CCT 245737

May 31, 2016 10:08 am / 1 Comment on CCT 245737

CCT 245737

CAS:1489389-18-5
M.Wt: 379.34
Formula: C16H16F3N7O

2-Pyrazinecarbonitrile, 5-[[4-[[(2R)-2-morpholinylmethyl]amino]-5-(trifluoromethyl)-2-pyridinyl]amino]-

(R)-5-(4-(Morpholin-2-ylmethylamino)-5-(trifluoromethyl)pyridin-2-ylamino)pyrazine-2-carbonitrile

(+)-5-[[4-[[(2R)-Morpholin-2-ylmethyl]amino]-5-(trifluoromethyl)pyridin-2-yl]amino]pyrazine-2-carbonitrile

Cancer Research Technology Limited INNOVATOR

SAREUM

IND Filed, Sareum FOR CANCER

Synthesis, Exclusive by worlddrugtracker

5-[[4-[[morpholin-2-yl]methylamino]-5- (trifluoromethyl)-2-pyridyl]amino]pyrazine-2-carbonitrile compounds (referred to herein as “TFM compounds”) which, inter alia, inhibit Checkpoint Kinase 1 (CHK1) kinase function. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to inhibit CHK1 kinase function, and in the treatment of diseases and conditions that are mediated by CHK1 , that are ameliorated by the inhibition of CHK1 kinase function, etc., including proliferative conditions such as cancer, etc., optionally in combination with another agent, for example, (a) a DNA topoisomerase I or II inhibitor; (b) a DNA damaging agent; (c) an antimetabolite or a thymidylate synthase (TS) inhibitor; (d) a microtubule targeted agent; (e) ionising radiation; (f) an inhibitor of a mitosis regulator or a mitotic checkpoint regulator; (g) an inhibitor of a DNA damage signal transducer; or (h) an inhibitor of a DNA damage repair enzyme.

Checkpoint Kinase 1 (CHK1)

Progression through the cell division cycle is a tightly regulated process and is monitored at several positions known as cell cycle checkpoints (see, e.g., Weinert and Hartwell,

1989; Bartek and Lukas, 2003). These checkpoints are found in all four stages of the cell cycle; G1 , S (DNA replication), G2 and M (Mitosis) and they ensure that key events which control the fidelity of DNA replication and cell division are completed correctly. Cell cycle checkpoints are activated by a number of stimuli, including DNA damage and DNA errors caused by defective replication. When this occurs, the cell cycle will arrest, allowing time for either DNA repair to occur or, if the damage is too severe, for activation of cellular processes leading to controlled cell death.

All cancers, by definition, have some form of aberrant cell division cycle. Frequently, the cancer cells possess one or more defective cell cycle checkpoints, or harbour defects in a particular DNA repair pathway. These cells are therefore often more dependent on the remaining cell cycle checkpoints and repair pathways, compared to non-cancerous cells (where all checkpoints and DNA repair pathways are intact). The response of cancer cells to DNA damage is frequently a critical determinant of whether they continue to proliferate or activate cell death processes and die. For example, tumour cells that contain a mutant form(s) of the tumour suppressor p53 are defective in the G1 DNA damage checkpoint. Thus inhibitors of the G2 or S-phase checkpoints are expected to further impair the ability of the tumour cell to repair damaged DNA. Many known cancer treatments cause DNA damage by either physically modifying the cell’s DNA or disrupting vital cellular processes that can affect the fidelity of DNA replication and cell division, such as DNA metabolism, DNA synthesis, DNA transcription and microtubule spindle formation. Such treatments include for example, radiotherapy, which causes DNA strand breaks, and a variety of chemotherapeutic agents including topoisomerase inhibitors, antimetabolites, DNA-alkylating agents, and platinum- containing cytotoxic drugs. A significant limitation to these genotoxic treatments is drug resistance. One of the most important mechanisms leading to this resistance is attributed to activation of cell cycle checkpoints, giving the tumour cell time to repair damaged DNA. By abrogating a particular cell cycle checkpoint, or inhibiting a particular form of DNA repair, it may therefore be possible to circumvent tumour cell resistance to the genotoxic agents and augment tumour cell death induced by DNA damage, thus increasing the therapeutic index of these cancer treatments.

CHK1 is a serine/threonine kinase involved in regulating cell cycle checkpoint signals that are activated in response to DNA damage and errors in DNA caused by defective replication (see, e.g., Bartek and Lukas, 2003). CHK1 transduces these signals through phosphorylation of substrates involved in a number of cellular activities including cell cycle arrest and DNA repair. Two key substrates of CHK1 are the Cdc25A and Cdc25C phosphatases that dephosphorylate CDK1 leading to its activation, which is a

requirement for exit from G2 into mitosis (M phase) (see, e.g., Sanchez et al., 1997). Phosphorylation of Cdc25C and the related Cdc25A by CHK1 blocks their ability to activate CDK1 , thus preventing the cell from exiting G2 into M phase. The role of CHK1 in the DNA damage-induced G2 cell cycle checkpoint has been demonstrated in a number of studies where CHK1 function has been knocked out (see, e.g., Liu et ai, 2000; Zhao et al., 2002; Zachos et al., 2003).

The reliance of the DNA damage-induced G2 checkpoint upon CHK1 provides one example of a therapeutic strategy for cancer treatment, involving targeted inhibition of CHK1. Upon DNA damage, the p53 tumour suppressor protein is stabilised and activated to give a p53-dependent G1 arrest, leading to apoptosis or DNA repair (Balaint and Vousden, 2001). Over half of all cancers are functionally defective for p53, which can make them resistant to genotoxic cancer treatments such as ionising radiation (IR) and certain forms of chemotherapy (see, e.g., Greenblatt et al., 1994; Carson and Lois, 1995). These p53 deficient cells fail to arrest at the G1 checkpoint or undergo apoptosis or DNA repair, and consequently may be more reliant on the G2 checkpoint for viability and replication fidelity. Therefore abrogation of the G2 checkpoint through inhibition of the CHK1 kinase function may selectively sensitise p53 deficient cancer cells to genotoxic cancer therapies, and this has been demonstrated (see, e.g., Wang et al., 1996; Dixon and Norbury, 2002). In addition, CHK1 has also been shown to be involved in S phase cell cycle checkpoints and DNA repair by homologous recombination. Thus, inhibition of CHK1 kinase in those cancers that are reliant on these processes after DNA damage, may provide additional therapeutic strategies for the treatment of cancers using CHK1 inhibitors (see, e.g., Sorensen et al., 2005). Furthermore, certain cancers may exhibit replicative stress due to high levels of endogenous DNA damage (see, e.g., Cavalier et al., 2009; Brooks et al., 2012) or through elevated replication driven by oncogenes, for example amplified or overexpressed MYC genes (see, e.g., Di Micco et al. 2006; Cole et al., 2011 ; Murga et al. 2011). Such cancers may exhibit elevated signalling through CHK1 kinase (see, e.g., Hoglund et al., 2011). Inhibition of CHK1 kinase in those cancers that are reliant on these processes, may provide additional therapeutic strategies for the treatment of cancers using CHK1 inhibitors (see, e.g., Cole et al., 2011 ; Davies et al., 2011 ; Ferrao et al., 2011).

Several kinase enzymes are important in the control of the cell growth and replication cycle. These enzymes may drive progression through the cell cycle, or alternatively can act as regulators at specific checkpoints that ensure the integrity of DNA replication through sensing DNA-damage and initiating repair, while halting the cell cycle. Many tumours are deficient in early phase DNA-damage checkpoints, due to mutation or deletion in the p53 pathway, and thus become dependent on the later S and G2/M checkpoints for DNA repair. This provides an opportunity to selectively target tumour cells to enhance the efficacy of ionising radiation or widely used DNA-damaging cancer chemotherapies. Inhibitors of the checkpoint kinase CHK1 are of particular interest for combination with genotoxic agents. In collaboration with Professor Michelle Garrett (University of Kent, previously at The Institute of Cancer Research) and Sareum (Cambridge) we used structure-based design to optimise the biological activities and pharmaceutical properties of hits identified through fragment-based screening against the cell cycle kinase CHK1, leading to the oral clinical candidate CCT245737. The candidate potentiates the efficacy of standard chemotherapy in models of non-small cell lung, pancreatic and colon cancer. In collaboration with colleagues at The Institute of Cancer Research (Professor Louis Chesler, Dr Simon Robinson and Professor Sue Eccles) and Newcastle University (Professor Neil Perkins), we have shown that our selective CHK1 inhibitor has efficacy as a single agent in models of tumours with high replication stress, including neuroblastoma and lymphoma.

The checkpoint kinase CHK2 has a distinct but less well characterised biological role to that of CHK1. Selective inhibitors are valuable as pharmacological tools to explore the biological consequences of CHK2 inhibition in cancer cells. In collaboration with Professor Michelle Garrett (University of Kent, previously at The Institute of Cancer Research), we have used structure-based and ligand-based approaches to discover selective inhibitors of CHK2. We showed that selective CHK2 inhibition has a very different outcome to selective CHK1 inhibition. Notably, while CHK2 inhibition did not potentiate the effect of DNA-damaging chemotherapy, it did sensitize cancer cells to the effects of PARP inhibitors that compromise DNA repair.

Synthesis

(R)-5-(4-(Morpholin-2-ylmethylamino)-5-(trifluoromethyl)pyridin-2-ylamino)pyrazine-2-carbonitrile

as a pale-yellow amorphous solid.

¹H NMR ((CD₃)₂SO, 500 MHz) δ 10.7 (br s, 1H), 9.10 (d, J = 1.4 Hz, 1H), 8.77 (d, J = 1.4 Hz, 1H), 8.20 (s, 1H), 7.19 (s, 1H), 6.32 (br t, J = 5.5 Hz, 1H), 3.75 (br d, J = 11.0 Hz, 1H), 3.64–3.59 (m, 1H), 3.43 (ddd, J = 10.7, 10.7, and 3.4 Hz, 1H), 3.22 (m, 2H), 2.82 (dd, J = 12.1 and 2.1 Hz, 1H), 2.67–2.59 (m, 2H), 2.42 (dd, J = 12.1 and 10.0 Hz, 1H).

¹³C NMR ((CD₃)₂SO, 125 MHz) δ 155.7, 151.9, 151.6, 147.2, 145.9 (q, J_CF = 6.3 Hz), 136.8, 124.8 (q, J_CF= 270.9 Hz), 118.9, 117.1, 104.4 (q, J_CF = 30.0 Hz), 93.2, 73.6, 67.2, 48.9, 45.4, 44.9.

LCMS (3.5 min) t_R = 1.17 min; m/z (ESI⁺) 380 (M + H⁺).

HRMS m/z calcd for C₁₆H₁₇F₃N₇O (M + H) 380.1441, found 380.1438.

PATENT

WO 2013171470

http://www.google.com/patents/WO2013171470A1?cl=enSynthesis 1 D

5-[[4-[[(2R)-Morpholin-2-yl]methylamino]-5-(trifluoromethyl)-2-pyridyl]amino]py

carbonitrile (Compound 1)

A solution of (S)-tert-butyl 2-((2-(5-cyanopyrazin-2-ylamino)-5-(trifluoromethyl)pyridin-4- ylamino)methyl)morpholine-4-carboxylate (1.09 g, 2.273 mmol) in dichloromethane (8 mL) was added dropwise over 10 minutes to a solution of trifluoroacetic acid (52.7 mL, 709 mmol) and tnisopropylsilane (2.61 mL, 12.73 mmol) in dry dichloromethane (227 mL) at room temperature. After stirring for 30 minutes, the mixture was concentrated in vacuo. The concentrate was resuspended in dichloromethane (200 mL) and

concentrated in vacuo, then resuspended in toluene (100 mL) and concentrated.

The above procedure was performed in triplicate (starting each time with 1.09 g (S)-tert- butyl 2-((2-(5-cyanopyrazin-2-ylamino)-5-(trifluoromethyl)pyridin-4- ylamino)methyl)morpholine-4-carboxylate) and the three portions of crude product so generated were combined for purification by ion exchange chromatography on 2 x 20 g Biotage NH2 Isolute columns, eluting with methanol. The eluant was concentrated and 10% methanol in diethyl ether (25 mL) was added. The resulting solid was filtered, washed with diethyl ether (30 mL), and dried in vacuo to give the title compound as a light straw coloured powder (2.30 g, 89%). H NMR (500 MHz, CD₃OD) δ 2.62 (1 H, J = 12, 10 Hz), 2.78-2.84 (2H, m), 2.95 (1 H, dd, J = 12, 2 Hz), 3.27-3.38 (2H, m), 3.63 (1 H, ddd, J = 14, 9.5, 3 Hz), 3.73-3.78 (1 H, m), 3.91 (1 H, ddd, J = 11 , 4, 2 Hz), 7.26 (1 H, s), 8.18 (1 H, s), 8.63 (1 H, s), 9.01 (1 H, s).

LC-MS (Agilent 4 min) R_t 1.22 min; m/z (ESI) 380 [M+H⁺]. Optical rotation [a]_D ²⁴ = +7.0 (c 1.0, DMF).

Synthesis 2B

(R)-tert- Butyl 2-((2-chloro-5-(trifluoromethyl)pyridin-4-ylamino)methyl)morpholine-

To a solution of 2-chloro-5-(trifluoromethyl)pyridin-4-amine (1 g, 5.09 mmol) in

dimethylformamide (32.6 mL) was added sodium hydride (60% by wt in oil; 0.407 g, 10.18 mmol) portionwise at room temperature followed by stirring for 10 minutes at 80°C. (S)- tert-Butyl 2-(tosyloxymethyl)morpholine-4-carboxylate (2.268 g, 6.1 1 mmol) was then added portionwise and the reaction mixture was stirred at 80°C for 2.5 hours. After cooling, the mixture was partitioned between saturated aqueous sodium

hydrogencarbonate solution (30 mL), water (100 mL) and ethyl acetate (30 mL). The organic layer was separated and the aqueous layer was further extracted with ethyl acetate (2 x 30 mL). The combined organic layers were washed with brine (2 x 70 mL), dried over magnesium sulfate, filtered, concentrated and dried thoroughly in vacuo. The crude material was purified by column chromatography on a 90 g Thomson SingleStep column, eluting with an isocratic mix of 2.5% diethyl ether / 2.5% ethyl acetate in dichloromethane, to give the title compound as a clear gum that later crystallised to give a white powder (1.47 g, 73%). H NMR (500 MHz, CDCI₃) δ 1.48 (9H, s), 2.71-2.83 (1 H, m), 2.92-3.05 (1 H, m), 3.18- 3.23 (1 H, m), 3.33-3.37 (1 H, m), 3.56-3.61 (1 H, m), 3.66-3.71 (1 H, m), 3.80-4.07 (3H, m), 5.32 (1 H, broad s), 6.61 (1 H, s), 8.24 (1 H, s). LC-MS (Agilent 4 min) R_t 3.04 min; m/z (ESI) 396 [MH⁺]. Svnthesis 2C

(R)-tert-Butyl 2-((2-(5-cyanopyrazin-2-ylamino)-5-(trifluoromethyl)pyridin-4-

(R)-tert-Butyl 2-((2-chloro-5-(trifluoromethyl)pyridin-4-ylamino)methyl)morpholine-4- carboxylate (1.44 g, 3.64 mmol), 2-amino-5-cyanopyrazine (0.612 g, 5.09 mmol, 1.4 eq.), tris(dibenzylideneacetone)dipalladium(0) (0.267 g, 0.291 mmol, 0.08 eq.), rac-2,2′- bis(diphenylphosphino)-1 ,1 ‘-binaphthyl (0.362 g, 0.582 mmol, 0.16 eq.) and caesium carbonate (2.37 g, 7.28 mmol) were suspended in anhydrous dioxane (33 ml_) under argon. Argon was bubbled through the mixture for 30 minutes, after which the mixture was heated to 100°C for 22 hours. The reaction mixture was cooled and diluted with dichloromethane, then absorbed on to silica gel. The pre-absorbed silica gel was added to a 100 g KP-Sil SNAP column which was eluted with 20-50% ethyl acetate in hexanes to give the partially purified product as an orange gum. The crude product was dissolved in dichloromethane and purified by column chromatography on a 90 g SingleStep Thomson column, eluting with 20% ethyl acetate in dichloromethane, to give the title compound (1.19 g, 68%). H NMR (500 MHz, CDCI₃) δ 1.50 (9H, s), 2.71-2.88 (1 H, m), 2.93-3.08 (1 H, m), 3.27- 3.32 (1 H, m), 3.40-3.44 (1 H, m), 3.55-3.64 (1 H, m), 3.71-3.77 (1 H, m), 3.82-4.11 (3H, m), 5.33 (1 H, broad s), 7.19 (1 H, s), 8.23 (1 H, s), 8.58 (1 H, s), 8.84 (1 H, s). LC-MS (Agilent 4 min) R_t 2.93 min;m/z (ESI) 480 [MH⁺].

Paper

Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic–pharmacodynamic (PK–PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition.

Multiparameter Lead Optimization to Give an Oral Checkpoint Kinase 1 (CHK1) Inhibitor Clinical Candidate: (R)-5-((4-((Morpholin-2-ylmethyl)amino)-5-(trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile (CCT245737)

James D. Osborne†, ET AL

^†Cancer Research UK Cancer Therapeutics Unit and ^‡Division of Radiotherapy and Imaging, The Institute of Cancer Research, London SM2 5NG, U.K.

^§ Sareum Ltd., Cambridge CB22 3FX, U.K.

J. Med. Chem., Article ASAP

DOI: 10.1021/acs.jmedchem.5b01938

Publication Date (Web): May 11, 2016

*Phone: +44 2087224000. Fax: +44 2087224126. E-mail: ian.collins@icr.ac.uk.

http://pubs.acs.org/doi/full/10.1021/acs.jmedchem.5b01938

///////////CCT 245737, IND, PRECLINICAL, Cancer Research Technology Limited, SAREUM

N#CC(C=N1)=NC=C1NC2=NC=C(C(F)(F)F)C(NC[C@@H]3OCCNC3)=C2

Supporting Info SEE NMR OF COMPD 4

Quisapride Hydrochloride

May 20, 2016 9:37 am / 1 Comment on Quisapride Hydrochloride

Quisapride Hydrochloride

(R) – quinuclidine-3-5 – ((S) -2 – (( ⁴ – amino-5-chloro-2-ethoxy benzoylamino) methyl) morpholino) hexanoate

A 5-HT4 agonist potentially for the treatment of gastrointestinal motility disorders.

SHR-116 958, SHR 116958

CAS 1132682-83-7 (Free)

Shanghai Hengrui Pharmaceutical Co., Ltd.

Shanghai Hengrui Pharmaceutical Co., Ltd.

CAS 1274633-87-2 (dihcl)

(3R)-1-Azabicyclo[2.2.2]oct-3-yl (2S)-2-[[(4-amino-5-chloro-2-ethoxybenzoyl)amino]methyl]-4-morpholinehexanoate hydrochloride (1:2)
SHR 116958
C₂₇ H₄₁ Cl N₄ O₅ . 2 Cl H,

4-Morpholinehexanoic acid, 2-[[(4-amino-5-chloro-2-ethoxybenzoyl)amino]methyl]-, (3R)-1-azabicyclo[2.2.2]oct-3-yl ester, hydrochloride (1:2), (2S)-

5-HT is a neurotransmitter Chong, widely distributed in the central nervous system and peripheral tissues, 5-HT receptor subtypes at least seven, and a wide variety of physiological functions of 5-HT receptor with different interactions related. Thus, the 5-HT receptor subtypes research is very necessary.

The study found that the HT-5 ₄ receptor agonists useful for treating a variety of diseases, such as gastroesophageal reflux disease, gastrointestinal disease, gastric motility disorder, non-ulcer dyspepsia, functional dyspepsia, irritable bowel syndrome, constipation, dyspepsia, esophagitis, gastroesophageal disease, nausea, postoperative intestinal infarction, central nervous system disorders, Alzheimer’s disease, cognitive disorder, emesis, migraine, neurological disease, pain, cardiovascular disease, heart failure , arrhythmias, intestinal pseudo-obstruction, gastroparesis, diabetes and apnea syndrome.

The HT-5 ₄ receptor agonists into benzamides, benzimidazole class and indole alkylamines three kinds, which benzamides derivatives act on the neurotransmitter serotonin in the central nervous system by modulation, It showed significant pharmacological effect. The role of serotonin and benzamides derivatives and pharmacologically related to many diseases. Therefore, more and more people will focus on the human body produce serotonin, a storage position and the position of serotonin receptors, and to explore the relationship between these positions with a variety of diseases.

Commonly used in clinical cisapride (cisapride) and Mosapride (Tony network satisfied) is one of the novel benzamides drugs.

These drugs mainly through the intestinal muscle between the excited 5-HT neurofilament preganglionic and postganglionic neurons ₄ receptor to promote the release of acetylcholine and enhancing cholinergic role in strengthening the peristalsis and contraction of gastrointestinal smooth muscle. In large doses, it can antagonize the HT-5₃ receptors play a central antiemetic effect, when typical doses, through the promotion of gastrointestinal motility and antiemetic effect. These drugs can increase the lower esophageal smooth muscle tension and promote esophageal peristalsis, improving the antrum and duodenum coordinated motion, and promote gastric emptying, but also promote the intestinal movement and enhanced features, increase the role of the proximal colon emptying, It is seen as the whole digestive tract smooth muscle prokinetic effect of the whole gastrointestinal drugs.

Mainly used for reflux esophagitis, functional dyspepsia, gastroparesis, postoperative gastrointestinal paralysis, functional constipation and intestinal pseudo-obstruction patients. Since there is slight antagonism cisapride the HT-5 ₃ and anti-D2 receptor, can cause cardiac adverse reactions, prolonged QT occurs, and therefore, patients with severe heart disease, ECG QT prolonged, low potassium, and low blood magnesium prohibited drug. Liver and kidney dysfunction, lactating women and children is not recommended. Due to increase between drug diazepam, ethanol, acenocoumarol, cimetidine and ranitidine the absorption of anticholinergic drugs may also antagonize the effect of this product to promote peristalsis of the stomach, should be aware of when using these, such as when diarrhea should reduce, anticoagulant therapy should pay attention to monitoring the clotting time. Mosapride selective gastrointestinal tract the HT-5 ₄ receptor agonists, there is no antagonism of D2 receptors, does not cause QT prolonged, reduce adverse reactions, mainly fatigue, dizziness, loose stools, mild abdominal pain , the efficacy of cisapride equivalent clinical effect broader Puka cisapride (prucalopride, Pru) of benzimidazole drugs, with high selectivity and specificity of the HT-5 ₄ receptor, increasing cholinergic neurotransmitters quality release, stimulate peristalsis reflex, enhance colon contraction, and accelerate gastric emptying, gastrointestinal motility to promote good effect, can effectively relieve the patient’s symptoms of constipation, constipation and for treatment of various gastrointestinal surgery peristalsis slow and weak, and intestinal pseudo-obstruction.

WO2005068461 discloses as the HT-5 ₄ receptor agonists benzamides compounds, particularly discloses compounds represented by the formula:

ATI-7505

ATI-7505 is stereoisomeric esterified. Cisapride analogs, safe and effective treatment of various gastrointestinal disorders, including gastroparesis, gastroesophageal reflux disease and related disorders. The drug can also be used to treat a variety of central nervous system disorders. ATI-7505 for the treatment or prevention of gastroesophageal reflux disease, also taking cisapride significantly reduced side effects. These side effects include diarrhea, abdominal cramps and blood pressure and heart rate rise.

Further, the compounds and compositions of this patent disclosure also useful in treating emesis and other diseases. Such as indigestion, gastroesophageal reflux, constipation, postoperative ileus, and intestinal pseudo-obstruction. In the course of treatment, but also taking cisapride reduce the side effects.

ΑΉ-7505 as the HT-5 ₄ receptor ligands may be mediated by receptors to treat the disease. These receptors are located in several parts of the central nervous system, modulate the receptor can be used to affect the CNS desired modulation.

ATI-7505 contained in the ester moiety does not detract from the ability of the compounds to provide treatment, but to make the compound easier to serum and / or cytosolic esterases degraded, so you can avoid the drug cytochrome P450 detoxification system, and this system with cisapride cause side effects related, thus reducing side effects.

The HT-Good 5 ₄ receptor agonists and should the HT-5 ₄ receptor binding powerful, while the other hardly shows affinity for the receptor, and show functional activity as agonists. They should be well absorbed from the gastrointestinal tract, metabolically stable and possess desirable pharmacokinetic properties. When targeting the receptor in the central nervous system, they should cross the blood-free, selectively targeting peripheral nervous system receptors, they should not pass through the blood-brain barrier. They should be non-toxic, and there is little proof of side effects. Furthermore, the ideal drug candidate will be a stable, non-hygroscopic and easily formulated in the form of physical presence.

Based on the HT-5 ₄ receptor agonists current developments, the present invention relates to a series of efficacy better, safer, less side effects of the benzamide derivatives.

Synthesis

PATENT

WO 2009033360

Example 3

(R) – quinuclidine-3-5 – ((S) -2 – (( ⁴ – amino-5-chloro-2-ethoxy benzoylamino) methyl) morpholino) hexanoate

REFERENCES

China Pharmaceuticals: Asia Insight: China Has R&D

pg.jrj.com.cn/acc/Res/CN_RES/…/cd837477-44e9-4f98-a2b9-97620cd64576.pdf

Nov 6, 2012 – levofolinate, sevoflurane inhalation, ambroxol hydrochloride, ioversol, etc ….. dextromethorphan hydrochloride 复方沙芬那敏. 3.2 …… quisapride.

Pharmazie (2011), 66(11), 826-830

//////SHR-116 958, SHR 116958, Quisapride Hydrochloride, preclinical

Cl.Cl.Clc1cc(c(OCC)cc1N)C(=O)NC[C@H]4CN(CCCCCC(=O)O[C@H]3CN2CCC3CC2)CCO4

PDE4 Inhibitors, Boehringer Ingelheim Pharmaceuticals

May 18, 2016 12:31 pm / Leave a comment

R CONF SHOWN

BI ?

(R)-2-(4-(4-Chlorophenoxy)piperidin-1-yl)-4-((tetrahydro-2H-pyran-4-yl)amino)-6,7-dihydrothieno[3,2-d]pyrimidine 5-Oxide

C₂₂ H₂₇ Cl N₄ O₃ S, 462.99

CAS 1910076-27-5

Thieno[3,2-d]pyrimidin-4-amine, 2-[4-(4-chlorophenoxy)-1-piperidinyl]-6,7-dihydro-N-(tetrahydro-2H-pyran-4-yl)-, 5-oxide, (5R)-

¹H NMR (400 MHz, CDCl₃) δ 1.49 (dq, J = 4.2, 11.8 Hz, 1H), 1.62 (dq, J = 4.2, 11.8 Hz, 1H), 1.74–1.89 (m, 3H), 1.90–2.02 (m, 3H), 2.96–3.07 (m, 2H), 3.29 (dt, J = 13.6, 8.4 Hz, 1H), 3.44 (ddd, J = 19.2, 11.2, 2.0 Hz, 2H), 3.62 (dt, J = 17.2, 7.8 Hz, 1H), 3.76 (m, 2H), 3.96 (dd, J = 15.6, 12.8 Hz, J = 2H), 4.09–3.99 (m, 3H), 4.51 (m, 1H), 6.21 (br d, J = 6.0 Hz, 1H), 6.86 (d, J = 8.8 Hz, 2H), 7.24 (d, J = 8.8 Hz, 2H);

¹³C NMR (100 MHz, CDCl₃) δ 30.4, 32.5, 32.7, 41.0, 47.2, 49.6, 66.9, 66.9, 72.9, 107.8, 117.5, 125.9, 129.5, 155.8, 158.9, 163.0, 174.6.

