EA040816B1 - GLP-1 RECEPTOR AGONISTS AND THEIR USE - Google Patents

Description

Field of invention

Proposed are 6-carboxylic acids of benzimidazoles as GLP-1R agonists, methods for preparing said compounds, and methods comprising administering said compounds to a mammal in need thereof.

Prior Art

Diabetes is a major health problem due to its increasing prevalence and associated health risks. This disease is characterized by high blood glucose levels due to impaired insulin production, insulin action, or both. Two main forms of diabetes are recognized: type 1 diabetes and type 2 diabetes. Type 1 diabetes (T1D) develops when the body's immune system destroys pancreatic beta cells, the only cells in the body that produce the hormone insulin, which regulates blood glucose levels. To survive, people with type 1 diabetes must inject insulin by injection or pump. Type 2 diabetes mellitus (generally referred to as T2DM) usually begins either with insulin resistance or when insulin production is insufficient to maintain acceptable glucose levels.

Various pharmacological approaches are currently available for the treatment of hyperglycemia and subsequently T2DM (Hampp, C. et al. Use of Antidiabetic Drugs in U.S., 2003-2012, Diabetes Care 2014, 37, 1367-1374). These approaches can be grouped into six major classes, each acting through different primary mechanisms: (A) insulin secretagogues, including sulfonylureas (eg, glipizide, glimepiride, glyburide), meglitinides (eg, nateglidin, repaglidin), dipeptidyl peptidase IV inhibitors ( DPP-IV) (eg, sitagliptin, vildagliptin, alogliptin, dutogliptin, linagliptin, saxogliptin) and glucagon-like protein-1 receptor (GLP-1R) agonists (eg, liraglutide, albiglutide, exenatide, lixisenatide, dulaglutide, semaglutide), which increase secretion insulin by acting on the beta cells of the pancreas. Sulfonylureas and meglitinides have limited efficacy and tolerability, cause weight gain and often induce hypoglycemia. DPP-IV inhibitors have limited efficacy. Commercially available GLP-1R agonists are peptides administered by subcutaneous injection. Liraglutide has been additionally approved for the treatment of obesity. (B) Biguanides (eg, metformin) are believed to act primarily by reducing hepatic glucose production. Biguanides often cause gastrointestinal disorders and lactic acidosis, which also limits their use. (C) Alpha-glucosidase inhibitors (eg, acarbose) reduce intestinal absorption of glucose. These agents frequently cause gastrointestinal disturbances. (D) Thiazolidinediones (eg, pioglitazone, rosiglitazone) act on a specific receptor (peroxisome proliferator-activated receptor-gamma) in liver, muscle, and adipose tissue. They regulate lipid metabolism, subsequently enhancing the response of these tissues to the action of insulin. Frequent use of these drugs can lead to weight gain and may cause swelling and anemia. (E) Insulin is used in more severe cases, either alone or in combination with the above agents, and their frequent use can also lead to weight gain and carry the risk of hypoglycemia. (F) Sodium-dependent glucose co-transporter type 2 (SGLT2) inhibitors (eg, dapagliflozin, empagliflozin, canagliflozin, ertugliflozin) inhibit glucose reabsorption in the kidneys and thereby lower blood glucose levels. This emerging class of drugs may be associated with ketoacidosis and urinary tract infections.

However, with the exception of GLP-1R agonists and SGLT2 inhibitors, these drugs have limited efficacy and do not address the critical problems of deterioration in β-cell function and exacerbation of associated obesity.

Obesity is a chronic disease that is highly prevalent in today's society and is associated with numerous medical problems, including hypertension, hypercholesterolemia, and coronary heart disease. It is also closely associated with T2DM and insulin resistance, which is usually accompanied by hyperinsulinemia or hyperglycemia, or both. In addition, T2DM is associated with a two to fourfold increase in the risk of coronary artery disease. Currently, only bariatric surgery is the only treatment that treats obesity with high efficiency, but this treatment is costly and risky. Pharmacological intervention is usually less effective and is associated with side effects. Therefore, there is a clear need for a more effective pharmacological intervention with fewer side effects and convenient administration.

Although T2DM is most commonly associated with hyperglycemia and insulin resistance, other diseases associated with T2DM include hepatic insulin resistance, impaired glucose tolerance, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, obesity, dyslipidemia, hypertension, hyperinsulinemia, and nonalcoholic fatty disease. liver (NAFLD).

NAFLD is a hepatic manifestation of the metabolic syndrome and is a spectrum of hepatic conditions encompassing steatosis, non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and potentially hepatocellular carcinoma. NAFLD and NASH are considered major fatty liver diseases as they occur in the largest proportion of individuals with elevated liver lipid levels. The severity of NAFLD/NASH depends on the presence of lipid, inflammatory cell infiltrate, hepatocyte ballooning, and degree of fibrosis. Although not all individuals with steatosis develop NASH, a significant proportion do.

GLP-1 is a 30 amino acid incretin hormone secreted by L cells in the intestine in response to ingestion of food. GLP-1 has been shown to stimulate insulin secretion in a physiological and glucose-dependent manner, decrease glucagon secretion, inhibit gastric emptying, reduce appetite, and stimulate beta cell proliferation. In non-clinical experiments, GLP-1 stimulates ongoing beta-cell competence by stimulating the transcription of genes important for glucose-dependent insulin secretion and by stimulating beta-cell neogenesis (Meier, et al. Biodrugs. 2003; 17(2): 93-102).

In a healthy individual, GLP-1 plays an important role in the regulation of post-prandial blood glucose levels by stimulating glucose-dependent pancreatic insulin secretion leading to increased peripheral glucose uptake. GLP-1 also suppresses the secretion of glucagon, which leads to a decrease in glucose production in the liver. In addition, GLP-1 delays gastric emptying and slows small intestinal motility, delaying food absorption. In people with T2DM, normal post-prandial growth of GLP-1 is absent or reduced (Vilsboll T, et al. Diabetes. 2001.50; 609-613).

Hoist (Physiol. Rev. 2007, 87, 1409) and Meier (Nat. Rev. Endocrinol. 2012, 8, 728) report that GLP-1 receptor agonists such as GLP-1, liraglutide and exendin-4 have 3 main pharmacological activities aimed at improving glycemic control in patients with T2DM by reducing fasting and postprandial glucose levels (FPG and PPG): (1) increasing glucose-dependent insulin secretion (improvement of the first and second phases), (2 ) glucagon suppression activity in hyperglycemic conditions, (3) slowing the rate of gastric emptying, resulting in a slower absorption of glucose from food.

There remains a need for easily implemented prevention and/or treatment of cardiometabolic and associated diseases.

Brief description of graphic materials

Fig. 1 is an observed X-ray powder diffraction pattern for the anhydrous (anhydrate) crystalline form (Form 1) of the Tris salt of the compound of Example 7.

