic layer was dried using Na2SO4, concentrated and the pure product 5 was obtained by triturating with 40% CH2Cl2/Hexane as a light brown solid. Yield: 3.71 g (76%, 2-steps).

[0272] Step 4: To a solution of methylmagnesium bromide (3 M in Et2O, 40 mL, 120 mmol, 4.0 equiv) at 0 °C under N2 was added a solution of methyl 2-(hydroxymethyl)pyridine-2-carboxylate (5.0 g, 29.9 mmol) in THF (70 mL, 0.4 M) over the course of 30 minutes. The resulting mixture was allowed to warm to room temperature and stirred for 3 h. The reaction mixture was quenched with NH4Cl aq (55 mL) and EtOAc (50 mL) was added. The organic phase was separated, and the aqueous phase was extracted with EtOAc (3 x 40 mL). The combined organic extracts were washed with saturated aqueous sodium bisulfite (7 x 20 mL), then dried (Na2SO4), filtered and concentrated in vacuo to give the title compound (3.45 g, 69% yield; 96% purity as judged by LCMS) as a pale yellow liquid. LCMS: Method A, retention time = 0.722 and 1.06 mm, ESI MS [M+H]+ for C9H13NO2, calcd 167.09, found 167.2

[0273] Step 5: To a solution of 2-hydroxymethyl-6-(1-hydroxy-1-methylethyl)pyridine (5 g, 29.9 mmol, 1.0 equiv) in PhMe (33 mL, 0.9 M) at 0 °C under N2 was added diphenylphosphoryl azide (7.73 mL, 35.9 mmol, 1.2 equiv.), followed by l,8-diazabicyclo[5.4.0]undec-7-ene (5.37 mL, 35.9 mmol, 1.2 equiv.). The resulting mixture was to warm to room temperature and stirred for 14 h. Upon completion, diluted with ethyl acetate and washed with water, the organic layer was dried (Na2SO4), filtered and concentrated. The residue was dissolved in 1N aq HCl (2 eq, 60 mmol) and extracted with MTBE in hexanes (3:7, 100 mL), the organic layer was washed with water (50 mL) and the combined aqueous layer was neutralized with 2N aqueous NaOH and extracted with ethyl acetate (3×75 mL), dried the organic layer (Na2SO4), filtered through a plug of cotton and concentrated the filtrate to afford the pure compound as pale yellow color liquid (3.75 g, 75%). LCMS: Method A, retention time = 2.67 mm, ESI MS [M+H]+ for C9H12N4O, calcd 193.1, found 193.2

[0274] Step 6: A mixture of azide (3.34 g, 17.4 mmol), alkyne (3.71 g, 15.8 mmol), copper(II) sulfate (39 mg; 0.158 mmol), and sodium ascorbate (156 mg, 0.790 mmol) in 2:1 t-BuOH/H2O (158 mL) was heated at 60 °C for 13 h. The solvent was removed in vacuo, the residue dry loaded onto silica gel, and purified by silica gel chromatography (0-100% EtOAc in hexanes) to afford the desired product as an off-white solid (6.08 g, 90%). 1H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.80 (t, J = 7.8 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.61 (d, J= 8.0 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.28 (s, 1H), 7.10 (d, J = 7.6 Hz, 2H), 6.90 (s, 2H), 5.81 (s, 2H), 5.23 (s, 1H), 2.55 (s, 3H), 1.38 (s, 6H). ESI MS [M+H]+ for C23H23N8O, calcd 427.2, found 427.3.

/////////ETRUMADENANT, AB-928, AB 928, PHASE 2

TILDACERFONT


Tildacerfont.png

img

TILDACERFONT

Synonyms:

Tildacerfont

1014983-00-6

3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-7-(1-ethyl-propyl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidine

7-(1-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolo[1,5-a]pyrimidine

MW/ MF 420 g/mol/ C20H26ClN5OS
  • Originator Spruce Biosciences
  • Class2 ring heterocyclic compounds; Morpholines; Pyrazoles; Pyrimidines; Small molecules; Thiazoles
  • Mechanism of Action Corticotropin receptor antagonists
  • Orphan Drug Status Yes – Congenital adrenal hyperplasia
  • New Molecular Entity Yes
  • Phase II Congenital adrenal hyperplasia
  • 09 Jul 2020 Spruce Biosciences initiates a phase II trial in Congenital adrenal hyperplasia in USA (PO) (NCT04457336)
  • 24 Sep 2019 Spruce Biosciences completes a phase II trial in Congenital adrenal hyperplasia in USA (NCT03687242)
  • 19 Sep 2019 Updated safety and efficacy data from a phase II trial in Congenital adrenal hyperplasia release by Spruce Biosciences

Deuterated pyrazolo[1,5-a]pyrimidine derivatives, particularly tildacerfont (SPR-001), useful as CRF antagonists for treating congenital adrenal hyperplasia.  Spruce Bioscience is developing tildacerfont under license from Lilly as an oral capsule formulation for the treatment of congenital adrenal hyperplasia; in July 2017, a phase II trial for CAH was initiated.

Corticotropin releasing factor (CRF) is a 41 amino acid peptide that is the primary physiological regulator of proopiomelanocortin (POMC) derived peptide secretion from the anterior pituitary gland. In addition to its endocrine role at the pituitary gland, immunohistochemical localization of CRF has demonstrated that the hormone has a broad extrahypothalamic distribution in the central nervous system and produces a wide spectrum of autonomic, electrophysiological and behavioral effects consistent with a neurotransmitter or neuromodulator role in the brain. There is also evidence that CRF plays a significant role in integrating the response in the immune system to physiological, psychological, and immunological stressors.

PATENT

Product case, WO2008036579 ,

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2008036579

Example 16
3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-7-(l-ethyl-propyl)-2,5-dimethyl- pyrazolo [ 1 ,5 -α]pyrimidine

Under a nitrogen atmosphere dissolve 3-(4-bromo-2-morpholin-4-yl-thiazol-5-yl)-7-(l-ethyl-propyl)-2,5-dimethyl-pyrazolo[l,5-α]pyrimidine (116 mg, 0.25 mmol) in THF (1.5 mL) and chill to -78 0C. Add n-butyl lithium (0.1 mL. 2.5 M in hexane, 0.25 mmol) and stir at -78 0C for 30 min. Add N-chlorosuccinimide (33.4 mg, 0.25 mmol) and stir for another 30 min, slowly warming to room temperature. After stirring overnight, quench the reaction by adding a solution of saturated ammonia chloride and extract with ethyl acetate. Wash the organic layer with brine, dry over sodium sulfate, filter, and concentrate to a residue. Purify the crude material by flash chromatography, eluting with hexanes:dichloromethane: ethyl acetate (5:5:2) to provide the title compound (54 mg). MS (APCI) m/z (35Cl) 420.6 (M+l)+1H NMR (400 MHz, CDCl3): 6.44 (s, IH), 3.79 (t, 4H, J=4.8 Hz), 3.63-3.56 (m, IH), 3.47 (t, 4H, J=4.8 Hz), 2.55 (s, 3H), 2.45 (s, 3H), 1.88-1.75 (m, 4H), 0.87 (t, 6H, J=7.5 Hz).
Alternate Preparation from Preparation 6:
Combine 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-α]pyrimidine, (9 g,

26.2 mmol) and 4-chloro-2-morpholino-thiazole (7.5 g, 36.7 mmol) in
dimethylformamide (90 mL) previously degassed with nitrogen. Add cesium carbonate (17.8 g, 55 mmol), copper iodide (250 mg, 1.31 mmol), triphenylphosphine (550 mg, 2.09 mmol) and palladium acetate (117 mg, 0.52 mmol). Heat the mixture to 125 0C for 16 h and then cool to 22 0C. Add water (900 mL) and extract with methyl-?-butyl ether (3 x 200 mL). Combine the organic portions and evaporate the solvent. Purify by silica gel chromatography eluting with hexanes/ethyl acetate (4/1) to afford the title compound (6.4 g, 62%). ES/MS m/z (35Cl) 420 (M+l)+.

Example 16a
3-(4-Chloro-2-morpholin-4-yl-thiazol-5-yl)-7-(l-ethyl-propyl)-2,5-dimethyl- pyrazolo[l,5-α]pyrimidine, hydrochloride
Dissolve 3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-7-(l-ethyl-propyl)-2,5-dimethyl-pyrazolo[l,5-α]pyrimidine (1.40 g, 3.33 mmol) in acetone (10 mL) at 50 0C and cool to room temperature. Add hydrogen chloride (2 M in diethyl ether, 2.0 mL, 4.0 mmol) and stir well in a sonicator. Concentrate the solution a little and add a minimal amount of diethyl ether to crystallize the HCl salt. Cool the mixture in a refrigerator overnight. Add additional hydrogen chloride (2 M in diethyl ether, 2.0 mL, 4.0 mmol) and cool in a refrigerator. Filter the crystalline material and dry to obtain the title compound (1.15 g, 75%). ES/MS m/z (35Cl) 420 (M+l)+1H NMR(CDCO): 9.18 (br, IH), 6.86 (s, IH), 3.72 ( m, 4H), 3.49(m, IH), 3.39 (m, 4H), 2.48 (s, 3H), 2.38(s, 3H), 1.79 (m, 4H), 0.79 (m, 6H).

PATENT

US-20200255436

https://patentscope.wipo.int/search/en/detail.jsf?docId=US301567348&tab=PCTDESCRIPTION&_cid=P22-KE0UZI-30504-1

PATENT

WO2019210266

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2019210266

claiming the use of CRF-1 antagonists (eg tildacerfont).

PATENT

WO 2010039678

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2010039678

EXAMPLES

Example 1 : 7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolori ,5-alpyrimidine nthroline 

Charge 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (1.03 g, 3.00 mmoles), K3PO4 (1.95 g, 9.00 mmoles), 2,4-dichlorothiazole (0.58 g, 3.75 mmoles), 1,10 phenanthroline (0.05 g, 0.30 mmoles) and anhydrous DMAC (5 mL) to a round bottom flask equipped with a magnetic stir bar, thermal couple and N2 inlet. Degas the yellow heterogeneous reaction mixture with N2 (gas) for 30 min. and then add CuI (0.06 g, 0.30 mmoles) in one portion followed by additional 30 min. degassing with N2 (gas). Stir the reaction mixture at 120 0C for about 6 hr. Cool the reaction mixture to room temperature overnight, add toluene (10 mL) and stir for 1 hr. Purify the mixture through silica gel eluting with toluene (10ml). Extract with 1 M HCl (10 mL), water (10 mL), brine (10 mL) and concentrate under reduced pressure to give a yellow solid. Recrystallize the solid from methanol (5ml) to yield the title compound as a yellow crystalline solid. (0.78 g, 70% yield, >99% pure by LC) MS(ES) = 369 (M+ 1). 1H NMR (CDCl3)= 6.5 (IH, s); 3.6 (IH, m); 2.6 (3H, s); 2.5 (3H, s); 1.9 (4H, m); 0.9 (6H, t).

Example 2: 7-(l-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolol! ,5-aipyrimidine

Charge 7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (0.37 g, 1.00 mmoles), K2CO3 (0.28 g, 2.00 mmoles) and anhydrous morpholine (3 mL) to a round bottom flask equipped with a magnetic stir bar and N2 inlet. Stir the yellow mixture at 100 0C for about 4 hr., during which time the reaction becomes homogeneous. Cool the reaction mixture to room temperature, add H2O (10 mL) and stir the heterogeneous reaction mixture overnight at room temperature. Collected the yellow solid by filtration, wash with H2O and allowed to air dry overnight to give the crude title compound (391mg). Recrystallize from isopropyl alcohol (3 mL) to yield the title compound as a light yellow crystalline solid (380 mg, 90.6% yield, >99% by LC). MS(ES) = 420 (M+l). 1H NMR (CDCl3)= 6.45 (IH, s); 3.81 (m, 4H); 3.62 (IH, m); 3.50 (m, 4H); 2.6 (3H, s); 2.45 (3 H, s); 1.85 (4H, m); 0.9 (6H, t).

Example 3 :

The reactions of Example 1 are run with various other catalysts, ligands, bases and solvents, which are found to have the following effects on yield of 7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine. (See Tables 1 – 4).

Table 1 : Evaluation of different li ands

(Reactions are carried out in parallel reactors with 1.2 mmol 2,4-dichlorothiazole, 1 mmol 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine, 0.5 mmol CuI, 0.5 mmol ligand and 2.1 mmol Cs2CO3 in 4 mL DMAC. The reactions are degassed under N2 for 30 min. and then heated at between 80 and

1000C overnight under N2. Percent product is measured as the percent of total area under the HPLC curve for the product peak. Longer reaction times are shown in parenthesis) Table 2: Evaluation of various solvents


(Reactions are carried out in parallel reactors with 1.2 mmol 2,4-dichlorothiazole 1 mmol 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine, 0.25 mmol CuI, 0.25 mmol 1,10-phenanthroline and 2.1 mmol Cs2CO3 in 3 mL specified solvent. The reactions are degassed under N2 for 30 minutes and then heated at 1000C overnight under N2. Percent product is measured as the percent of total area under the HPLC curve for the product peak.)

Table 3 : Evaluation of different copper sources

(Reactions are carried out in in parallel reactors with 1 mmol 2,4-dichlorothiazole 1 mmol 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine, 0.05 mmol CuX, 0.01 mmol 1,10-phenanthroline and 3 equivalents K3PO4 in 3 mL DMAC. The reactions are degassed under N2 for 30 minutes and then heated at 1000C overnight under N2. Percent product is measured as the percent of total area under the HPLC curve for the product peak.)

Table 4: Evaluation of various inorganic bases

(Reactions are carried out in in parallel reactors with 1 mmol 2,4-dichlorothiazole 1 mmol 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine, 0.1 mmol CuI, 0.1 mmol 1,10-phenanthroline and 2.1 mmol base and degassed for 30 minutes prior to the addition of 3 mL DMAC. The reactions are degassed under N2 for 10 minutes and then heated at 1000C overnight under N2. Percent product is measured as the percent of total area under the HPLC curve for the product peak.)

Example 4. Use of morpholine both as a reactant and base in 2-MeTHF as solvent.

solvent

7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-ajpyrimidine (15.2 g, 41.16 mmoles) is charged into a 250 mL 3-necked round bottomed flask, followed by addition of 2-MeTHF (61 mL, 4.0 volumes), the yellowish brown slurry is stirred at about 20 0C for 5 min. Then morpholine (19 g, 218.18 mmoles) is added over 2-5 minutes. Contents are heated to reflux and maintained at reflux for 12 hr. The slurry is cooled to 25 0C, followed by addition of 2-MeTHF (53 mL, 3.5 volumes) and water ( 38 mL 2.5 volumes). The reaction mixture is warmed to 40 0C, where upon a homogenous solution with two distinct layers formed. The layers are separated, the organic layer is filtered and concentrated to ~3 volumes at atmospheric pressure. Four volumes 2-propanol (61 mL) are added. The solution is concentrated to ~3 volumes followed by addition of 4 volumes 2-propanol (61 mL), re-concentrated to ~3 volumes, followed by addition of another 6 volumes 2-propanol (91 mL), and refluxed for 15 min. The clear solution is gradually cooled to 75 0C, seeded with 0.45 g 7-(l-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine slurried in 2 mL 2-propanol, rinsed with an additional 2 mL 2-propanol and transferred to a crystallization flask. The slurry is cooled to between 0-5 0C, maintained for 1 hr, filtered and the product rinsed with 2-propanol (30 mL, 2 volumes). The solid is dried at 60 0C in a vacuum oven to afford 16.92 g 7-(l-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine. Purity of product by HPLC assay is 100.00 %. XRPD and DSC data of product is consistant with reference sample. MS(ES) = 420 (M+ 1).