The use of phosphodiesterase type 4 (PDE4) inhibitors for the treatment of COPD (chronic obstructive pulmonary disease) by reducing inflammation and improving lung function is well documented. Given the potential therapeutic benefit offered by these compounds, a number of PDE4-selective inhibitors containing a dihydrothieno[3,2-d]pyrimidine core were identified as preclinical candidates in Boehringer Ingelheim Pharmaceuticals discovery laboratories

While the pathogenesis of chronic obstructive pulmonary disease (COPD) is incompletely understood, chronic inflammation is a major factor. In fact, the inflammatory response is abnormal, with CD8⁺ T-cells, CD68⁺ macrophages, and neutrophils predominating in the conducting airways, lung parenchyma, and pulmonary vasculature. Elevated levels of the second messenger cAMP can inhibit some inflammatory processes. Theophylline has long been used in treating asthma; it causes bronchodilation by inhibiting cyclic nucleotide phosphodiesterase (PDE), which inactivates cAMP. By inhibiting PDE, theophylline increases cAMP, inhibiting inflammation and relaxing airway smooth muscle. Rather than one PDE, there are now known to be more than 50, with differing activities, substrate preferences, and tissue distributions. Thus, the possibility exists of selectively inhibiting only the enzyme(s) in the tissue(s) of interest. PDE 4 is the primary cAMP-hydrolyzing enzyme in inflammatory and immune cells (macrophages, eosinophils, neutrophils). Inhibiting PDE 4 in these cells leads to increased cAMP levels, down-regulating the inflammatory response. Because PDE 4 is also expressed in airway smooth muscle and, in vitro, PDE 4 inhibitors relax lung smooth muscle, selective PDE 4 inhibitors are being developed for treating COPD. Clinical studies have been conducted with PDE 4 inhibitors;

Chronic obstructive pulmonary disease (COPD) is a serious and increasing global public health problem; physiologically, it is characterized by progressive, irreversible airflow obstruction and pathologically, by an abnormal airway inflammatory response to noxious particles or gases (MacNee 2005a). The COPD patient suffers a reduction in forced expiratory volume in 1 second (FEV₁), a reduction in the ratio of FEV₁ to forced vital capacity (FVC), compared with reference values, absolute reductions in expiratory airflow, and little improvement after treatment with an inhaled bronchodilator. Airflow limitation in COPD patients results from mucosal inflammation and edema, bronchoconstriction, increased secretions in the airways, and loss of elastic recoil. Patients with COPD can experience ‘exacerbations,’ involving rapid and prolonged worsening of symptoms (Seneff et al 1995; Connors et al 1996; Dewan et al 2000; Rodriguez-Roisin 2006; Mohan et al 2006). Many are idiopathic, though they often involve bacteria; airway inflammation in exacerbations can be caused or triggered by bacterial antigens (Murphy et al 2000; Blanchard 2002; Murphy 2006;Veeramachaneni and Sethi 2006). Increased IL-6, IL-1β, TNF-α, GRO-α, MCP-1, and IL-8 levels are found in COPD patient sputum; their levels increase further during exacerbations. COPD has many causes and significant differences in prognosis exist, depending on the cause (Barnes 1998; Madison and Irwin 1998).

COPD is already the fourth leading cause of death worldwide, according to the World Health Organization (WHO); the WHO estimates that by the year 2020, COPD will be the third-leading cause of death and the fifth-leading cause of disability worldwide (Murray and Lopez 1997). COPD is the fastest-growing cause of death in developed nations and is responsible for over 2.7 million deaths per year worldwide. In the US, there are currently estimated to be 16 million people with COPD. There are estimated to be up to 20 million sufferers in Japan, which has the world’s highest per capita cigarette consumption and a further 8–12 million in Europe. In 2000, COPD accounted for over 20 million outpatient visits, 3.4 million emergency room visits, 6 million hospitalizations, and 116,500 deaths in the US (National Center for Health Statistics 2002). Factors associated with COPD, including immobility, often lead to secondary health consequences (Polkey and Moxham 2006).

Risk factors for the development of COPD include cigarette smoking, and occupational exposure to dust and chemicals (Senior and Anthonisen 1998; Anthonisen et al 2002; Fabbri and Hurd 2003; Zaher et al 2004). Smoking is the most common cause of COPD and the underlying inflammation typically persists in ex-smokers. Oxidative stress from cigarette smoke is also an issue in COPD (Domej et al 2006). Despite this, relatively few smokers ever develop COPD (Siafakas and Tzortzaki 2002).

While many details of the pathogenesis of COPD remain unclear, chronic inflammation is now recognized as a major factor, predominantly in small airways and lung parenchyma, characterized by increased numbers of macrophages, neutrophils, and T-cells (Barnes 2000; Stockley 2002). As recently as 1995, the American Thoracic Society issued a statement defining COPD without mentioning the underlying inflammation (American Thoracic Society 1995). Since then, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines have made it clear that chronic inflammation throughout the airways, parenchyma, and pulmonary vasculature plays a central role (Pauwels et al 2001; GOLD 2003). The comparatively recent realization of the role of airway inflammation in COPD has altered thinking with regard to potential therapies (Rogers and Giembycz 1998; Vignola 2004).

Most pharmacological therapies available for COPD, including bronchodilator and anti-inflammatory agents, were first developed for treating asthma. The mainstays of COPD treatment are inhaled corticosteroids (McEvoy and Niewoehner 1998; Borron and deBoisblanc 1998; Pauwels 2002; Gartlehner et al 2006;D’Souza 2006), supplemental oxygen (Petty 1998; Austin and Wood-Baker 2006), inhaled bronchodilators (Costello 1998; Doherty and Briggs 2004), and antibiotics (Taylor 1998), especially in severely affected patients (Anthonisen et al 1987; Saint et al 1995; Adams et al 2001; Miravitlles et al 2002; Donnelly and Rogers 2003; Sin et al 2003; Rabe 2006), though the use of antibiotics remains controversial (Ram et al 2006). Long-acting β₂-agonists (LABAs) improve the mucociliary component of COPD. Combination therapy with LABAs and anticholinergic bronchodilators resulted in modest benefits and improved health-related quality of life (Buhl and Farmer 2005; Appleton et al 2006). Treatment with mucolytics reduced exacerbations and the number of days of disability (Poole and Black 2006). The combined use of inhaled corticosteroids and LABAs has been demonstrated to produce sustained improvements in FEV₁ and positive effects on quality of life, number of hospitalizations, distance walked, and exacerbations (Mahler et al 2002;Szafranski et al 2003; Sin et al 2004; Miller-Larsson and Selroos 2006; van Schayck and Reid 2006). However, all of these treatments are essentially palliative and do not impact COPD progression (Hay 2000;Gamble et al 2003; Antoniu 2006a).

A further complication in drug development and therapy is that it can be difficult to determine the efficacy of therapy, because COPD has a long preclinical stage, is progressive, and patients generally do not present for treatment until their lung function is already seriously impaired. Moreover, because COPD involves irreversible loss of elasticity, destruction of the alveolar wall, and peribronchial fibrosis, there is often little room for clinical improvement.

Smoking cessation remains the most effective intervention for COPD. Indeed, to date, it is the only intervention shown to stop the decline in lung function, but it does not resolve the underlying inflammation, which persists even in ex-smokers. Smoking cessation is typically best achieved by a multifactor approach, including the use of bupropion, a nicotine replacement product, and behavior modification (Richmond and Zwar 2003).

In COPD, there is an abnormal inflammatory response, characterized by a predominance of CD8⁺ T-cells, CD68⁺ macrophages, and neutrophils in the conducting airways, lung parenchyma, and pulmonary vasculature (Soto and Hanania 2005; O’Donnell et al 2006; Wright and Churg 2006). Inflammatory mediators involved in COPD include lipids, inflammatory peptides, reactive oxygen and nitrogen species, chemokines, cytokines, and growth factors. COPD pathology also includes airway remodeling and mucociliary dysfunction (mucus hypersecretion and decreased mucus transport). Corticosteroids reduce the number of mast cells, but CD8⁺ and CD68⁺ cells, and neutrophils, are little affected (Jeffery 2005). Inflammation in COPD is not suppressed by corticosteroids, consistent with it being neutrophil-, not eosinophil-mediated. Corticosteroids also do not inhibit the increased concentrations of IL-8 and TNF-α (both neutrophil chemoattractants) found in induced sputum from COPD patients. Neutrophil-derived proteases, including neutrophil elastase and matrix metalloproteinases (MMPs), are involved in the inflammatory process and are responsible for the destruction of elastin fibers in the lung parenchyma (Mercer et al 2005; Gueders et al 2006). MMPs play important roles in the proteolytic degradation of extracellular matrix (ECM), in physiological and pathological processes (Corbel, Belleguic et al 2002). PDE 4 inhibitors can reduce MMP activity and the production of MMPs in human lung fibroblasts stimulated with pro-inflammatory cytokines (Lagente et al 2005). In COPD, abnormal remodeling results in increased deposition of ECM and collagen in lungs, because of an imbalance of MMPs and TIMPs (Jeffery 2001). Fibroblast/myofibroblast proliferation and activation also occur, increasing production of ECM-degrading enzymes (Crouch 1990; Segura-Valdez et al 2000). Additionally, over-expression of cytokines and growth factors stimulates lung fibroblasts to synthesize increased amounts of collagen and MMPs, including MMP-1 (collagenase-1) and MMP-2 and MMP-9 (gelatinases A and B) (Sasaki et al 2000; Zhu et al 2001).

It is now generally accepted that bronchial asthma is also a chronic inflammatory disease (Barnes et al 1988;Barnes 1995). The central role of inflammation of the airways in asthma’s pathogenesis is consistent with the efficacy of corticosteroids in controlling clinical symptoms. Eosinophils are important in initiating and continuing the inflammatory state (Holgate et al 1987; Bruijnzeel 1989; Underwood et al 1994; Teixeira et al 1997), while other inflammatory cells, including lymphocytes, also infiltrate the airways (Holgate et al 1987;Teixeira et al 1997). The familiar acute symptoms of asthma are the result of airway smooth muscle contraction. While recognition of the key role of inflammation has led to an emphasis on anti-inflammatory therapy in asthma, a significant minority of patients remains poorly controlled and some exhibit accelerated declines in lung function, consistent with airway remodeling (Martin and Reid 2006). Reversal or prevention of structural changes in remodeling may require additional therapy (Burgess et al 2006).

There is currently no cure for asthma; treatment depends primarily on inhaled glucocorticoids to reduce inflammation (Taylor 1998; Petty 1998), and inhaled bronchodilators to reduce symptoms (Torphy 1994;Costello 1998; Georgitis 1999; DeKorte 2003). Such treatments, however, do not address disease progression.

COPD and asthma are both characterized by airflow obstruction, but they are distinct in terms of risk factors and clinical presentation. While both involve chronic inflammation and cellular infiltration and activation, different cell types are implicated and there are differences in the inflammatory states (Giembycz 2000;Fabbri and Hurd 2003; Barnes 2006). In COPD, neutrophil infiltration into the airways and their activation appear to be key (Stockley 2002); in asthma, the inflammatory response involves airway infiltration by activated eosinophils and lymphocytes, and T-cell activation of the allergic response (Holgate et al 1987;Saetta et al 1998; Barnes 2006). While macrophages are present in both conditions, the major controller cells are CD8⁺ T-cells in COPD (O’Shaughnessy et al 1997; Saetta et al 1998) and CD4⁺ T-cells in asthma. IL-1, IL-8, and TNF-α are the key cytokines in COPD, while in asthma, IL-4, IL-5, and IL-13 are more important. There are differences in histopathological features of lung biopsies between COPD patients and asthmatics; COPD patients have many fewer eosinophils in lung tissue than asthmatics.

While the early phases of COPD and asthma are distinguishable, there are common features, including airway hyper-responsiveness and mucus hypersecretion. MUC5AC is a major mucin gene expressed in the airways; its expression is increased in COPD and asthmatic patients. At least in vitro, epidermal growth factor stimulates MUC5AC mRNA and protein expression; this can be reversed by PDE 4 inhibitors, which may contribute to their clinical efficacy in COPD and asthma (Mata et al 2005). Similar structural and fibrotic changes make COPD and asthma much less distinguishable in extreme cases; the chronic phases of both involve inflammatory responses, alveolar detachment, mucus hypersecretion, and subepithelial fibrosis. The two conditions have been linked epidemiologically; adults with asthma are up to 12 times more likely to develop COPD over time than those without (Guerra 2005).

PAPER

A practical, safe, and efficient process for the synthesis of PDE4 (phosphodiesterase type 4) inhibitors represented by 1 and 2 was developed and demonstrated on a multi-kilogram scale. Key aspects of the process include the regioselective synthesis of dihydrothieno[3,2-d]pyrimidine-2,4-diol 9 and the asymmetric sulfur oxidation of intermediate 11.

Development of a Practical Process for the Synthesis of PDE4 Inhibitors

Rogelio P. Frutos^*, ET AL

Chemical Development US, Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, P.O. Box 368, Ridgefield, Connecticut 06877-0368, United States

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.6b00104

http://pubs.acs.org/doi/full/10.1021/acs.oprd.6b00104

PDE 4 in COPD

With regard to COPD, PDE 4 is the primary cAMP-hydrolyzing enzyme in inflammatory and immune cells, especially macrophages, eosinophils, and neutrophils, all of which are found in the lungs of COPD and asthma patients (Torphy et al 1992; Karlsson and Aldous 1997; De Brito et al 1997; Wang et al 1999;Torphy and Page 2000). Inhibition of PDE 4 leads to elevated cAMP levels in these cells, down-regulating the inflammatory response (Dyke and Montana 2002).

PDE 4 has also attracted much attention because it is expressed in airway smooth muscle (Ashton et al 1994;Undem et al 1994; Nicholson et al 1995; Kerstjens and Timens 2003; Mehats et al 2003; Lipworth 2005; Fan Chung 2006). In vitro, PDE 4 inhibitors relax lung smooth muscle (Undem et al 1994; Dent and Giembycz 1995). In COPD and asthma, a selective PDE 4 inhibitor with combined bronchodilatory and anti-inflammatory properties would seem desirable (Nicholson and Shahid 1994; Lombardo 1995; Palfreyman 1995; Cavalia and Frith 1995; Palfreyman and Souness 1996; Karlsson and Aldous 1997; Compton et al 2001; Giembycz 2002; Jacob et al 2002; Soto and Hanania 2005).

PDE 4 inhibitors in COPD

So, because PDE 4 inhibitors suppress inflammatory functions in several cell types involved in COPD and asthma (Huang and Mancini 2006) and because, at least in vitro, PDE 4 inhibitors relax lung smooth muscle, selective PDE 4 inhibitors, originally intended for use in treating depression (Renau 2004), have been developed for the treatment of COPD and asthma (Torphy et al 1999; Spina 2000; Huang et al 2001; Spina 2004; Giembycz 2005a, 2005b; Lagente et al 2005; Boswell-Smith, Spina et al 2006). PDE 4 enzymes are strongly inhibited by the antidepressant drug rolipram (Pinto et al 1993), which decreases the influx of inflammatory cells at sites of inflammation (Lagente et al 1994; Lagente et al 1995; Alves et al 1996). PDE 4 inhibitors down-regulate cytokine production in inflammatory cells, in vivo and in vitro (Undem et al 1994;Dent and Giembycz 1995). TNF-α is an important inflammatory cytokine in COPD; its release is reduced by PDE 4 inhibitors (Souness et al 1996; Chambers et al 1997; Griswold et al 1998; Gonçalves de Moraes et al 1998; Corbel, Belleguic et al 2002). Some PDE 4 inhibitors, including cilomilast and AWD 12-281, can inhibit neutrophil degranulation, a property not shared by theophylline (Ezeamuzie 2001; Jones et al 2005). PDE 4 inhibitors reduce overproduction of other pro-inflammatory mediators, including arachidonic acid and leukotrienes (Torphy 1998). PDE 4 inhibitors also inhibit cellular trafficking and microvascular leakage, production of reactive oxygen species, and cell adhesion molecule expression in vitro and in vivo (Sanz et al 2005). PDE 4 inhibitors, including cilomilast and CI-1044, inhibit LPS-stimulated TNF-α production in whole blood from COPD patients (Burnouf et al 2000; Ouagued et al 2005).

There are now thought to be at least four PDE 4s, A, B, C, and D, derived from four genes (Lobbam et al 1994; Muller et al 1996; Torphy 1998; Conti and Jin 1999; Matsumoto et al 2003). Alternative splicing and alternative promoters add further complexity (Manganiello et al 1995; Horton et al 1995; Torphy 1998). Indeed, the four genes encode more than 16 PDE 4 isoforms, which can be divided into short (∼65–75 kDa) and long forms (∼80–130 kDa); the difference between the short and long forms lies in the N-terminal region (Bolger et al 1997; Huston et al 2006). PDE 4 isoforms are regulated by extracellular signal-related protein kinase (ERK), which can phosphorylate PDE 4 (Houslay and Adams 2003).

The four PDE 4 genes are differentially expressed in various tissues (Silver et al 1988; Lobbam et al 1994;Manganiello et al 1995; Horton et al 1995; Muller et al 1996; Torphy 1998). PDE 4A is expressed in many tissues, but not in neutrophils (Wang et al 1999). PDE 4B is also widely expressed and is the predominant PDE 4 subtype in monocytes and neutrophils (Wang et al 1999), but is not found in cortex or epithelial cells (Jin et al 1998). Upregulation of the PDE 4B enzyme in response to pro-inflammatory agents suggest that it has a role in inflammatory processes (Manning et al 1999). PDE 4C is expressed in lung and testis, but not in circulating inflammatory cells, cortex, or hippocampus (Obernolte et al 1997; Manning et al 1999; Martin-Chouly et al 2004). PDE 4D is highly expressed in lung, cortex, cerebellum, and T-cells (Erdogan and Houslay 1997; Jin et al 1998). PDE 4D also plays an important role in airway smooth muscle contraction (Mehats et al 2003).

A major issue with early PDE 4 inhibitors was their side effect profile; the signature side effects are largely gastrointestinal (nausea, vomiting, increased gastric acid secretion) and limited the therapeutic use of PDE 4 inhibitors (Dyke and Montana 2002). The second generation of more selective inhibitors, such as cilomilast and roflumilast, have improved side effect profiles and have shown clinical efficacy in COPD and asthma (Barnette 1999; Spina 2000; Lagente et al 2005). However, even cilomilast and roflumilast, the most advanced clinical candidates, discussed below, cause some degree of emesis (Spina 2003).

It is now thought that the desirable anti-inflammatory properties and unwanted side effects of nausea and emesis are associated with distinct biochemical activities (Torphy et al 1992; Jacobitz et al 1996; Barnette et al 1996; Souness et al 1997; Souness and Rao 1997). Specifically, the side effects are believed to be associated with the so-called ‘high-affinity rolipram binding site’ (HARBS) (Barnette et al 1995; Muller et al 1996; Jacobitz et al 1996; Kelly et al 1996; Torphy 1998) and/or inhibition of the form of PDE 4 found in the CNS (Barnette et al 1996). The exact nature of HARBS remains unclear, although it has been described as a conformer of PDE 4 (Souness and Rao 1997; Barnette et al 1998). Using mice deficient in PDE 4B or PDE 4D, it appears that emesis is the result of selective inhibition of PDE 4D (Robichaud et al 2002; Lipworth 2005), which is unfortunate, because the most clinically advanced PDE 4 inhibitors are selective for PDE 4D. Also, from animal studies, it appears that the nausea and vomiting are produced via the CNS, though there may also be direct effects on the gastrointestinal system (Barnette 1999).

While beyond the scope of this review, it has been proposed that PDE 4 inhibitors may be useful in treating inflammatory bowel disease (Banner and Trevethick 2004), cystic fibrosis (Liu et al 2005), pulmonary arterial hypertension (Growcott et al 2006), myeloid and lymphoid malignancies (Lerner and Epstein 2006), Alzheimer’s disease (Ghavami et al 2006), rheumatoid arthritis and multiple sclerosis (Dyke and Montana 2002), infection-induced preterm labor (Oger et al 2004), depression (Wong et al 2006), and allergic disease (Crocker and Townley 1999). Varying degrees of in vitro, in vivo, and clinical data exist to support these claims.

So, after that theoretical buildup, we reach the proof of the pudding; clinical studies have been conducted with PDE 4 inhibitors. A potent, but not-very-selective, PDE 4 inhibitor is approved in Japan and is used clinically, including for treating asthma. Another is awaiting approval in the US. One is in advanced clinical development and others are at earlier stages.

REF

Pouzet, P.; Hoenke, C.; Martyres, D.; Nickolaus, P.; Jung, B.; Hamman, H. Dihydrothienopyrimidines for the treatment of inflammatory diseases. PatentWO 2006111549 A1, October 26, 2006.

Ohnacker, G.; Woitun, E. Novel dihydrothieno[3, 2-d]pyrimidines. U.S. Patent US 3,318,881, May 9, 1967.

/////PDE4 Inhibitors, Boehringer Ingelheim Pharmaceuticals, BI ?, PRECLINICAL, 1910076-27-5

Supporting Info

Clc1ccc(cc1)OC2CCN(CC2)c4nc(NC3CCOCC3)c5c(n4)CCS5=O

A New Antibiotic (E)-3-(3-Carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one, from University Of Notre Dame

May 6, 2016 9:57 am / 1 Comment on A New Antibiotic (E)-3-(3-Carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one, from University Of Notre Dame

(E)-3-(3-Carboxyphenyl)-2-(4-ethynylstyryl)quinazolin-4(3H)-one

(E)-3-(2-(4-Cyanostyryl)-4-Oxoquinazolin-3(4h)-Yl)benzoic Acid;

1624273-22-8 CAS

NA SALT 1624273-21-7 CAS

INNOVATORS

University Of Notre Dame

Mayland Chang, Shahriar Mobashery, Renee BOULEY INVENTORS

C₂₄H₁₅N₃O₃
Molecular Weight:	393.3942 g/mol

¹H NMR (500 MHz, DMSO-d₆) δ 4.32 (s, 1H), 6.34 (d, J = 15.55 Hz, 1H), 7.35 (d, J = 8.37 Hz, 2H), 7.44 (d, J = 8.37 Hz, 2H), 7.49 (d, J = 7.58 Hz, 1H), 7.55 (t, J = 7.98 Hz, 1H), 7.58 (t, J = 7.78 Hz, 1H), 7.78 (d, J = 8.17 Hz, 1H), 7.87 (m, 3H), 8.05 (d, J = 7.78 Hz, 1H), 8.13 (d, J = 7.98 Hz, 1H).

¹³C NMR (126 MHz, DMSO-d₆) δ 82.70, 83.24, 120.66, 121.04, 122.84, 126.51, 126.81, 127.31, 127.83, 129.98, 130.12, 132.33, 132.39, 133.49, 134.90, 135.21, 137.21, 137.99, 147.36, 151.04, 161.37, 166.58.

HRMS (m/z): [M + H]⁺, calcd for C₂₅H₁₇N₂O₃, 393.1234; found, 393.1250. HRMS (m/z): [M + Na]⁺, calcd for C₂₅H₁₆N₂NaO₃, 415.1053; found, 415.1054.

The emergence of resistance to antibiotics over the past few decades has created a state of crisis in the treatment of bacterial infections.Over the years, β-lactams were the antibiotics of choice for treatment of S. aureus infections. However, these agents faced obsolescence with the emergence of methicillin-resistant S. aureus (MRSA). Presently, vancomycin, daptomycin, linezolid, or ceftaroline are used for treatment of MRSA infections, although only linezolid can be dosed orally. Resistance to all four has emerged. Thus, new anti-MRSA antibiotics are sought, especially agents that are orally bioavailable. a new antibiotic (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)–one, with potent activity against S. aureus, including MRSA. We document that quinazolinones of our design are inhibitors of cell-wall biosynthesis in S. aureus and do so by binding to dd-transpeptidases involved in cross-linking of the cell wall. quinazolinones possess activity in vivo and are orally bioavailable. This antibiotic holds promise in treating difficult infections by MRSA.

PAPER

Journal of the American Chemical Society (2015), 137(5), 1738-1741.

http://pubs.acs.org/doi/abs/10.1021/jacs.5b00056

Discovery of Antibiotic (E)-3-(3-Carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one

Renee Bouley^†, Malika Kumarasiri^†, Zhihong Peng^†, Lisandro H. Otero^‡, Wei Song^†, Mark A. Suckow^§, Valerie A. Schroeder^§, William R. Wolter^§, Elena Lastochkin^†, Nuno T. Antunes^†, Hualiang Pi^†, Sergei Vakulenko^†, Juan A. Hermoso^‡, Mayland Chang^*^†, and Shahriar Mobashery^*^†

^† Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States

^‡ Department of Crystallography and Structural Biology, Instituto de Química-Física “Rocasolano”, Consejo Superior de Investigaciones Científicas, Madrid, Spain

^§ Freimann Life Sciences Center and Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States

J. Am. Chem. Soc., 2015, 137 (5), pp 1738–1741

DOI: 10.1021/jacs.5b00056

Publication Date (Web): January 28, 2015

*mchang@nd.edu, *mobashery@nd.edu

Abstract

In the face of the clinical challenge posed by resistant bacteria, the present needs for novel classes of antibiotics are genuine. In silico docking and screening, followed by chemical synthesis of a library of quinazolinones, led to the discovery of (E)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)–one (compound 2) as an antibiotic effective in vivo against methicillin-resistant Staphylococcus aureus (MRSA). This antibiotic impairs cell-wall biosynthesis as documented by functional assays, showing binding of 2 to penicillin-binding protein (PBP) 2a. We document that the antibiotic also inhibits PBP1 of S. aureus, indicating a broad targeting of structurally similar PBPs by this antibiotic. This class of antibiotics holds promise in fighting MRSA infections.

PATENT

WO 2014138302

http://www.google.com/patents/WO2014138302A1?cl=en

Staphylococcus aureus is a common bacterium found in moist areas of the body and skin. S. aureus can also grow as a biofilm, representing the leading cause of infection after implantation of medical devices. Approximately 29% (78.9 million) of the US population is colonized in the nose with S. aureus, of which 1.5% (4.1 million) is methicillin-resistant S. aureus (MRSA). In 2005, 478,000 people in the US were hospitalized with a S. aureus infection, of these 278,000 were MRSA infections, resulting in 19,000 deaths. MRSA infections have been increasing from 2% of S. aureus infections in intensive care units in 1974 to 64% in 2004, although more recent data report stabilization. Approximately 14 million outpatient visits occur every year in the US for suspected S. aureus skin and soft tissue infections. About 76% of these infections are caused by S. aureus, of which 78% are due to MRSA, for an overall rate of 59%. Spread of MRSA is not limited to nosocomial (hospital-acquired) infections, as they are also found in community-acquired infections. Over the years, β-lactams were antibiotics of choice in treatment of S. aureus infections. However, these agents faced obsolescence with the emergence of

MRSA. Presently, vancomycin, daptomycin or linezolid are agents for treatment of MRSA infections, although only linezolid can be dosed orally. Resistance to all three has emerged. Thus, new anti-MRSA therapeutic strategies are needed, especially agents that are orally bioavailable.

Clinical resistance to β-lactam antibiotics by MRSA has its basis predominantly in acquisition of the mecA gene, which encodes penicillin-binding protein 2a (PBP2a). PBP2a, a cell-wall DD- transpeptidase, is refractory to inhibition by essentially all commercially available β-lactams (ceftaroline is an exception), antibiotics that irreversibly acylate the active-site serine of typical PBPs. PBPs catalyze biosynthesis of the bacterial cell wall, which is essential for the survival of the bacterium. Accordingly, new ηοη-β-lactam antibiotics that inhibit PBP2a are needed to combat drug-resistant strains of bacteria. SUMMARY

Staphylococcus aureus is responsible for a number of human diseases, including skin and soft tissue infections. Annually, 292,000 hospitalizations in the US are due to S. aureus infections, of which 126,000 are related to methicillin-resistant Staphylococcus aureus (MRSA), resulting in 19,000 deaths. A novel structural class of antibiotics has been discovered and is described herein. A lead compound in this class shows high in vitro potency against Gram-positive bacteria comparable to those of linezolid and superior to vancomycin (both considered gold standards) and shows excellent in vivo activity in mouse models of MRSA infection.