Fig. 2 is an observed X-ray powder diffraction pattern for the anhydrous (anhydrate) crystalline form (Form A) of the Tris salt of the compound of Example 10.

Detailed description of the invention

The present invention relates to a compound of formula I

I or a pharmaceutically acceptable salt thereof, where R is F; p is 0 or 1; ring A is phenyl or pyridinyl;

m is 0, 1, 2, or 3;

each R ¹ is independently selected from halogen, -CN, -C _1-3 alkylα, or -OC _1-3 alkyl, where C _1-3 alkyl and OC _1-3 αalkyl are substituted with 0-3 F atoms;

R ² is H or -C _1-3 alkyl, where alkyl is substituted with 0-1 OH;

X-L is N-CH2 or cyclopropyl;

Y is CH or N;

R ⁴ is -C1-3 alkyl, -C0-3 alkylene-C3-6 cycloalkyl, -C0-3 alkylene-R ⁵ or -C 1-3 alkylene-R ⁶ , wherein said alkyl may be substituted when valency permits, with 0-3 substituents, independently selected from 0-3 F atoms and 0-1 substituent selected from -C0-1 alkylene-OR ^O , -SO2-N(R ⁿ ) ₂ , -C(O)N(R ⁿ )2, -N(C =O)(R ⁿ ) and -N(R ⁿ )2;

R ⁵ is a 4-6 membered heterocycloalkyl selected from oxetanyl, tetrahydrofuranyl, morpholinyl, 1,3-oxazolidinyl and pyrrolidinyl, wherein said heterocycloalkyl may be substituted, when valency permits, with 0-2 substituents independently selected from: 0-1 oxo (=O);

R ⁶ is a 5-6 membered heteroaryl selected from oxazolyl, imidazolyl, pyridinyl, pyrazolyl and triazolyl, wherein said heteroaryl may be substituted, when valency permits, with 0-2 substituents independently selected from:

0-2 -C _1-3 alkyl, where the alkyl may be substituted, when valency permits, with 0-3 substituents independently selected from:

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0-1 -OR ^O ;

each R ^O is independently H or —C _1-3 alkyl;

each RN is independently H or —C _1-3 alkyl;

each of Z ¹ , Z ² and Z ³ is -CR ^Z ; and each R ^Z is independently H, F, Cl or —CH ₃ ; or

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1 - {2[methyl(methylsulfonyl )amino]ethyl}-1H-benzimidazole-6-carboxylic acid or a pharmaceutically acceptable salt thereof.

Another embodiment relates to a compound of formula II

or its pharmaceutically acceptable salt, where

R is F;

p is 0 or 1;

ring A is phenyl or pyridinyl;

m is 0, 1 or 2;

each R ¹ is independently selected from halogen, -CN, -C _1-3 alkyl, or -OC _1-3 alkyl, where C _1-3 alkyl and OC _1-3 alkyl are substituted with 0-3 F atoms;

R ² is H or CH ₃ ;

X-L is N-CH2 or cyclopropyl;

Y is CH or N;

Z ³ is -CR ^Z ; And

R ^Z is H or -CH ₃ .

Another embodiment relates to a compound of formula III

or a pharmaceutically acceptable salt thereof, wherein Ring A is phenyl or pyridinyl;

m is 0, 1 or 2;

each R ¹ is independently selected from F, Cl or -CN;

R ² is H or CH ₃ ; And

Y is CH or N.

Another embodiment relates to a compound of formula IV

or its pharmaceutically acceptable salt, where m is 0, 1 or 2; each R ¹ is independently selected from F, Cl and -CN; R ² is H or CH ₃ ; and Y is CH or N.

Another embodiment relates to a compound of formula V

or its pharmaceutically acceptable salt, where m is 0 or 1;

R ¹ is F, Cl or -CN;

R ² is H or CH ₃ ; And

Y is CH or N.

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Another embodiment relates to compounds of formula IV or formula V, where the phenyl or pyridinyl ring A has one R ¹ para to the carbon atom of said phenyl or pyridinyl attached to the dioxolane to form:

or their pharmaceutically acceptable salts, where each R ¹ is independently selected from F, Cl and -CN;

R ² is H or CH ₃ ; And

Y is CH or N.

Another embodiment relates to compounds of other embodiments, for example compounds of formula I or II or their pharmaceutically acceptable salts, where XL represents N-CH2; and Y is CH or N. Of the embodiments described herein, in that case, X is N and L is CH ₂ .

Another embodiment relates to compounds of other embodiments, for example compounds of formula I or II or their pharmaceutically acceptable salts, where X-L represents cyclopropyl; and Y is N.

In embodiments where X-L is cyclopropyl, the compounds of formula I or II provide:

Another embodiment relates to compounds of formula I, II, III, IV or V, where R ⁴ represents -CH2CH2OCH3, C1-3alkylene- ^R5 or C1-3alkylene- ^R6 , or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula II, III, IV or V, where R ⁴ such as defined for the compounds of formula I, or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where R ⁴ represents -C 1-3 alkyl, where the specified alkyl can be substituted, when valence allows, 0-1 substituent selected from -C 0-1 alkylene-OR ^O and -N(R ⁿ )2, or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where R ⁴ represents -(CH ₂ ) ₂ OCH ₃ or -(CH ₂ ) ₂ N(CH ₃ ) ₂ , or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where R ⁴ represents -CH 2 -R ⁵ where R ⁵ represents a 4-5 membered heterocycloalkyl, or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where heterocycloalkyl is

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where heterocycloalkyl is

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where heterocycloalkyl is

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where heterocycloalkyl is

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where heterocycloalkyl is

where heterocycloalkyl is unsubstituted.

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Another embodiment relates to compounds of formula I, II, III, IV or V, where -CH2-R ⁵ and the nitrogen atom to which R ⁴ is attached provide:

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where R ⁴ represents -CH2-R ⁶ where R ⁶ represents a 5-membered heteroaryl, where the specified heteroaryl can be substituted when valence allows, 0- 2 substituents independently selected from: 0-1 -CH ₃ , -CH ₂ CH ₃ or -CH ₂ CH ₂ OCH ₃ ; or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where heteroaryl is

where the specified heteroaryl may be substituted, when valence allows, 0-2 substituents, for example replacing a hydrogen atom, independently selected from:

0-2 -C _1-3 alkyl, where the alkyl may be substituted, when valency permits, with 0-3 substituents independently selected from:

0-1 -ORO;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of formula I, II, III, IV or V, where heteroaryl is

wherein said heteroaryl may be substituted when valency permits with 0-1 substituent -C _1-2 alkyl, where alkyl may be substituted when valency permits with 0-3 substituents independently selected from: 0-1 -ORO; and each RO is independently H or —C _1-3 alkyl; or their pharmaceutically acceptable salts. It is understood that any substituent may replace H on a carbon or nitrogen that is substituted. Non-limiting examples of substituted heteroaryls are:

It is understood that H is replaced by a substituent, for example R ^6s (a substituent provided on any heteroaryl R ⁶ ), to form:

where R ^6s is -C _1-2 alkyl, where alkyl may be substituted, when valency permits, with 0-3 substituents independently selected from: 0-1 -ORO; and each RO is independently H or —C _1-3 alkyl.