Example 5. Use of morpholine as both reactant and base in 2-propanol as solvent.

7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-ajpyrimidine (11.64 mmoles) is charged into a 100 mL 3 -necked round bottomed flask followed by addition of 2-propanol ( 16 mL, 3.72 volumes). The yellowish brown slurry is stirred at about 20 0C for 5 min. Then morpholine (3.3 g, 37.84 mmoles) is added over 2-5 minutes. Contents are refluxed for 6 hr. The slurry is cooled to 25 0C. 2-Propanol ( 32 mL, 7.44 volumes) and water ( 8.6 mL, 2.0 volumes) are added and the mixture warmed to 70-75 0C, filtered and concentrated to ~ 9 volumes at atmospheric pressure. The clear solution is gradually cooled to 55 0C, seeded with 0.06 g of crystalline 7-(l-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine slurried in 0.5 mL 2-propanol, rinsed with additional 0.5 mL 2-propanol and added to crystallization flask. The slurry is cooled to 0-5 0C, maintained for 1 hr., filtered and the product rinsed with 2-propanol ( 9 mL, 2.1 volumes). Suctioned dried under vacuum at 60 0C to afford 4.6 g of dry 7-(l-ethyl-propyl)-3-(4-chloro-2-morpholin-4-yl-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (88.8 % yield, purity by HPLC assay is 99.88 % ). MS(ES) = 420 (M+ 1).

Example 6: 7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolori ,5-alpyrimidine

7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (10 g, 29.17 mmoles), 2, 4-dichlorothiazole (5.2 g , 33.76 mmoles), cesium carbonate(19.9g, 61.07 mmoles) and 1,10-phenanthroline (1 g, 5.5 mmoles) are charged into a 250 mL 3-necked round bottomed flask, followed by 2-MeTHF (36 mL, 3.6 volumes). The reaction mixture is degassed with nitrogen and then evacuated. Cuprous chloride (0.57 g, 5.7 mmoles), DMAC (10 mL, 1 volume) and 2-MeTHF (4 mL, 0.4 volumes) are added in succession. The reaction mixture is degassed with nitrogen and then evacuated. The contents are refluxed for 20 hr. The reaction mixture is cooled to -70 0C and 2-MeTHF (100 mL, 10 volumes) is added. The contents are filtered at ~70 0C and the residual cake is washed with 2-MeTHF (80 mL, 8 volumes) at about 65-72°C. The filtrate is transferred into a separatory funnel and extracted with water. The organic layer is separated and washed with dilute HCl. The resulting organic layer is treated with Darco G60, filtered hot (600C). The filtrate is concentrated at atmospheric pressure to -2.8 volumes. 25 mL 2-propanol is added, followed by re-concentration to -2.8 volumes. An additional 25 mL 2-propanol is added, followed again by re-concentration to -2.8 volumes. Finally, 48 mL 2-propanol is added. The contents are cooled to -7 0C, maintained at -7 0C for 1 hr., filtered and rinsed with 20 mL chilled 2-propanol. Product is suction dried and then vacuum dried at 60 0C to afford 9.41 g 7-(l-ethyl-propyl)-3-(2,4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (purity of product by HPLC assay is 95.88 %). MS(ES) = 369 (M+ 1).

Example 7. Synthesis of 7-(l-ethyl-propyl)-3-(2, 4-dichloro-thiazol-5-yl)-2,5-dimethyl-pyrazolori,5-a1pyrimidine using 1,4-Dioxane solvent and CuCl catalyst

Add dioxane (9.06X), Cs2CO3 (2.00X), 7-(l-ethyl-propyl)-3-iodo-2,5-dimethyl-pyrazolo[l,5-a]pyrimidine (1.0 equivalent), 2,4-dichlorothiazole (0.54 equivalent) to a reactor under N2. Purge the reactor with N2 three times, degas with N2 for 0.5-1 hr., and then add 1,10-phenanthroline (0.3 eq) and CuCl (0.3eq) under N2 , degassing with N2 for 0.5-1 hr. Heat the reactor to 1000C -1100C under N2 . Stir the mixture for 22-24 hr. at 100 0C -1100C. Cool to 10~20°C and add water (10V) and CH3OH (5V), stir the mixture for 1-1.5 hr. at 10~20°C. Filter the suspension, resuspend the wet cake in water, stirr for 1-1.5 hr. at 10~20°C, and filter the suspension again. Charge the wet cake to n-heptane (16V) and EtOAc (2V) under N2. Heat the reactor to 40 °C~500C under N2.

Active carbon (0. IX) is added at 40 °C~500C. The reactor is heated to 55°C~650C under N2 and stirred at 55 °C~650C for 1-1.5 hr. The suspension is filtered at 40~55°C through diatomite (0.4 X). The cake is washed with n-heptane (2.5V). The filtrate is transferred to another reactor. EtOAc (10V) is added and the the organic layer washed with 2 N HCl (10V) three times, followed by washing two times with water (10X, 10V). The organic layer is concentrated to 3-4V below 500C. The mixture is heated to 80-90 0C. The mixture is stirred at this temperature for 40-60 min. The mixture is cooled to 0~5°C, stirred for 1-1.5 hr. at 0~5°C and filtered. The cake is washed with n-heptane (IV) and vacuum dried at 45-500C for 8-10 hr. The crude product is dissolved in 2-propanol (7.5V) under N2, and re-crystallized with 2-propanol. The cake is dried in a vacuum oven at 45°C~50°C for 10-12 hr. (55-80% yield). 1H NMR56.537 (s, IH) 3.591-3.659 (m, IH, J=6.8Hz), 2.593 (s, 3H), 2.512 (s, 3H), 1.793-1.921(m, 4H), 0.885-0.903 (m, 6H).

REFERENCES

1: Zorrilla EP, Logrip ML, Koob GF. Corticotropin releasing factor: a key role in the neurobiology of addiction. Front Neuroendocrinol. 2014 Apr;35(2):234-44. doi: 10.1016/j.yfrne.2014.01.001. Epub 2014 Jan 20. Review. PubMed PMID: 24456850; PubMed Central PMCID: PMC4213066.

/////////////tildacerfont, SPR 001, Orphan Drug Status, Congenital adrenal hyperplasia, SPRUCE BIOSCIENCES, PHASE 2

CCC(CC)C1=CC(=NC2=C(C(=NN12)C)C3=C(N=C(S3)N4CCOCC4)Cl)C

SULCARDINE SULPHATE


Sulcardine.svg

ChemSpider 2D Image | HBI-3000 | C24H33N3O4S

sulcardine, HBI-3000

B 87823

  • Molecular FormulaC24H33N3O4S
  • Average mass459.602 Da

N-[[4-hydroxy-3,5-bis(pyrrolidin-1-ylmethyl)phenyl]methyl]-4-methoxybenzenesulfonamide

Benzenesulfonamide, N-[[4-hydroxy-3,5-bis(1-pyrrolidinylmethyl)phenyl]methyl]-4-methoxy-
N-[4-Hydroxy-3,5-bis(1-pyrrolidinylmethyl)benzyl]-4-methoxybenzenesulfonamide
343935-60-4 [RN]

heart arrhythmia

Sulcardine sulfate,343935-61-5 (Sulcardine sulfate)

CAS No. : 343935-61-5 (Sulcardine sulfate)

Synonyms: B-87823; HBI-3000; B87823; HBI3000; B 87823; HBI 3000;N-(4-hydroxy-3,5-bis(pyrrolidin-1-ylmethyl)benzyl)-4-methoxybenzenesulfonamide sulfate
Molecular Formula: C24H35N3O8S2
Molecular Weight: 557.67
  • Originator Jiangsu Furui Pharmaceuticals; Shanghai Institute of Materia Medica
  • Developer HUYA Bioscience International; Jiangsu Furui Pharmaceuticals
  • Class Antiarrhythmics; Small molecules
  • Mechanism of ActionIon channel antagonists
  • Phase I Atrial fibrillation
  • No development reported Arrhythmias
  • 13 Mar 2020 Chemical structure information added
  • 28 Feb 2020 No recent reports of development identified for preclinical development in Arrhythmias in USA (IV)
  • 16 Dec 2019 Adverse events data from a phase I trial in Atrial fibrillation (In volunteers) presented at the American Heart Association Scientific Sessions 2019 (AHA-2019)

HUYA Bioscience , under license from Shanghai Institute of Materia Medica (SIMM), is developing sulcardine (HBI-3000, oral, i.v, heart arrhythmia), a myocardial ion channel inhibitory compound, for the treatment of arrhythmia; In September 2016, the drug was still in phase II development, as of August 2020, the company website states that a phase II trial was pending in China.

HBI-3000 (sulcardine sulfate) is an experimental drug candidate that is currently in phase II of human clinical trials as an antiarrhythmic agent.[1][needs update] Clinical investigation will test the safety and efficacy of HBI-3000 as a treatment for both atrial and ventricular arrhythmias.[2]

The molecular problem

Anti-arrhythmic medication is taken to treat irregular beating of the heart. This irregular beating results from a deregulation of the initiation or propagation of the electrical stimulus of the heart. The most common chronic arrhythmia is atrial fibrillation.[3] There is an increased incidence of atrial fibrillation in the elderly and some examples of complications include heart failure exacerbation, hypotension and thrombembolic events.[3]

Most anti-arrhythmic medications exert their effects by decreasing the permeability of potassium ion channels (IKr) in heart cells. These potassium channel blockers delay ventricular repolarization and prolong action potential duration (APD; the prolongation of the electrical stimulus within heart cells). These changes can lower heart rate, eliminate atrial fibrillation, and ultimately sudden cardiac death.[4][5]

Mechanism of action in ventricular myocytes

Ventricular myocytes are heart muscle cells found in the lower chambers of the heart. Heart rate is dependent on the movement of an electrical stimulus through the individual heart cells. This is mediated by the opening of ion channels on cell surfaces. HBI-3000 exerts its effects on the heart by inhibiting multiple ion channels (INa-F, INa-L, ICa-L and IKr), but predominantly the INa-L ion channel . By decreasing the ion permeability of these channels, HBI-3000 slightly prolongs APD (due to IKr); however, unlike pure IKr channel blockers, it is self-limited (due to the decreased permeability of INa-L and ICa-L). This is similar to the medications ranolazine and amiodarone.[5] HBI-3000 suppresses early afterdepolarizations (EADs; a change in the normal net flow of ions during repolarization), does not produce any electrical abnormalities, and displays minimally pronounced prolongation of APD during a slow heart rate (i.e. stimulated at a slower frequency). Pronounced prolongation of APD during a slow heart rate can lead to proarrythmias. Overall, HBI-3000 seems to have a low proarrhythmic risk. The effect of HBI-3000 on contractility and cardiac conduction requires further investigation.[5]

Studies

Animal model

In a canine model, the intravenous injection of HBI-3000 demonstrated to be an effective anti-arrhythmic and anti-fribrillatory agent.[6]

Cellular isolation

The administration of HBI-3000 to isolated heart muscle cells demonstrated the potential to improve arrhythmias while having low proarrhythmic risk.[5]

Human studies

Jiangsu Furui Pharmaceuticals Co., Ltd is currently recruiting participants in their study.[1][

PAPER

 Acta Pharmacologica Sinica 2012

Discovery of N-(3,5-bis(1-pyrrolidylmethyl)-4-hydroxybenzyl)-4-methoxybenzenesulfamide (sulcardine) as a novel anti-arrhythmic agent

D. BaiWei-zhou Chen+6 authors Y. Wang

http://www.simm.cas.cn/wyp/wyp_lw/201804/W020180420480084769998.pdf

N-[3,5-bis(1-pyrrolidylmethyl)-4-hydroxybenzyl]-4-methoxybenzenesulfamide (sulcardine, 6f) and the sulfate (sulcardine sulfate) (1) To a suspension of 4-hydroxybenzylamine (133 g, 1.08 mol) in DMF (500 mL) was added dropwise 4-methoxybenzensul-fonyl chloride (206 g, 1.00 mol) in DMF (320 mL) over a period of 30 min at 0–10 °C with stirring, followed by the addition of triethylamine (158 mL, 1.12 mol) over 30 min at the same temperature. The stirring was continued for an additional 1.5 h at room temperature. The reaction mixture was poured into ice-water (5 L). After stirring for 10 min, the suspension was allowed to stand for 2 h. The solid was filtered, washed with water (300 mL×3), and dried in a desiccator over anhydrous calcium chloride, yielding N-(4-hydroxybenzyl)-4-methoxybenzenesulfamide (11) (248 g, 85%) as a white solid, mp 160–162 °C. The authentic sample was obtained by recrystallization from ethyl acetate, mp 161–162 °C. 1 H NMR (CD3OD) δ 3.70 (s, 3H), 3.76 (s, 2H), 6.48 (d, J=8.4 Hz, 2H), 6.82(d, J=8.4 Hz, 2H), 6.86 (d, J=8.7 Hz, 2H), 7.56 (d, J=8.7 Hz, 2H). EIMS (m/z): 293 (M+ ), 254, 195, 185, 171, 155, 149, 122 (100), 107, 99, 77, 65. Anal. (C14H15NO4S) C, H, N.

(2) A mixture of 11 (230 g, 0.78 mmol), pyrrolidine (200 mL, 2.44 mol) and 36% aqueous formaldehyde (250 mL, 3.30 mol) in ethanol (800 mL) was stirred under reflux for 8 h. The reaction mixture was concentrated under vacuum to dryness. The resulting oil residue was dissolved in chloroform (350 mL), and the solution was washed with water (300 mL×3). Under stirring, the organic layer was mixed with water (300 mL), and then concentrated hydrochloric acid (approximately 165 mL) was added portionwise at 0-10 °C to adjust the pH of the aqueous phase to ~2. The aqueous phase was washed with chloroform (200 mL) and then mixed with additional chloroform (300 mL). Under stirring, the two-phase mixture was treated portionwise with 25%–28% aqueous ammonia (~300 mL) to adjust the pH of the aqueous phase to 9–10. The organic layer was separated, and the aqueous layer was further extracted with chloroform (200 mL×2). The combined organic layer was dried over anhydrous sodium sulfate and concentrated under vacuum to dryness. The oily residue was treated with acetone (45 mL) and isopropyl ether (290 mL), and the mixture was heated under reflux until the suspension became a solution. The solution was cooled to room temperature, seeded with an authentic sample, and allowed to stand at 0°C overnight. The solid was filtered and dried under vacuum, yielding product 6f (290 g, 81%) as a yellowish solid, mp 96–98 °C. The authentic sample was obtained by preparative TLC or column chromatography (silica gel; CHCl3:MeOH:25% NH4OH=92:7:1). The compound could be recrystallized from ethanol-water, mp 101–102 °C. 1 H NMR (CDCl3) δ 1.77–1.86 (m, 8H), 2.53–2.63 (m, 8H), 3.68 (s, 4H), 3.86 (s, 3H), 3.97 (s, 2H), 6.86 (s, 2H), 6.95 (d, J=8.7 Hz, 2H), 7.78 (d, J=8.6 Hz 2H). EIMS (m/z): 459 (M+ ), 390, 388, 202, 171, 148, 107, 84, 70 (100). Anal. (C24H33N3O4S) C, H, N.