The invention thus provides a novel class of ηοη-β-lactam antibiotics, the quinazolinones, which inhibit PBP2a by an unprecedented mechanism of targeting both its allosteric and active sites. This inhibition leads to the impairment of the formation of cell wall in living bacteria. The quinazolinones described herein are effective as anti-MRSA agents both in vitro and in vivo. Furthermore, they exhibit activity against other Gram-positive bacteria. The quinazolinones have anti-MRSA activity by themselves. However, these compounds synergize with β-lactam antibiotics. The use of a combination of a quinazolinone with a β-lactam antibiotic can revive the clinical use of β-lactam antibacterial therapy in treatment of MRSA infections. The invention provides a new class of quinazolinone antibiotics, optionally in combination with other antibacterial agents, for the therapeutic treatment of methicillin- resistant Staphylococcus aureus and other bacteria.

The quinazolinone compounds described herein can be prepared using standard synthetic techniques known to those of skill in the art. Examples of such techniques are described by Khajavi et al. (J. Chem. Res. (S), 1997, 286-287) and Mosley et al. (J. Med. Chem. 2010, 53, 5476-5490). A general preparatory scheme for preparing the compounds described herein, for example, compounds of Formula

wherein each of the variables are as defined for one or more of the formulas described herein, such as Formula (A).

EXAMPLES

Example 1. Compound Preparation

Chemistry. Organic reagents and solvents were purchased from Sigma- Aldrich. ^lH and ¹³C NMR spectra were recorded on a Varian INOVA-500. High-resolution mass spectra were obtained using a Bruker micrOTOF/Q2 mass spectrometer.

2-Methyl-4H-benzo[</| [l,3]oxazin-4-one (3). Anthranilic acid (20 g, 146 mmol) was dissolved in triethyl orthoacetate (45 mL, 245 mmol) and refluxed for 2 h. The reaction mixture was cooled on ice for 4 h to crystallize the intermediate. The resulting crystals were filtered and washed with hexanes to give 3 (17 g, 72% yield). ^lH NMR (500 MHz, CDC1₃) δ 2.47 (s, 3H), 7.50 (t, J= 7.38 Hz, 1H), 7.54 (d, J = 7.98 Hz, 1H), 7.80 (t, J= 7.18 Hz, 1H), 8.18 (d, J= 7.78 Hz, 1H). ¹³C NMR (126 MHz, CDCI3) δ 21.59, 1 16.84, 126.59, 128.42, 128.66, 136.77, 146.61, 159.89, 160.45. HRMS (m/z): [M + H]⁺, calcd for C9H8NO2, 162.0550; found , 162.0555.

2-Methyl-3-(3-carboxyphenyl)-quinazolin-4(3//)-one (4). Compound 3 (2 g, 12.4 mmol) and 3- aminophenol (1.7 g, 12.4 mmol) were suspended in glacial acetic acid (8 mL, 140 mmol), and dissolved upon heating. The reaction was refluxed for 5 h, at which point 5 mL water was added to the cooled reaction mixture. The resulting precipitate was filtered and washed with water, followed by cold ethanol and hexane to give 4 (3.19 g, 92% yield). ^lH (500 MHz, DMSO-d₆) δ 2.87 (s, 3H), 7.52 (t, J= 7.38 Hz, 1H), 7.66-7.73 (m, 3H), 7.84 (t, J= 7.38 Hz, 1H), 8.01 (s, 1H), 8.09 (t, J= 7.58 Hz, 2H). ¹³C NMR (126 MHz, DMSO-de) δ 24.13, 120.48, 126.32, 126.47, 126.72, 129.52, 129.83, 130.01, 132.40, 133.07, 134.67, 138.18, 147.37, 154.13, 161.44, 166.58. HRMS (m/z): [M + H]⁺, calcd for C16H13N2O3 ,

281.0921 ; found, 281.0917.

Sodium (£^‘)-3-(3-carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one (2). Compound 4 (1.0 g, 3.6 mmol) and 4-formylbenzonitrile (0.56 g, 4.3 mmol) were suspended in glacial acetic acid (5 mL, 87 mmol), a suspension that dissolved upon heating. The reaction was refluxed for 18 h and 5 mL water was added to the cooled reaction mixture. The resulting precipitate was filtered and washed with water, followed by cold ethanol and hexanes to afford the carboxylic acid (0.77g, 75% yield). HRMS (m/z): [M + H]⁺, calcd for C24H16N3O3, 394.1 186; found 394.1214. The carboxylic acid (0.45 g, 1.1 mmol) was dissolved in hot ethanol, to which sodium 2-ethylhexanoate (0.28 g, 1.7 mmol) was added. The reaction mixture was stirred on ice for 2 h. The precipitate was filtered and washed with cold ethanol. The product was obtained by dissolving the precipitate in about 5 mL of water and subsequent lyophilization of the solution to give 2 as the sodium salt (0.4 g, 85% yield).

¾ NMR (500 MHz, DMSO- de) δ 6.47 (d, J= 15.55 Hz, 1H), 7.59 (m, 3H), 7.74 (d, J= 5.38 Hz, 2H), 7.79 (m, 3H), 7.91 (m, 2H), 8.05 (s, 1H), 8.14 (d, J= 7.78 Hz, 2H).

¹³C NMR (126 MHz, DMSO-de) δ 11 1.56, 1 18.61, 120.76, 123.42, 126.50, 127.01, 127.35, 128.26, 129.99, 130.06, 130.12, 132.33, 132.83, 133.46, 134.89, 136.95, 137.03, 139.25, 147.21, 150.74, 161.25, 166.52.

HRMS (m/z): [M + H]⁺, calcd for C24Hi₅N₃NaO₃, 416.1006; found, 416.0987.

PAPER

http://pubs.acs.org/doi/full/10.1021/acs.jmedchem.6b00372

Structure–Activity Relationship for the 4(3H)-Quinazolinone Antibacterials

Renee Bouley^†, Derong Ding^†, Zhihong Peng^†, Maria Bastian^†, Elena Lastochkin^†, Wei Song^†, Mark A. Suckow^‡,Valerie A. Schroeder^‡, William R. Wolter^‡, Shahriar Mobashery^*^†, and Mayland Chang^*^†

^† Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States

^‡ Freimann Life Sciences Center and Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States

J. Med. Chem., Article ASAP

DOI: 10.1021/acs.jmedchem.6b00372

Publication Date (Web): April 18, 2016

*S.M.: e-mail, mobashery@nd.edu; phone, 574-631-2933., *M.C.: e-mail, mchang@nd.edu; phone, 574-631-2965.

ACS Editors’ Choice – This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.

We recently reported on the discovery of a novel antibacterial (2) with a 4(3H)-quinazolinone core. This discovery was made by in silico screening of 1.2 million compounds for binding to a penicillin-binding protein and the subsequent demonstration of antibacterial activity againstStaphylococcus aureus. The first structure–activity relationship for this antibacterial scaffold is explored in this report with evaluation of 77 variants of the structural class. Eleven promising compounds were further evaluated for in vitro toxicity, pharmacokinetics, and efficacy in a mouse peritonitis model of infection, which led to the discovery of compound 27. This new quinazolinone has potent activity against methicillin-resistant (MRSA) strains, low clearance, oral bioavailability and shows efficacy in a mouse neutropenic thigh infection model.

NMR

INVENTORS

Renee Bouley

Renee Bouley selected to receive prestigious ACS Predoctoral Fellowship

Published: July 02, 2013

Renee Bouley

Renee Bouley, a third year graduate student in the Department of Chemistry and Biochemistry, has been selected to receive a prestigious American Chemical Society (ACS) Division of Medicinal Chemistry Predoctoral Fellowship. Bouley is one of only four recipients chosen for the 2013-2014 cycle.

This award supports doctoral candidates working in the area of medicinal chemistry who have demonstrated superior achievements as graduate students and who show potential for future work as independent investigators. These fellowships have been awarded annually since 1991 and include one year stipend support and an invitation to present the fellow’s research results at a special awards session at the ACS National Meeting.

Bouley’s work, conducted under the advisement of Shahriar Mobashery, Navari Family Professor in Life Sciences, and Mayland Chang, Research Professor and Director of the Chemistry-Biochemistry-Biology Interface (CBBI) Program, centers around the discovery of a new class of antibiotics that are selective against staphylococcal species of bacteria, including hard-to-treat methicillin-resistant Staphylococcus aureus (MRSA). She has already identified a class of compounds that has in vitro activity against bacteria and demonstrated efficacy in mice. Bouley spent three months in 2012 in the laboratory of Prof. Juan Hermoso at Consejo Superior de Investigaciones Cientificas in Madrid, Spain, where she solved the crystal structure of the lead compound in complex with its target protein. Her studies have shown an unprecedented mechanism of action that opens opportunities for clinical resurrection of β-lactam antibiotics in combination with the new antibiotics. Bouley’s work during her fellowship tenure will explore structural analogs of these compounds with the goal of optimizing their potency in vivo and improving their drug-like properties.

Bouley is already the recipient of a National Institutes of Health Ruth L. Kirschstein National Research Service Award – CBBI (Chemistry-Biochemistry-Biology Interface) Program, a CBBI Research Internship Award, and an American Heart Association Predoctoral Fellowship (declined)………..https://www.linkedin.com/in/renee-bouley-43243215

University of Notre Dame

MAYLAND CHANG

http://chemistry.nd.edu/people/mayland-chang/

MAYLAND CHANG

Research Professor; Director, Chemistry-Biochemistry-Biology Interface (CBBI) Program
Office: 247 NSH
Phone: (574) 631-2965

Dr. Chang obtained B.S. degrees in biological sciences and chemistry from the University of Southern California, and a Ph.D. in chemistry from the University of Chicago. Subsequently, she conducted postdoctoral research at Columbia University as a National Institutes of Health postdoctoral fellow. She joined the faculty of the University of Notre Dame in 2003. Previously, Dr. Chang was Chief Operating Officer of University Research Network, Inc., Senior Scientist with Pharmacia Corporation, and Senior Chemist at Dow Chemical Company. She has characterized the ADME properties of numerous drugs, as well as prepared NDAs, INDs, Investigator’s Brochures, product development plans, and candidate drug evaluations.

Shahriar Mobashery

Navari Professor at University of Notre Dame

The Mobashery research program integrates computation, biochemistry, molecular biology, and the organic synthesis of medically important molecules. Bringing together these different disciplines is required to produce both scientific and medical advances for very difficult, but critically important clinical problems.

http://chemistry.nd.edu/people/shahriar-mobashery/

https://www.linkedin.com/in/shahriar-mobashery-71b67b4b

/////// 1624273-22-8, Antibiotic, (E)-3-(3-Carboxyphenyl)-2-(4-cyanostyryl)quinazolin-4(3H)-one, methicillin-resistant S. aureus, MRSA, 1624273-21-7, PRECLINICAL

O=C(O)c1cc(ccc1)N3C(=Nc2ccccc2C3=O)/C=C/c4ccc(C#N)cc4

VX-? , an Azaindolyl-Pyrimidine Inhibitor of Influenza Virus Replication from Vertex

May 2, 2016 10:21 am / Leave a comment

VX-?

An Azaindolyl-Pyrimidine Inhibitor of Influenza Virus Replication from Vertex

SYNTHESIS COMING……..

CAS 1259498-06-0

MF C₂₃ H₂₇ F₂ N₇ O, MW, 455.50

1-Piperidinecarboxamide, N-[(1R,3S)-3-[[5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)-4-pyrimidinyl]amino]cyclohexyl]-

N-[(1R,3S)-3-[[5-Fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl]amino]cyclohexyl]morpholine-4-carboxamide

N-[(1R,3S)-3-[[5-Fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl]amino]cyclohexyl]morpholine-4-carboxamide , (1R,3S)-cis-diaminocyclohexane.

Specific Rotation

[α]²¹_D = −165.7° (c = 1 in MeOH).

¹H NMR (300 MHz, d₆-DMSO) δ 12.23 (s, 1H), 8.42 (dd, J = 9.8, 2.9 Hz, 1H), 8.34–8.18 (m, 2H), 8.14 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 6.33 (d, J= 7.6 Hz, 1H), 4.24–4.00 (m, 1H), 3.75–3.57 (m, 1H), 3.57–3.42 (m, 4H), 3.28–3.09 (m, 4H), 2.15 (d, J = 11.4 Hz, 1H), 2.01 (d, J = 11.2 Hz, 1H), 1.83 (d, J = 9.7 Hz, 2H), 1.60–1.07 (m, 4H).¹⁹F NMR (282.4 MHz, d₆-DMSO) −138.10, −158.25 ppm.

HRMS (ESI) [M + H]⁺ calculated for C₂₂H₂₆F₂N₇O₂ 458.2111, found 458.2110.

Influenza spreads around the world in seasonal epidemics, resulting in the deaths of hundreds of thousands annually – millions in pandemic years. For example, three influenza pandemics occurred in the 20th century and killed tens of millions of people, with each of these pandemics being caused by the appearance of a new strain of the virus in humans. Often, these new strains result from the spread of an existing influenza virus to humans from other animal species.

Influenza is primarily transmitted from person to person via large virus-laden droplets that are generated when infected persons cough or sneeze; these large droplets can then settle on the mucosal surfaces of the upper respiratory tracts of susceptible individuals who are near (e.g. within about 6 feet) infected persons. Transmission might also occur through direct contact or indirect contact with respiratory secretions, such as touching surfaces contaminated with influenza virus and then touching the eyes, nose or mouth. Adults might be able to spread influenza to others from 1 day before getting symptoms to approximately 5 days after symptoms start. Young children and persons with weakened immune systems might be infectious for 10 or more days after onset of symptoms. [00103] Influenza viruses are RNA viruses of the family Orthomyxoviridae, which comprises five genera: Influenza virus A, Influenza virus B, Influenza virus C, Isavirus and Thogoto virus.

The Influenza virus A genus has one species, influenza A virus. Wild aquatic birds are the natural hosts for a large variety of influenza A. Occasionally, viruses are transmitted to other species and may then cause devastating outbreaks in domestic poultry or give rise to human influenza pandemics. The type A viruses are the most virulent human pathogens among the three influenza types and cause the most severe disease. The influenza A virus can be subdivided into different serotypes based on the antibody response to these viruses. The serotypes that have been confirmed in humans, ordered by the number of known human pandemic deaths, are: HlNl (which caused Spanish influenza in 1918), H2N2 (which caused Asian Influenza in 1957), H3N2 (which caused Hong Kong Flu in 1968), H5N1 (a pandemic threat in the 2007-08 influenza season), H7N7 (which has unusual zoonotic potential), H1N2 (endemic in humans and pigs), H9N2, H7N2 , H7N3 and H10N7. [00105] The Influenza virus B genus has one species, influenza B virus. Influenza B almost exclusively infects humans and is less common than influenza A. The only other animal known to be susceptible to influenza B infection is the seal. This type of influenza mutates at a rate 2-3 times slower than type A and consequently is less genetically diverse, with only one influenza B serotype. As a result of this lack of antigenic diversity, a degree of immunity to influenza B is usually acquired at an early age. However, influenza B mutates enough that lasting immunity is not possible. This reduced rate of antigenic change, combined with its limited host range (inhibiting cross species antigenic shift), ensures that pandemics of influenza B do not occur.

The Influenza virus C genus has one species, influenza C virus, which infects humans and pigs and can cause severe illness and local epidemics. However, influenza C is less common than the other types and usually seems to cause mild disease in children. [00107] Influenza A, B and C viruses are very similar in structure. The virus particle is 80-120 nanometers in diameter and usually roughly spherical, although filamentous forms can occur. Unusually for a virus, its genome is not a single piece of nucleic acid; instead, it contains seven or eight pieces of segmented negative-sense RNA. The Influenza A genome encodes 11 proteins: hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), Ml, M2, NSl, NS2(NEP), PA, PBl, PB1-F2 and PB2.

HA and NA are large glycoproteins on the outside of the viral particles. HA is a lectin that mediates binding of the virus to target cells and entry of the viral genome into the target cell, while NA is involved in the release of progeny virus from infected cells, by cleaving sugars that bind the mature viral particles. Thus, these proteins have been targets for antiviral drugs. Furthermore, they are antigens to which antibodies can be raised. Influenza A viruses are classified into subtypes based on antibody responses to HA and NA, forming the basis of the H and N distinctions (vide supra) in, for example, H5N1. [00109] Influenza produces direct costs due to lost productivity and associated medical treatment, as well as indirect costs of preventative measures. In the United States, influenza is responsible for a total cost of over $10 billion per year, while it has been estimated that a future pandemic could cause hundreds of billions of dollars in direct and indirect costs. Preventative costs are also high. Governments worldwide have spent billions of U.S. dollars preparing and planning for a potential H5N1 avian influenza pandemic, with costs associated with purchasing drugs and vaccines as well as developing disaster drills and strategies for improved border controls.

Current treatment options for influenza include vaccination, and chemotherapy or chemoprophylaxis with anti-viral medications. Vaccination against influenza with an influenza vaccine is often recommended for high-risk groups, such as children and the elderly, or in people that have asthma, diabetes, or heart disease. However, it is possible to get vaccinated and still get influenza. The vaccine is reformulated each season for a few specific influenza strains but cannot possibly include all the strains actively infecting people in the world for that season. It takes about six months for the manufacturers to formulate and produce the millions of doses required to deal with the seasonal epidemics; occasionally, a new or overlooked strain becomes prominent during that time and infects people although they have been vaccinated (as by the H3N2 Fujian flu in the 2003-2004 influenza season). It is also possible to get infected just before vaccination and get sick with the very strain that the vaccine is supposed to prevent, as the vaccine takes about two weeks to become effective. [00111] Further, the effectiveness of these influenza vaccines is variable. Due to the high mutation rate of the virus, a particular influenza vaccine usually confers protection for no more than a few years. A vaccine formulated for one year may be ineffective in the following year, since the influenza virus changes rapidly over time, and different strains become dominant.

Also, because of the absence of RNA proofreading enzymes, the RNA- dependent RNA polymerase of influenza vRNA makes a single nucleotide insertion error roughly every 10 thousand nucleotides, which is the approximate length of the influenza vRNA. Hence, nearly every newly-manufactured influenza virus is a mutant — antigenic drift. The separation of the genome into eight separate segments of vRNA allows mixing or reassortment of vRNAs if more than one viral line has infected a single cell. The resulting rapid change in viral genetics produces antigenic shifts and allows the virus to infect new host species and quickly overcome protective immunity.

Antiviral drugs can also be used to treat influenza, with neuraminidase inhibitors being particularly effective, but viruses can develop resistance to the standard antiviral drugs.

PAPER

http://pubs.acs.org/doi/full/10.1021/acs.oprd.6b00063

Development of a Scalable Synthesis of an Azaindolyl-Pyrimidine Inhibitor of Influenza Virus Replication

Jianglin Liang^*, John E. Cochran, Warren A. Dorsch, Ioana Davies, and Michael P. Clark

Vertex Pharmaceuticals Incorporated, 50 Northern Avenue, Boston, Massachusetts 02210, United States

Org. Process Res. Dev., Article ASAP

DOI: 10.1021/acs.oprd.6b00063

Publication Date (Web): April 08, 2016

A scalable, asymmetric route for the synthesis of the influenza virus replication inhibitor 2 is presented. The key steps include an enzymatic desymmetrization of cis-1,3-cyclohexanediester in 99% yield and 96% ee, S_NAr displacement of a methanesulfinylpyrimidine, and a Curtius rearrangement to form a morpholinyl urea. This high-yielding route allowed us to rapidly synthesize hundreds of grams of 2 in 99% purity to support in vivo studies.

About Influenza

Often called “the flu,” seasonal influenza is caused by influenza viruses, which infect the respiratory tract.¹ The flu can result in seasonal epidemics² and can produce severe disease and high mortality in certain populations, such as the elderly.³ Each year, on average 5 to 20 percent of the U.S. population gets the flu⁴ resulting in more than 200,000 flu-related hospitalizations and 36,000 deaths.⁵The overall national economic burden of influenza-attributable illness for adults is $83.3 billion.⁵ Direct medical costs for influenza in adults totaled $8.7 billion including $4.5 billion for adult hospitalizations resulting from influenza-attributable illness.⁵ The treatment of the flu consists of antiviral medications that have been shown in clinical studies to shorten the disease and reduce the severity of symptoms if taken within two days of infection.⁶ There is a significant need for new medicines targeting flu that provide a wider treatment window, greater efficacy and faster onset of action.

About Vertex

Vertex is a global biotechnology company that aims to discover, develop and commercialize innovative medicines so people with serious diseases can lead better lives. In addition to our clinical development programs focused on cystic fibrosis, Vertex has more than a dozen ongoing research programs aimed at other serious and life-threatening diseases.

Founded in 1989 in Cambridge, Mass., Vertex today has research and development sites and commercial offices in the United States, Europe, Canada and Australia. For four years in a row, Science magazine has named Vertex one of its Top Employers in the life sciences. For additional information and the latest updates from the company, please visit www.vrtx.com.

Vertex’s press releases are available at www.vrtx.com.

SYNTHESIS COMING

WO-2010148197

http://www.google.co.in/patents/WO2010148197A1?cl=en

General Scheme 44 SIMILAR TO A POINT BUT NOT SAME

(a) Pd(PPh₃)₄ sodium carbonate, DME/water, reflux (b) meta-chloroperbenzoic acid, dichloromethane, rt. (c) 20a, tetrahydrofuran, 5O°C (d) trifluoroacetic acid, dichloromethane, rt.

SIMILAR NOT SAME

(e) morpholιne-4-carbonyl chloride, dimethylformamide, rt (f) sodium methoxide, methanol, rt.

Formation of 5-fluoro-3-[5-fluoro-4-(methylthio)pyrimidin-2-yl]-1-tosyl-lΗ- pyrrolo[2,3-b]pyridine (44b)

2-Chloro-5-fluoro-4-methylsulfanyl-pyrimidine (34.1 g, 191.0 mmol) , 5-fluoro-1-(p- tolylsulfonyl)-3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)pyrrolo[2,3-b]pyridine, 44a, (53.0 g, 127.3 mmol) and Na₂Cθ3 (40.5 g, 381.9 mmol) were dissolved in a mixture of DME (795 mL) and water (159 mL). The mixture was purged with nitrogen for 20 minutes and treated with Pd(PPh₃ )₄ (7.4 g, 6.6 mmol). After purging with nitrogen for another 20 minutes, the reaction was heated to reflux overnight, cooled to room temperature and diluted with water (60OmL). The resulting suspension was stirred at room temperature for 30 minutes and the precipitate was then collected by filtration, washed with water and acetonitrile and dried at 50 °C to afford 48.2 g of 5-fluoro-3-[5-fluoro-4-(methylthio)pyrimidin-2-yl]-1-tosyl-1H- pyrrolo[2,3-b]pyridine as a white solid.

¹H NMR (300 MHz, OMSO-d6) δ 8.70 – 8.58 (m, 2H), 8.54 – 8.41 (m, 2H), 8.09 (d, J = 8.4 Hz, 2H), 7.45 (d, J= 8.2 Hz, 2H), 2.76 (s, 3H), 2.36 (s, 3H).

Formation of 5-fluoro-3-[5-fluoro-4-(methylsulfinyl)pyrimidin-2-yl]-1- tosyl-1H-pyrrolo[2,3-b]pyridine (44c)

5-fluoro-3 – [5 -fluoro-4-(methylthio)pyrimidin-2-yl] – 1 -tosyl- 1 H-pyrrolo [2,3 – b]pyridine, 44b, (48.2 g, 111.5 mmol) was dissolved in dichloromethane (2.3 L) and treated portionwise with m-CPBA (27.5 g, 122.6 mmol) while keeping the temperature below 20 °C. After addition was complete, the reaction was stirred at room temperature for 2 hours, then treated with another portion of m-CPBA (1.9 g) and stirred for another hour. The reaction mixture was washed with 12% aqueuous K₂CO3 (2 x 1.0 L) and the organic layer was dried on Na₂SO₄ and concentrated in vacuo to provide 50 g of 5-fluoro-3-[5-fluoro-4- (methylsulfinyl)pyrimidin-2-yl]-1-tosyl-1H-pyrrolo[2,3-b]pyridine as a yellow solid.

¹H NMR (300 MHz, DMSO-rf<5) δ 9.11 (d, J= 1.5 Hz, 1H), 8.69 (s, 1H), 8.65 (dd, J = 9.0, 2.9 Hz, 1H), 8.52 (dd, J= 2.8, 1.2 Hz, 1H), 8.11 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 3.05 (s, 3H), 2.36 (s, 3H).

[001057] Formation of tert-butyl N-[(IR, 3S)-3-[[5-fluoro-2-[5-fluoro-1-(p- tolylsulfonyl)pyrrolo [2,3-b] pyridin-3-yl]pyrimidin-4-yl] amino] cyclohexyl] carbamate (44d)

5-fluoro-3-(5-fluoro-4-methylsulfinyl-pyrimidin-2-yl)-1-(p-tolylsulfonyl)pyrrolo[2,3- b]pyridine, 44c, (5.9 g, 10.5 mmol) and tert-butyl N-[(IR, 35^*)-3-aminocyclohexyl]carbamate (3 g, 12.60 mmol) were dissolved in THF (100 mL). The reaction mixture was heated to 50 °C for 6 hours, then cooled to room temperature. C₆ lite was added and the solvent was removed under reduced pressure. The C₆ lite-supported residue was purified by silica gel chromatography (20-80% EtOAc/hexanes gradient to provide 3.7 g of tert-butyl N-[(IR, 3S)- 3-[[5-fluoro-2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]pyrimidin-4- yl]amino]cyclohexyl]carbamate.

¹H NMR (300 MHz, CDCl₃) δ 8.51 (s, 1H), 8.46 – 8.41 (m, 1H), 8.29 (d, J = 1.6 Hz, 1H), 8.11 (s, 1H), 8.08 (s, 1H), 8.06 (d, J= 3.2 Hz, 1H), 7.27 (d, J= 8.4 Hz, 2H), 4.91 (d, J = 8.0 Hz, 1H), 4.41 (s, 1H), 4.29 – 4.01 (m, 1H), 3.64 (s, 1H), 2.47 (d, J= 11.5 Hz, 1H), 2.36 (s, 3H), 2.24 (d, J = 13.1 Hz, 1H), 2.08 (d, J= 10.9 Hz, 1H), 1.91 (d, J= 13.8 Hz, 1H), 1.43 (s, 9H), 1.30 – 1.03 (m, 4H).

Formation of (IS, SΛHVHS-fluoro^-β-fluoro-1-Cp- tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]pyrimidin-4-yl]cyclohexane-1,3-diamine (44e) tert-Butyl N-[(IR, 3S>3-[[5-fluoro-2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3- b]pyridin-3-yl]pyrimidin-4-yl]amino]cyclohexyl]carbamate, 44d, (3.7 g, 6.2 mmol) was dissolved in dichloromethane (105 mL) and treated with trifluoroacetic acid (31 mL). After 5 minutes, the volatiles were evaporated under reduced pressure, and the resulting residue was treated with IN NaOH (75 mL). The resulting precipitate was collected by filtration, washed with water (3 x 30 mL) and vacuum dried to provide 2.7 g of (IS, 3R)-Nl -[5-fluoro-2-[5- fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]pyrimidin-4-yl]cyclohexane-l,3-diamine as a white solid.