Another embodiment relates to compounds of formula I, II, III, IV or V, where heteroaryl is kN-Ci.xyl d/. ^OR or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of other embodiments herein, for example compounds of formula I, II, III, IV or V, where R ^Z represents H; or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of other embodiments herein, for example compounds of formula I, II, III, IV or V, where each R ¹ is independently F, Cl or -CN; or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of other embodiments herein, for example compounds of formula I, II, III, IV or V, where R ⁴ represents -CH ₂ -R ⁵ ; or their pharmaceutically acceptable salts.

Another embodiment refers to compounds of other embodiments herein, e.g.

- 5 040816 compounds of formulas I, II, III, IV or V, where R ⁴ represents -CH2-R ⁶ or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of other embodiments herein, for example compounds of formula I, II, III, IV or V, where the compound is a free acid.

Another embodiment refers to any embodiment of the compounds of formula I, II, III, IV or V, where ring A and R2 provide

or their pharmaceutically acceptable salts, where

R is F;

p is 0 or 1;

m is 0, 1 or 2; and each R ¹ is independently selected from halogen, -CN, -C ₁ . ₃ alkyl and -OC ₁ . ₃ alkyl, where alkyl C ₁ . ₃ alkyl and OC ₁ . ₃ alkyls are substituted with 0-3 F atoms.

Another embodiment relates to compounds of the invention wherein R ² is H; or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention, wherein the compound is

2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1 |[(28)-oxetan-2-ylmethyl ]-1H-benzimidazole-6-carboxylic acid or

2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7fluoro-1-[(2 8)-oxetan- 2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention, wherein the compound is

2-({4-((2 8)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl] piperidin-1 yl } methyl)-1 - [(2 8)- oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid or

2-( { 4-((2 8)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl] piperidin-1 yl } methyl)-7-fluoro-1 -[( 2 8)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention wherein R ² is CH ₃ ; or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention, wherein the compound is

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1 yl } methyl)-1 - [(2 8)-oxetane -2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid;

2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl} methyl)-1 - [(2 8)-oxetan- 2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid;

2-( { 4- (2- (5 -chloropyridin-2-yl) -2-methyl-1,3 - benzode ioxol-4-yl] piperidin-1 yl } methyl) -1 - [(2 8) - oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1 yl } methyl)-3 -(1,3-oxazol- 2-ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1 yl} methyl)-1-((1-ethyl-1Н -imide azol-5-yl)methyl]-1 H-benzimidazole-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1 yl}methyl)-1-(1,3-oxazol- 4-ylmethyl)-I R-benz and midazol-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperidin-1 yl } methyl)-1 -(pyridin-3-ylmethyl) - 1H-benzimid azole-6-carboxylic acid;

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2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzode ioxol-4-yl] piperidin-1 yl}methyl)-1-(1,3-oxazole -5-ylmethyl)- 1//-benzimidazole-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzode ioxol-4-yl] piperidin-1 yl } methyl)-1 - [(1 -ethyl- 1H-1,2,3-triazol-5-yl)methyl] - 1H-benzimid azole-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzode ioxol-4-yl] piperidin-1 yl } methyl)-1-(1,3-oxazole -2-ylmethyl)- 1//-benzimidazole-6-carboxylic acid;

2-({4-[2-(4-chloro-2-fluorophenyl)-7-fluoro-2-methyl-1,3-benzodioxol-4yl]piperide in-1-yl}methyl)-1 - [(25 )-oxetan-2-ylmethyl] - 1/7-benzimid azole-6-carboxylic acid;

2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl } methyl)-1-(1,3-oxazol-2 -ylmethyl)- 1H-benzimidazole-6-carboxylic acid or 2-({4-((2 8)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzode ioxol-4- yl] piper idin1 -yl } methyl)-7-fluoro-1 - [(2 8)-oxetan-2-ylmethyl] -1 H-benzimid azole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention wherein the compound is 2-({4-[(28)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine1 -yl } methyl)-1 -[(2 8)-oxetan-2-ylmethyl] -1 H-benzimidazole-6-carboxylic acid or

2-({4-[(28)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1 -yl } methyl)-7-fluoro-1 - [(2 8)-oxetan-2-ylmethyl]-1 H-benzimid azole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention wherein the compound is 2-({4-((28)-2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl] piperide in1 -yl } methyl)-1-[(2 8)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid;

2-({4-((2 8)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzode ioxol-4-yl]piperidin1-yl}methyl)-1-[ (2 8)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid or

2-( { 4-((2 8)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzode ioxol-4-yl] piper idin1 -yl } methyl)-1-( (1-Ethyl-1H-imide azol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention wherein the compound is 2-({4-[(2K)-2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine1 -yl } methyl)-1 -[(2 8)-oxetan-2-ylmethyl] -1 H-benzimidazole-6-carboxylic acid;

2-({4-[(2R)-2-(5-chloropyride in-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[( 2 8)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid or 2-({4-[(2K)-2-(4-chloro-2-fluorophenyl)-2-methyl-1, 3-benzodioxol-4-yl]piperidin1-yl } methyl)-1-((1-ethyl-1H-imide azol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid;

or their pharmaceutically acceptable salts.

Another embodiment relates to compounds of the invention wherein the compound is 2-({4-[2-(4-cyano-2-fluorophenyl)-2*-methyl-1,3-benzodioxol-4-yl]piperidin-1yl } methyl)-1 - [(2 8)-oxetan-2-ylmethyl] -1 H-benzimidazole-6-carboxylic acid, where the chirality 2* comes from C56;

2-( { 4- (2- (5 -chloropyridin-2-yl) -2 * -methyl-1,3 -benzodioxol-4-yl] piperidin-1 yl } methyl) -1 - [(2 8) - oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid, where the chirality 2* comes from P9;

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2-({4-[2-(4-chloro-2-fluorophenyl)-2*-methyl-1,3-benzodioxol-4-yl]piperidin-1yl} methyl)-1 - [(1-ethyl- 1H -imidazol-5-yl)methyl]-1 H-benzimidazole-6-carboxylic acid, where the chirality 2* comes from 17;

2-({4-[2-(4-chloro-2-fluorophenyl)-7-fluoro-2*-methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(28 )-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, where the chirality 2* comes from 96; or

2-({4-[2-(4-cyano-2-fluorophenyl)-2*-methyl-1,3-benzodioxol-4-yl] piperidin-1 yl} methyl)-1-(1,3-oxazole -2-ylmethyl)-1H-benzimidazole-6-carboxylic acid, where the chirality 2* comes from C82;

or their pharmaceutically acceptable salts.