(3) Under stirring, the Mannich base 6f (150.5 g, 0.327 mol) was mixed with 2 mol/L H2SO4 (172 mL, 0.344 mol), and the mixture was heated at 80 °C until the solid dissolved. The solution was cooled to room temperature, seeded with an authentic sample, and the sulfate of 6f was formed as crystals. To the stirred mixture was added anhydrous ethanol (520 mL), and the mixture was allowed to stand at 0°C for 24 h. The solid was filtered, washed with ethanol, and recrystallized with 80% ethanol (250 mL). The sulfate was dried over concentrated sulfuric acid in a desiccator, giving the sulfate of 6f (143 g, 71%) as a trihydrate, mp 125–140°C. 1 H NMR (D2O) δ 2.00–2.13 (m, 4H), 2.14–2.25 (m, 4H), 3.12–3.22 (m, 4H), 3.45– 3.55 (m, 4H), 3.90 (s, 3H), 4.20 (s, 2H), 4.33 (s, 4H), 7.06 (d, J=8.7 Hz, 2H), 7.28 (s, 2H), 7.66 (d, J=8.9 Hz, 2H). 13C NMR (D2O) δ 24.7, 47.6, 55.7, 56.1, 58.1, 116.6, 122.5, 131.3, 132.3, 133.3, 136.0, 155.8, 164.8. EIMS (m/z): 459, 390, 388, 202, 171, 148, 107, 84, 70 (100). Anal. (C24H33N3O4S∙H2SO4∙3H2O) C, H, N, S.

PATENT

Preparation of sulcardine sulfate salt has been reported in U.S. Patent No. 6,605,635.

https://patents.google.com/patent/US6605635

Synthesis and antiarrhythmic activities of changrolin (1) have been reported (Liangquan Li, et al., Scientia Sinica, 1979, 7, 723; Weizhou Chen, et al., Acta Pharmaceutica Sinica, 1979, 14, 710). Thereafter, investigations of the chemical structural modifications and the physiological activities have successively been carried out by domestic and foreign scientists (Cunji Sun, et al., Acta Pharmaceutica Sinica, 1981, 16, 564; 1986, 21, 692; Mulan Lin, et al., ibid., 1982, 17, 212; D. M. Stout, et al. J. Med. Chem., 1983, 26, 808; 1984, 27, 1347; 1985, 28, 295; 1989, 32, 1910; R. J. Chorvat, et al., ibid., 1993, 36, 2494).

Figure US06605635-20030812-C00001

Changrolin is an effective antiarrhythmic agent. Ventricular premature beats disappear 2-3 days after oral administration of changrolin to patients suffering from arrhythmia; I.v. injection or instillaton may result in significant reduction or even disappearence of ventricular premature beats and ventricular tachycardia. However, oral administration of changrolin for a period of over one month may cause a reversible pigmentation on the skin of patients, which gradually retrogresses after ceasing the administration. This pigmentation is associated to the subcutaneous oxidation of certain structural moieties in changrolin molecule or to its instability in solution.

EXAMPLE 1N-[3,5-bis(1-Piperidinomethyl)-4-hydroxy]phenyl-1-naphthalenesulfonamide (B-87836)

(1) To a solution of 4-aminophenol (4.5 g) in dioxane (20 ml) was added dropwise a solution of 1-naphthalenesulfonyl chloride (4.4 g) in dioxane (20 ml). The mixture was further stirred at room temperatue for 4.5 hours and poured into water. The precipitate was collected by filtration, recrystallized from ethanol and decolored with activated carbon to give N-(ρ-hydroxyphenyl)-1-naphthalenesulfonamide (4.2 g), mp 195-196° C.

(2) A mixture of N-(ρ-hydroxyphenyl)-1-naphthalenesulfonamide (2.0 g), 37% aqueous formaldehyde (4.5 g) and piperidine (5.6 g) in ethanol (100 ml) was heated to reflux for 50 hours. The ethanol was removed by evaporation in vacuo and chloroform was added to the residue. The organic layer was washed with water then dried over anhydrous Na2SO4. Then the chloroform was removed in vacuo and the residue was triturated in water to give a solid, which was then recrystallized from ethanol to give the titled product (1.4 g), mp 197-198° C.

1HNMR(CDCl3): 1.30-1.50(m, 12H), 2.10-2.21(m, 8H), 3.28(s, 4H), 6.45(s, 2H), 7.24-8.04(m, 6H), 8.56(m, 1H). Elemental analysis (C28H35N3O3S ): Calcd. (%): C, 68.12; H, 7.15; N, 8.51. Found (%): C, 67.96; H, 7.16; N, 8.56.

PATENT

WO-2020159959

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020159959&tab=PCTDESCRIPTION&_cid=P11-KDSBL9-99100-1

Novel crystalline forms of acid salts of sulcardine useful for treating arrhythmia and atrial fibrillation.

4-Methoxy-N-(3,5-bis-(l-pyrrolidinylmethyl)-4-hydroxybenzyl)benzene sulfonamide (or N-(4-hydroxy-3,5-bis(pyrrolidin-l-ylmethyl)benzyl)-4-methoxybenzenesulfonamide), also known as sulcardine, and its salts, such as sulcardine sulfate, constitute a group of compounds with potent anti -arrhythmic activity. Sulcardine is a multi-ion channel blocker that specifically inhibits iNa-Peak, iNa-Late, Ica,L, and Ixrwith similar in vitro potencies (and Ito and IKUT to a lesser degree) in human atrial cardiomyocytes and represents what may be the sole example of a substituted sulfonamide class of anti-arrhythmic. Sulcardine salts can be used as an intravenous injectable or as oral doses for the treatment of arrhythmias, including supraventricular tachyarrhythmia, premature ventricular contractions, ventricular tachycardia, ventricular fibrillation, and atrial fibrillation. See, e.g ., U.S. Patent Nos. 8,541,464 and 8,637,566. Preparation of sulcardine sulfate salt has been reported in U.S. Patent No. 6,605,635.

[0004] In addition, the evidence to date suggests that one advantage of sulcardine and its salts is that they lack significant pro-arrhythmic activity, as demonstrated in rigorous preclinical safety models, including a post-MI sudden-death conscious canine model and the validated rabbit ventricular wedge model. Additionally, it has been shown that they do not significantly increase defibrillation threshold, nor increase defibrillation failure risk in a post-MI canine model as was seen with flecainide. On the basis of these data, sulcardine and salts, with their very low apparent pro-arrhythmic potential, could potentially be used to treat acute and recurrent atrial fibrillation in the presence of organic heart disease, prolonged QR syndrome, and ventricular arrhythmias, including premature ventricular contractions (PVCs), ventricular tachycardia (VT), and ventricular fibrillation (VF), in either acute- or chronic-administration settings owing to their ability to be formulated into intravenous and oral dosing formulations.

Sulcardine has a chemical name of 4-methoxy-N-(3,5-bis-(l-pyrrolidinylmethyl)- 4-hydroxybenzyl)benzene sulfonamide (or N-(4-hydroxy-3,5-bis(pyrrolidin-l-ylmethyl)benzyl)-4-methoxybenzenesulfonamide), and has the following structure:

[0062] Sulcardine sulfate has the following structure:

[0063] Sulcardine sulfate can exist in a hydrated form. One such form is a trihydrate.

HPLC analysis was performed on a Dionex Ultimate 3000 instrument with the following parameters:

Column: Phenomenex Luna C18, 150×4.6mm, 5pm

Column Temperature: 30°C

Mobile Phase A: 0.2% Phosphoric Acid

Mobile Phase B: Methanol

Diluent: 50:50 MeOH:H20

Runtime: 12 minutes

Flow Rate: l.OmL/min

Injection Volume: 5pL

Detection: 237 nm

Gradient:

EXAMPLE 2 – PREPARATION OF FREE BASE AND SCREENING

[00348] Sulcardine sulfate trihydrate was dissolved in ethyl acetate (16 vol.) and saturated sodium bicarbonate solution (16 vol.). The biphasic solution was transferred to a separating funnel and the layers separated. The organic layer was dried over sodium sulfate and then the solvent was removed by rotary evaporation and the resulting oil dried under vacuum at ambient temperature for ca. 3 hr. FIG. 4 is an XRPD pattern of the resulted amorphous sulcardine free base. In all cases, the initial screening work detailed below was performed on 10 mg of sulcardine free base. All XRPD diffractograms were compared with sulcardine sulfate trihydrate, sulcardine free base and relevant counterions and found to be distinct.

Patent

WO2020123824

claiming treatment of atrial fibrillation (AF) by intravenously administering sulcardine sulfate .

PATENT

US6605635

References

  1. Jump up to:a b Jiangsu Furui Pharmaceuticals (November 5, 2010). “Efficacy and safety of sulcardine sulfate tablets in patients with premature ventricular contractions”ClinicalTrials.gov. U.S. National Library of Medicine. Retrieved 2019-12-20.
  2. ^ “HUYA Bioscience Int’l announces clinical trial milestones in China for promising new anti-arrhythmic compound; Data supports desirable safety profile” (Press release). San Francisco, California: HUYA Bioscience International. Retrieved 2019-12-20.
  3. Jump up to:a b Mashal, Abdallah; Katz, Amos; Shvartzman, Pesach (2011). “Atrial fibrillation: A primary care cross-sectional study”Israel Medical Association Journal13 (11): 666–671. PMID 22279699.
  4. ^ Farkas, András; Leprán, István; Papp, Julius Gy. (1998). “Comparison of the antiarrhythmic and the proarrhythmic effect of almokalant in anaesthetised rabbits”. European Journal of Pharmacology346 (2–3): 245–253. doi:10.1016/S0014-2999(98)00067-3PMID 9652366.
  5. Jump up to:a b c d Guo, Donglin; Liu, Que; Liu, Tengxian; Elliott, Gary; Gingras, Mireille; Kowey, Peter R.; Yan, Gan-Xin (2011). “Electrophysiological properties of HBI-3000: A new antiarrhythmic agent with multiple-channel blocking properties in human ventricular myocytes”. Journal of Cardiovascular Pharmacology57 (1): 79–85. doi:10.1097/FJC.0b013e3181ffe8b3PMID 20980921.
  6. ^ Lee, Julia Y.; Gingras, Mireille; Lucchesi, Benedict R. (2010). “HBI-3000 prevents sudden cardiac death in a conscious canine model”. Heart Rhythm7 (11): 1712. doi:10.1016/j.hrthm.2010.09.028.
HBI-3000
Sulcardine.svg
Names
IUPAC name

N-({4-Hydroxy-3,5-bis[(pyrrolidin-1-yl)methyl]phenyl}methyl)-4-methoxybenzene-1-sulfonamide
Identifiers
3D model (JSmol)
ChemSpider
PubChem CID
UNII
Properties
C24H33N3O4S
Molar mass 459.61 g·mol−1
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
Infobox references

////////////////sulcardine sulfate, phase 2, china, HBI 3000, atrial fibrillation, B 87823,

COC1=CC=C(C=C1)S(=O)(=O)NCC2=CC(=C(C(=C2)CN3CCCC3)O)CN4CCCC4

NARONAPRIDE


 

Thumb

Naronapride | C27H41ClN4O5 - PubChem

Naronapride | ATI-7505 | CAS#860174-12-5 | 860169-57-9 | 5-HT(4 ...

NARONAPRIDE

860174-12-5

Average: 537.1

C27H41ClN4O5

ATI 7505 / ATI-7505

(3R)-1-azabicyclo[2.2.2]octan-3-yl 6-[(3S,4R)-4-(4-amino-5-chloro-2-methoxybenzamido)-3-methoxypiperidin-1-yl]hexanoate

INGREDIENT UNII CAS
Naronapride dihydrochloride 898PE2W8US 860169-57-9

 860174-12-5 (free base)   860169-57-9 (HCl)

Naronapride (free base), also known as ATI-7505, is a highly selective, high-affinity 5-HT(4) receptor agonist for gastrointestinal motility disorders. ATI-7505 accelerates overall colonic transit and tends to accelerate GE and AC emptying and loosen stool consistency.

 

Investigated for use/treatment in gastroesophageal reflux disease (GERD) and gastroparesis.

Renexxion , presumed to have been spun-out from Armetheon , under license from ARYx Therapeutics is developing naronapride (ATI-7505; phase 2 clinical in February 2020), an analog of the gastroprokinetic 5-HT 4 agonist cisapride identified using ARYx’s RetroMetabolic platform technology (ARM), for the oral treatment of upper GI disorders. In September 2018, this was still the case . PATENT

WO2005068461

NEW PATENT

WO-2020096911

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020096911&tab=PCTDESCRIPTION&_cid=P21-KANOVN-53661-1

Process for preparing trihydrate salt of naronapride  hydrochloride as 5-HT 4 receptor agonist useful for treating gastrointestinal disorders such as dyspepsia, gastroparesis, constipation, post-operative ileus. Appears to be the first filing from the assignee and the inventors on this compound,

In some aspects, provided herein is a method of making a trihydrate form of (3S, 4R, 3’R)-6-[4-(4-amino-5-chloro-2-methoxy-benzoylamino)-3-methoxy-piperidin-l-yl]-hexanoic acid l-azabicyclo[2.2.2]oct-3’-yl ester di-hydrochloride salt, which has the following formula:

Example 5: NMR Characterization of the Trihydrate

[0282] ^-Nuclear Magnetic Resonance Spectroscopy (‘H-NMR) : Approximately 6 mg of the trihydrate was dissolved in in 1 g of deuterated solvent (dimethylsulfoxide (DMSO)-C45 99.9% d, with 0.05% v/v tetramethyl silane (TMS)). A Varian Gemini 300 MHz FT-NMR spectrometer was used to obtain the ¾-NMK spectrum. A list of the peaks is provided in Table 1 below. A representative ‘H-NMR spectrum is provided in FIG. 6.

Table 1. ‘H-NMR peak list for trihydrate

[0283] 13 C-Nuclear Magnetic Resonance Spectroscopy ( 13C-NMR ): Approximately 46 mg of the trihydrate was dissolved in 1 mL of deuterated solvent (deuterium oxide, Aldrich, 99.9% D, TPAS 0.75%). The 13C-NMR spectrum was obtained using a Varian Gemini 300 MHz FT-NMR spectrometer. A list of the peaks is provided in Table 2 below. A representative 13C-NMR spectrum is provided in FIG. 7.

Table 2. 13C-NMR peak list for trihydrate

 

 

PATENT

US10570127 claiming composition (eg tablet) comprising a trihydrate form of naronapride.

patent

ARYX THERAPEUTICS, WO2005/68461, A1, (2005)

Methods

titanium tetraethoxide; toluene;

Reactants can be synthesized in 1 step.
ARYX THERAPEUTICS, WO2005/68461, A1, (2005) The ester (1 part by weight) and (R)-3-Quinuclidinol (about 1.12 part by weight) were suspended in toluene before slowly adding titanium (IV) ethoxide (about 0.5 part by weight) to the stirred suspens ion. The mixture was heated to about 91 °C under a stream of nitrogen, and partial vacuum was applie d to the flask through a distillation apparatus in order to azeotropically remove the ethanol. Addit ional toluene was added as needed to maintain a minimum solvent volume in the flask. The reaction was considered complete after about 33 hours. The mixture was cooled to about room temperature and ext racted five times with water. The organic layer was concentrated under reduced pressure and the resulting residue was redissolved in EtOH/iPrOH (about 1: 1 v/v) and then filtered through a 0.45 micron membrane filter to remove any particulates. Concentrated hydrochloric acid was added slowly to the stirred filtrate to precipitate out the desired product as the dihydrochloride salt. The resulting s uspension was stirred for several hours at room temperature and collected under vacuum filtration and rinsed with EtOH/tPrOH (1: 1; v/v) to provide 0.53 part by weight of the crude product salt. Crude dihydrochloride salt was resuspended in ethanol and heated to reflux before cooling to room temperature over about 1 hour. The product was collected under vacuum filtration and rinsed with ethanol an d then air-dried. The solids were resuspended in ethanol and warmed to about 55 °C to give a clear s olution before adding warm isopropanol and the product was allowed to precipitate by slow cooling to room temperature. The resulting suspension was stirred for several hours before vacuum filtering and rinsing with, e. g., isopropanol. The product was vacuum dried, initially at room temperature for several hours and then at about 55 °C until a constant weight was achieved.