¹H NMR (300 MHz, MeOD) d 8.56 (dd, J = 8.0, 3.9 Hz, 2H), 8.35 – 8.26 (m, 1H), 8.12 (dd, J= 10.3, 6.1 Hz, 3H), 7.43 (d, J= 8.4 Hz, 2H), 4.36 – 4.21 (m, 1H), 3.28 – 3.13 (m, 1H), 2.48 (d, J= 12.3 Hz, 1H), 2.46 (s, 3H), 2.25 – 1.97 (m, J= 17.3, 10.6, 4.1 Hz, 4H), 1.76 – 1.28 (m, 3H).

Formation of N-[(IR, 3S>3-[[5-fluoro-2-[5-fluoro-1-(p- tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]pyrimidin-4-yl]amino]cyclohexyl] morpholine- 4-carboxamide (44f)

(15, 3R)-M-[5-fluoro-2-[5-fluoro-1-(p-tolylsulfonyl)pyrrolo[2,3-b]pyridin-3- yl]pyrimidin-4-yl]cyclohexane- 1,3 -diamine, 44e, (2.3 g, 4.6 mmol) was dissolved in DMF (5OmL) and treated with morpholine-4-carbonyl chloride (2.1 g, 13.8 mmol) and DIPEA (4.2 g, 5.6 mL, 32.3 mmol). After one hour, the resulting solution was diluted with water (400 mL) and stirred for an additional two hours. The resulting precipitate was collected by filtration, washed with water (3 x 50 mL) and dried to provide the crude product. This material was purified by flash chromatography on a 4Og column using EtOAc/DCM 20- 100%, to provide 2.0 g of N-[(1R, 35)-3-[[5-fluoro-2-[5-fluoro-1-(p- tolylsulfonyl)pyrrolo[2,3-b]pyridin-3-yl]pyrimidin-4-yl]amino]cyclohexyl]morpholine-4- carboxamide as a white solid.

¹H NMR (300 MHz, DMSO-Λ5) δ 8.53 – 8.43 (m, J = 11.9, 2.7 Hz, 3H), 8.22 (d, J = 3.9 Hz, 1H), 8.07 (d, J= 8.4 Hz, 2H), 7.44 (d, J= 8.3 Hz, 2H), 6.32 (d, J= 7.5 Hz, 1H), 4.05 (s, J= 19.4 Hz, 1H), 3.62 (s, 1H), 3.58 – 3.45 (m, 4H), 3.27 – 3.18 (m, 4H), 2.36 (s, 3H), 2.12 (d, J= 11.7 Hz, 1H), 1.99 (d, J= 9.5 Hz, 1H), 1.83 (d, J= 10.3 Hz, 2H), 1.53 – 1.11 (m, J = 32.3, 22.8, 10.9 Hz, 4H).

ormation of N-[(IR, 3S>3-[[5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3- b]pyridin-3-yl)pyrimidin-4-yl] amino] cyclohexyl]morpholine-4-carboxamide (706)

N- [( IR, 35)-3 – [ [5 -fluoro-2- [5 -fluoro- 1 -(p-tolylsulfonyl)pyrrolo [2,3 -b]pyridin-3 – yl]pyrimidin-4-yl]amino]cyclohexyl]morpholine-4-carboxamide, 44f, (2.0 g, 3.2 mmol) was suspended in methanol (50 mL) and treated with 25% sodium methoxide in methanol (19.9 mL, 92.3 mmol) . After stirring for 1 hour, the solvent was evaporated under reduced pressure, and the residue was partitioned between water (100 mL) and ethyl acetate (100 mL). The organic layer was collected, dried on Νa₂SO₄ and concentrated to provide the crude product as a yellow solid. This material was purified by silica gel chromatography on a 4Og column, using DCM/MeOH 1-6%. The purified fractions were treated with 2N HCl in ether and concentrated to provide 1.5 g of N-[(1R, 35)-3-[[5-fluoro-2-(5-fluoro-1H- pyrrolo[2,3-b]pyridin-3-yl)pyrimidin-4-yl]amino]cyclohexyl]-morpholine-4-carboxamide as a white solid.

HCI D DCM

44e

Formation of (IS, S^-M-^-fluoro-S-CS-fluoro-1H-pyrrolo^S-^pyridin- 3-yl)phenyl)cyclohexane-1,3-diamine (44e)

To a solution of tert-butyl (IR, 35)-3-(2-fluoro-5-(5-fluoro-1-tosyl-lH-pyrrolo-[2,3- &]pyridin-3-yl)phenylamino)cyclohexylcarbamate, 44d, (0.65 g, 1.09 mmol) in methylene chloride (22 mL) was added hydrogen chloride (2.71 mL of 4M solution in 1,4-dioxane, 10.86 mmol). The reaction was heated to 50 °C and stirred for 6 hours. The mixture was cooled to room temperature and concentrated in vacuo, producing a yellow solid. The crude residue was purified via silica gel chromatography (25-50% Ethyl Acetate/hexanes gradient). Desired fractions were combined and concentrated in vacuo to produce 350 mg of 44e as a yellow powder.

General Scheme 67 SIMILAR TO A POINT BUT NOT SAME

(a) Pd/C (wet, Degussa), hydrogen, EtOH (b) 2,4-dichloro-5-fluoropyrimidine, ¹Pr₂NEt, THF, reflux (c) LiOH, THF/water, 5O°C

SIMILAR BUT NOT SAME

(d) DPPA, Et₃N, THF, 85 °C (e) 5-fluoro-3-(4,4,5,5-tetramethyl-1,3 ,2-dioxaborolan-2-yl)-1- tosyl-l//-pyrrolo[2,3-i]pyridine, XPhos, Pd₂(dba)₃, K₃PO₄, 2-methylTHF, water, 125 °C (f)

Formation (IR, 35)-ethyl 3-aminocyclohexanecarboxylate (67b)

To a solution of (IR, 35)-ethyl 3-(benzyloxycarbonylamino)cyclohexane-carboxylate, 18b, (14.0 g, 45.9 mmol) in ethanol (3 mL) was added Pd/C (wet, Degussa (2.4 g, 2.3 mmol). The mixture was evacuated and then stirred under atmosphere of nitrogen at room temperature overnight. The reaction mixture was filtered through a pad of celite and the resulting filtrate concentrated in vacuo to provide an oil that was used without further purification.

Formation (IR, SS^-ethyl 3-(2-chloro-5-fluoropyrimidin-4-ylamino)cyclohexane- carboxylate (67c)

To a solution of (IR, 3«S)-ethyl S-aminocyclohexanecarboxylate, 67b, (5.1 g, 24.1 mmol) and 2,4-dichloro-5,-fluoropyrimidine (6.0 g, 36.0 mmol) in THF (60 mL) was added diisopropylethylamine (9.6 mL, 55.4 mmol). The mixture was heated to reflux overnight. The reaction was cooled to room temperature and concentrated in vacuo. The residue was diluted with water and extracted twice with ethyl acetate. The combined organic phases were dried (MgSO₄), filtered and concentrated in vacuo. The residue was purified by silica gel chromatography (0-40% EtOAc/hexanes gradient) to provide 6.7 g of (IR, 35^*)-ethyl 3-(2- chloro-5-fluoropyrimidin-4-ylamino)cyclohexane-carboxylate as a white solid: LCMS RT = 3.1 (M+H) 302.2.

Formation (IR, 35)-3-(2-chloro-5-fluoropyrimidin-4-ylamino)cyclohexanecarboxylic acid (67d)

To a solution of (IR, 35^*)-ethyl 3-(2-chloro-5-fluoropyrimidin-4- ylamino)cyclohexane-carboxylate, 67c, (20.0 g, 66.3 mmol) in THF (150 mL) was added added a solution of LiOH hydrate (8.3 g, 198.8 mmol) in 100ml water. The reaction mixture was stirred at 50 °C overnight, To the reaction mixture was added HCl (16.6 mL of 12 M solution, 198.8 mmol) and EtOAc. The organic phase was washed with brine and dried over MgSO₄ and the solvent was removed under reduced pressure to afford 17.5 g of product that was used without further purification: ¹H NMR (300 MHz, CDC13) δ 7.91 (d, J = 2.7 Hz, 2H), 5.24 (d, J = 7.3 Hz, 2H), 4.19 – 4.03 (m, 3H), 3.84 – 3.68 (m, 3H), 2.59 (ddd, J= 11.5, 8.2, 3.6 Hz, 2H), 2.38 (d, J = 12.4 Hz, 2H), 2.08 (d, J = 9.6 Hz, 6H), 1.99 – 1.76 (m, 5H), 1.63 – 1.34 (m, 6H), 1.32 – 1.15 (m, 4H).

Formation N-((1R, 35)-3-(2-chloro-5-fluoropyrimidin-4-ylamino)cyclohexyl)- pyrrolidine-1-carboxamide (67e)

A solution of (IR, 35)-3-(2-chloro-5-fluoropyrimidin-4-ylamino)cyclohexane- carboxylic acid, 67d, (8.2 g, 30.0 mmol), (azido(phenoxy)phosphoryl)oxybenzene (9.7 mL, 45.0 mmol) and triethylamine (5.8 mL, 42.0 mmol) in THF (200 mL) was degassed under nitrogen for 15 minutes. The reaction mixture was heated at 85 °C for 30 minutes until LC/MS indicated complete consumption of carboxylic acid, 67d. To the reaction mixture was added pyrrolidine (7.5 mL, 90.0 mmol) and the reaction was heated at 85 °C for an additional 15 min. The mixture was diluted into brine and extracted with EtOAc. The organic phase was separated, dried over MgSO₄. The product was isolated (6.25 g) by filtration after partial removal of solvent in vacuo: ¹H NMR (300 MHz, CDC13) δ 7.87 (d, J = 2.8 Hz, 2H), 5.04 (d, J = 8.1 Hz, 2H), 4.09 (ddd, J = 26.9, 13.4, 5.6 Hz, 4H), 3.91 – 3.71 (m, 2H), 3.32 (t, J= 6.5 Hz, 7H), 2.45 (d, J= 11.5 Hz, 2H), 2.08 (dd, J= 22.1, 12.0 Hz, 4H), 1.96- 1.82 (m, 9H), 1.54 (dd, J= 18.6, 8.5 Hz, 2H), 1.22 – 1.01 (m, 6H).

Formation N-((IR, 3S>3-(5-fluoro-2-(5-fluoro-1-tosyl-1H-pyrrolo[2,3-b]pyridm-3- yl)pyrimidin-4-ylamino)cyclohexyl)pyrrolidine-1-carboxamide (67f)

A solution of N-((1R, 3«S)-3-(2-chloro-5-fluoropyrimidin-4-ylamino)cyclohexyl)- pyrrolidine-1-carboxamide, 67e, (6.8 g, 20.0 mmol), 5-fluoro-1-(p-tolylsulfonyl)-3-(4,4,5,5- tetramethyl-l,3,2-dioxaborolan-2-yl)pyrrolo[2,3-b]pyridine, 44a, (12.5 g, 30.0 mmol) and K₃PO₄ (17.0 g, 80.0 mmol) in 2-methyl TΗF (180 mL) and water (20 mL) was degassed under nitrogen for 30 min. To the mixture was added dicyclohexyl-[2-(2,4,6- triisopropylphenyl)phenyl]phosphane (XPhos) (1.1 g, 2.4 mmol) and Pd₂(dba)₃ (0.5 g, 0.5 mmol). The reaction mixture was heated in a pressure bottle at 125 °C for 2.5 hr. The reaction mixture was filtered through celite, the solvent was removed under reduced pressure. The resulting residue was purified by silica gel chromatography (8%MeOΗ/CΗ₂Cl₂) to afford 11.5 g of the desired product: ¹H ΝMR (300 MHz, CDC13) δ 8.54 (s, 1H), 8.49 (dd, J= 9.0, 2.8 Hz, 1H), 8.32 (d, J= 2.1 Hz, 1H), 8.13 (d, J= 8.3 Hz, 2H), 8.07 (d, J= 3.2 Hz, 1H), 7.30 (d, J = 8.5 Hz, 2H), 4.98 (d, J = 6.3 Hz, 1H), 4.37 – 4.16 (m, 1H), 4.08 (d, J = 7.3 Hz, 1H), 3.99 – 3.80 (m, 1H), 3.33 (t, J= 6.5 Hz, 4H), 2.52 (d, J= 11.6 Hz, 1H), 2.39 (s, 3H), 2.29 (d, J= 11.3 Hz, 1H), 2.12 (d, J= 11.1 Hz, 1H), 1.99 – 1.81 (m, 5H), 1.70 – 1.55 (m, 1H), 1.22 – 1.08 (m, 2H).

Formation N-((IR, 3S>3-(5-fluoro-2-(5-fluoro-1H-pyrrolo[2,3-b]pyridin-3-yl)- pyrimidin-4-ylamino)cyclohexyl)pyrrolidine-1-carboxamide (895)

A solution of N-((1R, 35)-3-(5-fluoro-2-(5-fluoro-1-tosyl-lH-pyrrolo[2,3-b]pyridin-3- yl)pyrimidin-4-ylamino)cyclohexyl)pyrrolidine-1-carboxamide, 67f, (11.5 g, 19.3 mmol) in TΗF (150 mL) was added sodium methoxide (4.173 g, 19.31 mmol). After stirring the reaction mixture for 2 minutes, the mixture was poured into an aqueous saturated solution of NaHCO₃. The organic phase was washed with brine, dried over MgSO₄ and the solvent was removed under reduced pressure. The resulting residue was purified by silica gel chromatography (10%MeOH/CH₂Cl₂) to afford 6.5g of the desired product. The product was converted to an HCl salt by dissolving in MeOH (100 mL) and adding 2.4 mL of 12M HCl solution at room temperature. The solution was stirred at for lhour and the HCl salt precipitated out and filtered to provide 7.05g of the HCl salt: ¹H NMR (300 MHz, DMSO) δ 9.36 (s, 2H), 9.05 (d, J= 3.0 Hz, 2H), 8.49 (d, J= 5.6 Hz, 2H), 8.41 (dd, J= 2.6, 1.4 Hz, 2H), 8.31 (d, J= 9.5 Hz, 2H), 5.92 (s, 3H), 4.24 (s, 3H), 3.64 (s, 2H), 3.18 (t, J= 6.6 Hz, 7H), 2.07 (dt, J = 22.7, 11.5 Hz, 4H), 1.87 (t, J = 12.6 Hz, 4H), 1.77 (dd, J = 8.0, 5.3 Hz, 7H), 1.65 – 1.13 (m, 8H).

PATENT

US-20120171245-A1 / 2012-07-05

INHIBITORS OF INFLUENZA VIRUSES REPLICATION

Charifson, Paul S. [US] ET AL

/////////VX-? , an Azaindolyl-Pyrimidine Inhibitor, Influenza Virus Replication, Vertex, preclinical, 1259498-06-0

O=C(NC1CCC[C@@H](C1)Nc2nc(ncc2F)\C\4=C\N=C3\N\C=C(\F)/C=C3/4)N5CCCCC5

ND 0126

April 19, 2016 10:29 am / Leave a comment

ND 0126

CAS 1240322-54-6

Molecular Formula:	C₂₉H₂₅F₃N₆O₃
Molecular Weight:	562.54241 g/mol

methyl 5-[[2-methyl-5-[[3-(4-methylimidazol-1-yl)-5-(trifluoromethyl)benzoyl]amino]phenyl]methylamino]-1H-pyrrolo[2,3-b]pyridine-2-carboxylate

5-{2-Methyl-5-[3-(4-methyl-imidazol-1-yl)-5-trifluoromethyl-benzoylamino]-benzylamino}-1H-pyrrolo[2,3-b]pyridine-2-carboxylic Acid Methyl Ester

Oribase Pharma

Nova Decision, Azasynth

Potent dual ABL/SRC inhibitors based on a 7-azaindole core with the aim of developing compds. that demonstrate a wider activity on selected oncogenic kinases. Multi-Targeted Kinase Inhibitors (MTKIs) were then derived, focusing on kinases involved in both angiogenesis and tumorigenesis processes.

Dysfunction/deregulation of protein kinases (PK) is the cause of a large number of pathologies including oncological, immunological, neurological, metabolic and infectious diseases. This has generated considerable interest in the development of small molecules and biological kinase inhibitors for the treatment of these disorders.

Numerous PK are particularly deregulated during the process of tumorigenesis. Consequently protein kinases are attractive targets for anticancer drugs, including small molecule inhibitors that usually act to block the binding of ATP or substrate to the catalytic domain of the tyrosine kinase and monoclonal antibodies that specifically target receptor tyrosine kinases (RTK) and their ligands. In solid malignancies, it is unusual for a single kinase abnormality to be the sole cause of disease and it is unlikely that tumors are dependent on only one abnormally activated signaling pathway. Instead multiple signaling pathways are dysregulated. Furthermore, even single molecular abnormalities may have multiple downstream effects. Multi targeted therapy using a single molecule (MTKI = “Multi-Targeted Kinase Inhibitors”) which targets several signaling pathways simultaneously, is more effective than single targeted therapy. Single targeted therapies have shown activity for only a few indications and most solid tumors show deregulation of multiple signaling pathways. For example, the combination of a vascular endothelial growth factor receptor (VEGFR) inhibitor and platelet derived growth factor receptor (PDGFR) inhibitor results in a cumulative antitumor efficacy (Potapova et al, Mol Cancer Ther 5, 1280-1289, 2006).

Tumors are not built up solely of tumor cells. An important part consists of connective tissue or stroma, made up of stromal cells and extracellular matrix, which is produced by these cells. Examples of stromal cells are fibroblasts, endothelial cells and macrophages. Stromal cells also play an important role in the carcinogenesis, where they are characterized by upregulation or induction of growth factors and their receptors, adhesion molecules, cytokines, chemokines and proteolytic enzymes (Hofmeister et al., Immunotherapy 57, 1-17, 2007; Raman et al, Cancer Letters 256, 137-165, 2007; Fox et al, The Lancet Oncology 2, 278-289, 2001) The receptor associated tyrosine kinase VEGFR on endothelial and tumor cells play a central role in the promotion of cancer by their involvement in angiogenesis (Cebe-Suarez et al, Cell Mol Life Sci 63, 601-615, 2006). In addition, the growth factors TGF-β, PDGF and FGF2 secreted by cancer cells transform normal fibroblasts into tumor associated fibroblasts, which make their receptors a suitable target for inhibition by kinase inhibitors (Raman et al, 2007).

Moreover, increasing evidence suggests a link between the EGF receptor (EGFR) and HER2 pathways and VEGF-dependent angiogenesis and preclinical studies have shown both direct and indirect angiogenic effects of EGFR signaling (Pennell and Lynch, The Oncologist 14, 399-411, 2009). Upregulation of tumor pro -angiogenic factors and EGFR- independent tumor-induced angiogenesis have been suggested as a potential mechanism by which tumor cells might overcome EGFR inhibition. The major signaling pathways regulated by EGFR activation are the PI3K, MAPK and Stat pathways that lead to increased cell proliferation, angiogenesis, inhibition of apoptosis and cell cycle progression. EGFR is overexpressed in a wide variety of solid tumors, such as lung, breast, colorectal and cancers of the head and neck (Cook and Figg, CA Cancer J Clin 60, 222-243 2010). Furthermore, higher expression of EGFR has been shown to be associated with metastasis, decreased survival and poor prognosis.

c-Src, a membrane-associated non receptor tyrosine kinase, is involved in a number of important signal transduction pathways and has pleiotropic effects on cellular function. c-Src integrates and regulates signaling from multiple transmembrane receptor-associated tyrosine kinases, such as the EGFR, PDGFR, IGF1R, VEGFR, HER2. Together, these actions modulate cell survival, proliferation, differentiation, angiogenesis, cell motility, adhesion, and invasion (Brunton and Frame, Curr Opin Pharmacol 8, 427-432, 2008). Overexpression of the protein c-Src as well as the increase in its activity were observed in several types of cancers including colorectal, gastrointestinal (hepatic, pancreatic, gastric and oesophageal), breast, ovarian and lung (Yeatman, Nat Rev Cancer 4, 470-480, 2004).

The activation in EGFR or KRAS in cancers leads to a greatly enhanced level of Ras- dependent Raf activation. Hence, elimination of Raf function is predicted to be an effective treatment for the numerous cancers initiated with EGFR and KRAS lesions (Khazak et al, Expert Opin. Ther. Targets 11, 1587-1609, 2007). Besides activation of Raf signaling in tumors, a number of studies implicate the activation of the Ras-Raf-MAPK signaling pathway as a critical step in vasculo genesis and angiogenesis. Such activation is induced by growth factor receptors such as VEGFR2, FGFR2 and thus inhibition of Raf activation represents a legitimate target for modulation of tumor angiogenesis and vascularization.

Although VEGFR, PDGFR, EGFR, c-Src and Raf are important targets on both tumor cells and tumor stroma cells, other kinases such as FGFR only function in stromal cells and other oncogenes often only function in tumor cells.

Protein kinases are fundamental components of diverse signaling pathways, including immune cells. Their essential functions have made them effective therapeutic targets. Initially, the expectation was that a high degree of selectivity would be critical; however, with time, the use of “multikinase” inhibitors has expanded. Moreover, the spectrum of diseases in which kinase inhibitors are used has also expanded to include not only malignancies but also immune-mediated diseases / inflammatory diseases. The first step in signaling by multi-chain immune recognition receptors is mediated initially by Src family protein tyrosine kinases. MTKI targeting kinases involved in immune function are potential drugs for autoimmune diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel diseases (Kontzias et al. , F 1000 Medicine Reports 4, 2012)

Protein kinases mentioned previously are also key components of many other physiological and pathological mechanisms such as neurodegeneration and neuroprotection (Chico et al, Nature Reviews Drug Discovery 8, 892-909, 2009), atherosclerosis, osteoporosis and bone resorption, macular degeneration, pathologic fibrosis, Cystogenesis (human autosomal dominant polycystic kidney disease…).

In WO2010/092489 and related patents/patent applications, we identified several compounds which exhibited interesting properties for such applications. However, we have discovered that some of these compounds could be enhanced in their properties by selectively working on particular regions of their structures. However, the mechanism of action of these structures on kinases was not precisely elucidated at the time of WO2010/092489’s filing and thus it was unexpectedly that we found the high activities of the structures disclosed in the present application. The subject matter of the present invention is to offer novel multi-targeted kinase inhibitors, having an original backbone, which can be used therapeutically in the treatment of pathologies associated with deregulation of protein kinases including tumorigenesis, human immune disorders, inflammatory diseases, thrombotic diseases, neurodegenerative diseases, bone diseases, macular degeneration, fibrosis, cystogenesis. The inhibitors of the present invention can be used in particular for the treatment of numerous cancers and more particularly in the case of liquid tumors such hematological cancers (leukemias) or solid tumors including but not limited to squamous cell cancer, small- cell lung cancer, non-small cell lung cancer, gastric cancer, pancreatic cancer, glial cell tumors such as glioblastoma and neurofibromatosis, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, melanoma, colorectal cancer, endometrial carcinoma, salivary gland carcinoma, renal cancer, prostate cancer, vulval cancer, thyroid cancer, sarcomas, astrocytomas, and various types of hyperproliferative diseases.

Efforts were made to improve a series of potent dual ABL/SRC inhibitors based on a 7-azaindole core with the aim of developing compounds that demonstrate a wider activity on selected oncogenic kinases. Multi-targeted kinase inhibitors (MTKIs) were then derived, focusing on kinases involved in both angiogenesis and tumorigenesis processes. Antiproliferative activity studies using different cellular models led to the discovery of a lead candidate (6z) that combined both antiangiogenic and antitumoral effects. The activity of 6z was assessed against a panel of kinases and cell lines including solid cancers and leukemia cell models to explore its potential therapeutic applications. With its potency and selectivity for oncogenic kinases, 6z was revealed to be a focused MTKI that should have a bright future in fighting a wide range of cancers.

5-{2-Methyl-5-[3-(4-methyl-imidazol-1-yl)-5-trifluoromethyl-benzoylamino]-benzylamino}-1H-pyrrolo[2,3-b]pyridine-2-carboxylic Acid Methyl Ester (6z)

The reaction was carried out as described in general procedure A using 4a (170 mg, 0.63 mmol), 3-(4-methyl-imidazol-1-yl)-5-trifluoromethyl-benzoic acid 5z (200 mg, 0.63 mmol), HATU (735 mg, 1.93 mmol), DIEA (0.56 mL, 3.22 mmol), and anhydrous DMF (16 mL). Purification by flash chromatography on silica gel (EtOAc/EtOH, 100/0 to 90/10) yielded 6z (108 mg, 30%).

¹H NMR (300 MHz, DMSO-d₆, δ) 12.05 (s, 1H), 10.41 (s, 1H), 8.42–8.34 (m, 2H), 8.20 (s, 1H), 8.16–8.04 (m, 2H), 7.670–7.62 (m, 3H), 7.22 (d, J = 8.2 Hz, 1H), 6.97 (d, J = 2.3 Hz, 1H), 6.90 (d, J = 1.9 Hz, 1H), 6.11 (t, J = 5.0 Hz, 1H), 4.25 (d, J = 5.0 Hz, 2H), 3.83 (s, 3H), 2.34 (s, 3H), 2.17 (s, 3H). MS (ESI) m/z 563.2 [M + H]⁺ and 561.2 [M – H]⁻.

Rational Design, Synthesis, and Biological Evaluation of 7-Azaindole Derivatives as Potent Focused Multi-Targeted Kinase Inhibitors

Bénédicte Daydé-Cazals, Bénédicte Fauvel, Mathilde Singer, Clémence Feneyrolles, Benoit Bestgen, Fanny Gassiot, Aurélia Spenlinhauer, Pierre Warnault, Nathalie Van Hijfte, Nozha Borjini, Gwénaël Chevé, and Abdelaziz Yasri^*

OriBase Pharma, Cap Gamma, Parc Euromédecine, 1682 rue de la Valsière, CS 17383, Montpellier 34189 CEDEX 4,France

J. Med. Chem., Article ASAP

DOI: 10.1021/acs.jmedchem.6b00087

Publication Date (Web): March 24, 2016

*E-mail: ayasri@oribase-pharma.com. Phone: (+33) 467 727 670.

http://pubs.acs.org/doi/full/10.1021/acs.jmedchem.6b00087

PATENT

WO 2010092489

https://www.google.com/patents/WO2010092489A1?cl=en

Example 91: Preparation of methyl 5-(5-(3-(trifluoromethγl)-5~(4-methyl-1 H-imidazol-1 – yl)benzamido)-2-methγlbenzylamino)-1H-pyrrolo[2,3-blpyridine-2-carboχylate (ND0126)

Step 1 : preparation of methyl 5-(3-(trifluoromethyl)-5-(4-methyl-1 H-imidazol-1 – yl)benzamido)-2-methylbenzoate

The compound is obtained using the procedures of example 88 (step 4) replacing the 4-((3-(dimethylamino)pyrrolidin-1-yl)methyl)-3-(trifluoromethyl)-benzoic acid

(Shakespeare W. C, WO2007133562) by the 3-(trifluoromethyI)-5-(4-methyl-1H- imidazol-1-yl)benzoic acid.

Step 2: preparation of 3-(tπϊluoromethyl)-N-(3-formyl-4-methylphenyl)-5-(4- methyl-1H-imidazol-1-yl)benzamide

The compound is obtained by using the procedures of examples 83 (steps 1 and 2) replacing the methyl 5-(4-((4-methylpiperazin-1-yl)methyl)benzamido)-2- methylbenzoate with the methyl 5-(3-(trifluorometny))-5-(4-metbyl-1H-imidazol-1- yl)benzamido)-2-methylbenzoate.

Step 3: preparation of methyl 5-(5-(3-(trifluoromethyl)-5-(4-methyl-1 H-imidazol- 1-yl)benzamido)-2-methylbenzylamino)-1H-pyrrolo[2,3-bJpyridine-2-carboxylate (ND0126)

The composed is obtained according to example 83 (step 3) replacing N-(3-formyl-4- methylphenyl)-4-((4-methylpiperazin~1-yl)methyl)-benzamide with the 3- (trifluoromethyl)-N-(3-formyl-4-methylphenyl)-5-(4-methyl-1 H-imidazol-1-yl)benzamide.