Another embodiment includes a compound which is 2-({4-[(2S)-2-(4-chloro2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid or a pharmaceutically acceptable salt thereof, wherein the salt is a tris salt.

Another embodiment includes a compound which is 2-({4-[(2S)-2-(4-chloro2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid as the free acid.

Another embodiment includes a compound that is

or a pharmaceutically acceptable salt thereof.

Another embodiment includes a compound which is 2-({4-[(2S)-2-(4-chloro2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl }methyl)-1-[(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylic acid or a pharmaceutically acceptable salt, wherein the salt is a tris salt {the tris salt of this compound is also known as 2-({4 -[(2S)-2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2- ylmethyl]-1H-benzimidazole-6-carboxylate 1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium}.

In some embodiments, the present invention provides a crystalline form of anhydrous 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine tris salt -1-yl}methyl)-1[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid. In some additional embodiments, the crystalline form of the anhydrous (anhydrate) tris salt of 2-({4-[(2S)-2-(4-chloro-2fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine- 1 -yl} methyl)-1 -[(2S)-oxetan-2-ylmethyl] -1Hbenzimidazole-6-carboxylic acid is designated form I, which is characterized by its unique solid state characteristics in relation to, for example, powder X-ray diffraction (XRD ) described in this document (essentially as shown in Fig. 1). In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing at least two characteristic peaks selected from peaks at 3.7±0.2°; 7.3±0.2°; 8.5±0.2°; 10.1±0.2°; 14.7±0.2° and 16.9±0.2° in 2θ units. In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing at least three characteristic peaks selected from peaks at 3.7±0.2°; 7.3±0.2°; 8.5±0.2°; 10.1±0.2°; 14.7±0.2° and 16.9±0.2° in 2θ units. In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing at least four characteristic peaks selected from peaks at 3.7±0.2°; 7.3±0.2°; 8.5±0.2°; 10.1±0.2°; 14.7±0.2° and 16.9±0.2° in 2θ units. In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing at least five characteristic peaks selected from peaks at 3.7±0.2°; 7.3±0.2°; 8.5±0.2°; 10.1±0.2°; 14.7±0.2° and 16.9±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing characteristic peaks at 3.7 ± 0.2° and 7.3 ± 0.2° in units of 2θ.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing peaks at 3.7±0.2°; 7.3±0.2° and 14.7±0.2° in 2θ units. In some additional embodiments, Form I exhibits an X-ray powder diffraction pattern that further comprises at least one peak selected from peaks at 8.5±0.2°; 10.1±0.2° and 16.9±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing peaks at 3.7±0.2°; 7.3±0.2°; 14.7±0.2° and 16.9±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing peaks at 3.7±0.2°; 7.3±0.2°; 8.5±0.2°; 10.1±0.2°; 14.7±0.2° and 16.9±0.2° in 2θ units.

- 8 040816

In some embodiments, Form I exhibits an X-ray powder diffraction pattern substantially as shown in FIG. 1. The list of diffraction peaks, expressed in units of degrees 2θ, and relative intensities with a relative intensity of >3.0% is presented below in table. x1.

As is well known in the field of powder diffraction, the relative intensities of the peaks (reflections) can vary depending on the sample preparation method used, the sample setup procedure, and the particular instrument. Moreover, variations in instrument readings and other factors can affect 2-theta values. Therefore, the values of the XRD peaks can vary in the range of plus or minus about 0.2°.

Another embodiment includes a compound which is 2-({4-[(2S)-2-(4-chloro2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl } methyl)-1-[(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylic acid as the free acid.

Another embodiment includes a compound that is

or a pharmaceutically acceptable salt thereof.

Another embodiment includes a compound which is 2-({4-[2-(5chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1 -[(2S)-oxetan-2-ylmethyl] 1H-benzimidazole-6-carboxylic acid, 2-({4-[(2S)-2-(5-chloropyridin-2-yl)-2-methyl-1 ,3-benzodioxol4-yl]piperidin-1-yl}methyl)- 1-[(2S)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid or 2({4-[(2R)- 2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H- benzimidazole-6-carboxylic acid as the free acid.

Another embodiment includes a compound which is 2-({4-[2-(5chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1 -[(2S)-oxetan-2-ylmethyl] 1H-benzimidazole-6-carboxylic acid, 2-({4-[(2S)-2-(5-chloropyridin-2-yl)-2-methyl-1 ,3-benzodioxol4-yl]piperidin-1-yl}methyl)- 1-[(2S)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylic acid or 2({4-[(2R)- 2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H- benzimidazole-6-carboxylic acid; or a pharmaceutically acceptable salt thereof, wherein the salt is a tris salt.

Another embodiment includes a compound which is 2-({4-[2-(5chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1-[(2S)-oxetan-2-ylmethyl] 1H-benzimidazole-6-carboxylic acid, DIAST-X2:

or a pharmaceutically acceptable salt thereof. In some additional embodiments, the present invention provides a compound which is 2-({4-[2-(5-chloropyridin2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl} methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2, or its tris salt [i.e. salt of 1,3-dihydroxy-2(hydroxymethyl)propan-2-amine]. The chiral center on the left side of the compound structure is labeled abs to show that the chiral center has only one stereoconfiguration (i.e. not a racemate at that chiral center).

In some embodiments, the present invention provides a crystalline form of anhydrous tris salt of 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl } methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2. In some additional embodiments, the crystalline form of the anhydrous (anhydrate) tris salt of 2-({4-[2-(5-chloropyridin-2-yl)2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl }methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2, is designated Form A, which is characterized by its unique solid state performance in relation to, for example, X-ray powder diffraction (DRLP) described in this document (essentially as shown in Fig. 2). In some embodiments, Form A exhibits an X-ray powder diffraction pattern containing at least two characteristic peaks selected from peaks at 7.7±0.2°; 15.2±0.2°; 15.7±0.2° and 17.6±0.2° in 2θ units. In some embodiments, Form A exhibits an X-ray powder diffraction pattern containing at least three characteristic peaks selected from peaks at 7.7±0.2°; 15.2±0.2°; 15.7±0.2° and 17.6±0.2° in 2θ units. In some embodiments, Form A exhibits an X-ray powder diffraction pattern containing characteristic peaks selected from peaks at 7.7±0.2°; 15.2±0.2°; 15.7±0.2° and 17.6±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing characteristic peaks at 7.7±0.2° and 17.6±0.2° in units of 2θ.