Patent

Methods

dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; DMFA;

Reactants can be synthesized in 2 steps.
ARYX THERAPEUTICS, WO2007/28073, A2, (2007) Production of Compound IV and Compound VI[0394] A mixture of (+)-Comrhoound II (1 eq.), (R)-(-)-3-quinuclidinol HCl salt (1 eq.), EDAC (1 eq.) and DMAP (1 eq.) in DMF is heated at around 5OC overnight . After cooling and diluting with water, the mixture is purified by chromatography or by crystallization to provide Compound IV. Similarly, using (S)-(+)-quinuclidinol, Compound VI is obtained

REFERENCES

1: Jiang C, Xu Q, Wen X, Sun H. Current developments in pharmacological therapeutics for chronic constipation. Acta Pharm Sin B. 2015 Jul;5(4):300-9. doi: 10.1016/j.apsb.2015.05.006. Epub 2015 Jun 6. Review. PubMed PMID: 26579459; PubMed Central PMCID: PMC4629408.

2: Buchwald P, Bodor N. Recent advances in the design and development of soft drugs. Pharmazie. 2014 Jun;69(6):403-13. Review. PubMed PMID: 24974571.

3: Mozaffari S, Didari T, Nikfar S, Abdollahi M. Phase II drugs under clinical investigation for the treatment of chronic constipation. Expert Opin Investig Drugs. 2014 Nov;23(11):1485-97. doi: 10.1517/13543784.2014.932770. Epub 2014 Jun 24. Review. PubMed PMID: 24960333.

4: Shin A, Camilleri M, Kolar G, Erwin P, West CP, Murad MH. Systematic review with meta-analysis: highly selective 5-HT4 agonists (prucalopride, velusetrag or naronapride) in chronic constipation. Aliment Pharmacol Ther. 2014 Feb;39(3):239-53. doi: 10.1111/apt.12571. Epub 2013 Dec 5. Review. PubMed PMID: 24308797.

5: Stevens JE, Jones KL, Rayner CK, Horowitz M. Pathophysiology and pharmacotherapy of gastroparesis: current and future perspectives. Expert Opin Pharmacother. 2013 Jun;14(9):1171-86. doi: 10.1517/14656566.2013.795948. Epub 2013 May 11. Review. PubMed PMID: 23663133.

6: Tack J, Camilleri M, Chang L, Chey WD, Galligan JJ, Lacy BE, Müller-Lissner S, Quigley EM, Schuurkes J, De Maeyer JH, Stanghellini V. Systematic review: cardiovascular safety profile of 5-HT(4) agonists developed for gastrointestinal disorders. Aliment Pharmacol Ther. 2012 Apr;35(7):745-67. doi: 10.1111/j.1365-2036.2012.05011.x. Epub 2012 Feb 22. Review. PubMed PMID: 22356640; PubMed Central PMCID: PMC3491670.

7: Hoffman JM, Tyler K, MacEachern SJ, Balemba OB, Johnson AC, Brooks EM, Zhao H, Swain GM, Moses PL, Galligan JJ, Sharkey KA, Greenwood-Van Meerveld B, Mawe GM. Activation of colonic mucosal 5-HT(4) receptors accelerates propulsive motility and inhibits visceral hypersensitivity. Gastroenterology. 2012 Apr;142(4):844-854.e4. doi: 10.1053/j.gastro.2011.12.041. Epub 2012 Jan 4. PubMed PMID: 22226658; PubMed Central PMCID: PMC3477545.

8: Bowersox SS, Lightning LK, Rao S, Palme M, Ellis D, Coleman R, Davies AM, Kumaraswamy P, Druzgala P. Metabolism and pharmacokinetics of naronapride (ATI-7505), a serotonin 5-HT(4) receptor agonist for gastrointestinal motility disorders. Drug Metab Dispos. 2011 Jul;39(7):1170-80. doi: 10.1124/dmd.110.037564. Epub 2011 Mar 29. PubMed PMID: 21447732.

9: Tack J. Current and future therapies for chronic constipation. Best Pract Res Clin Gastroenterol. 2011 Feb;25(1):151-8. doi: 10.1016/j.bpg.2011.01.005. Review. PubMed PMID: 21382586.

10: Manabe N, Wong BS, Camilleri M. New-generation 5-HT4 receptor agonists: potential for treatment of gastrointestinal motility disorders. Expert Opin Investig Drugs. 2010 Jun;19(6):765-75. doi: 10.1517/13543784.2010.482927. Review. PubMed PMID: 20408739.

11: Sanger GJ. Translating 5-HT receptor pharmacology. Neurogastroenterol Motil. 2009 Dec;21(12):1235-8. doi: 10.1111/j.1365-2982.2009.01425.x. Review. PubMed PMID: 19906028.

12: Vakil N. New pharmacological agents for the treatment of gastroesophageal reflux disease. Rev Gastroenterol Disord. 2008 Spring;8(2):117-22. Review. PubMed PMID: 18641594.

13: Bayés M, Rabasseda X, Prous JR. Gateways to clinical trials. Methods Find Exp Clin Pharmacol. 2007 Jun;29(5):359-73. PubMed PMID: 17805439.

14: Camilleri M, Vazquez-Roque MI, Burton D, Ford T, McKinzie S, Zinsmeister AR, Druzgala P. Pharmacodynamic effects of a novel prokinetic 5-HT receptor agonist, ATI-7505, in humans. Neurogastroenterol Motil. 2007 Jan;19(1):30-8. PubMed PMID: 17187586.

////////////NARONAPRIDE, ATI 7505, ATI 7505,PHASE 2

CO[C@H]1CN(CCCCCC(=O)O[C@H]2CN3CCC2CC3)CC[C@H]1NC(=O)C1=C(OC)C=C(N)C(Cl)=C1

NIDUFEXOR


Nidufexor Chemical Structure

Nidufexor.png

NIDUFEXOR

LMB763

4-[[benzyl-(8-chloro-1-methyl-4H-chromeno[4,3-c]pyrazole-3-carbonyl)amino]methyl]benzoic acid

Nidufexor is a farnesoid X receptor (FXR) agonist.

Molecular Weight

487.93

Formula

C₂₇H₂₂ClN₃O₄

CAS No.

1773489-72-7

PHASE 2 Treatment of Liver and Biliary Tract Disorders,
Agents for Diabetic Nephropathy, NOVARTIS

Nidufexor

1773489-72-7LMB-763UNII-CJ1PL0TE6JCJ1PL0TE6JBCP28929EX-A1854

Nidufexor pound LMB-763 pound(c)

ZINC584641402

4-((N-benzyl-8-chloro-1-methyl-1,4-dihydrochromeno[4,3-c]pyrazole-3-carboxamido)methyl)benzoic acid

HY-109096

CS-0039398

https://pubs.acs.org/doi/pdf/10.1021/acs.jmedchem.9b01621

1 (7.6 g, 89% yield) as a white solid. Melting point: 232.6 °C.

1 H NMR (400 MHz, DMSO): δ 12.93 (s, 1H), 7.96−7.85 (m, 2H), 7.71 (dd, J = 7.1, 2.5 Hz, 1H), 7.42−7.20 (m, 8H), 7.06 (dd, J = 8.7, 1.9 Hz, 1H), 5.45 (d, J = 3.9 Hz, 2H), 5.25 (d, J = 9.2 Hz, 2H), 4.58 (d, J = 12.1 Hz, 2H), 4.12 (d, J = 16.6 Hz, 3H).

13C NMR (101 MHz, DMSO-d6): δ 167.07, 162.21, 151.98, 142.65, 139.18, 132.20, 132.67, 129.70, 129.50, 129.50, 128.53, 128.53, 127.43, 127.43, 127.43, 127.43, 127.43, 125.53, 122.24, 119.0, 117.09, 116.64, 64.51, 50.68, 48.24. LC-MS m/z: 488.2/490.2 (M +H)+ ; chlorine pattern; method 3; RT = 1.41 min.

Elemental Analysis calcd for C27H22ClN3O4: C 66.46, H 4.54, N 8.61; found: C 66.43, H 4.56, N 8.62.

TRIS Salt Formation. Methanol (400 mL) was added to a mixture of 1 (4.0 g, 8.2 mmol) and 2-amino-2-hydroxymethylpropane-1,3-diol (TRIS, 1.0 g, 8.2 mmol). The mixture was heated to 70 °C for 0.5 h. After cooling to room temperature, the solvent was removed in vacuum. The residue was sonicated in dichloromethane (10 mL) and concentrated again. The resulting white solid was dried under vacuum overnight. The crude material was crystallized by slurring the solid residue in a 4:1 mixture of acetonitrile and methanol (5 mL). The mixture was stirred at room temperature for 24 h to give 4-((N-benzyl-8-chloro-1-methyl-1,4-dihydrochromeno- [4,3-c]pyrazole-3-carboxamido)methyl)benzoic acid TRIS salt as a white salt (3.7 g, 73% yield). Melting point: 195.6 °C. 1 H NMR (400 MHz, DMSO): δ 7.92−7.80 (m, 2H), 7.78−7.64 (m, 1H), 7.41− 7.19 (m, 8H), 7.13−7.00 (m, 1H), 5.44 (s, 2H), 5.25−5.14 (m, 2H), 4.61−4.48 (m, 2H), 4.18−4.03 (m, 3H), 3.39 (s, 7H). TRIS OH masked by water peak. LC-MS m/z: 488.0/490.0 (M+H)+ ; chlorine pattern, method 3. RT = 1.58 min. Elemental Analysis calc for C31H33ClN4O7: C 61.00, H 5.36, N 9.15; found: C 60.84, H 5.34, N 9.13.

https://pubs.acs.org/doi/suppl/10.1021/acs.jmedchem.9b01621/suppl_file/jm9b01621_si_001.pdf

Patent

WO 2015069666

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015069666&tab=PCTDESCRIPTION

///////NIDUFEXOR, LMB 763, Phase II, PHASE 2, Liver and Biliary Tract Disorders,  Diabetic Nephropathy, NOVARTIS

CN1C(C2=CC(Cl)=CC=C2OC3)=C3C(C(N(CC4=CC=CC=C4)CC5=CC=C(C(O)=O)C=C5)=O)=N1

LNP 023


LNP023

4-[(2S,4S)-4-Ethoxy-1-[(5-methoxy-7-methyl-1H-indol-4-yl)methyl]piperidin-2-yl]benzoic acid.png

LNP 023

CAS 1644670-37-0

ROTATION +

4-((2S,4S)-4-ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoic acid

M.Wt 422.525
Formula C25H30N2O4

4-[(2S,4S)-4-ethoxy-1-[(5-methoxy-7-methyl-1H-indol-4-yl)methyl]piperidin-2-yl]benzoic acid

LNP023

RENRQMCACQEWFC-UGKGYDQZSA-N

PATENT US9682968, Example-26a

BDBM160475

ZINC223246892

HY-127105

CS-0093107

4-((2S,4S)-(4-ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl))benzoic acid

4-[(2~{S},4~{S})-4-ethoxy-1-[(5-methoxy-7-methyl-1~{H}-indol-4-yl)methyl]piperidin-2-yl]benzoic acid

LNP023 (LNP-023) is a highly potent, reversible, selective inhibitor of factor B (IC50=10 nM), the proteolytically active component of the C3 and C5 convertases.

LNP023 (LNP-023) is a highly potent, reversible, selective inhibitor of factor B (IC50=10 nM), the proteolytically active component of the C3 and C5 convertases; shows direct, reversible, and high-affinity binding to human FB with Kd of 7.9 nM in SPR assays, demonstrates potent inhibition of AP-induced MAC formation in 50% human serum with IC50 of 0.13 uM; shows no inhibition of factor D (FD), as well as classical or lectin complement pathway activation (up to 100 uM), and no significant effects (up to 10 μM) in a broad assay panel of receptors, ion channels, kinases, and proteases; blocks zymosan-induced MAC formation membrane attack complex (MAC) with IC50 of 0.15 uM, prevents KRN-induced arthritis in mice and is effective upon prophylactic and therapeutic dosing in an experimental model of membranous nephropathy in rats afer oral adminstration; also prevents complement activation in sera from C3 glomerulopathy patients and the hemolysis of human PNH erythrocytes.

Other Indication

Phase 2 Clinical

PATENT

WO 2015009616

https://patents.google.com/patent/WO2015009616A1/en

PATENT

https://patents.google.com/patent/US9682968B2/en

Example-26Example-26a4-((2S,4S)-(4-ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl))benzoic acid ((+) as TFA Salt)

Figure US09682968-20170620-C00315

A mixture of methyl 4-((2S,4S)-4-ethoxy-1-((5-methoxy-7-methyl-1H-indol-4-yl)methyl)piperidin-2-yl)benzoate, Intermediate 6-2b peak-1 (tr=1.9 min), (84 mg, 0.192 mmol) and LiOH in H2O (1 mL, 1 mmol) in THF (1 mL)/MeOH (2 mL) was stirred at room temperature for 16 h, and then concentrated. The resulting residue was purified by RP-HPLC (HC-A) to afford the title compound. Absolute stereochemistry was determined by comparison with enantiopure synthesis in Example-26c. 1H NMR (TFA salt, 400 MHz, D2O) δ 8.12 (d, J=8.19 Hz, 2H), 7.66 (br. d, J=8.20 Hz, 2H), 7.35 (d, J=3.06 Hz, 1H), 6.67 (s, 1H), 6.25 (d, J=3.06 Hz, 1H), 4.65 (dd, J=4.28, 11.49 Hz, 1H), 4.04 (d, J=13.00 Hz, 1H), 3.87-3.98 (m, 2H), 3.53-3.69 (m, 5H), 3.38-3.50 (m, 1H), 3.20-3.35 (m, 1H), 2.40 (s, 3H), 2.17-2.33 (m, 2H), 2.08 (br. d, J=15.70 Hz, 1H), 1.82-1.99 (m, 1H), 1.28 (t, J=7.03 Hz, 3H); HRMS calcd. for C26H31N2O(M+H)423.2284, found 423.2263.