PATENT

WO 2014102376

REFERENCES

WO2005063747A1 *	Dec 23, 2004	Jul 14, 2005	Pfizer Italia S.R.L.	PYRROLO[2,3-b] PYRIDINE DERIVATIVES ACTIVE AS KINASE INHIBITORS, PROCESS FOR THEIR PREPARATION AND PHARMACEUTICAL COMPOSITION COMPRISING THEM
WO2008028617A1 *	Sep 4, 2007	Mar 13, 2008	F. Hoffmann-La Roche Ag	Heteroaryl derivatives as protein kinase inhibitors
WO2008124849A2 *	Apr 10, 2008	Oct 16, 2008	Sgx Pharmaceuticals, Inc.	Pyrrolo-pyridine kinase modulators
WO2008144253A1 *	May 9, 2008	Nov 27, 2008	Irm Llc	Protein kinase inhibitors and methods for using thereof

WO2014102376A1 *	Dec 30, 2013	Jul 3, 2014	Oribase Pharma	Protein kinase inhibitors
WO2014102377A1 *	Dec 30, 2013	Jul 3, 2014	Oribase Pharma	Azaindole derivatives as multi kinase inhibitors
WO2014102378A1 *	Dec 30, 2013	Jul 3, 2014	Oribase Pharma	Azaindole derivatives as inhibitors of protein kinases
US20150353540 *	Dec 30, 2013	Dec 10, 2015	Oribase Pharma	Azaindole derivatives as inhibitors of protein kinases

US2011312959

2011-12-22

Derivatives of Azaindoles as Inhibitors of Protein Kinases ABL and SRC

///////ND 0126, 1240322-54-6, PRECLINICAL

O=C(OC)c1cc2cc(cnc2n1)NCc3cc(ccc3C)NC(=O)c4cc(cc(c4)n5cc(C)nc5)C(F)(F)F

CC1=C(C=C(C=C1)NC(=O)C2=CC(=CC(=C2)N3C=C(N=C3)C)C(F)(F)F)CNC4=CN=C5C(=C4)C=C(N5)C(=O)OC

IACS -9571

March 30, 2016 11:16 am / Leave a comment

IACS-9571

TRIM24/BRPF1 bromodomain inhibitor

IACS-9571; IACS 9571; IACS9571.

Molecular Formula:	C₃₂H₄₂N₄O₈S
Molecular Weight:	642.76288 g/mol

N-[6-[3-[4-(dimethylamino)butoxy]-5-propoxyphenoxy]-1,3-dimethyl-2-oxobenzimidazol-5-yl]-3,4-dimethoxybenzenesulfonamide

BOARD OF REGENTS, UNIVERSITY OF TEXAS SYSTEM

IACS-9571 is a potent and selective inhibitor TRIM24 and BRPF1. The bromodomain containing proteins TRIM24 (Tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). IACS-9571 has low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and 14 nM, respectively). With its excellent cellular potency (EC50 = 50 nM) and favorable pharmacokinetic properties (F = 29%), IACS-9571 is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo. (J Med Chem. 2015 Jun 10. [Epub ahead of print] )

PAPER

http://pubs.acs.org/doi/abs/10.1021/acs.jmedchem.5b00405

Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor

^†Institute for Applied Cancer Science, and ^‡Core for Biomolecular Structure and Function, The University of Texas MD Anderson Cancer Center, 1881 East Road, Unit 1956, Houston, Texas 77054, United States

^§ Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center,

1515 Holcombe Boulevard

, Houston, Texas 77030, United States

J. Med. Chem., 2016, 59 (4), pp 1440–1454

DOI: 10.1021/acs.jmedchem.5b00405

Publication Date (Web): June 10, 2015

*E-mail: wpalmer@mdanderson.org. Telephone: (001) 713-745-3022. Fax: (001) 713-745-8865.

The bromodomain containing proteins TRIM24 (tripartite motif containing protein 24) and BRPF1 (bromodomain and PHD finger containing protein 1) are involved in the epigenetic regulation of gene expression and have been implicated in human cancer. Overexpression of TRIM24 correlates with poor patient prognosis, and BRPF1 is a scaffolding protein required for the assembly of histone acetyltransferase complexes, where the gene of MOZ (monocytic leukemia zinc finger protein) was first identified as a recurrent fusion partner in leukemia patients (8p11 chromosomal rearrangements). Here, we present the structure guided development of a series of N,N-dimethylbenzimidazolone bromodomain inhibitors through the iterative use of X-ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor 8i (IACS-9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC K_d = 31 nM and ITC K_d = 14 nM, respectively). With its excellent cellular potency (EC₅₀ = 50 nM) and favorable pharmacokinetic properties (F = 29%), 8i is a high-quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo.

TFA salt of 8i (106 mg, 57%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, J = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, J = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12–3.05 (m, 2H), 2.78 (d, J = 4.7 Hz, 6H), 1.77–1.63 (m, 6H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (600 MHz, DMSO-d6) δ 160.3, 160.0, 159.3, 154.1, 152.0, 148.4, 143.9, 131.8, 128.2, 126.0, 121.9, 120.5, 110.4, 109.4, 106.4, 100.6, 95.9, 95.8, 95.2, 68.9, 66.7, 56.3, 55.6, 55.4, 42.1, 27.1, 27.0, 25.6, 21.9, 20.7, 10.4. MS (ESI) m/z 644 [M + H]+.

NMR

IACS -9571

STR2

N-(6-(3-(4-(dimethylamino)butoxy)-5- propoxyphenoxy)-l,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4- dimethoxybenzenesulfonamide 2,2,2-trifluoroacetate

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ABSTRACT

251st ACS National Meeting & Exposition

13–17 March 2016

San Diego, United States

MEDI 5

Discovery and development of a potent dual TRIM24/BRPF1 bromodomain inhibitor, IACS -9571, using structure- based drug design Wylie S. Palmer 1 , wpalmer@mdanderson.org, Guillaume Poncet -Montagne 1 , Gang Liu 1 , Alessia Petrocchi 1 , N aphtali Reyna 1 , Govindan Subramanian 1 , Jay Theroff 1 , Maria Kost -Alimova 1 , Jennifer Bardenhagen 1 , Elisabetta Leo 1 , Hannah Sheppard 1 , Trang Tieu 1 , Shi Xi 1 , Yanai Zhan 1 , Shuping Zhao 1 , Michelle Barton 2 , Giulio Draetta 1 , Carlo Toniatti 1 , Philip Jones 1 , Mary Ge ck Do 1 , Jannik Andersen 1 . (1) Institute for Applied Cancer Science, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States (2) Department of Epigenetics and Molecular Carcinogenesis, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States

Bromodomains are an important class of chromatin remodeling proteins that recognize acetylated lysine residues on histone tails. As epigenetic targets they regulate gene transcription and offer a new way to treat diseas es, particularly in inflammation and oncology. The bromodomain and extra- terminal (BET) family has emerged as an important and druggable example of this class of proteins with the successful entry of small- molecule inhibitors into the clinic. Other families of bromodomains are only starting to be explored, such as the Tripartite Motif -containing 24 protein (TRIM24) and bromodomain- PHD finger protein 1 (BRPF1). Both proteins contain a dual PHD -bromo motif which have a role in recognizing specific histone mar ks. TRIM24 recognizes the dual histone marks of unmodified H3K4 and acetylated- H3K23 within the same histone tail. TRIM24 is a potent co- activator of ER -alpha and overexpression of TRIM24 has been linked to poor survival rates in breast cancer patients.

This presentation will describe the structure guided development of a series of N,N- dimethyl -benzimidazolones through the iterative use of X -ray cocrystal structures. A unique binding mode enabled the design of a potent and selective inhibitor (IACS -9571) with low nanomolar affinities for TRIM24 and BRPF1 (ITC Kd = 31 nM and ITC Kd = 14 nM, respectively). With its excellent cellular potency (EC 50 = 50 nM) and favorable pharmacokinetic properties, IACS -9571 is a high- quality chemical probe for the evaluation of TRIM24 and/or BRPF1 bromodomain function in vitro and in vivo

PATENT

WO-2016033416-A1

Synthesis of Intermediates:

N-(6-bromo-l ,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-2,2,2- trifluoroacetamide (Intermediate 1):

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Step 1 : 5-nitro-lH-benzo[d]imidazol-2(3H)-one:

To a 0 °C solution of 4-nitrobenzene- 1 ,2-diamine (44 g, 285 mmol) in 80 mL of DMF was added l, l’-carbonyldiimidazole (70 g, 428 mmol). The reaction mixture was stirred at RT for 4 h, then water (250 mL) was added. The resulting suspension was filtered, and the collected solids were washed with water (200 mL) and dried to give 5-nitro-lH- benzo[d]imidazol-2(3H)-one as a yellow solid (45 g, 88%). MS (ES+) C7H5N3O3 requires: 179, found: 180 [M+H]⁺.

Step 2: l,3-dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one:

To a solution of 5-nitro-lH-benzo[d]imidazol-2(3H)-one (55 g, 309 mmol) in 150 mL of DMF was added K2CO3 (85 g, 618 mmol), the reaction mixture was cooled to 0 °C, then iodomethane (109 g, 772 mmol) was slowly added. The reaction mixture was stirred at RT overnight, then water was added to the reaction mixture. The resulting suspension was filtered and the collected solids were washed with water (200 mL) and dried to give 1,3- dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one as a yellow solid (55 g, 86%). MS (ES+) C9H9N3O3 requires: 207, found: 208 [M+H] ⁺.

Step 3: 5-amino-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

To a solution of l,3-dimethyl-5-nitro-lH-benzo[d]imidazol-2(3H)-one (50 g, 240 mmol) in 200 mL of EtOAc under an inert atmosphere was added 10% palladium on activated carbon (5 g, 24 mmol). The reaction mixture was then charged with hydrogen and stirred at RT under an ¾ atmosphere overnight. The reaction mixture was filtered through a pad of celite then concentrated to give 5-amino-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)- one as a yellow solid (32 g, 68%). MS (ES+) C9H11N3O requires: 177, found: 178 [M+H]⁺.

Step 4: 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

To a 0 °C solution of 5-amino-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (4 g, 22.6 mmol) in 25 mL of CHCI3 and 25 mL of AcOH was slowly added drop wise bromine (3.5 g, 22.6mmol). The mixture was stirred at RT for 30 min, then concentrated and purified by silica gel chromatography (1 : 1 EtOAc/ hexanes) to afford 5-amino-6-bromo-l ,3-dimethyl- lH-benzo[d]imidazol-2(3H)-one as a yellow solid (3.2 g, 69%). MS (ES+) C₉HioBrN₃0 requires: 256, found: 257 [M+H]⁺.

Step 5: N-(6-bromo-l ,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-2,2,2- trifluoroacetamide:

To a 0 °C solution of 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol- 2(3H)-one (1.50 g, 5.9 mmol) in DCM (45 ml) was added DMAP (72 mg, 0.59 mmol), triethylamine (1.63 ml, 11.7 mmol) and trifluoroacetic anhydride (0.91 ml, 6.4 mmol). The reaction mixture was stirred for 2 h and warmed to RT. The reaction mixture was then quenched with water and the organic phase was washed with brine, dried over sodium sulfate, filtered and concentrated to give N-(6-bromo-l,3-dimethyl-2-oxo-2,3-dihydro-lH- benzo[d]imidazol-5-yl)-2,2,2-trifluoroacetamide (Intermediate 1) as a yellow solid (2.20 g, 100%). MS (ES+) CiiH₉BrF₃N302 requires: 352, found 353 [M+H]⁺.

5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (Intermediate 2, Route A):

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To a mixture of 5-amino-6-(3-(benzyloxy)phenoxy)-l,3-dimethyl-lH- benzo[d]imidazol-2(3H)-one (400 mg, 1.07 mmol) in DCM (20 mL) at -78 °C was added tribromoborane (5.3 mL, 5.3 mmol). The mixture was warmed up to room temperature gradually, then quenched by methanol dropwise, concentrated, and purified by column chromatography (20-100% EtOAc/hexanes and then 0-40% methanol/EtOAc) to give 5- amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one as a solid (240 mg, 79%). MS (ES+) C15H15N3O3 requires: 285, found: 286 [M+H]⁺.

5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (Intermediate 2, Route B):

Step 2

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Step 1: 3-[(ieri-butyldimethylsilyl)oxy]phenol:

A mixture of lH-imidazole (2.25 g, 33.1 mmol), ieri-butylchlorodimethylsilane (3.83 g, 25.4 mmol) and resorcinol (5.6 g, 51 mmol) in THF (30 ml) was stirred at 80 °C for 5 h. The resulting suspension of the cooled reaction mixture was filtered and the collected filtrate was concentrated and purified by silica-gel chromatography (20:80 to 0:100, EtOAc/hexanes) to give 3-((ieri-butyldimethylsilyl)oxy)phenol (2.78 g, 49%). MS (ES+) C12H20O2S1 requires: 224, found 225 [M+H]⁺.

Step 2: 5-amino-6-(3-((ier^butyldimethylsilyl)oxy)phenoxy)-l ,3-dimethyl-lH- benzo[d]imidazol-2(3H)-one:

A mixture of 3-((ieri-butyldimethylsilyl)oxy)phenol (1.39 g, 6.20 mmol), quinolin-8-ol (79 mg, 0.55 mmol), copper(I) chloride (20 mg, 0.21 mmol), potassium phosphate (526 mg, 2.48 mmol) and 5-amino-6-bromo-l ,3-dimethyl-lH-benzo[d]imidazol- 2(3H)-one (529 mg, 2.07 mmol) in diglyme (20 ml) in a 100 mL round-bottom flask was degassed under a nitrogen atmosphere and heated to 120 °C for 24 h. To the cooled reaction mixture was added silica gel, stirred for 2 min, then the mixture was filtered through a pad of silica gel. The collected filtrate was concentrated and purified by column chromatography (20:80 to 0: 100, EtOAc/hexanesthen 0: 100 to 40:60, MeOH/EtOAc) to give 5-amino-6-(3- ((ieri-butyldimethylsilyl)oxy)phenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (521 mg, 63%). MS (ES+) C21H29N3O3S1 requires: 399, found 400 [M+H]⁺.

Step 3: 5-amino-6-(3-hydroxyphenoxy)-l,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one:

To a 0 °C solution of 5-amino-6-(3-((ieri-butyldimethylsilyl)oxy)phenoxy)-l,3- dimethyl-lH-benzo[d]imidazol-2(3H)-one (623 mg, 1.56 mmol) in THF was added a solution of ieira-butylammonium fluoride (0.90 mL, 3.1 mmol) in THF, the reaction mixture was allowed to warm up to RT and then stirred for 1-2 h. The reaction mixture was quenched with 1 M hydrogen chloride (0.10 mL, 3.1 mmol) and then partitioned between EtOAc and water. The seperated organic layer was washed with water twice, then concentrated and purified by column chromatography (20-80% EtOAc/hexanes and 0-40% MeOH/DCM) to give 5-amino-6-(3-hydroxyphenoxy)-l ,3-dimethyl-lH-benzo[d]imidazol-2(3H)-one (120 mg, 27%) as a solid. MS (ES+) C15H15N3O3 requires: 285, found 286 [M+H]⁺.

EXAMPLE 10: N-(6-(3-(4-(dimethylamino)butoxy)-5-propoxyphenoxy)-l,3-dimethyl-2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4-dimethoxybenzenesulfonamide 2,2,2-

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To a solution of N-(6-(3-(4-aminobutoxy)-5-propoxyphenoxy)-l ,3-dimethyl-2- oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4-dimethoxybenzenesulfonamide 2,2,2- trifluoroacetate (180 mg, 0.247 mmol) in methanol (3.0 ml) was added triethylamine (0.034 ml, 0.25 mmol), acetic acid (0.028 ml, 0.49 mmol), formaldehyde (0.054 ml, 2.0 mmol), and sodium triacetoxyborohydride (131 mg, 0.618 mmol). The reaction mixture was stirred at room temperature and checked by LCMS every 30 minutes. After 3 h the reaction was complete by LCMS. The reaction was quenched with a few drops of TFA and concentrated under reduced pressure. The residue was purified by prep-HPLC using a gradient of 20-60% ACN/water containing 0.1% TFA to afford N-(6-(3-(4-(dimethylamino)butoxy)-5- propoxyphenoxy)-l,3-dimethyl-2-oxo-2,3-dihydro-lH-benzo[d]imidazol-5-yl)-3,4- dimethoxybenzenesulfonamide 2,2,2-trifluoroacetate (106 mg, 57%) as a white solid. MS (ES+) C32H42N4O8S requires: 642, found 643 [M+H]⁺. ¾ NMR (600 MHz, DMSO-ifc) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, 7 = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, 7 = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, 7 = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12-3.05 (m, 2H), 2.78 (d, 7 = 4.7 Hz, 6H), 1.77-1.63 (m, 6H), 0.95 (t, 7 = 7.3 Hz, 3H)

References

1: Palmer WS, Poncet-Montange G, Liu G, Petrocchi A, Reyna N, Subramanian G, Theroff J, Yau A, Kost-Alimova M, Bardenhagen JP, Leo E, Shepard HE, Tieu TN, Shi X, Zhan Y, Zhao S, Draetta G, Toniatti C, Jones P, Geck Do M, Andersen JN. Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor. J Med Chem. 2015 Jun 10. [Epub ahead of print] PubMed PMID: 26061247.

US-20160060260-A1

Institute for Applied Cancer Science, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States

The University of Texas MD Anderson Cancer Center | University of Texas System

The new Institute for Applied Cancer Science will be located at the south campus of M.D.

Draetta arrived at MD Anderson in 2011 to direct the Institute for Applied Cancer Science. He oversees the moon shots platforms

Department of Epigenetics and Molecular Carcinogenesis, The University of Texas, MD Anderson Cancer Center, Houston, Texas, United States

///////IACS-9571, TRIM24, BRPF1 bromodomain inhibitor, IACS-9571, IACS 9571, IACS9571, BOARD OF REGENTS, UNIVERSITY OF TEXAS SYSTEM
CAS BASE 1800477-30-8
CAS OF 1:1 TRIFLUOROACETATE 1883598-69-3

c1(cc(cc(c1)OCCC)Oc3cc2N(C(N(c2cc3NS(=O)(=O)c4cc(c(cc4)OC)OC)C)=O)C)OCCCCN(C)C

CCCOC1=CC(=CC(=C1)OC2=C(C=C3C(=C2)N(C(=O)N3C)C)NS(=O)(=O)C4=CC(=C(C=C4)OC)OC)OCCCCN(C)C

TFA salt of 8i (106 mg, 57%) as a white solid. 1H NMR (600 MHz, DMSO-d6) δ 9.46 (s, 1H), 9.30 (br-s, 1H), 7.19 (m, 2H), 7.07 (s, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.75 (s, 1H), 6.13 (t, J = 2.2 Hz, 1H), 5.71 (t, J = 2.0 Hz, 1H), 5.67 (t, J = 2.0 Hz, 1H), 3.84 (t, J = 5.9 Hz, 2H), 3.77 (m, 5H), 3.62 (s, 3H), 3.29 (s, 3H), 3.20 (s, 3H), 3.12–3.05 (m, 2H), 2.78 (d, J = 4.7 Hz, 6H), 1.77–1.63 (m, 6H), 0.95 (t, J = 7.3 Hz, 3H). 13C NMR (600 MHz, DMSO-d6) δ 160.3, 160.0, 159.3, 154.1, 152.0, 148.4, 143.9, 131.8, 128.2, 126.0, 121.9, 120.5, 110.4, 109.4, 106.4, 100.6, 95.9, 95.8, 95.2, 68.9, 66.7, 56.3, 55.6, 55.4, 42.1, 27.1, 27.0, 25.6, 21.9, 20.7, 10.4. MS (ESI) m/z 644 [M + H]+.

AVORALSTAT

March 23, 2016 11:50 am / Leave a comment

2D chemical structure of 918407-35-9

Avoralstat, BCX4161,

CAS 918407-35-9
UNII: UX17773O15

513.5513, C28-H27-N5-O5

2-Pyridinecarboxylic acid, 3-(2-(((4-(aminoiminomethyl)phenyl)amino)carbonyl)-4-ethenyl-5-methoxyphenyl)-6-(((cyclopropylmethyl)amino)carbonyl)-

3-(2-((4-Carbamimidoylphenyl)carbamoyl)-4-ethenyl-5-methoxyphenyl)-6-((cyclopropylmethyl)carbamoyl)pyridine-2-carboxylic acid

Hereditary angioedema (HAE)

Kallikrein inhibitor

BioCryst Pharmaceuticals

Biocryst Logo

BioCryst is also investigating second-generation plasma kallikrein inhibitors to avoralstat, for treating HAE (in February 2016, this program was listed as being in preclinical development).

2D chemical structure of 918407-35-9

Prevent acute attacks in patients with hereditary angioedema (HAE); Treat hereditary angioedema (HAE)

U.S. – Fast Track (Treat hereditary angioedema (HAE));
U.S. – Orphan Drug (Prevent acute attacks in patients with hereditary angioedema (HAE))

26 Feb 2016Clinical trials in Hereditary angioedema (Prevention) in USA (PO, Hard-gelatin capsule) before February 2016

24 Feb 2016Discontinued – Phase-III for Hereditary angioedema (Prevention) in France (PO, Soft-gelatin capsule)

24 Feb 2016Discontinued – Phase-III for Hereditary angioedema (Prevention) in Germany (PO, Soft-gelatin capsule)

Conditions	Interventions	Phases	Recruitment	Sponsor/Collaborators
Hereditary Angioedema\|HAE	Drug: BCX4161\|Drug: Placebo	Phase 2\|Phase 3	Recruiting	BioCryst Pharmaceuticals
Hereditary Angioedema	Drug: BCX4161\|Drug: Placebo	Phase 2	Completed	BioCryst Pharmaceuticals
Hereditary Angioedema	Drug: BCX4161	Phase 1	Completed	BioCryst Pharmaceuticals
Hereditary Angioedema	Drug: BCX4161	Phase 1	Completed	BioCryst Pharmaceuticals

Avoralstat, also known as BCX-4161, is a potent and orally active Kallikrein inhibitor and Bradykinin inhibitor. Avoralstat may be potentially useful for treatment for Hereditary angioedema. Avoralstat inhibits plasma kallikrein and suppresses bradykinin production. Bradykinin is the mediator of acute swelling attacks in HAE patients.

Selective inhibitor of plasma kallikrein that subsequently suppresses bradykinin production

Hereditary angioedema (HAE) is a serious and potentially life-threatening rare genetic illness, caused by mutations in the C1-esterase inhibitor (C1 INH) gene, located on chromosome 11q. HAE is inherited as an autosomal dominant condition, although one quarter of diagnosed cases arise from a new mutation. HAE has been classed as an orphan disease in Europe, with an estimated prevalence of 1 in 50,000. Individuals with HAE experience recurrent acute attacks of painful subcutaneous or submucosal edema of the face, larynx, gastrointestinal tract, limbs or genitalia which, if untreated, may last up to 5 days. Attacks vary in frequency, severity and location and can be life-threatening. Laryngeal attacks, with the potential for asphyxiation, pose the greatest risk. Abdominal attacks are especially painful, and often result in exploratory procedures or unnecessary surgery. Facial and peripheral attacks are disfiguring and debilitating.

HAE has a number of subtypes. HAE type I is defined by C1 INH gene mutations which produce low levels of C1 -inhibitor, whereas HAE type II is defined by mutations which produce normal levels of ineffective C1 protein. HAE type III has separate pathogenesis, being caused by mutations in the F12 gene which codes for the serine protease known as Factor XII. Diagnostic criteria for distinguishing the subtypes of HAE, and distinguishing HAE from other angioedemas, can be found in Ann Allergy Asthma Immunol 2008; 100(Suppl 2): S30-S40 and J Allergy Clin Immunol 2004; 114: 629-37, incorporated herin by reference.

Current treatments for HAE fall into two main types. Older non-specific treatments including androgens and antifibrinolytics are associated with significant side effects, particularly in females. Newer treatments are based on an understanding of the molecular pathology of the disease, namely that C1 INH is the most important inhibitor of kallikrein in human plasma and that C1 INH deficiency leads to unopposed activation of the kallikrein-bradykinin cascade, with bradykinin the most important mediator of the locally increased vascular permeability that is the hallmark of an attack.

Approved therapies include purified plasma-derived C1 INH (Cinryze®, Berinert), the recombinant peptide kallikrein inhibitor ecallantide (Kalbitor®), and the bradykinin receptor B2 inhibitor iticabant (Firazyr®). All of the currently available targeted therapies are administered by intravenous or subcutaneous injection. There is currently no specific targeted oral chronic therapy for HAE.

There are many delivery routes for active pharmaceutical ingredients (APIs). Generally, the oral route of administration is favored. Oral administration provides a number of advantages, such as, but not limited to, patient convenience, flexibility of timing of administration, location of administration and non-invasiveness. Oral administration also provides more prolonged drug exposure compared with intermittent intravenous infusion, which may be important for drugs with schedule-dependent efficacy. For example, a drug with a short half-life can achieve a greater exposure time by either continuous infusion or by continuous oral dosing. The use of oral therapy further has the potential to reduce the cost of healthcare resources for inpatient and ambulatory patient care services.

In the pharmaceutical arts, it is known that a number of APIs cannot be administered effectively by the oral route. The main reasons why these compounds cannot be administered by the oral route are: a) rapid enzymatic and metabolic degradation; b) chemical and/or biological instability; c) low solubility in aqueous medium; and/or d) limited permeability in the gastrointestinal tract. For such compounds, non-oral routes of delivery, such as parenteral administration, mainly via intramuscular or subcutaneous injections, may be developed. However, non-oral administration poses a disadvantage for the patient as well as healthcare providers, and for this reason, it is important to develop alternative routes of administration for such compounds, such as oral routes of administration.

While the oral route of administration is the most convenient for the patient and the most economical, designing formulations for administration by the oral route involves many complications. Several methods are available to predict the ease by which an API may be formulated into a formulation suitable for administration by the oral route. Such methods include, but are not limited to, and Lipinski rule (also referred to as the Rule of Five) and the Biopharmaceutical Drug Disposition Classification System (BDDCS).

The BDDCS divides APIs into four classifications, depending on their solubility and permeability. Class I APIs have high solubility and high permeability; Class II APIs have low solubility and high permeability; Class III APIs have high solubility and low permeability; and Class IV APIs have low solubility and low permeability. APIs in higher classes in the BDDCS face greater challenges in formulating into an effective, pharmaceutically acceptable product than those in lower classes. Of the four classes, APIs falling into Class IV are the most difficult to formulate into a formulation for administration by the oral route that is capable of delivering an effective amount of the API as problems of both solubility and permeability must be addressed (note the BDDCS does not inherently address chemical stability). The role of BDDCS in drug development is described generally in L.Z. Benet J Pharm Sci. 2013, 102(1), 34-42.

Lipinski’s rule (described in Lipinski et al. Adv. Drug Deliv. Rev. 46 (1-3): 3-26) states, in general, that in order to develop a successful formulation for administration by the oral route, an API can have no more than one violation of the following criteria:

i) not more than 5 hydrogen bond donors (nitrogen or oxygen atoms with one or more hydrogen atoms)

ii) not more than 10 hydrogen bond acceptors (nitrogen or oxygen atoms) iii) a molecular mass less than 500 daltons

iv) an octanol-water partition coefficient log P not greater than 5.

J. Zhang et al. Medicinal Chemistry, 2006, 2, 545-553, describes a number of small molecule amidine compounds which have activity as inhibitors of kallikrein. The molecules described in this document fall into Class IV of the BDDCS as described above. The compounds are poorly soluble in aqueous and physiological fluids, and are poorly permeable as demonstrated by oral dosing in rats and in vitro experiments with Caco-2 cells.