In some embodiments, form A shows an x-ray powder diffraction pattern containing peaks at 7.7±0.2°; 15.2±0.2° and 17.6±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing peaks at 7.7±0.2°; 15.2±0.2° and 15.7±0.2° in 2θ units.

In some embodiments, Form I exhibits an X-ray powder diffraction pattern containing peaks at 7.7±0.2°; 15.2±0.2°; 15.7±0.2° and 17.6±0.2° in 2θ units.

In some embodiments, Form A exhibits an X-ray powder diffraction pattern substantially as shown in FIG. 2. The list of diffraction peaks, expressed in units of degrees 20, and relative intensities with a relative intensity of >3.0% is presented above in table. X2.

As is well known in the field of powder diffraction, the relative intensities of the peaks (reflections) can vary depending on the sample preparation method used, the sample setup procedure, and the particular instrument. Moreover, variations in instrument readings and other factors can affect 2-theta values. Therefore, the values of the XRD peaks can vary in the range of plus or minus about 0.2°.

In another embodiment, the invention provides a pharmaceutical composition comprising a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, in admixture with at least one pharmaceutically acceptable excipient. This would include a pharmaceutical composition containing a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, in admixture with at least one pharmaceutically acceptable excipient and one or more other therapeutic agent discussed in this document.

This document also discloses: a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, for use as a drug; a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, for use in the prevention and/or treatment of the cardiometabolic and associated diseases discussed herein, including T2DM, prediabetes, NASH and cardiovascular disease; a method of treating a disease in which a GLP-1R agonist is indicated in a subject in need of such prevention and/or treatment, comprising administering to that subject a therapeutically effective amount of a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein; the use of a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, for the manufacture of a medicament for the treatment of a disease or condition for which a GLP-1R agonist is indicated; a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, for use in the treatment of a disease or condition for which a GLP-1R agonist is indicated; or a pharmaceutical composition for the treatment of a disease or condition in which a GLP-1R agonist is indicated, comprising a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein.

Each exemplified compound or pharmaceutically acceptable salt thereof may be claimed individually or grouped together in any combination with any number of each and every embodiment described herein.

Also disclosed is a pharmaceutical composition containing a compound of formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein, for use in the treatment and/or prevention of cardiometabolic and associated diseases discussed in this document, including T2DM, prediabetes, NASH, and cardiovascular disease.

Also disclosed is the use of compounds of formula I, II, III, IV, or V, or pharmaceutically acceptable salts thereof, as defined in any of the embodiments described herein, in the treatment and/or prevention of cardiometabolic and associated diseases, including diabetes (T1D and/or T2DM, including prediabetes), idiopathic T1D (type 1b), latent autoimmune diabetes in adults (LADA), early onset T2DM (EOD), atypical diabetes of youth (YOAD), diabetes of adulthood in young adults (MODY), diabetes associated with insufficiency nutrition, gestational diabetes, hyperglycemia, insulin resistance, hepatic insulin resistance, impaired glucose tolerance, diabetic neuropathy, diabetic nephropathy, kidney disease (eg, acute kidney injury, renal tubular dysfunction, proinflammatory changes in the proximal renal tubules), diabetic retinopathy, adipocyte dysfunction, visceral fat deposition, sleep apnea, obesity (including hypothalamic eskoe obesity and monogenic

- 10 040816 obesity) and associated comorbidities (eg, osteoarthritis and urinary incontinence), eating disorders (including compulsive overeating syndrome, bulimia nervosa, and syndromic obesity such as Prader-Willi and Bardet-Biedl syndromes), weight gain due to for the use of other agents (for example, due to the use of steroids and antipsychotics), excessive sugar cravings, dyslipidemia (including hyperlipidemia, hyperglyceridemia, elevated total cholesterol, high LDL (low-density lipoprotein) cholesterol, and low HDL (high-density lipoprotein) cholesterol , hyperinsulinemia, NAFLD (including associated diseases such as steatosis, NASH, fibrosis, cirrhosis, and hepatocellular carcinoma), cardiovascular disease, atherosclerosis (including coronary artery disease), peripheral vascular disease, hypertension, endothelial dysfunction, vasodilatation disorder , congestive heart failure, myocardial infarction a (eg, necrosis and apoptosis), stroke, hemorrhagic stroke, ischemic stroke, traumatic brain injury, pulmonary hypertension, restenosis after angioplasty, intermittent claudication, postprandial lipemia, metabolic acidosis, ketosis, arthritis, osteoporosis, Parkinson's disease, hypertrophy left ventricular disease, peripheral arterial disease, macular degeneration, cataracts, glomerulosclerosis, chronic renal failure, metabolic syndrome, syndrome X, premenstrual syndrome, angina pectoris, thrombosis, atherosclerosis, transient ischemic attacks, vascular restenosis, impaired glucose metabolism, impaired fasting glucose conditions , hyperuricemia, gout, erectile dysfunction, skin and connective tissue disorders, psoriasis, foot ulceration, ulcerative colitis, hyperapolipoprotein B-lipoproteinemia, Alzheimer's disease, schizophrenia, cognitive impairment, inflammatory bowel disease, co colon, Crohn's disease, colitis, irritable bowel syndrome, in the prevention or treatment of polycystic ovary syndrome and in the treatment of addiction (eg, alcohol and/or drug addiction).

The term alkyl as used herein means a straight or branched monovalent hydrocarbon group of the formula -C _n H( _2n+ i). Non-limiting examples include methyl, ethyl, propyl, butyl, 2-methyl-propyl, 1,1-dimethylethyl, pentyl, and hexyl.

The term alkylene as used herein means a divalent straight chain or branched hydrocarbon group of the formula -C _n H _2n -. Non-limiting examples include ethylene and propylene.

The term cycloalkyl as used herein means a cyclic monovalent hydrocarbon group of the formula -C _n H( _2n - ₁ ) containing at least three carbon atoms. Non-limiting examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

The term halogen as used herein refers to fluoride, chloride, bromide, or iodide.

The term heterocycloalkyl as used herein refers to a cycloalkyl group in which one or more ring methylene groups (-CH2-) is substituted with a group selected from -O-, -S- or a nitrogen atom, where the nitrogen atom may provide an attachment point or may be substituted as suggested in each embodiment. In cases where the nitrogen atom provides the point of attachment, the image of the heterocycloalkyl structure will have a hydrogen on the specified nitrogen atom. Generally, heterocycloalkyl may be substituted, when valency permits, with 0-2 substituents independently selected from oxo, -CN, halo, alkyl, and -Oalkyl, and alkyl may be further substituted. It should be noted that in the case of zero substitution heterocycloalkyl is unsubstituted.