PATENT

WO 2020016749

https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=D7DA400C5FC41AD0EA9F0AB9B74A1D86.wapp1nB?docId=WO2020016749&tab=PCTDESCRIPTION

The present invention relates to a process for the preparation of phenylpiperidinyl indole derivatives. More particularly, the present invention relates to a process for the preparation of the compound of formula (I)

also referred to as 4-((2S,4S)-(4-ethoxy-1 -((5-methoxy-7-methyl-1 /-/-indol-4-yl)methyl)piperidin-2-yl))benzoic acid, or a pharmaceutically acceptable salt thereof, which is capable of inhibiting the activation of the alternative pathway of the complement system. The complement system plays a major role in the innate and adaptive immunity system and comprises a group of proteins that are normally present in an inactive state. These proteins are organized in three activation pathways: the classical, the lectin, and the alternative pathways (Holers, In Clinical Immunology: Principles and practice, ed. R.R. Rich, Mosby Press; 1996, 363-391 ). Molecules from microorganisms, antibodies or cellular components can activate these pathways resulting in the formation of protease complexes known as the C3-convertase and the C5-convertase. The classical pathway is a calcium / magnesium-dependent cascade, which is normally activated by the formation of antigen-antibody complexes. It can also be activated in an antibody-independent manner by the binding of C-reactive protein complexed to

ligand and by many pathogens including gram-negative bacteria. The alternative pathway is a magnesium-dependent cascade, which is activated by deposition and activation of C3 on certain susceptible surfaces (e.g. cell wall polysaccharides of yeast and bacteria, and certain biopolymer materials). The alternative pathway (AP) utilizes C3 fragments (C3b) to opsonize the pathogens hence targeting them for phagocytosis without the need for antibodies. Hyperactivity of the complement system, and in particular in its AP, plays a role in a large number of complement-driven diseases, such as C3 glomerulopathy (C3G), paroxysmal nocturnal hemoglobinuria (PNH) and IgA nephropathy (IgAN). Phenylpiperidinyl indole derivatives, such as compound of formula (I), or a pharmaceutically acceptable salt thereof, play a role in the inhibition of complement factor B, a known critical enzyme for activation of the alternative complement pathway (Lesavre et al J. Exp. Med. 1978, 148, 1498-1510; Volanakis et al New Eng. J. Med. 1985, 312, 395-401 ), which may also be a suitable target for the inhibition of the amplification of the complement pathways. The phenylpiperidinyl indole derivatives, such as compound of formula (I), or a pharmaceutically acceptable salt thereof, and a method for preparing such derivatives, are described in WO2015/009616. In particular, compound of formula (I) is described in example 26, of WO2015/009616. One of the drawbacks of the synthesis was the use of hazardous chemicals (such as sodium hydride, or dimethylacetamide, which represent safety concerns on a larger scale) and the poor enantio- and diastereo-selectivity of the steps, leading to unwanted stereoisomers.

Thus, there is a need to provide an alternative reaction route in a process for producing compound of formula (I), or a pharmaceutically acceptable salt thereof, generating less by products, and easier to handle on a large scale.

Scheme 1 , vide infra.

Compound of fformula (II)


ormu a ( )


formula (1)

Scheme 1

1. Asymmetric synthesis of compound of formula (II): .

One aspect of the present invention relates to an asymmetric process for preparing a compound of formula (II), or salt thereof, as outlined in Scheme 2 below, wherein the stereocenters in position 2 and in position 4 on the piperidine are obtained in high enantio- and diastereo-selectivity.

formula (ii)

Scheme 2

Example 1 : Synthesis of Benzyl-2-r4-(methoxycarbonyl)phenyl1-4-oxopiperidine-1 -carboxylate according to the following sequence:

R = Methyl R = Methyl R =: Methyl

Step 1 : Synthesis of Benzyl-2-[4-(methoxycarbonyl)phenyl]-4-oxo-3, 4-dihydro pyridine-1(2W)-carboxylate (C3, wherein Pi = Cbz and R = methyl)

iPrMgCI (2N THF, 109.96 g, 54.98 ml_, 2.0 eq) was charged in a reactor. A solution of bis[2 -(N,N-dimethylaminoethyl)] ether (2.5 eq, 22.03 g, 137.46 mmol) in THF (24 ml.) was added at 15 – 25 °C. The mixture was stirred for 1 hour. A solution of C1 (20.17 g, 76.98 mmol, 1 .4 eq) in THF (102 ml.) was added slowly at 15 – 25 °C. The mixture was heated to 25 – 30 °C, stirred for more than 1 hour, and checked by HPLC. The mixture was cooled to -30 °C. A solution of C2 (methyl 4-iodobenzoate, 6.0 g, 54.98 mmol, 1 .0 eq) in THF (20 ml.) was added, followed by a solution of benzyl chloroformate (1 .15 eq, 10.79 g, 63.23 mmol) in THF (36 ml_). The mixture was stirred for 2 hours and quenched with AcOH (6.60 g, 109.96 mmol, 2 eq). Isopropyl acetate (60 ml.) was added. Hydrogen chloride (15%, 90 g) was added to adjust the pH = 1 – 2. The organic layer was separated and washed with brine (15%, 100 g), and concentrated. Isopropyl acetate (160 ml.) was added and concentrated to remove the THF. The crude product was recrystallized in Isopropyl

acetate (1 14 ml.) and n-heptane (120 ml_). The product was dried at 60 °C to provide C3 as light yellow solid (16.0 g, 79.65 % yield). 1 H-NMR (400 MHz, DMSO-d6) d (ppm) = 8.1 1 (dd, J=8.39, 1.01 Hz, 1 H), 7.91 (d, J=8.39 Hz, 2H), 7.33 – 7.37 (m, 6H), 5.82 (d, J= 7.20 Hz, 1 H), 5.20 – 5.35 (m, 3H) , 3.83 (s, 3H), 3.41 (br. s, 1 H), 3.31 (dd, J=16.64, 7.52 Hz, 1 H), 2.66 (br. d, J=16.55 Hz, 1 H).

Step 2: Synthesis of Benzyl-2-[4-(methoxycarbonyl)phenyl]-4-oxopiperidine-1 -carboxylate (C4, wherein Pi = Cbz and R = methyl)

A solution of C3 (25 g, 68.42 mmol, 1 .0 eq) in AcOH (200 ml.) was heated to 50 – 60 °C to form a clear solution. The solution was then cooled to 35 °C. Zn powder (13.42 g, 205.26 mmol, 3.0 eq) was added portionwise while keeping the inner temperature at 35 – 40 °C. After addition, the mixture was stirred for more than 8 hours and checked by HPLC. THF (250 ml.) was added. The mixture was cooled to 25 °C, filtered, and the filter cake was washed with THF (125 volume). The filtrate was concentrated to dryness. Isopropanol (375 ml.) was added. The solution was cooled to 0 – 5 °C. EDTA-4Na.2H20 (40 g) in water (200 ml.) was added. The mixture was neutralized to pH = 9 – 10 with 30% sodium hydroxide solution and stirred for 2 hours. The organic layer was collected, washed with brine (15%, 250 g) and concentrated to about 50 ml_. MTBE (100 ml.) was added and concentrated to about 50 ml_. MTBE (80 ml.) was added followed by n-heptane (20 ml.) dropwise. Then the mixture was cooled to 0 °C gradually. The mixture was filtered and the filter cake was dried to afford C4 as a light yellow solid (20.1 1 g, 80.0 % yield). 1 H NMR (400 MHz, CDCIs) d (ppm)= 7.99 (d, J=8.31 Hz, 2H), 7.27 – 7.39 (m, 7H), 5.83 (br. s, 1 H), 5.14 – 5.28 (m, 2H), 4.20 – 4.42 (m, 1 H), 3.92 (s, 3H), 3.12 – 3.33 (m, 1 H), 2.84 – 3.04 (m, 2H), 2.46 – 2.65 (m, 1 H), 2.23 – 2.45 (m, 1 H).

Example 2: Synthesis of Benzyl -4-hvdroxy-2-(4-(methoxycarbonyl)phenyl)piperidine-1- 

carboxylate (C5. wherein Pi = Cbz and R = methyl)

P1 = Cbz P i = Cbz

R = Methyl R = Methyl

A 0.1 M pH = 7.0 PBS was prepared with disodium phosphate dodecahydrate (22.2 g), sodium dihydrogen phosphate dihydrate (6.2 g) and purified water (999 g). To a reactor equipped with a pH meter 0.1 M pH = 7.0 PBS (499 g), D-glucose (40.2 g, 233.14 mmol, 2.0 eq), NADP (EnzymeWorks, 0.72 g), GDH (EnzymeWorks, 0.41 g) and KRED-EW124 (EnzymeWorks, 2.05 g)

were added, followed by addition of emulsion of C4 (41 g, 1 1 1 .60 mmol, 1 .0 eq) in DMSO (102.5 ml_). The mixture was heated to JT < 45 °C, IT 41 ± 3 °C and stirred at IT 41 ± 3 °C for > 16 h while controlling pH 6.9-7.2 by adding 1 M sodium hydroxide solution. A mixture of NADP (0.29 g), GDH (0.16 g) and KRED-EW124 (0.82 g, #Enzyme Works Inc. China) in 0.1 M pH = 7.0 PBS (1 1 g) were charged and stirred at IT 41 ± 3 °C for > 20 hours. The reaction was monitored by HPLC.

The reaction was filtered to afford white wet cake. To a 1 .0 L Radleys reactor equipped with anchor agitator crude C5 wet cake (80 g) and acetonitrile (500 ml.) were charged. The mixture was stirred to form a light yellow suspension (700 RPM). The suspension was heated to IT = 70 ± 5 °C and stirred for 4 hours, and then cooled to IT = 25 ± 5 °C. The suspension was filtered and the cake was washed with acetonitrile (75 ml_). To a clean 500 ml. Radleys reactor equipped with anchor agitator the resulting mother liquor was charged. The mother liquid was concentrated to about 95 g, solvent exchanged with three portions of toluene (105 g) to 95 g residue. Toluene (170 g) was charged and the reaction was checked by GC (acetonitrile / (toluene + acetonitrile) < 1 .2%). The suspension was heated to IT = 80 ± 5 °C, held for 1 hour, cooled to IT = 45 ± 3 °C and adjusted the agitation speed to low mode. Sequential operations of seeding and aging for 2 hours, charging n-heptane (10.2 g) in 0.5 hours and aging for 1 hour, charging n-heptane (34 g) over 1 .5 hours and aging for 0.5 hours were carried out. The mixture was cooled to IT = 10 ± 3 °C over 7 hours and maintained at 10 ± 3 °C for 2 hours. The mixture was filtered and the cake was washed with cold mixed solvents of toluene (50 ml.) and n-heptane (10 ml.) to afford a light yellow solution of C5 (330 g, trans/cis = 90/10, assay 6.8%, yield 52%). The mother liquor was telescoped to the next step. 1 H-NMR (400 MHz, CDCI3, mixture of two isomers, data for the minor isomer is shown in brackets): d (ppm) = 7.99 (d, J=8.44 Hz, 2H) [7.92 (d, J=8.44 Hz, 0.04H)], 7.23 – 7.39 (m, 7H) [7.10 – 7.18 (m, 0.21 H)], 5.69 (br. s, 1 H) [5.40-5.42 (m, 0.1 1 H)], 5.19 (s, 2H) [5.14 (s, 0.23H)], 4.26 (br. d, J=13.33 Hz, 1 H) [4.18-4.20(m, 0.13H)], 3.91 (s, 3H) [3.90 (s, 0.4H)], 3.67 – 3.79 (m, 1 H) [3.38-3.45 (m, 0.1 1 H)], 2.83 (td, J=13.51 , 2.81 Hz, 1 H), 2.64 (br. d, J=13.33 Hz, 1 H) [2.41 -2.47 (m, 0.12H)], 1 .81-1 .91 (m, 2H) [2.17-2.22 (m, 0.12H)], 1 .72 – 1 .77 (m, 1 H), 1 .45 – 1 .56 (m, 1 H). HRMS: Calcd for C21 H24NO5 (M+H): 370.1654m, found 370.1662.

Example 3: Synthesis of Methyl 4-r(2S,4S)-4-ethoxypiperidin-2-yl1benzoate (Compound of formula according to the following sequence:

R = Methyl R = Methyl R = Methyl

Step 1 : Synthesis of Benzyl (4S)-4-((tert-butyldimethylsilyl)oxy)-2-(4-(methoxycarbonyl) phenyl)piperidine-1 -carboxylate (C8, wherein Pi = Cbz, P2 = TBS and R = methyl).

To a 500 ml. Radleys Reactor charged with C5 in a toluene/heptane solution (1 .0 eq, 145.67 g from previous step, assay 6.07%, 23.94 mmol). The solution was concentrated to about 25 g. Then dichloromethane (1 17.1 g) was charged and the solution was cooled to 23 ± 4 °C. To the clear solution, imidazole (3.42 g, 50.26 mmol, 2.1 eq) and TBS-CI (6.13 g, 40.69 mmol, 1 .7 eq) were introduced. The yellow suspension was stirred at 23 ± 4 °C for 10 hours. The reaction was monitored by HPLC. Then 10% Na2CC>3 (70.7 g) was charged and the mixture was stirred for 1 hours. The organic phase was washed with 5% brine (53 g) and concentrated to about 30 g. Then the solvent was exchange with toluene (45 g) to about 25 g. The residue was diluted with dichloromethane (66 g) and the mixture was filtered through a pad of 200-300 mesh silica gel (1 .66 g). The silica gel was eluted with another portion of dichloromethane (17.5 g). The eluent was concentrated and the residue was subjected to solvent exchange with acetonitrile (71 .1 g + 98.2 g) to 90 g (yield 100%). C8 in acetonitrile solution was used in the next step. 1 H-NMR (400 MHz, CDCI3, mixture of two isomers, data for the minor isomer is shown in brackets): d (ppm) = 8.01 (d, J=8.44 Hz, 2H) [7.94 (d, J=8.44 Hz, 0.17H)], 7.26 – 7.34 (m, 7H) [7.09 – 7.18 (m, 0.13H)], 5.65 (br. d, J=2.04 Hz, 1 H) [5.41 (br. d, J=2.04 Hz, 0.08H)], 5.19 (s, 2H) [5.13 (s, 0.16H)], 4.22 (br. d, J=13.69 Hz, 1 H) [4.10-4.14(m, 0.19H)], 3.92 (s, 3H) [3.90 (s, 0.3H)], 3.62 – 3.69 (m, 1 H) [3.43-3.50 (m, 0.08H)], 2.81 (td, J=13.54, 2.87 Hz, 1 H), 2.49 (br. d, J=13.57 Hz, 1 H) [2.31 -2.35 (m, 0.1 OH)], 1.84-1 .92 (m, 1 H) [2.08-2.14 (m, 0.07H)], 1 .74 – 1 .75 (m, 1 H), 1 .48 – 1 .59 (m, 1 H), 0.86 (s, 9H) [0.56 (s, 0.65H)], 0.03 (s, 3H) [0.09 (s, 0.27H)].