Furthermore, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, one of the compounds described in Zhang et al., is a Class IV API and violates criteria iii) and iv) as set forth in the Lipinski Rule.

Furthermore, the compounds described in Zhang et al., including 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, exhibit poor stability with respect to oxidation in air, to light

(photodegradation) and in aqueous and physiological fluids, as well as to elevated temperatures.

Therefore, the compounds described by Zhang et al. including, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid, not only exhibit poor solubility and permeability characteristics, but also poor stability characteristics. As a result, such compounds are predicted to be especially difficult to formulate into an effective, orally deliverable

pharmaceutical composition that is capable of delivering an effective amount of the compound to a subject.

Polymorphism, the occurrence of different crystal forms, is a property of some molecules. A single molecule may give rise to a variety of polymorphs having distinct crystal structures and physical properties, such as, but not limited to, melting point, thermal behaviors (e.g. measured by thermogravimetric analysis (TGA), or differential scanning calorimetry (DSC), x-ray diffraction pattern, infrared absorption fingerprint, and solid state NMR spectrum. One or more of these techniques may be used to distinguish different polymorphic forms of a compound.

Discovering new polymorphic forms and solvates of a pharmaceutical product can provide alternate forms of the compound that display a number of desirable and advantageous properties, such as, but not limited to, ease of handling, ease of processing, ease of formulation, storage stability, and/or ease of purification. Further, new polymorphic forms and solvates of a pharmaceutically useful compound or salts thereof may further provide for improved pharmaceutical products, by providing compounds that are more soluble in a set of pharmaceutical excipients. Still further, the provision of new polymorphic forms and solvates of a pharmaceutically useful compound or salts thereof enlarges the repertoire of compounds that a formulation scientist has available for formulation optimization, for example by providing a pharmaceutical product with different properties, such as, but not limited to, improved processing characteristics, improved handling characteristics, improved solubility profiles, improved dissolution profile and/or improved shelf-life. Therefore, there is a need for additional polymorphs of pharmaceutically useful compounds, such as, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6- (cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid and the compounds disclosed herein.

In one aspect, the present invention provides an oral formulation that is capable of delivering an effective amount of the amidine compounds described by Zhang et al. to a subject. In particular, the present invention provides an oral formulation that is capable of delivering an effective amount of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid to a subject. In one specific aspect, the 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid is present in a particular crystal form designated Form A. In light of the art suggesting the difficulties in formulating such an oral formulation, this result was unexpected.

As described herein, the amidine compounds described in Zhang et al., including, but not limited to, 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6- (cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (specifically including particular crystal Form A), may now be conveniently used in oral administration and further used in oral administration for the treatment of a number of diseases and conditions in a subject, such as, but not limited to, HAE as described herein.

Avoralstat

May 16 is HAE awareness day
See BioCryst’s video regarding HAE to learn more

Avoralstat is being developed as an oral prophylactic treatment for patients suffering from Hereditary Angioedema (HAE). Avoralstat inhibits plasma kallikrein and suppresses bradykinin production. Bradykinin is the mediator of acute swelling attacks in HAE patients.

In May 2014 BioCryst, announced that the OPuS-1 (OralProphylaxiS-1) Phase 2a proof of concept clinical trial met its primary efficacy endpoint, several secondary endpoints and all other objectives established for the trial. OpuS-1 enrolled 24 HAE patients with a history of HAE attack frequency of at least 1 per week. Treatment with avoralstat demonstrated a statistically significant mean attack rate reduction of 0.45 attacks per week versus placebo, p<0.001. The mean attack rate per week was 0.82 on BCX4161 treatment, compared to 1.27 on placebo.

In December 2014, BioCryst initiated enrollment in OPuS-2 (Oral ProphylaxiS-2). OPuS-2 is a blinded, randomized, 12-week, three-arm, parallel cohort design trial evaluating the efficacy and safety of two different dose regimens of avoralstat administered three-times daily, 300 mg and 500 mg, compared with placebo. The primary efficacy endpoint for the trial will be the mean angioedema attack rate, which will be reported for each avoralstat dose group compared to placebo. The trial is being conducted in the U.S., Canada and Europe. On October 8, 2015, announced that it has completed enrollment of approximately 100 HAE patients with a history of moderately frequent to very frequent attacks in OPuS-2. BioCryst expects to report the OPuS-2 trial results in early 2016.

PATENT

WO200234711

http://www.google.com/patents/WO2002034711A1?cl=en

PATENT

WO2015134998

PATENT

WO2016029214

Examples

Example 1 – Synthesis of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl- phenyll-6-(cvclopropylmethyl-carbarnoyl)-pyridine-2-carboxylic acid

The synthesis of the above compound and intermediates is described below. In this section, the following abbreviations are used:

The synthesis of starting material, (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) is described in Scheme 1.

f ^0HCY ° ^ΒΓΥΥ°

Preparation of 6-bromobenzofdl[1,3ldioxole-5-carbaldehvde (1b)

1a 1b

To a mixture of piperonal (1a) (498 g, 3.32 mol) in glacial acetic acid (1000 mL) was added a solution of bromine (200 mL, 3.89 mol) in glacial acetic acid (500 mL) over a period of 30 min and stirred at room temperature for 24h. The reaction mixture was poured into water (2000 mL) and the solid that separated was collected by filtration. The solid was dissolved in boiling ethanol (4000 mL) and cooled to room temperature. The solid obtained on cooling was collected by filtration to furnish 6-bromobenzo[d][1 ,3]dioxole-5-carbaldehyde (lb) (365 g, 48 %) as a white solid, MP 126 °C; HNMR (300 MHz, DMSO-d₆): δ 10.06 (s, 1 H), 7.42 (s,1 H), 7.29 (s, 1 H), 6.20 (d, J=12.3, 2H); IR (KBr) 3434, 2866, 1673,1489, 1413, 259, 1112, 1031 , 925 cm^“1; Analysis calculated for CeH5BrO3.O 25H C, 41.15; H, 2.37; Found: C, 41.07; H, 2.11.

Preparation of 2-bromo-5-hvdroxy-4-methoxybenzaldehyde (1c)

1c

A solution of potassium tert-butoxide (397 g, 3.36 mol) in DMSO (1.5 L) was heated at 50 °C for 30 min. Methanol (1.5 L) was added to it and continued heating at 50 °C for additional 30 min. To the hot reaction mixture was added 6-bromo-benzo[d][1,3]dioxole-5-carbaldehyde (1 b) (350g, 1.53 mol) and continued heating at 50 °C for 30 min. The reaction mixture was cooled to room temperature and quenched with water (2.3 L) and sodium hydroxide (61.2 g, 1.53 mol). The reaction mixture was washed with ether (2 x 1.5 L), acidified to pH 2 using cone. HCI and extracted with ethyl acetate ( 1 L). The ethyl acetate layers were combined and concentrated under vacuum to dryness. The residue obtained was treated with water (1.5 L) and ethyl acetate (1 L). The solid obtained was collected by filtration to furnish 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (97 g, 27.5% as a first crop). The layers from the filtrate were separated and aqueous layer was extracted with ethyl acetate (200 ml_). The ethyl acetate layers were combined dried over MgS0₄ and concentrated under vacuum to dryness to furnish 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (192 g, 54.4%, second crop) as an orange solid, MP 108 °C; ‘HNMR (300MHz, DMSO-cfe): S 10.00 (s, 1 H), 9.92 (s,1 H), 7.27 (s, 1 H), 7.26 (s, 1 H), 3.93 (s, 3H); IR (KBr) 3477, 2967, 2917,

2837, 2767, 2740, 1657, 1595, 1428, 1270, 1210, 1164, 1022 cm^“‘; Analysis calculated for C₈H₇Br03_.H₂0: C, 38.58; H, 3.64: Found: C, 38.60; H, 3.60.

Preparation of 5-(benzyloxy)-2-bromo-4-methoxybenzaldehvde ( d)

To a solution 2-bromo-5-hydroxy-4-methoxybenzaldehyde (1c) (120 g, 520 mmol) in DMF (1000 mL) was added potassium carbonate (79 g, 572 mmol) and benzyl bromide (68 mL, 572 mmol). The reaction mixture was stirred at room temperature overnight and quenched with water (3000 mL). The solid obtained was collected by filtration, washed with ether and dried under vacuum to furnish 5-(benzyloxy)-2-bromo-4-methoxybenzaldehyde (1d) (113.19 g, 67.9%) as a white solid, MP 144 °C;¹HNMR (300 MHz, DMSO-c/₆): δ 10.06 (s, 1H), 7.47-7.34 (m, 7H), 5.17 (s, 2H), 3.92 (s, 3H); IR (KBr) 2898, 2851 , 1673, 1592, 1502, 1437, 1402, 1264, 1210, 1158, 1017, 754 cm^“1; Analysis calculated for C ₅H₁₃Br0₃: C, 56.10; H, 4.08; Found: C, 55.44; H, 4.08.

Preparation of 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e)

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1d 1e

To a solution of 5-(benzyloxy)-2-bromo-4-methoxybenzaldehyde (1d) (100 g, 311 mmol) in

ethanol (1500 mL) was added triethyl orthoformate (103 mL, 622 mmol), ammonium nitrate

(7.5 g, 93.3 mmol) and stirred at room temperature overnight. The reaction mixture was

treated with ether (1200 mL) and stirred for 15 min before filtration. The filtrate was

concentrated under vacuum to dryness to give 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e) (134 g) as a brown syrup; The product was used in the next step

without further purification; ¹H N R (300 MHz, DMSO-cf₆) δ 7.45 – 7.37 (m, 4H), 7.36 – 7.33

(m, 1 H), 7.17 – 7.14 (m, 1 H), 7.10 (s, 1 H), 5.10 (s, 2H), 3.80 (s, 3H), 3.58 – 3.33 (m, 5H),

1.13 – 1.07 (m, 6H); IR (KBr) 2974, 2879, 1601 , 1503, 1377, 1260, 1163, 1060 cm^“1;

Analysis calculated for C₁₉H₂₃Br0₄: C, 57.73; H, 5.86; Found: C, 57.21 ; H, 5.94.

acid (1fi

To a solution of 1-(benzyloxy)-4-bromo-5-(diethoxymethyl)-2-methoxybenzene (1e) (120 g,

300 mmol) in dry ether (1000 mL) at -78 °C was added n-butyllithium (1.6 M solution in

hexanes, 244 mL, 390 mmol) over a period of 30 min and further stirred at -78 °C for 30 min.

A solution of tri-n-butylborate (110 mL, 405 mmol) in dry ether (300 mL) was added to this

solution at -78 °C over a period of 30 min. The reaction mixture was further stirred for 2 h at -78 °C and warmed to 0 °C. The reaction mixture was quenched with 3N HCI (300 mL) at 0

°C and heated at reflux for 1 h. After cooling to room temperature, the solid obtained was

collected by filtration washed with water (250 mL) dried in vaccum to afford (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (30.85 gm, 37.6% as a white solid. The organic

layer from above filtrate was extracted with 1.5 N NaOH (3 x 200 mL). The combined basic

extracts were acidified with cone. HCI (pH about 4). The solid obtained was collected by

filtration, washed with water and dried under vacuum to furnish a second crop of (4-(benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (22.3 g, 26%) as a light orange solid

MP 158 °C; ¹H NMR (300 MHz, DMSO-cfe) δ 10.08 (s, 1 H), 7.52 (s, 1 H), 7.48 – 7.33 (m, 5H),

7.24 (s, 1H), 5.18 (s, 2H), 3.89 (s, 3H); ¹H NMR (300 MHz, DMSO-d₆/D₂0) δ 10.06 (s, 1H),

7.52 (s, 1H), 7.49 – 7.32 (m, 5H), 7.23 (s, 1 H), 5.18 (s, 2H), 3.89 (s, 3H); MS (ES+) 309.1 (M+Na); IR (KBr) 3335, 2937, 1647, 1545, 1388, 1348, 1268, 1146, 1095 cm^-1; Analysis calculated for C₁₅H₁₅BO₅.0.25H₂O: C, 62.00; H, 5.38; Found: C, 61.77; H, 5.19.

Synthesis of methyl-6-(cvclopropylmethylcarbamoyl¾-3-ftrifluoromethylsulfonyloxyVpicolinate

The synthesis of the intermediate methyl 6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethyl sulfonyloxy)picolinate (2h) is described in Scheme 2.

Preparation of 2-bromo-3-hvdroxy-6-methylpyridine (2b)

H₃C N Br

2a 2b

To a solution of 3-hydroxy-6-methylpyridine (2a) (3000 g, 27.5 mol) in pyridine (24 L) cooled to 15 °C was added a solution of bromine (4.83 kg, 1.55 L, 30.2 mol) in pyridine (3 L) over a period of 50 min maintaining the internal temperature between 20 to 25 ^DC. After stirring for 19 h at room temperature the solvent was removed under vacuum and the residue was triturated with water. The solid separated was collected by filtration, washed with water and dried under vacuum to give 2-bromo-3-hydroxy-6-methylpyridine (2b) (3502 g, 67.7 %) as a light brown solid which was used as such without further purification; ¹H NMR (300 MHz, DMSO-d₆) δ 10.43 (s, 1H), 7.18 (d, J = 8.0 Hz, 1 H), 7.08 (d, J

MS (ES+) 188.35, 186.36 (M+1).

(2c)

2b ^2c

A mixture of 2-bromo-3-hydroxy-6-methylpyridine (2b) (3000 g, 15.96 mol), anhydrous potassium carbonate (3308 g, 23.94 mol), and iodomethane (2.491 kg, 1.09 L, 17.556 mol) in 30 L of acetone was heated at 40 °C overnight. The reaction mixture was cooled to room temperature and filtered through Celite. Evaporation of the solvent followed by silica gel chromatography (Hexane: ethyl acetate = 7:3) afforded the desired compound, 2-bromo-3-methoxy-6-methylpyridine (2c) which was used as such for the next step; ¹H NMR (300 MHz, DMSO-cfe) δ 7.42 (dd, J = 8.3, 1.5 Hz, 1H), 7.29 – 7.19 (m, 1H), 3.84 (d, J = 1.6 Hz, 3H), 2.37 (d, J = 1.7 Hz, 3H).

2c

2d

To a solution of 2-bromo-3-methoxy-6-methylpyridine (2c) (310 g, 1.53 mol) in 6000 mL of water at 60 °C was added KMnO, (725 g, 4.59 mol) in small portions over a 90 min period with vigorous mechanical stirring. A dark purple solution resulted. This solution was kept at 90 °C for a further 3 h and filtered through Celite while still hot to give a colourless filtrate.

After cooling, the aqueous solution was acidified to pH 1-2 by adding 6 N HCI. The white solid obtained was collected by filtration to give on drying 6-bromo-5-methoxy-2-pyridinecarboxylic acid (2d) (302g, 85%) of product, which was used as such in the next reaction without further purification. An analytical sample was obtained by recrystallization from methanol to give 6-bromo-5-methoxy-2-pyridinecarboxylic acid; ¹H NMR (300 MHz, DMSO-tfe) δ 7.40 – 7.28 (m, 1H), 7.17 (d, J = 8.3 Hz, 1 H), 3.83 (d, J = 1.7 Hz, 3H).

Preparation of 6-bromo-N-(cvclopropylmethyl)-5-methoxypicolinamide (2e)

To a solution of 6-bromo-5-methoxy-2-pyridinecarboxylic acid (2d) (12 g, 52 mol) in pyridine (70 mL) was added EDCI (11.5 g, 59 mmol) and cyclopropylmethylamine (3.6 g, 52 mmol). The reaction mixture was stirred at room temperature overnight and then concentrated under vacuum. The reaction mixture was diluted with water (100 mL) and ethyl acetate (100 mL). The organic layer was separated and the water layer was extracted with ethyl acetate (2 x 100 mL). The organic layers were combined and washed with water (2 x 50 mL), brine (500 mL), dried over magnesium sulphate, filtered and concentrated under vacuum to furnish 10.43g of crude product. The crude product was converted into a slurry (silica gel 20 g) and purified by flash column chromatography (silica gel 230 g, eluting with 0-100% ethyl acetate in hexane) to yield compound 6-bromo-N-(cyclopropylmethyl)-5-methoxypicolinamide (2e) (8.02 g, 54%) as off white solid, mp 67-70 °C; ¹HNMR (300 MHz, DMSO-d₆) δ 8.51 (t, J = 5.8, 1 H), 8.02 (d, J = 8.4, 1 H), 7.65 (d, J = 8.5, 1 H), 3.96 (s, 3H), 3.14 (t, J = 6.5, 2H), 1.11 -0.99 (m, 1 H), 0.47 – 0.36 (m, 2H), 0.27 – 0.20 (m, 2H); MS (ES+) 307.0, 309.0 (100%

M+Na)

Preparation of methyl 6-(cvclopropylmethylcarbamoyl)-3-methoxypicolinate (2f)

To a solution of 6-bromo-N-(cyclopropylmethyl)-5-methoxypicolinamide (2e) (7.5 g, 27.6 mol) in methanol (300 mL) in a 2-L stainless steel bomb was added Pd(OAc)₂(750 mg), 1 ,1-bis(diphenylphosphino)-ferrocene (750 mg), and triethylamine (3.9 mL, 27.6 mmol). The reaction mixture was vacuum flushed and charged with CO gas to 150 psi. The reaction mixture was and heated with stirring at 150°C overnight and cooled to room temperature. The catalyst was filtered through a pad of celite, and concentrated to dryness to furnish crude product. The crude was purified by flash column chromatography (silica gel 150 g,

eluting with, 0%, 5%, 10%, 20%, 30%, 50% ethyl acetate/hexanes (250 mL each) as eluents to give methyl 6-(cyclopropylmethyl-carbamoyl)-3-methoxypicolinate (2f) (6.29 g, 86.1 %) as a salmon coloured solid, MP 107 °C; ¹HNMR (300 MHz, DMSO-cfe) δ 8.28 (t, J = 6.0, 1H), 7.91 (d, J = 8.8, 1H), 7.55 (d, J = 8.8, 1 H), 3.68 (s, 3H), 3.64 (s, 3H), 2.90 (t, J = 6.5, 2H), 0.89 – 0.68 (m, 1 H), 0.26 – 0.09 (m, 2H), 0.08 – 0.00 (m, 2H); MS (ES+) 287.1 (M+Na); IR (KBr) 3316, 2921 , 1730, 1659, 1534, 1472, 1432, 1315, 1272, 1228, 1189, 1099, 1003, 929, 846, 680 cm^“1; Analysis calculated for C₁₃H_{16 2}0₄: C, 59.08; H, 6.10; N, 10.60; Found: C, 58.70; H, 5.97; N, 10.23.

Preparation of 6-(cvclopropylmethylcarbamoyl 3-hvdroxypicolinic acid (2q)

2f 2g

Aluminium chloride method:

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-methoxypicolinate (2f) (0.16 mmol) in dichloromethane (840 mL) was added AICI₃ (193 g, 1.5 mol). The reaction mixture was heated at reflux for 12 h under nitrogen. After slowly adding ~2L of 1 N HCI, the organic layer was separated. The aqueous layer was re-extracted several times with ethyl acetate/DME. The combined organic layer was washed with brine, dried (MgSO.4), and evaporated in vacuo to furnish crude 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid. To a solution of 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid was added a solution of acetyl chloride (1 10 mL) in methanol (1.1 L). The reaction mixture was stirred for 12 h at room temperature and then concentrated to dryness in vacuo. After co-evaporating once with methanol, the compound was purified by flash-column chromatography (silica gel, 500 g, eluted with chloroform and 3% methanol in chloroform) to furnish 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g).

Boron tribromide method:

To a stirring solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-ethoxypicolinate (2f) (58.0 g, 208 mmol) was added BBr₃ (79 mL, 834 mmol) in CH₂CI₂ (1.3 L) at 0-5 °C. The reaction mixture was allowed to warm to room temperature and stirred for 18h. The reaction mixture was evaporated to dryness and anhydrous methanol (1 L) was added to the light yellowish solid residue. Insoluble solid was collected by filtration (36 g). Mother liquor was evaporated and co-evaporated with MeOH (2 x 200 mL). The insoluble solid (36 g) was treated with MeOH (500 mL) and acetyl chloride (50 mL) and stirred at room temperature for 18 h (at this point reaction mixture was clear). The mixture was evaporated to dryness and diluted with water and extracted with EtOAc. White solid that separated out from EtOAc layer was collected by filtration, washed with water (2 x 20 mL), dried in vacuo at 50 °C to afford 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g) (5.36 g, 10 %) as a white solid, MP 92-95 °C. ¹HNMR (DMSO-cfe) δ 11.04 (s, 1 H, exchangeable with D₂0), 8.37 (t, J = 6.0, 1 H, exchangeable with D₂0), 8.12 (d, J = 8.7 Hz, 1 H), 7.57 (d, J = 8.7 Hz, 1 H), 3.90 (m, 3 H), 3.15 (m, 2 H), 1.04 ( m, 1 H), 0.41 (m, 2 H), 0.24 (m, 2 H). IR (KBr): 3346, 3205, 1684 cm^“1; MS (ES+): 251.1 (M+1); Analysis calculated for C₁₂H₁₄N2O4.0.1 H₂O: C, 57.18; H, 5.67; N, 11.14; Found: C, 57.11 ; H, 5.61; N, 11.09.

Preparation of methyl-6-(cvclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy) picolinate (2h

To a solution of 6-(cyclopropylmethylcarbamoyl)-3-hydroxypicolinic acid (2g) (28 mmol) in DMF (200 mL) were added triethylamine (12 mL, 84 mmol) and N-phenyl-bis(trifluoromethanesulfonimide) (12 g, 34 mmol). The reaction mixture was stirred for 1.5 h at room temperature and then poured into ice. After diluting with water and extracting with ethyl acetate, the aqueous phase was re-extracted, and then the combined organic layer was washed with water and concentrated under vacuum to give methyl-6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy)picolinate (2h), which was used in the next step without purification.

¹H NMR (300 MHz, CDCI₃) δ 8.50 (d, J = 8.6, 1 H), 8.07 (s, 1 H), 7.88 (d, J = 8.6, 1 H), 4.09 (d, J = 12.6, 3H), 3.48 – 3.24 (m, 2H), 1.18 – 1.01 (m, 1 H), 0.69 – 0.44 (m, 2H), 0.42 – 0.20 (m, 2H). MS (ES*): 405.17, 100%, M+Na.

Synthesis of 3-f2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyll-6-(cvclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid:

The synthesis of 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (3i) is described as shown in Scheme 3.

3-f4-Benzyloxy-2-formyl-5-methoxy-phenylV6-(cvcloDroDvlmethvl-carbarnovn-pyridine-2-carboxylic acid methyl ester (3a)

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3a

To a solution of methyl-6-(cyclopropylmethylcarbamoyl)-3-(trifluoromethylsulfonyloxy)

picolinate (2h) (24.3g, 63 mmol) in DME (225 mL) were added water (25 mL), (4- (benzyloxy)-2-formyl-5-methoxyphenyl)boronic acid (1f) (27.3 g, 95 mmol), NaHC0₃(15.9 g,

5 189 mmol), and bis(triphenylphosphine)palladium(ll) chloride (0.885 g). The reaction

mixture was stirred at 70°C overnight under nitrogen. After extracting with ethyl acetate, the organic layer was washed with water and brine and dried (MgSO^), and then concentrated

under vacuum. The compound was purified by flash-column chromatography (silica gel, 300 g, eluting with 10%, 20%, 30% and 40% ethyl acetate in hexane) to furnish 3-(4-benzyloxy- 10 2-formyl-5-methoxy-phenyl)-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid

methyl ester (3a) (25 g, 83%) as off white solid, MP 48-50°C: ¹H NMR (300 MHz, DMSO-cfe) δ 9.61(s, 1 H), 8.40 (d, J= 7.9 Hz, 1H), 8.14 (t, J= 5.0 Hz, 1H), 7.87 (d, J= 8.1 Hz, 1 H), 7.58

(s, 1H), 7.54-7.30 (m, 5H), 6.71 (s, 1 H), 5.24 (s, 2H), 3.93 (s, 3H), 3.70 (s, 3H), 3.45-3.34 (m,

2H), 1.19-1.05 (m, 1 H), 0.64-0.54 (m, 2H), 0.37-0.30 (m, 2H); IR ( Br) 1735, 1678, 1594,

15 1513, 1437, 1283, 1217, 1141, 1092 cm^“1; MS (ES+) 497.29 (M+Na); Analysis calculated for

C₂₇H_2eN₂0₆: C, 68.34; H, 5.52; N, 5.90; Found; C, 68.16; H, 5.62; N, 5.80.

2-(6-(Cvclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-vn-4-methoxy-5- vinylbenzoic acid (3b)

To a solution of 3-(4-benzyloxy-2-formyl-5-methoxy-phenyl)-6-(cyclopropylmethyl- carbamoyl)-pyridine-2-carboxylic acid methyl ester (3a) (24g, 50.6 mmol) in acetonitrile (50

mL), 2-methyl-2-propanol (350 mL), and water (125 mL) were added sodium dihydrogen

phosphate (12.5 g) and 2-methyl-2-butene (55 mL, 519 mmol). The reaction mixture was cooled in an ice bath and then sodium chlorite (28 g) was added. After stirring for 1 h, the reaction mixture was extracted with ethyl acetate and washed with water. The aqueous layer was re-extracted and then the combined organic layers were dried (MgS0₄). The solvent was evaporated in vacuo to furnish 5-(benzyloxy)-2-(6- ((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxybenzoic acid (3b) (29 g) which was used for the next step. MS (ES⁺): 513.24, (M+Na(; (ES ): 489.26, M-1.

Methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxytoarbonyltohenyl)-6-(cvclopropylmethylcarbamovnpicolinate (3c)

To a mixture of 5-(benzyloxy)-2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxy-carbonyl)pyridin-3-yl)-4-methoxybenzoic acid (3b) (31 g, 63.2 mmol), and triethylamine (17.7 mL, 126.4 mmol) in dichloromethane (300 mL), was added MEM-chloride (9.03 mL, 79 mmol), and stirred at room temperature overnight. The reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was washed with water and dried over MgS04, filtered and concentrated in vacuo. The residue was purified by flash column chromatography (silica gel, 40 g) to furnish methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)phenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3c) (32.8 g, 89%) as a thick gum; H NMR (300 MHz, CDCI₃) δ 8.35 (d, J = 8.0 Hz, 1 H), 8.15 (t, J = 5.7 Hz, 1 H), 7.78 (d, J = 8.0 Hz, 1H), 7.71 (s, 1H), 7.49 (d, J = 6.8 Hz, 2H), 7.36 (ddd, J = 7.5, 14.8, 22.4 Hz, 3H), 6.66 (s, 1 H), 5.37-5.13 (m, 4H), 3.90 (s, 3H), 3.69 (s, 3H), 3.60-3.49 (m, 2H), 3.49 (s, 2H), 3.39 (dd, J = 4.4, 8.4 Hz, 2H), 3.34 (s, 3H), 1.19-1.00 (m, 1H), 0.57 (q, J = 5.8 Hz, 2H), 0.38-0.25 (m, 2H). MS (ES⁺): 601.24 (M+Na); (ES^“): 577.27 (M-1);¹H NMR (300 MHz, DMSO-cfe) δ 8.69 (t, 7 = 6.1 Hz, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1 H), 7.63 (s, 1H), 7.41 (m, 5H), 6.92 (s, 1 H), 5.20 (m, 4H), 3.83 (s, 3H), 3.57 (s, 3H), 3.44 (m, 2H), 3_:33 (m, 2H), 3.21 (m, 5H), 1.14 (m, 1H), 0.44 (m, 2H), 0.27 (m, 2H). IR (KBr):

1732, 1671 cm^“1. MS (ES+): 601.1(M+Na); Analysis calculated for C₃₁H ₂Oe: C, 64.35; H, 5.92; N, 4.84; Found: C, 64.27; H, 6.04; N, 4.79.