The term heteroaryl as used herein refers to a monocyclic aromatic hydrocarbon containing 5 to 6 carbon atoms in which at least one ring carbon atom is replaced by a heteroatom selected from an oxygen atom, a nitrogen atom and a sulfur atom. Such a heteroaryl group may be attached through a ring carbon atom or, if valency permits, through a ring nitrogen atom. Generally, heteroaryl may be substituted, when valency permits, with 0-2 substituents independently selected from halo, OH, alkyl, Oalkyl, and amino (e.g., NH2, NH-alkyl, N(alkyl) ₂ ), and alkyl may be additionally replaced. It should be noted that in the case of zero substitution, heteroaryl is unsubstituted.

Room temperature: r.t. (from 15 to 25°C).

Methanol: MeOH.

Ethanol: EtOH.

Isopropanol: iPrOH.

Ethyl acetate: EtOAc.

Tetrahydrofuran: THF.

Toluene: PhCH ₃ .

Cesium carbonate: Cs ₂ CO ₃ .

Lithium bis(trimethylsilyl)amide: LiHMDS.

sodium tert-butoxide: NaOtBu.

- 11 040816 Potassium tert-butoxide: KOtBu.

Lithium diisopropylamide: LDA.

Triethylamine: Et ₃ N.

N,N-Diisopropylethylamine: DIPEA.

Potassium carbonate: K2CO3.

Dimethylformamide: DMF.

Dimethylacetamide: DMAc.

Dimethyl sulfoxide: DMSO.

N-methyl-2-pyrrolidinone: NMP.

Sodium hydride: NaH.

Trifluoroacetic acid: TFA.

Trifluoroacetic anhydride: TFAA.

Acetic anhydride: Ac2O.

Dichloromethane: DCM.

1,2-Dichloroethane: DCE.

Hydrochloric acid: HCl.

1,8-Diazabicyclo[5.4.0]undec-7-ene: DBU.

Borane-dimethylsulfide complex: BH ₃ -DMS.

Borane-tetrahydrofuran complex: BH ₃ -THF.

Lithium aluminum hydride: LAH.

Acetic acid: AcOH.

Acetonitrile: MeCN.

p-Toluenesulfonic acid: pTSA.

Dibenzylidineacetone: DBA.

2,2'-Bis(diphenylphosphino)-1,1'-binaphthalene: BINAP.

1,1'-Ferrocenediyl-bis(diphenylphosphine): dppf.

1,3-Bis(diphenylphosphino)propane: DPPP.

3-Chloroperbenzoic acid: m-CPBA.

tert-Butyl methyl ether: MTBE.

Methansulfonyl: Ms.

N-Methylpyrrolidinone: NMP.

Thin layer chromatography: TLC.

Supercritical Fluid Chromatography: SFC.

4-(Dimethylamino)pyridine: DMAP.

tert-Butyloxycarbonyl: Vos.

1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate: HATU.

Petroleum ether: RE.

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate: HBTU.

2-Amino-2-(hydroxymethyl)propane-1,3-diol: tris.

Tris(dibenzylideneacetone)dipalladium: Pd ₂ (dba) ₃ .

The 1H nuclear magnetic resonance (NMR) spectra in all cases agreed with the proposed structures. Characteristic chemical shifts (δ) are given in parts per million (ppm) relative to the residual proton signal in deuterated solvent (CHCl ₃ at 7.27 ppm; CD2HOD at 3.31 ppm; MeCN at 1.94 ppm; DMSO at 2.50 ppm) and are listed using the standard abbreviations for major peaks: eg s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broadened. The symbol ^Λ means that the area of the 1H NMR peak was inferred because the peak was partially hidden by the water peak. The symbol λλ means that the area of the 1H NMR peak was inferred because the peak was partially obscured by the solvent peak.

Used in this document, the wavy line “ ^h '” indicates the point of attachment of the substituent to another group.

The compounds and intermediates described below have been named using the naming convention provided by ACD/ChemSketch 2012, File Version C10H41, Build 69045 (Advanced Chemistry Development, Inc., Toronto, Ontario, Canada). The naming convention provided by ACD/ChemSketch 2012 is well known to those skilled in the art, and the naming convention provided by ACD/ChemSketch 2012 is generally considered to be consistent with the IUPAC (International Union of Pure and Applied Chemistry) recommendations for Nomenclature organic chemistry and CAS (Chemical Abstract Service) identifier rules. It should be noted that chemical names may have parentheses only, or may have parentheses and square brackets. Stereochemical descriptors may also appear in different places within the name itself, depending on the naming convention. One skilled in the art will be aware of these placements and will appreciate that they provide for the same chemical structure.

Pharmaceutically acceptable salts of compounds of formula I include acid addition salts and base addition salts.

Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulfate/sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, glyceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride salts. , hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfate, naphthylate, 2-napsilate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrophosphate/dihydrogen phosphate, pyroglutamate, saccharate , stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate, salts of 1,5-naphthalenedisulfonic acid and xinafoate.

Suitable base addition salts are formed from bases which form non-toxic salts. Examples include aluminum salts, salts with arginine, benzathine, calcium salts, salts with choline, diethylamine, bis(2-hydroxyethyl)amine (diolamine), glycine, lysine, magnesium salts, salts with meglumine, 2-aminoethanol (olamine), potassium , sodium salts, salts with 2-amino-2(hydroxymethyl)propane-1,3-diol (tromethamine), and zinc salts.

Hemisalts of acids and bases can also be formed, for example hemisulfates and hemicalcium salts. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002).

Pharmaceutically acceptable salts of the compounds of formula I can be obtained by one or more of three methods:

(1) by reacting a compound of formula I with the desired acid or desired base;

(2) by removing an acid- or base-labile protecting group from a suitable precursor of a compound of formula I, or by ring-opening a suitable cyclic precursor, eg lactone or lactam, using the desired acid or desired base; or (3) by converting one salt of a compound of formula I to another by reaction with a suitable acid or a suitable base, or using a suitable ion exchange column.

All three reactions are usually carried out in solution. The resulting salt may precipitate and may be collected by filtration or may be isolated by evaporating the solvent. The degree of ionization of the resulting salt can vary from fully ionized to almost non-ionized.

The compounds of formula I and their pharmaceutically acceptable salts may exist in unsolvated form and in solvated forms. The term solvate is used herein to describe a molecular complex containing a compound of formula I, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable solvent molecules, such as ethanol.

The term hydrate is used when said solvent is water.