Step 2: Synthesis of Benzyl (4S)-4-ethoxy-2-(4-(methoxycarbonyl)phenyl)piperidine-1 -carboxylate (C9, wherein Pi = Cbz, R = methyl)

To a 250 ml. Radleys Reactor equipped with impeller agitator C8 in acetonitrile solution (135.5 g, assay 12.53%, 35.10 mmol) was charged and rinsed with acetonitrile (with 8.5 g). Et3SiH (12.25 g, 105.31 mmol, 3.0 eq) was charged. The reactor was cooled to IT = 4 ± 5 °C. TESOTf (1 .392 g,

5.265 mmol, 0.15 eq) was charged. A solution of 2,4,6-trimethyM ,3,5-trioxane (4.64 g, 35.10 mmol, 1 .0 eq) in acetonitrile (7.9 g) was added to the mixture in 60 min at IT = 4 ± 5 °C. After addition, the mixture was stirred for 15 min and followed by HPLC. To the reaction mixture was charged 5% aqueous Na2CC>3 (21 .22 g) and water (30 g). Followed by n-heptane (20.4 g) and the mixture was stirred at 25 ± 5 °C for 30 min. Phase cut and the bottom acetonitrile phase was collected. The acetonitrile phase was concentrated to about 65 g. MTBE (100.6 g) and 5% aqueous Na2CC>3 (43.44 g) were charged to the residual acetonitrile solution. The mixture was stirred for 30 min. The upper MTBE phase was collected and filtered via Charcoal film. The charcoal film was washed with MTBE (7.4 g). The mother liquor was concentrated to about 35 g. To the residue methanol (79.2 g) was charged and the solution was concentrated to 70 g. The solution was telescoped to the next step. 1 H NMR (400 MHz, CDCI3, mixture of two isomers, data for the minor isomer is shown in brackets) d (ppm) = 8.01 (d, J= 8.31 Hz, 2H) [7.96 (d, J= 8.31 Hz, 0.21 H)], 7.29 – 7.32 (m, 7H) [7.07 – 7.22 (m, 0.40H)], 5.68 (br. s, 1 H) [5.32 – 5.34 (m, 0.10H)], 5.19 (s, 2H) [5.1 1 (s, 0.19H)], 4.27 (br. d, J=13.08 Hz, 1 H) [4.05 – 4.14 (m, 0.15H)], 3.91 (s, 3H) [3.89 (s, 0.15H)], 3.41 – 3.54 (m, 2H) [3.14 – 3.25 (m, 0.21 )], 3.30 – 3.40 (m, 1 H) [3.86 – 3.75 (m, 0.13H)], 2.84 (td, J=13.51 , 2.81 Hz, 1 H), 2.66 (br. d, J=13.20 Hz, 1 H), 1 .62 – 1 .95 (m, 2H), 1 .40 – 1 .53 (m, 1 H), 1 .18 (t, J= 6.97 Hz, 3H).

Step3: Synthesis of Methyl 4-((4S)-4-ethoxypiperidin-2-yl)benzoate (removal of the protecting group Pi = Cbz – R = methyl)

To a 500 ml. autoclave charged with 10% Pd/C (50% wet, 3.83 g), C9 solution in methanol (assay 19.97%, 192 g, 96.46 mmol) and methanol (28 g). The reactor was purged with vacuum/H2, three times. The mixture was hydrogenated at 3 bar and at a temperature of 25 ± 4 °C for 4 hours. The mixture was filtered and the Pd/C cake was washed with methanol (20 g). The mother liquor was concentrated to 48 g, solvent swapped twice with 142 g isopropyl acetate to 106 g, cooled to 8 ± 5 °C, and 3% hydrogen chloride solution (90.2 g) was added. After phase separation, the aqueous phase was collected and washed with isopropyl acetate (86.4 g). To the aqueous phase MTBE (72 g) and 10% Na2C03 (99.2 g) were added. After phase separation, the aqueous phase was extracted with MTBE (72 g). The combined MTBE phase was washed with water (40 g). The MTBE solution was introduced into the next step. 1 H NMR (400 MHz, CDCI3, mixture of two isomers, data for the minor isomer is shown in brackets) d (ppm) = 7.96 (m, J= 8.31 Hz, 2H), 7.40 – 7.46 (m, 2H), 4.06 (dd, J=1 1 .62, 2.45 Hz, 1 H), 3.88 (s, 3H), 3.70 – 3.79 (m, 1 H) [3.64 – 3.69 (m, 0.12H)], 3.48 -3.56 (m, 2H) [3.38 – 3.45(m, 0.1 1 H)], 3.1 1 – 3.18 (m, 1 H) [3.21 – 3.26 (m, 0.1 1 H)], 2.88 – 2.97 (m, 1 H) [2.73 – 2.80 (m, 0.12H )], 1 .94 – 2.00 (m, 1 H) [ 2.14 – 2.19 (m, 0.10H)], 1.84 – 1 .89 (m, 1 H) [2.02 – 2.07 (m, 0.12H)], 1 .75 (S, 1 H), 1 .65 – 1 .70 (m, 1 H) [1 .45 – 1 .49 (m, 0.10H)], 1 .59 – 1 .64 (m, 1 H) [1 .36 – 1 .42 (m, 0.1 1 H)], 1 .22 – 1 .25 (t, 3H) [1 .17 – 1 .20 (t, J= 6.97, 0.24H)].

Step 4: Synthesis of Methyl 4-[(2S,4S)-4-ethoxypiperidin-2-yl]benzoate (Compound of formula (II) – R = methyl).

To a 500 ml. one neck flask was added the crude solution of step 3 (above) in MTBE (telescoped from last step, 1 10 g, assay 10.52%, light yellow solution, 43.95 mmol). The solution was concentrated to 18.4 g and the solvent was exchanged (JT = 60 °C) with 55 g of n-heptane twice to get 35 g yellow solution. The solution was transferred to 100 ml. Easy Max equipped with impeller agitator. The solution was heated to 50 °C with 300 RPM , aged for 30 min, cooled to 41 ± 2 °C and seed was added. The agitation was adjusted to low speed. The mixture was aged at 41 ± 2 °C for 2 hours, cooled to 35 ± 2 °C in 8 – 10 hours and then aged at 35 ± 2 °C for 1 – 2 hours n-heptane (7.9 g) was added dropwise. The agitation was adjusted to medium speed. The mixture was cooled to IT = 25 ± 2 °C in 1 hour and aged at 25 ± 2 °C for 10 – 20 minutes. The mixture was filtered. The filtrate was re-charged to the reactor for rinsing the solid on the reactor wall. The mixture was filtered and the filter cake was washed with pre-cooled (-5 °C) n-heptane (7.9g). The cake was dried at 40 °C for > 10 hours to afford 6.4 g of white solid (50% yield). 1H NMR (400 MHz, CDCIs) d (ppm) = 7.99 (m, J=8.31 Hz, 2H), 7.45 (m, J=8.19 Hz, 2H), 4.09 (dd, J=1 1 .62, 2.20 Hz, 1 H), 3.90 (s, 3H), 3.75 (t, J=2.81 Hz, 1 H), 3.53 (q, J= 6.97 Hz, 2H), 3.17 (td, J=12.13, 2.63 Hz, 1 H), 2.91 – 2.99 (m, 1 H), 1.99 (dd, J=13.57, 2.69 Hz, 1 H), 1 .88 (dt, J=13.79, 2.58 Hz, 1 H), 1 .69 – 1 .79 (m, 1 H), 1 .57 – 1 .68 (m, 2H), 1 .25 (t, J= 7.03 Hz, 3H).

Example 4: Enantioselective synthesis of compound according to the following

sequence:

Step 1 : Synthesis of Benzyl 4-oxo-3,4-dihydropyridine-1 (2H)-carboxylate (C6, wherein Pi = Cbz and R = methyl)

To a 2.0 L reactor, 4-methoxypyridine (C1 , 45.0 g, 412.39 mmol, 1 .0 eq) and methanol (900 ml.) were added. The mixture was cooled to -75 °C with dry ice/acetone bath. A solution of benzyl

chloroformate (73.86 g, 432.99 mmol, 1 .05 eq) in THF (90 ml.) was charged dropwise while keeping IT < -70 °C. The reaction was stirred for 1 hour to afford a white suspension at -70 °C. Sodium borohydride (16.38 g, 432.99 mmol, 1 .05 eq) was added in portions while keeping IT < -70 °C. The reaction was stirred at -70 °C for 2 hours. Water (200 g) was added and the cooling bath was removed. A solution of 36% hydrogen chloride (16.72 g, 164.95 mmol, 0.4 eq) in water (50 ml.) was added in 10 min at 0 – 5 °C and stirred for 1 hour. Then 20% Na2CC>3 (85.5 g) was added to adjust pH = 7 while maintained IT < 5 °C. Organic solvents were removed under vacuum. The resulting residue was extracted with dichloromethane (450 ml_). The dichloromethane phase was washed with 3wt% hydrogen chloride (151 ml.) and 3 wt% Na2C03 (151 ml_). After solvent exchange with MTBE, about 4 volume (180 ml) of the MTBE mixture was obtained. The mixture was heated to 50 °C to afford a solution and then cooled to 45 °C. Crystal seed of C6 was charged and the mixture was aged at 40 – 45 °C for 7 hours. The mixture was cooled to 10 – 15 °C in 3 hours. The white suspension was filtered and the wet cake was rinsed with cold MTBE (45 ml_). The cake was dried under vacuum at 40 – 50 °C for 2 hours to afford C6 as a white powder (91.56 g, 60% yield). 1H NMR (400 MHz, CDCI3): d (ppm) = 7.85 (br. s, 1 H), 7.37 – 7.43 (m, 5H), 5.43 (br. s, 1 H), 5.26 (s, 2H), 4.05 (t, J=7.34 Hz, 2H), 2.54 – 2.58 (m, 2H).

Step 2: Synthesis of Benzyl (S)-2-(4-(methoxycarbonyl)phenyl)-4-oxopiperidine-1 -carboxylate ((S)-C4, wherein Pi = Cbz and R = methyl)

Method 1 : A 500 ml Radleys reactor was purged 3 times with vacuum/N2. C6 (8 g, 34.60 mmol, 1.0 eq), C7 (9.34 g, 51.89 mmol, 1 .5 eq), tert- Amyl alcohol (160 ml.) and deionized water (16 ml.) were added. The mixture was stirred for > 40 minutes to give a clear colorless solution. The solution was purged 4 times with vacuum / N2 and bubbled with N2 via a syringe needle for 1 hour. To the colorless solution was charged the mixed solid of (S)-XylBINAP (0.381 g, 0.519 mmol, 0.015 eq) and Rh(Acac)(C2H4)2 (0.134 g, 0.519 mmol, 0.015 eq). The mixture was continued to bubble with N2 for 15 minutes and purged 4 times with vacuum / N2. The suspension was stirred for another 2 hours to dissolve (S)-XylBINAP. The reaction mixture was stirred at 55 ± 4 °C for 15 hours. The reaction was followed by HPLC. The mixture was cooled and treated with 7.7% sodium hypochlorite (1 g, 1 .04 mmol, 0.03 eq) for 1 .5 hours at 40 ± 4 °C. tert- Amyl alcohol was distilled off. The residue was extracted with isopropyl acetate (64 ml.) and ethyl acetate (8 ml.) and filtered. The organic phase was washed with 5% NaHC03 (50 g) then with 15% brine (40 g) at 50 ± 5 °C. Some solvents were removed and ethyl acetate (21 .6 g) was added. The solution was treated with Smopex-234 (1 .2 g) at IT =55 ± 5 °C for 2 hours then filtered via 200 – 300 mesh silica gel (1 .6 g). After solvent exchange with n-heptane, MTBE (44.4 g) was added. The mixture was cooled to IT = 42 ± 3 °C. (S)-C4 seed (10 mg) was added. The mixture was aged for 2 hours and cooled to IT =

31 ± 3 °C in 3 hours n-heptane (23.2 g) was then charged in 1 – 2 hours. The mixture was aged for 2 hours and cooled to IT = 20 ± 3 °C in 2 hours. The mixture was filtered and the cake was washed with a mixed solvent of MTBE (4.4 g) and n-heptane (4.1 g). Dried the wet cake at 60 °C for > 5 hours to afford (S)-C4 (7.63 g, 60% yield) as yellow powder. 1H NMR (400 MHz, CDCI3): d (ppm) = 7.99 (d, J=8.44 Hz, 2 H), 7.28 – 7.37 (m, 7H), 5.82 (br. s, 1 H), 5.14 – 5.28 (m, 2H), 4.30 (br. s, 1 H), 3.91 (s, 3H), 3.22 (br. d, J=8.31 Hz, 1 H), 2.84 – 3.03 (m, 2H), 2.46 – 2.64 (m, 1 H), 2.38 (br. d, J=16.26 Hz, 1 H).

Method 2: To a 500 ml Radleys reactor purged 3 times with vacuum/N2, C6 (8 g, 34.60 mmol, 1 .0 eq), C7 (9.34 g, 51 .89 mmol, 1 .5 eq), fe/f-Amyl alcohol (160 ml.) and deionized water (16 ml.) were added. The mixture was stirred for roughly 40 minutes to give a clear colorless solution. The solution was purged 4 times with vacuum / N2 and bubbled with N2 via a syringe needle for 1 hour. To the colorless solution, was charged the mixed solid of (R, R)-Ph-BPE-Rh(Acac) (0.005 eq., 0.122 g, 0.173 mmol). The mixture was continued to bubble with N2 for 15 minutes and purged with vacuum / N2. The reaction mixture was stirred at 55 ± 4 °C for 15 hours. The reaction was followed by HPLC. Tert- amyl alcohol was distilled off. The residue was extracted with isopropyl acetate (64 ml.) and ethyl acetate (8 ml_), and then filtered. The organic phase was washed with 5% NaHC03 (50 g), then with 15% brine (40 g) at 50 ± 5 °C. Some solvents were removed and ethyl acetate (21 .6 g) was added. The solution was treated with Smopex-234 (1 .2 g) at IT = 55 ± 5 °C for 2 hours then filtered via 200 – 300 mesh silica gel (1 .6 g). After solvent exchange with n-heptane, MTBE (44.4 g) was added. The mixture was cooled to IT = 42 ± 3 °C. (S)-C4 seed (10 mg) was added. The mixture was aged for 2 hours and cooled to IT = 31 ± 3 °C in 3 hours n-heptane (23.2 g) was then charged in 1 – 2 hours. The mixture was aged for 2 hours and cooled to IT = 20 ± 3 °C in 2 hours. The mixture was filtered and the cake was washed with a mixed solvent of MTBE (4.4 g) and n-heptane (4.1 g). The wet cake was dried at 60 °C for roughly 5 hours to afford (S)-C4 (10.17 g, 80% yield) as yellow powder. 1 H NMR (400 MHz, CDCI3) d (ppm) = 7.99 (d, J=8.44 Hz, 2 H), 7.28 – 7.37 (m, 7H), 5.82 (br. s, 1 H), 5.14 – 5.28 (m, 2H), 4.30 (br. s, 1 H), 3.91 (s, 3H), 3.22 (br. d, J=8.31 Hz, 1 H), 2.84 – 3.03 (m, 2H), 2.46 – 2.64 (m, 1 H), 2.38 (br. d, J=16.26 Hz, 1 H).

Method 3: To a 500 ml Radleys reactor purged 3 times with vacuum/N2. C6 (8 g, 34.60 mmol, 1 .0 eq), C7 (9.34 g, 51 .89 mmol, 1 .5 eq), tert- amyl alcohol (160 ml.) and deionized water (16 ml.) were added. The mixture was stirred for roughly 40 minutes to give a clear colorless solution. The solution was purged 4 times with vacuum / N2, and bubbled with N2 via a syringe needle for 1 hour. To the colorless solution was charged the mixed solid of (S)-XylBINAP-Rh(Acac) (0.01 eq., 0.324

g, 0.346 mmol). The mixture was continued to bubble with N2 for 15 minutes and purged with vacuum / N2. The reaction mixture was stirred at 55 ± 4 °C for 15 hours. The reaction was followed by HPLC. Tert- amyl alcohol was distilled off. The residue was extracted with isopropyl acetate (64 mL) and ethyl acetate (8 mL), and then filtered. The organic phase was washed with 5% NaHC03 (50 g), then with 15% brine (40 g) at 50 ± 5 °C. Some solvents were removed and ethyl acetate (21 .6 g) was added. The solution was treated with Smopex-234 (1 .2 g) at IT =55 ± 5 °C for 2 hours then filtered via 200 – 300 mesh silica gel (1 .6 g). After solvent exchange with n-heptane, MTBE (44.4 g) was added. The mixture was cooled to IT = 42 ± 3 °C. (S)-C4 seed (10 mg) was added. The mixture was aged for 2 hours and cooled to IT = 31 ± 3 °C in 3 hours n-heptane (23.2 g) was then charged in 1 – 2 hours. The mixture was aged for 2 hours and cooled to IT = 20 ± 3 °C in 2 hours. The mixture was filtered, and the cake was washed with a mixed solvent of MTBE (4.4 g) and n-heptane (4.1 g). The wet cake was dried at 60 °C for roughly 5 hours to afford (S)-C4 (10.30 g, 81 % yield) as yellow powder. 1H NMR (400 MHz, CDCI3) d (ppm) = 7.99 (d, J=8.44 Hz, 2 H), 7.28 – 7.37 (m, 7H), 5.82 (br. s, 1 H), 5.14 – 5.28 (m, 2H), 4.30 (br. s, 1 H), 3.91 (s, 3H), 3.22 (br. d, J=8.31 Hz, 1 H), 2.84 – 3.03 (m, 2H), 2.46 – 2.64 (m, 1 H), 2.38 (br. d, J=16.26 Hz, 1 H).