Methyl 6-(cvclopropylmethylcarbamoyl)-3-(4-hvdroxy-5-methoxy-2-(((2-methoxyethoxy¾methoxy)carbonyl)phenyl)picolinate (3d)

3c 3d

To a solution of methyl 3-(4-(benzyloxy)-5-methoxy-2-(((2-methoxyethoxy)methoxy)-carbonyl)phenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3c) (32.8 g, 56.68 mmol) in ethanol (650 mL) was added 10% Pd/C (4 g) and hydrogenated at 45 psi for 5 h. The catalyst was removed by filtration through Celite and the filtrate was concentrated under vacuum to yield methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)phenyl)picolinate (3d) (31.87 g, 86%), which was pure enough to be used as such for the next step. An analytical sample of methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy) methoxy)carbonyl)phenyl)picolinate (3d) was obtained by purification of 350 mg of above crude using flash column chromatography (silica gel, eluting with ethyl acetate in hexane) to afford methyl 6-(cyclopropylmethyl-carbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)-phenyl)picolinate (3d) as a clear gum; ¹HNMR (300 MHz, DMSO-d₆) δ 9.74 (s, 1 H), 8.68 (t, J = 6.1 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1 H), 7.95 (d, J = 8.0 Hz, 1H), 7.47 (s, 1H), 6.83 (s, 1H), 5.19 (s, 2H), 3.77 (m, 3H), 3.58 (s, 3H), 3.44 (m, 2H), 3.34 (m, 2H), 3.21 (m, 5H), 1.04 (m, 1 H), 0.44 (m, 2H), 0.27 (m, 2H); IR (KBr): 1731 , 1664 cm^‘1. MS (ES^*): 489.0 (M+1); Analysis calculated for C^e^O,,: C, 59.01; H, 5.78; N, 5.73; Found: C, 58.92; H, 6.15; N, 5.29.

6-(Cvclopropylmethylcarbamovn-3-(5-methoxy-2-(((2-methoxyethoxy^methoxy)-carbonyl)-4- (trifluoromethylsulfonyloxy)phenyl)picolinate (3e)

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-(4-hydroxy-5-methoxy-2-(((2- methoxyethoxy) methoxy)carbonyl)phenyl)picolinate (3d) (14.3 g, 29.3 mmol) in dichloromethane (150 mL) were added pyridine (12 mL, 146 mmol) and triflic anhydride (7.5 mL g, 44 mmol). After stirring overnight at room temperature under N₂. the reaction mixture was poured into ice water and then extracted twice with dichloromethane. After washing the combined organic extracts with water and drying (MgS0 ), the solvent was evaporated in vacuo. The compound was purified by flash chromatography over silica gel column using ethyl acetate: hexane to afford methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2- methoxyethoxy)methoxy)-carbonyl)-4-(trifluoromethylsulfonyloxy)phenyl)picolinate (3e) (1 g, 93%); H NMR (300 MHz, CDCy a 8.41 (d, J = 8.0, 1H), 8.17 (s, 1H), 8.03 (s, 1H), 7.79 (d, J = 8.0, 1 H), 6.82 (s, 1H), 5.32 (q, J = 6.1, 2H), 3.97 (s, 3H), 3.74 (s, 3H), 3.67 – 3.57 (m, 2H), 3.55 – 3.45 (m, 2H), 3.41 (dd, J = 8.2, 14.5, 2H), 3.34 (s, 3H), 1.36 – 1.17 (m, 1H), 0.58 (d, J = 7.1 , 2H), 0.33 (d, J = 5.1 , 2H).

Methyl 6-(cvclopropylmethylcarbamoyl)-3-(5-methoxy-2-f((2-methoxyethoxy)- methoxy)carbonvn-4-vinylphenyl)picolinate (3f)

To a solution of methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2- methoxyethoxy)methoxy)carbonyl)-4-(trifluoromethylsulfonyloxy)phenyl)picolinate (3e) (37.4

g, 60.30 mmol) and potassium vinyltrifluoroborate (16.87 g, 120.6 mmol) in DMF (450 mL) and water (45 mL) was bubbled N₂ for 5 min. To this mixture was added NaHC0₃ (20.26 g, 241.2 mmol) and dichloro-bis(triphenylphosphine)palladium (II) (6.34 g, 9.0 mmol). The reaction mixture was stirred at 70 °C for 20 h under N₂(reaction progress was checked by ¹H N R because product and starting material had same R_f in TLC). The reaction mixture was cooled down to room temperature and diluted with ethyl acetate. The organic layer was separated, washed with water, brine, dried ( gS0₄) and filtered. The filtrate was concentrated under vacuum to yield crude methyl 6-(cyclopropylmethyl-carbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy)carbonyl)-4-vinylphenyl)-picolinate (3f). The crude product was purified by flash column chromatography (silica gel, 1 kg, eluting with 0-100% ethyl acetate in hexane) to afford methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy) carbonyl)-4-vinylphenyl)picolinate [31) (26.54 g, 88%) as an amber gum; H NMR (300 MHz, DMSO-c¾ δ 8.70 (t, J = 6.1 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1 H), 8.12 (s, 1 H), 8.00 (d, J = 8.0 Hz, 1 H), 6.98 (m, 2H), 5.94 (dd, J = 1.2, 17.8 Hz, 1H), 5.43 (d, J = 12.5 Hz, 1 H), 5.21 (d, J = 6.5 Hz, 2H), 3.88 (s, 3H), 3.64 (s, 3H), 3.48 (d, J = 3.1 Hz, 2H), 3.35 (m, 5H), 3.22 (m, 2H), 1.11 (s, 1H), 0.44 (dt, J = 4.9, 5.5 Hz, 2H), 0.28 (q, J = 4.8 Hz, 2H). IR (KBr); 1732, 1670 cm^“1. MS (ES+) 499.1 (M+1).

2-(6-(cvclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzolc acid (3g)

A mixture of methyl 6-(cyclopropylmethylcarbamoyl)-3-(5-methoxy-2-(((2-methoxyethoxy)methoxy) carbonyl)-4-vinylphenyl)picolinate (3f) (27.4 mmol) in DME (160 mL) and 6N HCI (40 mL) was stirred at room temperature for 6 h or till TLC showed complete conversion. The solvent was removed under vacuum. The residue obtained was suspended in water, the solid separated out was collected by filtration, washed with water and dried under vacuum to give 2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (3g) (7.0 g, 63%) as a white

solid MP 40 – 42 °C; H NMR (300 MHz, DMSO-d_e) δ 8.69 (t, J= 6.0 Hz, 1H, NH), 8.20 (d, J= 7.9 Hz, 1H), 8.09 (s, 1 H), 7.95 (d, J= 8.1 Hz, 1H), 6.97 (dd, J= 18.0, 11.3 Hz, 1H), 6.88 (s, 1H), 5.92 (d, J= 7.9 Hz, 1H), 5.38 (d, J= 11.1 Hz, 1H), 3.85 (s, 3H), 3.63 (s, 3H), 3.27-3.17 (m, 2H), 1.15-1.05 (m, 1 H), 0.48-0.40 (m, 2H), 0.31-0.24 (m, 2H); IR (KBr): 3084, 1728, 1650, 1533, 1212, 1143 cm-1; MS (ES+) 433.26 (M+Na); (ES-): 409.28 (M-1); Analysis calculated for θ₂2Η22Ν₂Ο₆.0.25Η₂Ο; C, 63.68; H, 5.47; N, 6.75; Found C, 63.75; H, 5.56; N, 6.65

Methyl-3-(2-(4-carbamimidoylprienylcarbamoyl)-5-metrioxy-4-vinylphenyl)-6- (cvclopropylmethylcarbamoyl)picolinate (3h)

To a solution of 2-(6-(cyclopropylmethylcarbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (3g) (2.35 g, 5.7 mmol) and 4-aminobenzimidamide dihydrochloride (3j) (1.79 g, 8.6 mmol) in DMF (20 mL) and pyridine (30 mL) at 0 °C was added EDCI (1.65 g, 8.6 mmol) and allowed to warm to room temperature overnight. The reaction mixture was quenched with 6N HCI (60 mL) and extracted with chloroform (3 x 60 mL). The organic layer was dried over MgS04, filtered and purified by flash column chromatography (silica gel, 110 g, eluting with 0 to 100% chloroform in CMA 80 in CMA 50) yielding methyl-3-(2-(4-carbamimidoylphenyl-carbamoyl)-5-methoxy-4-vinylphenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3h) (2.2 g, 65%) as a white solid MP 266 °C; ¹H NMR (300 MHz, DMSO-c/₆) δ 10.78 (s, 1 H), 9.26 (s, 2H), 9.03 (s, 2H), 8.67 (t, J = 6.1 , 1 H), 8.22 (d, J = 8.0, 1 H), 8.06 (d, J = 8.0, 1 H), 7.96 (s, 1 H), 7.89 – 7.74 (m, 4H), 7.13 – 6.96 (m, 2H), 6.07 (d, J = 17.7, 1H), 5.45 (d, J = 12.4, 1 H), 3.91 (s, 3H), 3.61 (s, 3H), 3.20 (s, 2H), 1.09 (dd, J = 4.7, 8.2, 1H), 0.43 (dt, J = 4.9, 5.4, 2H), 0.34 – 0.21 (m, 2H); MS (ES+) 528.1 (M+1); Analysis calculated for
C, 58.93; H, 5.63; N,11.85; Found: C, 58.75; H, 5.65; N, 11.92.

46578

159

3-r2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy -vinyl-phenyll-6-(cvclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (3i)

3h 3i

To a solution of methyl-3-(2-(4-carbamirriidoylphenylcarbarnoyl)-5-methoxy-4-vinylphenyl)-6-(cyclopropylmethylcarbamoyl)picolinate (3h) (1 g, 1.9 mmol) in methanol (10 mL) and THF

(10 mL) was added 2 N NaOH (10 mL). The reaction mixture was stirred at room

temperature for 3 h, and concentrated in vacuo to remove methanol and THF. The aqueous layer was acidified with 6N HCI to pH 6-7 and the solid obtained was collected by filtration

washed with water and ether to furnish on drying 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid

(3i)(0.775 g, 80%) as the hydrochloride salt as an off white solid.

¹H NMR (300 MHz, DMSO-d₆) δ 12.67 (s, 1 H), 9.11 (s, 2H), 8.97 (s, 2H), 8.74 (s, 1 H), 7.90

(d, J = 7.8, 1 H), 7.80 (s, 1 H), 7.72 – 7.58 (m, 4H), 6.99 (dd, J = 11.3, 17.7, 1 H), 6.78 (s, 1H),

5.95 (d, J = 17.2, 1H), 5.38 (d, J = 11.9, 1H), 3.82 (s, 3H), 3.18 (s, 2H), 1.06 (s, 1 H), 0.43 (d,

J = 7.9, 2H), 0.25 (d, J = 4.7, 2H); MS (ES+) 514.0 (M+1 ); Analysis calculated for

C2eH27N5O5.HCI.H₂O: C, 59.21; H, 5.32; N, 12.33; Found: C, 59.43; H, 5.21; N, 12.06.

Example 1A- Preparation of 3-f2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyll-6-(cvclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride in Form

C

The jacket of a 10 L glass reactor was set to -5 °C. To the reactor was charged 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) prepared in Step (11) of Example 1 (500 g, 1.22 mol), 4-amino-benzamidine-2HCI (280 g, 1.34 mol), and 2-propanol (4.05 kg). The mixture was cooled to 0.3 °C, and pyridine (210 g, 2.62 mol) followed by EDCI HCI (310 g, 1.61 mol) was added. The mixture was stirred at -1.1 to -0.3 °C for 22 hrs followed by addition of the second portion of EDCI HCI (58 g, 0.30 mol). The temperature of jacket was set to 14.0 °C, and the mixture was stirred for 89 hrs. The precipitate was filtered, and washed with 1.32 kg of 2-propanol.

The wet product (8a) was recharged to the reactor followed by addition of acetonitrile (1.6 kg) and water (0.57 kg). The mixture was heated to 46 °C. Smopex-234 (21 g) and Acticarbone 2SW (10 g) were added and the mixture was stirred at this temperature for 1 hr. The solution was filtered, and filtrate was returned back to the reactor. The jacket of the reactor was set to -5 °C, and the mixture was cooled to -0.2 “C. NaOH solution (256 g 46% NaOH, 2.95 mol, in 960 g water) was added in 25 min keeping the temperature ❤ °C. The mixture was stirred at 0.2-2.0 °C for 1 hr 40 min and then quenched with cone, acetic acid (40 g, 0.66 mol). Diluted acetic acid (80 g, 1.33 mol AcOH in 1000 g water) was added during 1 hr 20 min (temperature 1.7-3.0 °C), followed by 1250 g water (30 min). The

suspension was stirred at 0-3.0 “for 1 hr, and filtered at 0-5 °C (ice mantle around the filter). The reactor and product (8d) was rinsed with 3.5 kg water.

The wet product (8d) was recharged to the reactor followed by 0.65 kg water and 1.69 kg acetonitrile. The mixture was heated to 57-60 °C, and stirred at this temperature for 14.5 hrs. The mixture was cooled to -2.2 °C (T_jackel= -5 °C), and a solution of NaOH (163 g 46%, 1.87 mol, in 580 g water) was added during 15 min. The temperature rose to -0.4 °C. Hydrochloric acid (407 g 37% HCI, 4 mol) was added in 10 min, the temperature rose to 7.5 °C. The suspension was agitated at -3 – 0 °C for 19 hrs. The product was filtered and the filter cake was rinsed with 2.87 kg water, compressed and pulled dry. The wet product (1.30 kg) was dried at 40-43 °C and 50 mbar for 11 hrs to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (484 g) as Form C.

Example-1 B: Preparation of 3-f2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyll-6-(cvclopropylmethylcarbartiovQpyridine-2-carboxylic acid hydrochloride in Form A

The procedure was carried out in an identical manner to Example 1 A, with the exception that after the final filtration the filter cake was rinsed with 2.87 kg methyl ierf-butyl ether instead of 2.87 kg water, and pulled dry. The product was dried at 40-43 °C and 50 mbar to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) as Form A.

PATENT

WO 2016029216

Methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (compound 6a) is (I) (pages 85 and 86). Avoralstat hydrochloride (compound of formula XVIII) is (II) (claim 40, page 109). A Markush structures is presented (claim 1, page 99).

The synthesis of (II) via intermediate (I) is described (example 1, pages 80-93).

A synthesis of the compound 3-[2-(4-carbamimidoyl-phenylcarbamoyl)-5-methoxy-4-vinyl-phenyl]-6-(cyclopropylmethyl-carbamoyl)-pyridine-2-carboxylic acid (Compound 3i) is described in Schemes A-C.

O y OHCk n ^Br^ ^OCH₃

B Brr₂₂,, AAccOOHH Y^ V”^“ \ \ tt–BBuuOOKK

OHC^^^{^}O ” _Br^\^0 MeOH ” OHC

1a 1b 66%

1d 95% ^{1 e}

1f

Scheme A



3h 31

Scheme C

Examples. In this section, the following abbreviations are used:

Example-1 : Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b)

7b

Step (1): Preparation of 6-Bromobenzo 1 ,3]dioxole-5-carbaldehyde (1 b):

1b

A solution of bromine (33.0 kg, 206.49 mol) in acetic acid (27.5 L) was added slowly to a solution of piperonal (1a) (29.9 kg, 199.16 mol) in acetic acid (105 L) at room

temperature over a period of 50 min and the reaction mixture was stirred at room temperature for 14.2 h. Additional solution of bromine (33 kg, 206.49 mol) in acetic acid (27.5 L) was added slowly to the reaction mixture over a period of 2 h and the reaction mixture was stirred for 22 h. The reaction mixture was quenched by addition of ice water (500 L) with stirring over a period of 6 h and continued stirring for additional 1.25 h. The mixture was allowed to settle and most of the supernatant liquid was decanted to a waste container using nitrogen pressure. Water (600 L) was added to the solid, stirred, mixture was allowed to settle and then most of the supernatant liquid was decanted to a waste container using nitrogen pressure. Water (100 L) was added to the decanted mixture, stirred for 15 min and the solid obtained was collected by filtration using a centrifuge. The solid was washed with water (2 x 100 L) and air-dried in a tray drier for 3.75 h to afford the crude product 1 b (52 kg). The crude product (51.2 kg) was stirred in n-hexane (178 L) for 3 h, collected by filtration, washed with n-hexane (25 L) and dried to afford 6-bromobenzo[1 ,3]dioxole-5-carbaldehyde (1b) (40.1 1 kg, 87.9%) as a light brown solid. MP: 109-112°C. ¹H NMR (300 MHz, CDCI₃) δ 10.21 (s, 1 H), 7.37 (s, 1 H), 7.07 (s, 1 H), 6.10 (s, 2H); HNMR (DMSO-cf₆): δ 10.06 (s, 1 H), 7.42 (s, 1 H), 7.29 (s, 1 H), 6.20 (d, J =12.3 Hz, 2H)

The process is also illustrated in Fig. 1.

Average yield of isolated 1 b from step-1 is 78 – 88%.

Step (2): Preparation of 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c)

A solution of potassium terf-butoxide (10.7 kg, 95.36 mol) in DMSO (49 L) was stirred at 50 °C for 30 min. Methanol (49 L) was added slowly over a period of 4.25 h and stirred at 50 °C for 30 min. 6-Bromobenzo[1 ,3]dioxole-5-carbaldehyde (1 b) (9.91 kg, 43.27 mol) was added to the reaction mixture in small portions over a period of 45 min and stirred at 50 °C for 1 h. The reaction mixture was cooled to room temperature and split into two equal portions. Each portion was quenched with water (50.9 L) and basified with 50% aqueous NaOH solution (2.4 L). Each portion was extracted with MTBE (4 x 36 L) to remove impurities. The aqueous layer was acidified with cone. HCI to pH ~ 3 to obtain

product as a yellow solid. The solid was collected by filtration using a centrifuge, washed with water (2 x 35 L) and air-dried to afford 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) (4.37 kg, 40.7%, contains 7 % water); Mp: 100-102°C; ¹HNMR (300MHz, DMSO-d₆): δ 10.00 (s, 1 H), 9.92 (s,1 H), 7.27 (s, 1 H), 7.26 (s, 1 H), 3.93 (s, 3H).

The process is also illustrated in Fig. 2.

Average yield of isolated product 2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) from step-2 is 40-50%.

Step (3): 5-Hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxaborolan-2-y benzaldehyde (4a)

2-Bromo-5-hydroxy-4-methoxy-benzaldehyde (1c) [1.3 kg (93%, 7% water content), 5.25 mol] was dissolved in toluene (13 L) in a reaction flask equipped with a Dean Stark apparatus. The solution was heated at reflux with stirring to distil off about 25% of the toluene along with water (90 ml_). The solution was cooled to 90 °C then

bis(pinacolato)diboron (1.5 kg, 5.82 mol), KOAc (772.6 g, 7.87 mol) and Pd(PPh₃) (24.3 g, 0.02 mol) were added and the reaction mixture was heated at reflux for 10h. After confirming the completion of reaction by TLC (mobile phase: 100% DCM), the reaction mixture was cooled to room temperature and was kept standing overnight. The reaction mixture was filtered through celite and the celite cake was washed with toluene (4 L). The filtrate of this batch was mixed with the filtrate of another batch (batch size 1.3 kg obtained from an identical reaction). The mixed filtrate was washed with water (17.5 L), brine (17.5 L), dried over Na₂S0₄, filtered and the solution was passed through a pad of silica gel (2 kg, mesh size 230-400). The silica gel pad was washed with toluene. The combined filtrate and washing was concentrated under reduced pressure and the residual crude product was stirred with n-hexane (23 L) for 1 h to obtain a solid product. The solid was collected by filtration, washed with n-hexane (5 L) and dried to afford 5-hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxaborolan-2-yl)benzaldehyde (4a) (2.47 kg, 84.6%). H NMR (300 MHz, CDCI₃) δ 10.54 (s, 1 H), 7.57 (s, 1 H), 7.33 (s, 1 H), 5.89 (s, 1 H), 4.01 (s, 3H), 1.37 (s, 12H); ¹H NMR (300 MHz, DMSO-d₆) δ 10.35 (s, 1 H), 9.95 (s, 1 H), 7.33 (s, 1 H), 7.23 (s, 1 H), 3.87 (s, 3H), 1.33 (s, 12H); MS (ES+) 301.1 (M+Na); 579.1 (2M+Na); Analysis calculated for C₁₄H₁₉B0₅: C, 60.46; H, 6.89; Found: C, 60.60; H, 6.87

The average yield of 5-hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxa-borolan-2-yl)benzaldehyde (4a) from step (3) is 78 – 90%.

The process is also illustrated in Fig. 3.

Step (4): Preparation of 3-Bromo-2,6-dimethylpyridine (5b)

2,6-lutidine (5a) (115 kg, 1073.3 mol) was added into pre-chilled oleum (20-23%, 1015 kg, 2276.7 mol) at 0 °C over a period of 4.5 h (temperature r6ached 14 °C during the addition). Bromine (88.18 kg, 1103.6 mol) was then added at 5-10 °C over a period of 1 h. The reaction mixture was slowly heated to 150 °C over a period of 12h. TLC analysis indicated about 40-50% conversion to product and the formation of a dimer by-product (5%). The reaction mixture was cooled to room temperature and then additional bromine (88.18 kg, 1103.6 mol) was added slowly. The reaction mixture was slowly heated to maintain a temperature of 65-75 °C over a period of 15h. TLC analysis indicated a 65-70 % conversion to product and the formation of 5% dimer by product. The reaction mixture was quenched by addition of water (500L) while maintaining the reaction temperature below 20 °C. The mixture was basified with 6.6 M NaOH (3800 L) while maintain the temperature at < 40 °C. EtOAc (220 L) was added and the mixture was stirred for 1 h then allowed to settle over a period of 2 h. The layers were separated and the aqueous layer was treated with NaOH (10 kg) in water (10 L) and extracted with EtOAc (160 L). The organic extracts were combined washed with brine (100 L), dried over Na₂S0₄ (50.0 kg), filtered and the solvent was evaporated under atmospheric pressure. The residue was vacuum distilled and the desired product 3-bromo-2,6-dimethylpyridine (5b) was collected at 58-60 °C, 2 mmHg (98.45 kg, 49.2 %) as a colorless liquid.

The process is also illustrated in Fig. 4.

Step (5): Preparation of 3-Bromopyridine-2,6-dicarboxylic acid (5c)

5b 5c

To a stirred solution of 3-bromo-2,6-dimethylpyridine (5b) (98 kg, 5326 mol) in water (1310 L) was added KMn0 (225 kg, 1423.6 mol) in 5 equal portions in 1 h intervals at 70 °C. After stirring for 1 h at 70 °C, additional KMn0₄ (225 Kg, 1423.6 mol) was added in 5 equal portion in 1 h intervals at 90 °C. The reaction mixture was stirred for 12 h at 90 °C. The suspension was filtered hot through celite to obtain a clear solution. The solvent was distilled off to remove about 30% of the total volume. The remaining concentrated solution was chilled to 0 °C and made acidic (to pH 3-4) by the addition of cone. HCI (120 L). The white precipitate obtained was collected by filtration and dried at 70 °C to afford 3-bromopyridine-2,6-dicarboxylic acid (5c) as a white solid (109 kg, 84%).

The process is also illustrated in Fig. 5.

Step (6): Preparation of Dimethyl 3-Bromopyridine-2,6-dicarboxylate (5d)

To a stirred solution of 3-bromopyridine-2,6-dicarboxylic acid (5c) (20.0 kg, 81.29 mol) in methanol (100 L) was added cone. H₂S0₄ (4.4 L) over a period of 30 min. The reaction mixture was heated to 65 °C and maintained at that temperature for 5 h (the reaction was monitored by TLC analysis to determine completion of reaction). The reaction mixture was cooled to room temperature basified by careful addition of aqueous NaHC0₃ solution (prepared from 10 kg NaHC0₃ in 120 L of water) and further diluted with water (120 L). The white solid obtained was collected by filtration, washed with plenty of water and then oven-dried at 40 °C to obtain dimethyl 3-bromopyridine-2,6-dicarboxylate (5d) (9.2 kg, 41.3%) as a white solid; ¹HNMR (300 MHz, DMSO-cf₆) δ 8.47 (d, J = 8.4, 1 H), 8.08 (dd, J = 4.5, 8.4, 1 H), 3.95 (s, 3H), 3.91 (s, 3H); MS (ES+) 570.6 (2M+Na); Analysis calculated for C₉H₈BrN0₄: C_, 39.44; H, 2.94; Br, 29.15 N, 5. 1 ;

Found: C, 39.52; H, 2.92; Br, 29.28; N, 5.03.

The process is also illustrated in Fig. 6.

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Step (7): Preparation of Methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (

To a stirred solution of dimethyl 3-bromopyridine-2,6-dicarboxylate (5d) (27 kg, 98.52 mol) in ierf-butanol (135 L) was added at room temperature cyclopropylmethanamine (7.83 kg, 110.1 mol). The reaction mixture was heated at 65 °C for 17 h. The progress of reaction was monitored by TLC and HPLC (HPLC analysis showed the formation of 74% of the product 5e after 17 h. The reaction mixture was cooled to room temperature and then cone. HCI (2.7 L) was added slowly and the mixture was stirred for 15 min. The reaction mixture was concentrated under reduced pressure to obtain the crude product. The crude product was dissolved in hot /-PrOH (54 L) filtered through a celite pad. The filtrate was cooled with stirring to 10 °C to obtain a white precipitate. The solid obtained was collected by filtration, washed with cold

i-PrOH (13 kg), n-hexane (15 L) and dried to provide pure methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (5e) (15.7 kg, 50.9%). The filtrate was concentrated under reduced pressure and the crude product can be purified by silica gel column chromatography eluting with tert-butanol in hexanes to furnish additional 10% methyl 3-bromo-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate (5e). HNMR (300 MHz, DMSO-cf₆) δ 8.83 (t, J = 5.9, 1 H), 8.47 – 8.41 (m, 1 H), 8.06 (d, J = 8.4, 1 H), 3.96 (s, 3H), 3.16 (t, J = 6.5, 2H), 1.14 – 0.99 (m, 1 H), 0.42 (m, 2H), 0.30 -0.19 (m, 2H); MS (ES+) 337.0 (M+23), 650.8 (2M+23); Analysis calculated for

C₁₂H₁₃BrN₂0₃: C, 46.03; H, 4.18; N, 8.95; Br, 25.52; Found: C, 46.15; H, 4.17; N, 8.72; Br, 25.26.

The average isolated yield for step (7) is 50% to 60%.

The process is also illustrated in Fig. 7.