The currently accepted classification system for organic hydrates is one that defines unbound hydrates, channel type hydrates, or metal ion coordinated hydrates (see Polymorphism in Pharmaceutical Solids by K. R. Morris (Ed. H. G. Brittain, Marcel Dekker, 1995)). Unbound hydrates are hydrates in which water molecules are isolated from direct contact with each other by organic molecules lying between them. In channel-type hydrates, water molecules lie in lattice channels, where they are adjacent to other water molecules. In hydrates coordinated by metal ions, water molecules are bound to the metal ion.

When the solvent or water is strongly bound, then the complex will have a well-defined stoichiometry independent of humidity. However, when the solvent or water is weakly bound, as in the case of channel-type solvates and hygroscopic compounds, then the water/solvent content may depend on humidity and drying conditions. In such cases, the absence of stoichiometry will be the norm.

Also within the scope of this invention are multicomponent complexes (other than salts and solvates) in which the drug and at least one other component are present in stoichiometric or non-stoichiometric amounts. Complexes of this type include clathrates (drug-host inclusion complexes) and co-crystals. The latter are usually defined as crystalline complexes of neutral molecular moieties that are bonded together through non-covalent interactions, but may also be a complex of a neutral molecule with a salt. Co-crystals can be obtained by crystallization from a melt, recrystallization from solvents, or co-physical grinding of the components (see Chem. Commun., 17, 1889-1896, by O. Almarsson and M. J. Zaworotko (2004)). For a general overview of multicomponent complexes, see J Pharm. Sci, 64(8), 1269-1288, Haleblian (August 1975).

The compounds of the invention may exist in a continuum of solid states ranging from fully amorphous to fully crystalline. The term amorphous refers to the state, in

- 13 040816 in which the substance has no long-range order at the molecular level and, depending on the temperature, can exhibit the physical properties of a solid or liquid. Typically, such substances do not give characteristic X-ray powder diffraction patterns and, while exhibiting the properties of a solid, are more formally characterized as a liquid. When heated, there is a transition from the properties of a solid to the properties of a liquid, which is characterized by a change of state, usually of the second order (glass transition). The term crystalline refers to a solid phase in which the substance has a regular ordered internal structure at the molecular level and gives a characteristic X-ray powder diffraction pattern with defined peaks. Such substances, when sufficiently heated, will also exhibit the properties of a liquid, but the transition from solid to liquid is characterized by a phase transition, usually of the first order (melting point).

The compounds of formula I may also exist in a mesomorphic state (mesophase or liquid crystal) when exposed to appropriate conditions. The mesomorphic state is an intermediate state between the true crystalline state and the true liquid state (either melt or solution). Mesomorphism resulting from a change in temperature is classified as thermotropic, and mesomorphism resulting from the addition of a second component such as water or other solvent is classified as lyotropic. Compounds that can form lyotropic mesophases are classified as amphiphilic and consist of molecules that contain an ionic (such as -COO ^- Na ⁺ , -COO-K + , or -SO3 ^- Na ⁺ ) or non-ionic (such as -N ^- N ⁺ ( CH3) ₃ ) polar end group. For more information, see Crystals and the Polarizing Microscope by NH Hartshorne and A. Stuart, ^4th Edition (Edward Arnold, 1970).

Compounds of formula I may exhibit polymorphism and/or one or more types of isomerism (eg optical, geometric or tautomeric isomerism). The compounds of formula I may also be labeled with isotopes. Such variants are intended for the compounds of formula I, defined as they are, based on their structural features, and, therefore, are included in the scope of the invention.

Compounds of formula I containing one or more asymmetric carbon atoms may exist as two or more stereoisomers. If the compound of formula I contains an alkenyl or alkenylene group, geometric cis/trans (or Z/E) isomers are possible. If structural isomers are capable of interconversion due to a low energy barrier, then tautomeric isomerism (tautomerism) may occur. It may take the form of proton tautomerism in compounds of formula I containing, for example, an imino, keto or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety. It follows that a single compound can exhibit more than one type of isomerism.

Pharmaceutically acceptable salts of the compounds of formula I may also contain a counterion that is optically active (eg d-lactate or l-lysine) or racemic (eg dl-tartrate or dl-arginine).

The cis/trans isomers can be separated by standard methods known to those skilled in the art, such as chromatography and fractional crystallization.

Standard methods for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). Alternatively, a racemic precursor containing a chiral ester can be resolved by enzymatic resolution (see, for example, Int J Mol Sci 29682-29716 by A. C. L. M. Carvaho et al. (2015)). Where a compound of formula I contains an acidic or basic moiety, a salt may be formed with an optically pure base or an optically pure acid, for example l-phenylethylamine or tartaric acid. The resulting diastereomeric mixture may be separated by fractional crystallization and one or both diastereomeric salts may be converted to the corresponding pure enantiomer(s) by methods known to those skilled in the art. Alternatively, the racemate (or racemic precursor) may be subjected to a covalent interaction with a suitable optically active compound, such as alcohol, amine or benzyl chloride. The resulting diastereomeric mixture can be separated by chromatography and/or fractional crystallization by methods known to those skilled in the art to give the individual diastereomers as single enantiomers with 2 or more chiral centers. Chiral compounds of formula I (and their chiral precursors) can be obtained in an enantiomerically enriched form using chromatography, typically HPLC, on an asymmetric resin with a mobile phase consisting of a hydrocarbon, usually heptane or hexane, containing from 0 to 50% by volume of isopropanol, typically 2 to 20%, and 0 to 5% by volume alkylamine, typically 0.1% diethylamine. After concentration of the eluate, an enriched mixture is obtained. You can use chiral chromatography using sub - and supercritical fluids. Chiral chromatography techniques useful in some embodiments of the present invention are known in the art (see, for example, Smith, Roger M., Loughborough University, Loughborough, UK; Chromatographic Science Series (1998), 75 (Supercritical Fluid Chromatography

- 14 040816 with Packed Columns), pp. 223-249, and sources cited there). In some relevant examples given in this specification, the columns are from Chiral Technologies, Inc., West

Chester, Pennsylvania, USA, a subsidiary of Daicel® Chemical Industries, Ltd., Tokyo, Japan.

When any racemate crystallizes, two different types of crystals are possible. The first type is the racemic compound (true racemate) mentioned above, when a single homogeneous crystal form is formed containing both enantiomers in equimolar amounts. The second type is the racemic(s) mixture or conglomerate, when two crystal forms are formed in equimolar amounts, each containing a single enantiomer. Although both crystalline forms present in the racemic mixture have identical physical properties, they may have different physical properties compared to the true racemate. Racemic mixtures can be separated by standard methods known to those skilled in the art (see, for example, Stereochemistry_of Organic Compounds by E. L. Eliel and S. H. Wilen (Wiley, 1994)).

It must be emphasized that although the compounds of formula I are depicted in this description in a single tautomeric form, all possible tautomeric forms are included in the scope of the invention.