Example 5: Synthesis of Benzyl -4-hvdroxy-2-(4-(methoxycarbonyl)phenyl)piperidine- 

1-carboxylate f(S)-C5, wherein Pi = Cbz and R = methyl)

R = Methyl R = Methyl

Preparation of 0.1 M PBS, pH 7.0, with 0.1 % TPGS buffer solution: To a 500 ml. Radleys reactor equipped with impeller agitator was charged Na2HP04.12H20 (8.63 g), NaH2P04.2H20 (2.41 g), Tap Water (388.6 g) and TPGS-750-M.001 (0.388 g). The mixture was stirred for > 3 hours at IT = 60 ± 5 °C and then cooled to IT = 51 ± 3 °C. 80 g of the buffer solution was taken from the reactor to a flask and cooled to < 35 °C. Check pH value of the buffer solution (7.0 ± 0.5). To the above Radleys reactor (S)-C4 (20.0 g, 54.4 mmol, 1 .0 eq), Isopropanol (16.36 g, 272.2 mmol, 5.0 eq) and 0.1 % TPGS buffer solution (60 g) were added. To a 25 mL flask was charged KRED-P3-G09 (0.4 g, #Codexis), NADP+ (0.1 g) and 0.1 % TPGS buffer solution (60 g) from the above flask. All the solid was dissolved. The solution of enzyme was charged to the 500 mL Reactor at IT =50 ± 5 °C. Rinsed the 25 mL flask with 0.1 % TPGS buffer (10 g) and transferred the solution to the 500 mL reactor at IT =50 ± 5 °C. The mixture was stirred with agitation speed > 500 RPM at 51 ± 3 °C for >

8 hours. The reaction was followed by HPLC. To the reactor 2-MeTHF (200 mL) was added and the mixture was stirred for > 60 minutes at 50 ± 5 °C. The mixture was held for > 50 minutes without agitation and the bottom aqueous phase was separated. The organic phase was washed twice with another 200 g of water at 50 ± 5 °C. The organic phase was concentrated to about 70 g. After solvent exchange with twice 158 g acetonitrile to give about 80 g solution, which was cooled to < 30 °C then filtered via MCC. MCC cake was washed with isopropyl acetate (40 mL/35.5 g) to afford (S)-C5 in a light color solution (1 14.3 g, assay 16.95% 96.34% yield). The acetonitrile / isopropyl acetate solution was telescoped to the next step directly. 1 H NMR (400 MHz, CDCI3): d (ppm) = 7.98 (d, J=8.44 Hz, 2H), 7.23 – 7.38 (m, 7H), 5.61 – 5.72 (m, 1 H), 5.18 (s, 2H), 4.23 (br. d, J=13.33 Hz, 1 H), 3.90 (s, 3H), 3.62 – 3.75 (m, 1 H), 2.81 (td, J=13.51 , 2.81 Hz, 1 H), 2.62 (br. d, J=13.33 Hz, 1 H), 2.45 (br. s, 1 H), 1 .79 – 1 .91 (m, 2H), 1 .41 – 1 .56 (m, 1 H).

Example 6: Asymmetric synthesis of Methyl 4-r(2S.4S)-4-ethoxypiperidin-2-yl1benzoate

(Compound of formula . or a salt thereof. – R= methyl) according to the following

sequence:

(S)-C5 (S)-C9 Compound of (Pi = Cbz) (Pi = Cbz, P2 = TBS) (Pi = Cbz) formula (II) R = Methvl R = Methyl R = Methyl R = Methyl

Step 1 : Synthesis of Benzyl (2S,4S)-4-{[tert-butyl(dimethyl)silyl]oxy}-2-[4-(methoxy carbonyl) phenyl]piperidine-1 -carboxylate ((S)-(C8), wherein Pi = Cbz, P2 = TBS, and R = methyl).

To a 500 ml Radleys Reactor was charged with (S)-C5 solution (in acetonitrile / isopropyl acetate, 271 .8 g, assay 14.72%, contained 40.0 g of (S)-C5, 108.31 mmol, 1 .0 eq) from the previous step. After solvent exchange with isopropyl acetate (159.8 g / 180 ml_), 100 g clear solution was obtained. Isopropyl acetate (176 g /198 ml_), imidazole (26.54 g, 389.90 mmol, 3.6 eq) and TBS-CI (27.75 g, 184.12 mmol, 1 .7 eq) were added. The yellow suspension was stirred at 55 ± 4 °C for 7 hours. The reaction was followed by HPLC. The reaction mixture was cooled to 23 ± 4 °C and filtered through MCC (2 g). The cake was washed with isopropyl acetate (88.8 g / 100 ml_). 6% NaHC03 (240 g) was added and the mixture was stirred for 20 minutes. The organic phase was washed with 5% brine (2×240 g) and concentrated to about 105 g. After solvent exchange with toluene (120 g / 135.4 ml_), 105 g solution was obtained. Dichloromethane (298 g / 224.5 ml.) was added and the solution was filtered via 200-300 mesh silica gel (4.4 g). The silica gel was eluted with another portion of dichloromethane (44 g / 33 ml_). The mother liquor was concentrated and the solvent was exchanged with acetonitrile (2×280 ml_, 442.4 g in total) to 100 g. The residue was diluted with acetonitrile (105 g / 132.9 ml.) to afford a light yellow solution (205 g, assay 25.55%, 100% yield), which was used for the next step directly. 1 H NMR (400 MHz, CDCI3) d (ppm) = 8.01 (d, J=8.44 Hz, 2 H), 7.23 – 7.37 (m, 7 H), 5.60 – 5.70 (m, 1 H), 5.18 (s, 2H), 4.22 (br. d, J=13.45 Hz, 1 H), 3.90 (s, 3H), 3.62 – 3.71 (m, 1 H), 2.82 (td, J=13.51 , 2.81 Hz, 1 H), 2.49 (br. d, J=13.45 Hz, 1 H), 1.83 – 1 .96 (m, 1 H), 1 .75 – 1 .80 (m, 1 H), 1 .47 – 1.60 (m, 1 H), 0.86 (s, 9H), 0.03 (s, 3H), 0.00 (s, 3H).

Step 2: Synthesis of Benzyl (2S, 4S)-4-ethoxy-2-[4-(methoxycarbonyl)phenyl]piperidine-1 -carboxylate ((S)-C9, wherein Pi = Cbz amd R = methyl)

To a 500 ml. Radleys Reactor equipped with impeller agitator (S)-C8 in an acetonitrile solution (170.8 g, assay 29.28%, 103.38 mmol, 1 .0 eq) and fresh acetonitrile (220 g) were charged, followed by Et3SiH (36.06 g, 310.13 mmol, 3.0 eq). The mixture was cooled to IT =4 ± 5 °C and TESOTf (5.47 g, 20.68 mmol, 0.2 eq) was charged. To the mixture was charged a solution of 2,4,6-trimethyl-1 ,3,5-trioxane (13.66 g, 103.38 mmol, 1 .0 eq) in acetonitrile (23 g) over 60 minutes at IT =4 ± 5 °C. Upon addition, the mixture was stirred for 15 minutes. The reaction was followed by HPLC. To the reaction mixture was charged 5% aqueous sodium hydroxide (16.54 g, 20.68 mmol, 0.2 eq) and 20 g water, followed by n-heptane (60 g). The mixture was stirred for 30 minutes at 20 ± 5 °C. The bottom acetonitrile phase was collected. To the acetonitrile phase was charged with MTBE (1 1 1 g) and 10% brine (300 g). The mixture was stirred for 30 minutes. The upper MTBE phase was washed with 10% brine (2×300 g), concentrated to 90 g. MTBE (185 g) and water (150 g) were charged. After phase separation at 38 ± 4 °C and solvent exchange of the organic layer with isopropyl acetate (2×266.4 g), 205 g solution was obtained, which was filtered through Charcoal film slowly. The charcoal film was washed with isopropyl acetate (22.2 g) to afford as a light yellow solution (223 g, 100% yield). The solution was telescoped to the next step directly. 1 H NMR (400 MHz, CDCI3) d (ppm) = 8.01 (d, J=8.44 Hz, 2H), 7.25 – 7.38 (m, 7H), 5.68 (br. s, 1 H), 5.19 (s, 2H), 4.27 (br. d, J=13.33 Hz, 1 H), 3.92 (s, 3H), 3.42 – 3.54 (m, 2H), 3.34 (ddd, J=10.88, 6.91 , 4.22 Hz, 1 H), 2.84 (td, J=13.51 , 2.81 Hz, 1 H), 2.66 (br. d, J=13.20 Hz, 1 H), 1 .96 (br. d, J=10.51 Hz, 1 H), 1 .75 – 1 .90 (m, 1 H), 1 .33 – 1 .53 (m, 1 H), 1 .18 (t, J= 6.97 Hz, 3H).

Step 3: Synthesis of Methyl 4-((2S,4S)-4-ethoxypiperidin-2-yl)benzoate (compound of Formula (II), or a salt thereof – R= methyl)

To a 500 ml. autoclave which was purged with vacuum / N2 (S)-C9 in an isopropyl acetate solution (278.4 g, assay 17.96%, 50 g of (S)-C9, 125.80 mmol) and 10% Pd/C (5.0 g, 50% wet) were

charged. The reactor was purged with vacuum / H2 and stirred for > 7 hours at 25 ± 5 °C. The reaction was followed by HPLC analysis. Filtered the reaction mixture via MCC (7.7 g) which was pre-washed with isopropyl acetate . Rinsed the reactor and MCC with isopropyl acetate (39 g). The mother liquor was combined to afford compound of formula (II) as a light yellow solution (315 g, assay 10.0%, 95.1 % yield). 1 H NMR (400 MHz, CDCI3) d (ppm) = 7.99 (m, J=8.31 Hz, 2H), 7.45 (m, J=8.19 Hz, 2H), 4.09 (dd, J=1 1 .62, 2.20 Hz, 1 H), 3.90 (s, 3H), 3.75 (t, J=2.81 Hz, 1 H), 3.53 (q, J= 6.97 Hz, 2H), 3.17 (td, J=12.13, 2.63 Hz, 1 H), 2.91 – 2.99 (m, 1 H), 1 .99 (dd, J=13.57, 2.69 Hz, 1 H), 1 .88 (dt, J=13.79, 2.58 Hz, 1 H), 1 .69 – 1 .79 (m, 1 H), 1 .57 – 1 .68 (m, 2H), 1 .25 (t, J= 7.03 Hz, 3H).

Step 4: Synthesis of the maleic salt of compound of formula (II) (R = methyl)

To a 500 ml. Radleys Reactor equipped with impeller agitator a solution of methyl 4-((2S,4S)-4-ethoxypiperidin-2-yl)benzoate (381 g, assay 10.03%, 145.12 mmol, 1 .0 eq) from the previous step was charged. The solution was concentrated to 281 g and fresh isopropyl acetate (28.6 g) was added. Then a solution of maleic acid (8.45 g, 72.56 mmol, 0.5 eq) in acetone (30.5 ml.) was added at 51 ± 3 °C in 30 minutes. After stirring for 15 minutes, a seed of the maleic salt of compound of formula (II) was added and the mixture was aged for 2 hours. A solution of maleic acid (8.45 g, 72.56 mmol, 0.5 eq) in acetone (30.5 ml.) was charged at 51 ± 3 °C in 60 minutes and the mixture was aged for 2 hours. The mixture was cooled to IT = 10 ± 3 °C in 6 hours and stirred for > 120 minutes. The mixture was filtered and the filter cake was washed with pre-cooled isopropyl acetate (44.4 g). The cake was dried under high vacuum at 55 °C for 5 – 12 hours to afford maleic salt of compound of formula (II) as white solid (49.8 g, Yield 90.4%). 1 H NMR (400 MHz, CDCIs) d (ppm) 9.35 – 9.78 (m, 2H), 8.02 (m, J=8.31 Hz, 2H), 7.58 (m, J=8.31 Hz, 2H), 6.17 (s, 2H), 4.56 (br. d, J=1 1.13 Hz, 1 H), 3.90 (s, 3H), 3.86 (s, 1 H), 3.48 – 3.57 (m, 2H), 3.38 – 3.44 (m, 2H), 2.42 (br. t, J=13.57 Hz, 1 H), 1 .98 – 2.20 (m, 3H), 1 .24 (t, J= 6.97 Hz, 3H).

The maleic salt of compound of formula (II) may be characterized by a x-ray powder diffraction pattern (XRPD) comprising four or more 2Q values (CuKa l=1 .5418 A) selected from the group consisting of 5.893, 6.209, 1 1 .704, 13.014, 16.403, 17.295, 17.592, 18.629, 18.942, 21 .044, 21 .733, 21 .737, 22.380, 23.528, 24.195, 26.013, 26.825, 29.017, 29.515, 32.250, 35.069, 35.590, and 37.932, measured at a temperature of about 22 °C and an x-ray wavelength, l, of 1 .5418 A.

Example 7: Synthesis of fert-butyl 4-formyl-5-methoxy-7-methyl-1 H-indole-1 -carboxylate

(Compound of formula (III), or a salt thereof) according to the following seguence:

Step 1 : Synthesis of 7-methyl-1 H-indol-5-ol (C11 )

To a 250 ml. flask equipped with a thermometer 3.4% Na2HP04 (100 g, pH = 8.91 ) was charged, followed by addition of Fremy’s salt (4.84 g, 2.4 eq). The mixture was stirred at 20 ± 5 °C until a clear solution was formed. A solution of 7-methylindoline in acetone (9.1 g, 1 1 %) was added in one portion. The mixture was stirred at 20 ± 5 °C for 1 .5 hours. Then sodium sulfite (0.38 g) was added. The mixture was extracted with ethyl acetate (100 ml. x 2) The combined organic extracts were dried over anhydrous sodium sulfate, filtered and concentrated. To the residue 20ml_ acetonitrile was added. The solution was used directly in the next step.