Step (8): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a)

2

6a

THF (37.5 L) was charged to a 100 L reactor followed by ethyl 3-bromo-6- (cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate (5e) (2.5 kg, 7.98 mol) under a nitrogen atmosphere. The reaction mixture was degassed twice by applying alternate vacuum and nitrogen. 5-Hydroxy-4-methoxy-2-(4,4,5,5-tetramethyl-[1 ,3,2]dioxa-borolan-2-yl)benzaldehyde (4a) (2.88 kg, 10.36 mol) was added, followed by the addition of PPh₃ (53.13 g, 0.20 mol), PdCI₂(PPh₃)₂ (120.4 g, 0.17 mol) and a solution of Na₂C0₃(2.12 kg, 20.00 mol) in demineralized water (10.0 L) under nitrogen atmosphere. The reaction mixture was degassed again two times by applying alternate vacuum and nitrogen. The reaction mixture was heated at reflux for 6.5 h, cooled to room temperature and filtered through a Celite bed. Water (75 L) was added to the filtrate and the product was extracted with ethyl acetate (75 L). The aqueous layer was back extracted with ethyl acetate (2 ^χ 60 L). The combined ethyl acetate extract was divided into two equal portions and each portion was washed with brine (37 L), dried over Na₂S0₄, filtered and concentrated under reduced pressure to give crude methyl 6- ((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a) as a reddish viscous material (-4.5 Kg) which was used as such for the next step without further purification. An analytical sample was prepared by purification of a small sample by flash column chromatography (silica gel, eluting with 0-100% ethyl acetate in hexane) to furnish methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)-picolinate (6a) as an off-white solid; HNMR (300 MHz, DMSO-d₆) δ 9.89 (s, 1 H), 9.52 (s, 1 H), 8.79 (t, J = 6.1 Hz, 1 H), 8.23 (d, J = 8.0 Hz, 1 H), 8.09 (d, J = 8.0 Hz, 1 H), 7.34 (s, 1 H), 6.90 (s, 1 H), 3.85 (s, 3H), 3.62 (s, 3H), 3.22 (m, 2H), 1.16 -1.02 (m, 1 H), 0.49 – 0.38 (m, 2H), 0.32 – 0.22 (m, 2H); MS (ES+) 791.0 (2M+Na), (ES-) 382.7 (M-1), 767.3 (2M-1); Analysis calculated for C₂₀H₂₀N₂O₆.0.25 H₂0: C, 61.77; H, 5.31 ; N, 7.20; Found: C, 61.54; H, 5.13; N, 7.05.

The process is also illustrated in Fig. 8.

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Step (9): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-(((trifluoromethyl)sulfonyl)oxy)phenyl)picolinate (6b)

6a 6b

A solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-4-hydroxy-5-methoxyphenyl)picolinate (6a) (2.11 kg, estimated about 3.83 mol from step-8) in dichloromethane (16.0 L) and pyridine (1.4 L, 17.4 mol) cooled to -10°C and maintained at that temperature for 1 h was added a solution of triflic anhydride (980.0 ml_, 5.8 mol) in dichloromethane (6.0 L) drop wise over a period of 3 h at -10 °C. The reaction mixture was stirred at -5°C for 1.3 h, quenched with saturated aqueous NaHCO₃(10.4 L) and stirred for 30 mins. The organic layer was separated, washed successively with saturated aqueous NaHC0₃ (10.4 L), 1 HCI (2 x 16.6 L), water (13.2 L), brine (13.2 L), dried over MgS0₄, filtered and concentrated under reduced pressure to give the crude product. The crude product was stirred with 15% ethyl acetate in n-hexane (7.0 L) for 1 h. The solid obtained was collected by filtration washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was stirred again with 15% ethyl acetate in n-hexane (7.0 L) for 1 h, was collected by filtration and washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was stirred again with 15% ethyl acetate in n-hexane (8.0 L) for 1 h, collected by filtration washed with 15% ethyl acetate in n-hexane (3.0 L). The solid was dried to afford methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-(((trifluoromethyl)sulfonyl)-oxy)phenyl)picolinate (6b) as a light brown solid (1.7 kg, 86% yield, for combined steps 8 & 9). Average isolated yield for combined steps 8 and 9 was 70% to 86%; Ή NMR (300 MHz, DMSO-cf₆): δ 9.64 (s, 1 H), 8.78 (t, J = 6.1 , 1 H), 8.29 (d, J = 8.0, 1 H), 8.16 (d, J = 8.0, 1 H), 8.03 (s, 1H), 7.39 (s, 1 H), 4.00 (s, 3H), 3.63 (s, 3H), 3.22 (m, 2H), 1.11 (m, 1 H), 0.52 – 0.39 (m, 2H), 0.28 (m, 2H); MS (ES+) 538.9 (M+Na). The process is also illustrated in Fig. 9.

Step (10): Preparation of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c)

A solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4- (((trifluoromethyl)sulfonyl)oxy)phenyl)picolinate (6b) (12 kg, 23.24 mol) in DME (106 L) was charged into reactor under nitrogen. The reaction mixture was degassed twice by applying alternate vacuum and nitrogen. Potassium trifluoro(vinyl)borate (3.9 kg, 29.1 1 mol), PdCI₂(PPh₃)₂ (815 g, 1.13 mol), KHC0₃ (4.65 g, 46.44 mol) and demineralized water (12 L) was then added under a N₂ atmosphere. The reaction mixture was degassed by applying alternate vacuum and nitrogen. The reaction mixture was heated at reflux for 5 h. The reaction mixture was cooled to room temperature and then filtered through a Celite bed. Demineralized water (118 L) was added to the filtrate followed by ethyl acetate (124 L). The mixture was stirred for 20 min and then the organic layer was separated. The aqueous layer was back-extracted with ethyl acetate (2 x 95 L). The combined organic extract was washed with brine (95 L), dried over Na₂S0₄, and filtered. The solvent was evaporated under reduced pressure to give the crude product. The crude product was purified by column chromatography (silica gel, 120 kg, 230-400 mesh size, eluting with ethyl acetate in n-hexane) to obtain methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c) (6 kg, 72%). ¹H NMR (300 MHz, CDCI₃): δ (ppm) 9.64 (s, 1 H), 8.35 (d, J = 7.8 Hz, 1 H), 8.06-8.03 (m, 2H), 7.78(d, J = 7.8 Hz, 1 H), 7.02-6.92 (m, 1 H), 6.61 (s, 1 H), 5.86 (d, J = 17.7 Hz, 1 H), 5.38 (d, J = 1 1.4 Hz, 1 H), 3.84 (s, 3H), 3.67 (s, 3H), 3.35-3.29 (m, 2H),1.08-1.03 (m, 1H), 0.55-0.49 (m, 2H), 0.29-0.2 4(m, 2H). ¹HNMR (300 MHz, DMSO-d₆) 6 9.68 (s, 1 H), 8.77 (t, J = 6.1 , 1 H), 8.35 – 8.21 (m, 1 H), 8.16 – 8.01 (m, 2H), 7.14 -6.87 (m, 2H), 6.01 (dd, J = 1.2, 17.8, 1 H), 5.45 (dd, J = 1.1 , 1 1.3, 1 H), 3.91 (s, 3H), 3.64 (s, 3H), 3.23 (m, 2H), 1.21 – 1.01 (m, 1H), 0.51 – 0.40 (m, 2H), 0.34 – 0.20 (m, 2H). MS

(ES+) 417.0 (M+Na); Analysis calculated for C₂2H₂₂N₂0₅: C, 66.99; H, 5.62; N, 7.10;

Found: C, 66.75; H, 5.52; N, 7.06.

The process is also illustrated in Fig. 10.

Step (1 1): Preparation of 2-(6-((cyclopropylmethyl)carbamoyl)-2- (methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d)

To a stirred solution of methyl 6-((cyclopropylmethyl)carbamoyl)-3-(2-formyl-5-methoxy-4-vinylphenyl)picolinate (6c) (1.57 kg, 3.80 mol) in acetonitrile (15.4 L) was added ferf-butyl alcohol (22.2 L), demineralized water (3.2 L) and sodium dihydrogen phosphate monohydrate (323.74 g, 2.346 mol). The reaction mixture was cooled to 0 °C and added 2-methyl-2-butene (5.3 L, 50.0 mol) and stirred at 0 °C for 30 min. A solution of 80% sodium chlorite (1.36 kg, 12.0 mol) in demineralized water (5.2 L) was added to the reaction mixture over a period of 2.5 h at 0 °C [temperature rises to 7 °C during the addition]. The reaction mixture was stirred at 0 °C for 2 h, diluted with water (40 L) and ethyl acetate (24 L). After stirring the mixture, it was allowed to settle and the organic layer was separated. The aqueous layer was back-extracted with ethyl acetate (2 x 20 L) then acidified with 5.9 % aqueous acetic acid (2 L) and extracted once with ethyl acetate (10 L). The organic extracts were combined washed with water (2 x 20 L), a solution of acetic acid (125 mL) in water (20.0 L), brine (2 ^χ 20 L), dried over Na₂S0₄, filtered and concentrated under reduced pressure (vapor temperature below 40 °C). The residue obtained was dissolved in acetone (7 L) (residue didn’t dissolve completely). The solution was poured slowly into a reactor containing stirred n-hexane (70.0 L) to precipitate the solid product and the mixture was stirred for 2 h. The solid obtained was collected by filtration, washed with 10% acetone in n-hexane (6.3 L), AJ-hexane (6.3 L), dried to afford 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4- methoxy-5-vinylbenzoic acid (6d) as an off-white solid (1.29 Kg, yield: 79.0%). Average isolated yield for step 1 1 is 74% to 84%. ¹H NMR (300 MHz, DMSO-d₆): δ (ppm) 12.50 (brs, 1 H), 8.69(t, J= 6.0 Hz, 1 H, NH), 8.20 (d, J= 7.9 Hz, 1 H), 8.09 (s, 1 H), 7.95 (d, J= 8.1 Hz, 1 H), 6.97 (dd, J= 18.0, 1 1.3 Hz, 1 H), 6.88 (s, 1 H), 5.92 (d, J= 7.9 Hz, 1 H), 5.38 (d, J= 1 1.1 Hz, 1 H), 3.85 (s, 3H), 3.63 (s, 3H), 3.27-3.17 (m, 2H), 1.15-1.05 (m, 1 H), 0.48-0.40 (m, 2H), 0.31-0.24 (m, 2H); MS (ES+) 433.26, (M+Na); (ES-) 409.28 (M-1). The process is also illustrated in Fig. 1 1.

Step (12): Preparation of Methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylate methanesulfonate (7a

Pyridine (3.8 L, 47.17 mol) and EDCI (5.31 kg, 27.66 mol) were sequentially added to a cooled solution (0 °C) of 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) (9 kg, 21.92 mol) and 4-aminobenzamidine dihydrochloride (5.13 kg, 24.65 mol) in /-PrOH (90 L). The reaction mixture was allowed to warm to room temperature and stirred for 2 h. TLC analysis indicated incomplete reaction. Additional EDCI (1.08 kg, 5.6 mol) was added and the reaction mixture was stirred for 8 h. The reaction was still incomplete as indicated by TLC analysis, additional EDCI (0.54 kg, 2.8 mol) was added and the reaction mixture was stirred for 5 h. TLC analysis indicated there was trace amount of unreacted starting material remaining. The reaction mixture was cooled to 0 °C and a solution of

methanesulfonic acid (MSA) (9.13 kg, 95 mol) in MeOH (38.7 L) was added to the cooled mixture over a period of 4 h. The reaction mixture was allowed to warm to room temperature and stirred for 15 h. The product was collected by filtration, washed with a mixture of /^‘-PrOH and MeOH (4:1 , 45 L). The wet cake was slurried in a mixture of /-PrOH and MeOH (2:1 , 135 L) stirred for 1 h and the product was collected by filtration and washed with a mixture of /^‘-PrOH and MeOH (4:1 , 46.8 L). The product was dried in

2015/046582

a vacuum oven at 45 °C to afford methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate methanesulfonate (7a) as a pink-colored solid (12.71 kg, 93%). Average isolated yield for this step: >90%.

¹H NMR (300 MHz, DMSO-c/₆) δ 10.71 (s, 1 H), 9.16 (s, 2H), 8.80 (s, 2H), 8.68 (t, J = 6.1 Hz, 1 H), 8.22 (d, J = 8.0 Hz, 1H), 8.06 (d, J = 8.1 Hz, 1 H), 7.93 (s, 1H), 7.84 – 7.72 (m, 4H), 7.12 – 6.97 (m, 2H), 6.04 (dd, J = 17.8, 1.3 Hz, 1 H), 5.45 (d, J = 12.6 Hz, 1H), 3.91 (s, 3H), 3.60 (s, 3H), 3.25 – 3.16 (m, 2H), 2.32 (s, 3H), 1.10 – 1.01 (m, 1 H), 0.48 – 0.37 (m, 2H), 0.30 – 0.22 (m, 2H); MS (ES+) 528.0 (M+1); Analysis calculated for

C₂₉H₂₉N₅O5.CH₃SO₃H.2H₂O. C, 54.62; H, 5.65; N, 10.62; S, 4.86; Found: C, 54.95; H, 5.55; N, 10.61 ; S, 4.87.

The process is also illustrated in Fig. 12.

Step (13): Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-rnethoxy-4- vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrate

(3i) ^{,a 3i}

A pre-cooled (0-5 °C) aq. NaOH solution [prepared from solid NaOH (4 kg, 100 mol) in water (86 L)] was added to a suspension of methyl 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethyl-carbamoyl)pyridine-2-carboxylate methanesulfonate (7a) (28.7 kg, 46 mol) in acetonitrile (86 L) cooled to 0 to 5 °C over a period of 25 mins. The reaction mixture was stirred at 0 to 5 °C for 2.5 h (TLC analysis showed the reaction was complete). The reaction mixture was filtered through a sparkler filter, washed with a mixture of 1 :1 CH₃CN / H₂0 ( 57.4 L). Acetic acid (3.2 L, 55.9 mol) in water (56 L) was added to the filtrate at room temperature over a period of 25 mins and the resulting mixture was stirred at room temperature for 2.5 h. The solid product obtained was collected by filtration, washed with a 1 :4 mixture of CH₃CN / H₂0 (57.5 L). The solid was dried at 45°C in a vacuum oven to afford 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrate (3i) as an off-white solid (12,77 kg, 54.1%). Average yield for this step is 50% to 75%. Mp: >200°C; H NMR (300 MHz, DMSO-d₆): δ 13.49 (s, 1 H), 8.94 (bs, 4H), 8.56 (t, 1 H), 7.82 – 7.71 (m, 2H), 7.67 -7.56 (m, 4H), 7.51 (d, J = 7.8, 1 H), 6.98 (dd, J = 11.3, 17.8, 1 H), 6.68 (s, 1 H), 5.92 (d, J = 16.6, 1 H), 5.36 (d, J = 12.4, 1 H), 3.80 (s, 3H), 3.16 (m, 2H), 1.05 (m, 1 H), 0.43 (m, 2H), 0.24 (m, 2H); MS (ES+) 514.1 (M+1), 536.1 (M+Na), (ES-) 512.1 ; Analysis calculated for C28H27N5O5.3H2O: C, 59.25; H, 5.86; N, 12.34; Found C, 59.50; H,

5.75; N, 12.05. If needed this material can be crystallized from a mixture of acetone and water.

The process is also illustrated in Fig. 13.

Step 14: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b

A pre-cooled (5-8 °C) aqueous NaOH solution (prepared from solid NaOH (1.97 kg, 49.25 mol) in demineralized water (41 L) was added to a pre-cooled (0-5 °C) suspension of (3i) (13.8 kg, 26.9 mol) in acetonitrile (41 L). The reaction mixture was stirred at 0-5 °C for 30 min (until the reaction mixture becomes homogeneous). The reaction mixture was filtered through a sparkler filter washed with 50% acetonitrile in demineralized water (4.4 L). The filtrate was charged into a reactor and cooled to 0-5 °C. Aqueous HCI [prepared from cone. HCI (9.3 L) in demineralized water (36 L)] was added slowly with stirring to keep the reaction temperature at or below 15 °C, the resulting mixture was stirred at 10-15 °C for 13 h. The reaction mixture was cooled to 0-5 °C and stirred for 1 h. The solid obtained was collected by filtration and washed with demineralized water (36 L). The solid product was suspended in water (69 L) stirred for 30 mins and collected by filtration washed twice with water (20 L each). The solid product was dried in a vacuum oven at 45°C to afford 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-

(cyclopropylmethyl carbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (1 1.21 Kg, 75.77%). Mp: >200°C; ¹H NMR (300 MHz, DMSO-ci₆): δ 12.98 (br s, 1 H), 10.86 (s, 1 H), 9.24 (s, 3H), 9.04 (s, 2H), 8.22 (d, J = 7.8 Hz, 1 H), 7.96 (d, J = 5.7 Hz, 2H), 7.78 (s, 4H), 7.09-6.99 (m, 2H), 6.07 (d, J = 17.7 Hz, 1 H), 5.45(d, J = 11.4 Hz, 1 H), 3.88 (s, 3H), 3.26-3.24 (m, 2H), 1.09 (m, 1 H), 0.47 (m, 2H), 0.28 (m, 2H).

Average isolated yield for this step varies from 63% to 80%.

The process is also illustrated in Fig. 14.

Example-2: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b)

6d 8a

To a solution of 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) (2.35 g, 5.7 mmol) and 4-aminobenzamidine dihydrochloride (1.79 g, 8.6 mmol) in DMF (20 mL) and pyridine (30 ml_) at 0 °C was added EDCI (1.65 g, 8.6 mmol) and allowed to warm to room temperature overnight. The

reaction mixture was quenched with 6N HCI (60 mL) and extracted with chloroform (3 x 60 mL). The organic layer was dried over MgS0₄, filtered and concentrated in vacuum. The residue obtained was purified by flash column chromatography (silica gel, 110 g, eluting with 0 to 100% chloroform in CMA 80 and 0-100% chloroform in CMA 50) to furnish methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)-carbamoyl)picolinate hydrochloride (8a) (2.2 g, 65%) as a white solid; MP 266 °C; ¹HNMR (300 MHz, DMSO-d₆) δ 10.78 (s, 1 H), 9.26 (s, 2H), 9.03 (s, 2H), 8.67 (t, J = 6.1 , 1 H), 8.22 (d, J = 8.0, 1 H), 8.06 (d, J = 8.0, 1 H), 7.96 (s, 1 H), 7.89 -7.74 (m, 4H), 7.13 – 6.96 (m, 2H), 6.07 (d, J = 17.7, 1 H), 5.45 (d, J = 12.4, 1 H), 3.91 (s, 3H), 3.61 (s, 3H), 3.20 (s, 2H), 1.09 (dd, J = 4.7, 8.2, 1 H), 0.43 (dt, J = 4.9, 5.4, 2H), 0.34 – 0.21 (m, 2H); MS (ES+) 528.1 (M+1); Analysis calculated for C₂₉H₂₉N₅0₅ (H₂0)_{1 5} (HCI): C, 58.93; H, 5.63; N, 1 1.85; Found: C, 58.75; H, 5.65; N, 1 1.92.

Step-2: preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b)

8a 8b j₀ a solution of methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)carbamoyl)picolinate hydrochloride (8a) (1.128 g, 2 mmol) in acetonitrile (5 ml), was added 1 N aqueous sodium hydroxide (5.00 ml, 5.00 mmol) and stirred at room temperature for 2 h, TLC [CMA80/CMA50 (7/3)] shows reaction was complete. The reaction mixture was neutralized with a solution of sulfuric acid (0.483 ml, 9.00 mmol) in water (5 mL) and stirred for 10 min at room temperature. To this cold water (5 ml) was added and stirred at room temperature until product crystallized out. Cold water (5 mL) was added to the slurry and stir for additional 20 min, additional cold water (5 mL) was added prior to filtration of solid. The solid obtained was collected by filtration washed with water (5 mL and 2.5 mL), dried under vacuum overnight to afford 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-

(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid sulfate salt (8b) (1.103 g, 90 % yield) as a white solid; MP 221.7 °C; H NMR (300 MHz, DMSO-d₆) δ 12.30 – 10.91 (bs, 1 H, D₂0 exchangeable), 10.69 (bs, 1 H, D₂0 exchangeable), 9.24 (t, J = 6.0 Hz, 1 H), 9.16 (s, 2H, D2O exchangeable), 8.78 (s, 2H, D2O exchangeable), 8.24 (d, J = 8.0 Hz, 1 H), 8.04 – 7.91 (m, 2H), 7.84 – 7.67 (m, 4H), 7.13 – 6.94 (m, 2H), 6.03 (dd, J = 17.8, 1 .4 Hz, 1 H), 5.51 – 5.37 (m, 1 H), 3.88 (s, 3H), 3.24 (t, J = 6.4 Hz, 2H), 1.16 – 1.01 (m, 1 H), 0.52 – 0.41 (m, 2H), 0.32 – 0.22 (m, 2H); MS (ES+) 514.0 (M+1); Analysis calculated for: C₂₈H₂7N₆0₅ 1.0H₂SO₄ 1.5H₂0: C, 52.66; H, 5.05; N, 10.97; S, 5.02; Found: C, 52.81 ; H, 4.95; N, 10.94; S, 4.64.

Example-3: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid methane s

To a solution of methyl 3-(2-((4-carbamimidoylphenyl)carbamoyl)-5-methoxy-4-vinylphenyl)-6-((cyclopropylmethyl)carbamoyl)picolinate hydrochloride (8a) (1.128 g, 2 mmol) in acetonitrile (5 ml) was added 1 N aqueous sodium hydroxide (5.00 ml, 5.00 mmol) and stirred at room temperature for 2 h, TLC [CMA80/CMA50 (7/3)] shows reaction was complete. The reaction mixture was neutralized with methanesulfonic acid (0.584 ml, 9.00 mmol) and stirred for 1 h at room temperature. Cold water (5.00 ml) was added to the reaction mixture and stirred at room temperature until product crystallized out. To the slurry was added water (5 ml) of water stirred for additional 20 min, followed by the addition of water (5 ml) prior to filtration. The solid obtained was collected by filtration washed with water (5 ml and 2.5 ml), dried under vacuum to afford 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6- (cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid methane sulfonate salt (8c)

(1 .138 g, 1.867 mmol, 93 % yield) as a white solid; MP 221.2 °C; ¹ H NMR (300 MHz,

DMSO-d₆) δ 12.89 (s, 1 H, D2O exchangeable), 10.69 (s, 1 H, D2O exchangeable), 9.24

(t, J = 6.0 Hz, 1 H), 9.16 (s, 2H,), 8.85 (s, 2H), 8.24 (d, J = 8.0 Hz, 1 H), 8.06 – 7.91 (m, 2H), 7.86 – 7.70 (m, 4H), 7.15 – 6.96 (m, 2H), 6.03 (dd, J = 17.8, 1.4 Hz, 1 H), 5.52 – 5.35 (m, 1 H), 3.88 (s, 3H), 3.25 (t, J = 6.3 Hz, 2H), 2.34 (s, 3H), 1.17 – 1.01 (m, 1 H), 0.53 -0.43 (m, 2H), 0.32 – 0.23 (m, 2H); MS (ES+) 514.0 (M+1); Analysis calculated for:

CzeH^NsOsCHsSOsH 1.5H₂0: C, 54.71 ; H, 5.38; N, 11.00; S, 5.04; Found: C, 54.80; H, 5.14; N, 10.94; S, 4.90.

Example-4: Preparation of 3-[2-(4-Carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) in Form C (Compound XX)

The jacket of a 10 L glass reactor was set to -5 °C. To the reactor was charged 2-(6-((cyclopropylmethyl)carbamoyl)-2-(methoxycarbonyl)-pyridin-3-yl)-4-methoxy-5-vinylbenzoic acid (6d) prepared in Step (11) of Example 1 (500 g, 1.22 mol), 4-amino-benzamidine-2HCI (280 g, 1.34 mol), and 2-propanol (4.05 kg). The mixture was cooled

46582

to 0.3 °C, and pyridine (210 g, 2.62 mol) followed by EDCI HCI (310 g, 1.61 mol) was added. The mixture was stirred at -1.1 – -0.3 °C for 22 hrs followed by addition of the second portion of EDCI HCI (58 g, 0.30 mol). The temperature of jacket was set to 14.0 °C, and the mixture was stirred for 89 hrs. The precipitate was filtered, and washed with 1.32 kg of 2-propanol.

The wet product (8a) was recharged to the reactor followed by addition of acetonitrile (1 .6 kg) and 0.57 kg water. The mixture was heated to 46 °C. 21 g of Smopex-234 and 10 g Acticarbone 2SW were added and the mixture was stirred at this temperature for 1 hr. The solution was filtered, and filtrate was returned back to the reactor. The jacket of the reactor was set to -5 °C, and the mixture was cooled to -0.2 °C. NaOH solution (256 g 46% NaOH, 2.95 mol, in 960 g water) was added in 25 min keeping the temperature ❤ °C. The mixture was stirred at 0.2-2.0 °C for 1 hr 40 min and then quenched with cone, acetic acid (40 g, 0.66 mol). Diluted acetic acid (80 g, 1.33 mol AcOH in 1000 g water) was added during 1 hr 20 min (temperature 1.7-3.0 °C), followed by 1250 g water (30 min). The suspension was stirred at 0-3.0 °for 1 hr, and filtered at 0-5 °C (ice mantle around the filter). The reactor and product (8d) was rinsed with 3.5 kg water.

The wet product (8d) was recharged to the reactor followed by 0.65 kg water and 1.69 kg acetonitrile. The mixture was heated to 57-60 °C, and stirred at this temperature for 14.5 hrs. The mixture was cooled to -2.2 °C (T_jacke,= -5 °C), and a solution of NaOH (163 g 46%, 1.87 mol, in 580 g water) was added during 15 min. The temperature rose to -0.4 °C. Hydrochloric acid (407 g 37% HCI, 4 mol) was added in 10 min, the temperature rose to 7.5 °C. The suspension was agitated at -3 – 0 °C for 19 hrs. The product was filtered and the filter cake was rinsed with 2.87 kg water, compressed and pulled dry. The wet product (1.30 kg) was dried at 40-43 °C and 50 mbar for 1 17 hrs to furnish 3-[2-(4-carbamimidoylphenylcarbamoyl)-5-methoxy-4-vinylphenyl]-6-(cyclopropylmethylcarbamoyl)pyridine-2-carboxylic acid hydrochloride (7b) (484 g) as Form C (Compound XX).

/////avoralstat, BCX4161, Fast Track, Treat hereditary angioedema (HAE), Orphan Drug, PRECLINICAL

COc1cc(c(cc1C=C)C(=O)Nc2ccc(cc2)C(=N)N)c3cc(ncc3C(=O)O)C(=O)NCC4CC4

Avoralstat

March 4, 2016 3:57 am / Leave a comment

Avoralstat, BCX4161,

CAS 918407-35-9
UNII: UX17773O15

513.5513, C28-H27-N5-O5

2-Pyridinecarboxylic acid, 3-(2-(((4-(aminoiminomethyl)phenyl)amino)carbonyl)-4-ethenyl-5-methoxyphenyl)-6-(((cyclopropylmethyl)amino)carbonyl)-

3-(2-((4-Carbamimidoylphenyl)carbamoyl)-4-ethenyl-5-methoxyphenyl)-6-((cyclopropylmethyl)carbamoyl)pyridine-2-carboxylic acid

Hereditary angioedema (HAE)

Kallikrein inhibitor

BioCryst Pharmaceuticals

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BioCryst is also investigating second-generation plasma kallikrein inhibitors to avoralstat, for treating HAE (in February 2016, this program was listed as being in preclinical development).

2D chemical structure of 918407-35-9

Prevent acute attacks in patients with hereditary angioedema (HAE); Treat hereditary angioedema (HAE)

U.S. – Fast Track (Treat hereditary angioedema (HAE));
U.S. – Orphan Drug (Prevent acute attacks in patients with hereditary angioedema (HAE))

26 Feb 2016Clinical trials in Hereditary angioedema (Prevention) in USA (PO, Hard-gelatin capsule) before February 2016

24 Feb 2016Discontinued – Phase-III for Hereditary angioedema (Prevention) in France (PO, Soft-gelatin capsule)

24 Feb 2016Discontinued – Phase-III for Hereditary angioedema (Prevention) in Germany (PO, Soft-gelatin capsule)

Conditions	Interventions	Phases	Recruitment	Sponsor/Collaborators
Hereditary Angioedema\|HAE	Drug: BCX4161\|Drug: Placebo	Phase 2\|Phase 3	Recruiting	BioCryst Pharmaceuticals
Hereditary Angioedema	Drug: BCX4161\|Drug: Placebo	Phase 2	Completed	BioCryst Pharmaceuticals
Hereditary Angioedema	Drug: BCX4161	Phase 1	Completed	BioCryst Pharmaceuticals
Hereditary Angioedema	Drug: BCX4161	Phase 1	Completed	BioCryst Pharmaceuticals