Step 2: Synthesis of fert-butyl 5-hydroxy-7-methyl-1 H-indole-1 -carboxylate (C12, wherein P3 = Boc)

The above as prepared solution was cooled to 0 ± 5 °C. DMAP (0.34 g, 0.4 eq) was charged followed by addition of (Boc)20 (4.9 g, 3.0 eq). The mixture was warmed to 20 ± 5 °C, stirred at 20 ± 5 °C for 30 minutes and concentrated. To the residue was added methanol (40 ml_). The mixture was cooled to 0 ± 5 °C. Potassium carbonate (5.1 g, 5.0 eq) was added. The mixture was stirred at 0 ± 5 °C for 4 hours, warmed to 20 ± 5 °C and stirred for additional 2 hours. The mixture was cooled to 0 ± 5 °C. Acetic acid (2 g) was added. pH was 7-8. The mixture was filtered and the filter cake was washed with methanol (10 mL x 2). The filtrate was concentrated and ethyl acetate (30 ml.) was added. The mixture was washed with water (20 ml.) and 5% brine (20 ml_). The organic layer was concentrated to afford a dark oil, which was slurried with (3:2) n-heptane: Ethyl acetate (5 g) to afford a yellow solid. The solid was collected by filtration and dried to give C12 as yellow solid. 27.4% isolate yield from C10. 1 H-NMR (400 MHz, DMSO-d6): d (ppm) = 9.13 (s, 1 H), 7.52 (d, J= 3.67 Hz, 1 H), 6.74 (d, J= 2.2 Hz, 1 H), 6.56 (m, 1 H), 6.50 (d, J= 3.67 Hz, 1 H), 2.45 (s, 3 H), 1.57 (s, 9 H). LCMS (m/z): positive mode 248.1 [M]+, LCMS (m/z): negative mode 246.1 [M-1 ]-.

Step 3: Synthesis of fert-butyl 4-formyl-5-hydroxy-7-methyl-1 H-indole-1 -carboxylate (C13, wherein P3 = Boc)

To a solution of fe/f-butyl 5-hydroxy-7-methyl-1 H-indole-1 -carboxylate (C12) (53.8% assay, 1 .0 g, 2.2 mmol) in THF (20 ml.) was added dropwise the solution of CH3MgBr in THF (1 N, 2.2 ml_, 2.2 mmol). The resulting mixture was stirred at 20 – 25 °C for 10 minutes. (CHO)n (0.2 g, 6.53 mmol)

was added to the mixture. The reaction mixture was heated to 65 – 70 °C and stirred for 1 hours. The reaction mixture was cooled to 20 – 25 °C. Saturated NH4CI (20 ml.) and MTBE (20 ml.) were added. The mixture was separated and the aqueous layer was extracted with MTBE (20 ml_). The organic layers were combined and concentrated to give compound C13 as yellow solid (0.7 g, 79% assay, 92% yield). 1H-NMR (400 MHz, DMSO-d6) d (ppm) = 10.74 (s, 1 H), 10.54 (s, 1 H), 7.82 (d, J= 4.0 Hz, 1 H), 7.34 (d, J= 4.0 Hz, 1 H), 6.81 (s, 1 H), 2.59 (s, 3H), 1 .65 (s, 9H). LCMS (m/z): positive mode 290.1 [M]+.

Step 4: Synthesis of fert-Butyl 4-formyl-5-methoxy-7-methyl-1 H-indole-1 -carboxylate (Compound of formula (III)).

To a solution of compound C13 (50 mg, 0.182 mmol) in dry DMF (3 ml.) was added K2CO3 (50.2 mg, 0.363 mmol). The mixture was stirred for 10 minutes and then dimethyl sulfate (25.2 mg, 0.20 mmol) was added. The reaction mixture was stirred for 1 hours and poured into ice-water (12 ml_). The mixture was filtered and the filter cake was washed with water. The cake was dried under vacuum to give tert- Butyl 4-formyl-5-methoxy-7-methyl-1 H-indole-1 -carboxylate (Compound of formula (III)) as pale solid (48 mg, 91 % yield). 1 H-NMR (400 MHz, DMSO-d6) d (ppm) = 10.51 (s, 1 H), 7.80 (d, J= 4.0 Hz, 1 H), 7.31 (d, J= 4.0 Hz, 1 H), 6.81 (s, 1 H), 3.95 (s, 3H), 2.61 (s, 3H), 1 .59 (s, 9H). LCMS (m/z): negative mode 274.1 [M-1 ]-.

Example 8: Synthesis of fert-butyl 4-formyl-5-methoxy-7-methyl-1 H-indole-1 -carboxylate

(Compound of formula (III), or a salt thereof) according to the following sequence:


f available (P3 = Boc) 
formula (ill)

Step 1 : Synthesis of 5-(benzyloxy)-1 ,3-dimethyl-2 -nitrobenzene

To a solution of commercially available 3,5-dimethyl-4-nitrophenol (100.0 g, 590.4 mmol) in DMF (500 ml_), CS2CO3 (230.8 g, 708.5 mmol) was added and the resulting mixture was stirred for 10 minutes. Then, (bromomethyl)benzene (104.1 g, 590.4 mmol) was added dropwise to the mixture within 30 minutes. The reaction mixture was stirred at 20-25 °C for 1 hour, and then poured into ice-water (1800 ml_). The solid separated out was collected by filtration and washed with water (500 ml_). The cake was dissolved in ethyl acetate (500 ml.) and the solution was washed with a saturated solution of NaCI (50 ml_), was separated, and the solution was concentrated to give 5-(benzyloxy)-l ,3-dimethyl-2-nitrobenzene 2 (147 g, 97.8% yield) as brown solid. HPLC purity

99.7%. 1H-NMR (400 MHz, DMSO-d6) d (ppm) = 7.42 (m, 5 H), 6.94 (s, 2H), 5.16 (s, 2 H), 2.25 (s, 6 H); LCMS (m/z): negative mode 256.2 [M-1 ]-

Step 2: Synthesis of fert-butyl 5-hydroxy-7-methyl-1 H-indole-1 -carboxylate (C12, wherein P3 = Boc)

To a solution of 5-(benzyloxy)-1 ,3-dimethyl-2-nitrobenzene (60.0 g, 233.2 mmol, from Step 1) in DMF (300 ml.) were added DMF-DMA (87.8 g, 699.6 mmol) and pyrrolidine (50.3 g, 699.6 mmol). The solution was heated to 85-90 °C and stirred for 19 hours under nitrogen, then the mixture was cooled to 20-25 °C. The volatile components (DMF-DMA, pyrrolidine and DMF) were removed at 65-70 °C on a rotary evaporator. The crude mixture was dissolved in ethyl acetate (300 ml_), and Raney Nickel (6.0 g) was added. The reaction mixture was subjected to catalytic hydrogenation under atmospheric pressure, overnight. Then, the reaction mixture was put under nitrogen. The mixture was filtrated and the filtrate was concentrated to provide 5-(benzyloxy)-7-methyM H-indole as a black oil. 5-(benzyloxy)-7-methyl-1 H-indole was used without further purification into the next step.

5-(benzyloxy)-7-methyl-1 H-indole was dissolved in acetonitrile (300 ml_), (Boc)20 (53.6 g, 233.2 mmol) and DMAP (5.7 g, 46.6 mmol) were added. The reaction mixture was stirred at 20-25 °C for 1 hour. Acetonitrile was removed on a rotary evaporator, and the residual mixture was dissolved in ethyl acetate (300 ml_). The solution was washed with a saturated aqueous solution of NaHC03 and then concentrated to give a crude oil which was purified by column chromatography (Si02, 500 g) using a mixture of heptane / MTBE (1 :10) to provide the intermediate tert-butyl 5-(benzyloxy)-7-methyl-1 H-indole-1 -carboxylate as a brown oil (42.1 g, 49.2% yield). HPLC purity 93.5%. 1 H-NMR (400 MHz, DMSO-d6) d (ppm) = 7.59 (d, J= 3.67 Hz, 1 H), 7.40 (m, 5 H), 7.04 (d, J= 2.45 Hz, 1 H), 6.81 (d, J= 2.2 Hz, 1 H), 6.57 (d, J= 3.67 Hz, 1 H), 5.1 1 (s, 2 H), 2.51 (s, 3 H), 1.58 (s, 9 H). LCMS (m/z): negative mode 336.2 [M-1 ]- To a solution of intermediate tert-butyl 5-(benzyloxy)-7-methyl-1 H-indole-1-carboxylate (36.7 g, 100 mmol) in ethanol (250 mL), under nitrogen, 10% Pd/C (10.6 g, 10 mmol) and ammonium formate (6.8 g, 105 mmol) were added. The solution was heated to 45-50 °C and stirred for 5 hours under nitrogen. Then the mixture was cooled to room temperature, filtered, and the filtrate was concentrated to give a residue oil. The residual oil was dissolved in ethyl acetate (250 mL), the solution was washed with a saturated aqueous solution of NaCI (100 mL), the phases were separated. The organic layers were collected and concentrated. The obtained crude mixtures was slurried with a (1 :15) mixture of MTBE / Heptane (160 mL) for 2 hours. The precipitate was filtered and washed with heptane (50 mL). The cake was dried under vacuum to give tert-butyl 5-hydroxy-7-methyl-1 H-indole-1 -carboxylate (C12) as a tawny solid (21 .8 g, 87.2% yield). HPLC purity 97.7%. 1 H-NMR (400 MHz, DMS0-d6) d (ppm) = 9.13 (s, 1 H), 7.52 (d, J= 3.67 Hz, 1 H), 6.74 (d, J= 2.2 Hz, 1 H), 6.56 (m, 1 H), 6.50 (d, J= 3.67 Hz, 1 H), 2.45 (s, 3 H), 1 .57 (s, 9 H). LCMS (m/z): negative mode 246.2 [M-1 ]-

Step 3: Synthesis of fert-butyl 4-formyl-5-hydroxy-7-methyl-1 H-indole-1 -carboxylate (C13, wherein P3 = Boc)

To a mixture of MgCI2 (1 1 .6 g, 1 19.7 mmol) and (CHO)n (5.0 g, 159.6 mmol), in THF (150 ml), under nitrogen, triethylamine (17.8 ml_, 127.7 mmol) was added dropwise and the resulting mixture was stirred at 20-25 °C for 10 minutes. Then, tert-butyl 5-hydroxy-7-methyl-1 H-indole-1 -carboxylate (C12) (10.0 g, 39.9 mmol) was added to the mixture. The reaction mixture was heated to 65-70 °C and stirred for 3 hours. The reaction mixture was cooled to 20-25 °C, followed by addition of 2N HCI (70 ml) and isopropyl acetate (150 ml). The mixture was separated and the organic layer was washed with a 5% NaCI solution. Then, the solution was concentrated to give a crude solid. The solid was slurried with ethanol (100 ml.) for 1 hour. The solid precipitate was filtrated, and washed with ethanol (20 ml_). The cake was dried under vacuum to give tert-butyl 4-formyl-5-hydroxy-7-methyl-1 H-indole-1 -carboxylate (C13) as a tawny solid (7.2 g, 63.9% yield). HPLC purity 96.5%. The filtrate solution was concentrated to 20 mL, then stirred for 1 hour. The solid was filtrated, and washed with ethanol (5 mL). The cake was dried by vacuum to give an additional amount of tert-butyl 4-formyl-5-hydroxy-7-methyl-1 H-indole-1 -carboxylate (C13) as a tawny solid (1 .1 g, 95.3% assay, 9.5% yield.). HPLC purity 90.5%. 1 H-NMR (400 MHz, DMSO-d6) d (ppm) = 10.69 (s, 1 H), 10.47 (s, 1 H), 7.75 (d, J= 3.35 Hz, 1 H), 7.27 (d, J= 3.55 Hz, 1 H), 6.74 (s, 1 H), 2.51 (s, 3 H), 1 .59 (s, 9 H); LCMS (m/z): negative mode 274.2 [M-1 ]-.

Step 4: Synthesis of fert-Butyl 4-formyl-5-methoxy-7-methyl-1 H-indole-1 -carboxylate (Compound of formula (III)).

To a suspension of tert-butyl 4-formyl-5-hydroxy-7-methyl-1 H-indole-1 -carboxylate (C13) (6.0 g, 21 .3 mmol) in MeCN (60 mL), 50% K2C03 solution (20 mL) and dimethyl sulfate (2.26 mL, 23.4 mmol) were added. The resulting mixture was stirred at 35-40 °C for 3 hours. The reaction mixture was cooled to 20-25 °C and isopropyl acetate (30 mL) was added. The mixture was then extracted; the water layer was extracted with isopropyl acetate (15 mL), the organic layers were combined and concentrated to give a crude residual. The crude residual was dissolved in isopropyl acetate (60 mL), the solution was washed with a statured NH4CI solution, and then concentrated to give a crude product (6.6 g). The crude was slurried with ethyl acetate / Heptane (100 mL, 1/50) for 3 hours. The solid was filtrated, washed with heptane (20 mL). The cake was dried under vacuum to give tert-butyl 4-formyl-5-methoxy-7-methyl-1 H-indole-1 -carboxylate (Compound of formula (III))

as a pink solid (5.5 g, 87.8% yield). HPLC purity 99.3%. 1 H-NMR (400 MHz, DMSO-d6) d (ppm) = 10.52 (s, 1 H), 7.79 (d, J= 3.67 Hz, 1 H), 7.31 (d, J= 3.67 Hz, 1 H), 7.02 (s, 1 H) , 3.95 (s, 3 H), 2.61 (s, 3 H), 1 .60 (s, 9 H); LCMS (m/z): positive mode 290 [M]+.

Example 9: Synthesis of Compound of formula , or salt thereof (R = methyl).

Method 1 (Pa = Boc and R = methyl): To a vessel were added lr(CO)2acac (1 mg, 0.1 mol%), compound of formula (II) (maleic salt, 3 mmol, 1 .137g), compound of formula (III) (3 mmol, 0.867g) in 9 ml. of degassed ethanol. The autoclave was purged 3 times with nitrogen and 3 times with H2 under stirring (250 RPM). The reactions were run for 24 hours at 75 °C under 20 bar of H2 at 700 RPM. An aliquot of the reaction was diluted in methanol and was analyzed by HPLC. Compound of formula (C15) was obtained after 24 hours in 88% conversion.

Method 2 (Pa = Boc and R = methyl): To a vessel were added lrCI3, xH20 (0.05 mol%, 0.9 mg, anhydrous), compound of formula (II) (maleic salt, 6 mmol, 2.274 g ), compound of formula (III) (6 mmol, 1 .735g) in 12 ml. of degassed ethanol. The autoclave was purged 3 times with nitrogen and 3 times with carbon monoxide (CO) (250 RPM). The autoclave was pressurized with 1 bar of CO and 19 bar of H2 and run for 24 hours at 75 °C under 20 bar of H2 / CO at 700 RPM. An aliquot of the reaction was diluted in methanol and was analyzed by HPLC. Compound of formula (C15) was obtained after 24 hours in 62% conversion.

1H NMR (400 MHz, DMSO-d6) d ppm 8.13 (d, J=8.16 Hz, 2H), 7.77 (br. d, J=7.84 Hz, 2H), 7.62 -7.68 (m, 1 H), 6.85 (s, 1 H), 6.80 (d, J= 3.76 Hz, 1 H), 4.01 (s, 3H), 3.92 (s, 3H), 3.73 (br. s, 1 H), 3.55 – 3.67 (m, 4H), 3.39 – 3.42 (m, 1 H), 2.60 – 2.70 (m, 5H), 1 .99 – 2.02(br. d, 1 H), 1 .82 – 1.90 (m, 2H), 1.74 (s, 9H), 1 .64 – 1 70(m, 1 H), 1 .35 (t, J= 6.97 Hz, 3H).

1. Schubart A, et al. Proc Natl Acad Sci U S A. 2019 Mar 29. pii: 201820892.

 Proceedings of the National Academy of Sciences of the United States of America (2019), 116(16), 7926-7931.

//////LNP 023, BDBM160475, ZINC223246892HY-127105CS-0093107, LNP023

O=C(O)c1ccc(cc1)[C@@H]4C[C@H](CCN4Cc2c(OC)cc(C)c3nccc23)OCC

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