The present invention encompasses all pharmaceutically acceptable isotopically labeled compounds of formula I in which one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number that predominates in nature.

Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen such as ² H and ³ H, carbon such as ⁿ C, ¹³ C and ¹⁴ C, chlorine such as ³⁶ Cl, fluorine such as ¹⁸ F, iodine such as ¹²³ I and ¹²⁵ I, nitrogen such as ¹³ N and ¹⁵ N, oxygen such as ¹⁵ O, ¹⁷ O and ¹⁸ O, phosphorus such as ³² P, and sulfur such as ³⁵ S.

Certain isotopically labeled compounds of formula I, for example those with a radioactive isotope included, are useful in tissue distribution studies of drugs and/or substrates. Radioactive isotopes of tritium, i.e. ³ H, and carbon-14, i.e. ¹⁴ C are particularly useful for this purpose due to their ease of inclusion and ready-made detection tools.

Substitution by heavier isotopes such as deuterium, i.e. ² H may confer some therapeutic benefits through greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements.

Substitution with positron emitting isotopes such as ⁿ C, ¹⁸ F, ¹⁵ O and ¹³ N can be useful in positron emission tomography (PET) studies to study substrate receptor occupancy.

Isotopically labeled compounds of formula I can generally be prepared by standard methods known to those skilled in the art, or by methods analogous to those described in the accompanying examples and preparations, using an isotopically labeled reagent instead of the unlabeled reagent used previously.

Pharmaceutically acceptable solvates according to the invention include those in which the crystallization solvent can be replaced by an isotope, for example D2O, d ₆ -acetone, d ₆ -DMSO.

One way of carrying out the invention is the introduction of the compounds of formula I in the form of a prodrug. Thus, certain derivatives of a compound of formula I, which themselves may have little or no pharmacological activity, when administered into or on the body, can be converted into a compound of formula I having the desired activity, for example by hydrolytic cleavage, in particular hydrolytic cleavage, stimulated by the enzyme esterase or peptidase. Such derivatives are called prodrugs. Additional information on the use of prodrugs can be found in Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (Ed. E. B. Roche, American Pharmaceutical Association). Reference may also be made to Nature Reviews/Drug Discovery, 2008, 7, 355, and Current Opinion in Drug Discovery and Development, 2007, 10, 550.

Prodrugs in accordance with the invention can be obtained, for example, by substitution of the appropriate functional groups present in the compounds of formula I, some groups known to experts in this field as progroups, which are described, for example, in Design of Prodrugs by H. Bundgaard (Elsevier, 1985) and Y. M. Choi-Sledeski and C. G. Wermuth, Designing Prodrugs and Bioprecursors in Practice of Medicinal Chemistry, (Fourth Edition), Chapter 28, 657-696 (Elsevier, 2015).

Thus, a prodrug according to the invention is (a) an ester or amide derivative of a carboxyl group in a compound of formula I; (b) an ester, carbonate, carbamate, phosphate or ether derivative of the hydroxyl group in a compound of formula I; (c) an amide, imine, carbamate or amine derivative of an amino group in a compound of formula I; (d) an oxime or imine derivative of a carbonyl group in a compound of formula I; or (e) a methyl, primary alcohol or aldehyde group which can be metabolically oxidized to a carboxylic acid group in the compound of formula I.

Some specific examples of prodrugs according to the invention include:

(1) if a compound of formula I contains a carboxylic acid functional group (-COOH), then its ester, such as a compound in which the hydrogen of the carboxylic acid functional group of the compound of formula I is replaced by a C1- _C8 alkyl (e.g., ethyl) or group (C1 -C ₈ alkyl)C(=O)OCH ₂ (eg 'BuC(=O)OCH ₂ -);

(2) if the compound of formula I contains an alcohol functional group (-OH), then its ester, such as a compound in which the hydrogen of the alcohol functional group of the compound of formula I is replaced by a group -CO(C1-C ₈ alkyl) (for example, methylcarbonyl), or the alcohol is esterified with an amino acid;

(3) if the compound of formula I contains an alcohol functional group (-OH), then its ether, such as a compound in which the hydrogen of the alcohol functional group of the compound of formula I is replaced by a group (C ₁ -C ₈ alkyl)C (=O) OCH ₂ - or -CH ₂ OP(=O)(OH) ₂ ;

(4) if the compound of formula I contains an alcohol functional group (-OH), then its phosphate, such as a compound in which the hydrogen of the alcohol functional group of the compound of formula I is replaced by a group -P(=O)(OH) ₂ , or -P( =O)(ONa) ₂ , or -P(=O)(O ^- ) ₂ Ca ²⁺ ;

(5) if the compound of formula I contains a functional primary or secondary amino group (-NH2 or -NHR, where R ^ H), then its amide, for example a compound in which, as the case may be, one or both hydrogen atoms of the functional amino group of the compound of formula I is substituted /substituted with (C1-C ₁₀ )alkanoyl, -COCH2NH2, or the amino group is derivatized with an amino acid;

(6) if a compound of formula I contains a functional primary or secondary amino group (-NH2 or -NHR, where R ^ H), then its amine, for example a compound in which, as the case may be, one or both hydrogen atoms of the functional amino group of the compound of formula I is substituted /substituted with a -CH2OP(=O)(OH)2 group;

(7) where the carboxyl group in the compound of formula I is substituted with a methyl group, a -CH2OH group, or an aldehyde group.

Some compounds of formula I may themselves act as prodrugs of other compounds of formula I. It is also possible that two compounds of formula I are linked together to form a prodrug. In some circumstances, a prodrug of a compound of formula I can be created by internal linking of two functional groups in a compound of formula I, for example by formation of a lactone.

References to compounds of formula I include the compounds themselves and their prodrugs. The invention covers such compounds of formula I, as well as pharmaceutically acceptable salts of such compounds and pharmaceutically acceptable solvates of these compounds and salts.

Introduction and dosage

Typically, a compound of the invention is administered in an amount effective to treat the condition as described herein. The compounds of the invention may be administered as the compound itself or, alternatively, as a pharmaceutically acceptable salt. For purposes of administration and dosage, the compound itself or a pharmaceutically acceptable salt thereof will be referred to simply as the compounds of the invention.

The compounds of the invention are administered by any suitable route in the form of a pharmaceutical composition suitable for such route and at a dose effective for the intended treatment. The compounds of the invention may be administered orally, rectally, vaginally, parenterally or topically.

The compounds of the invention may be administered orally. Oral administration may include ingestion, such that the compound enters the gastrointestinal tract, or buccal or sublingual administration may be used, in which the compound enters the blood stream directly from the oral cavity.

In another embodiment, the compounds of the invention may be administered directly into the bloodstream, into a muscle, or into an internal organ. Suitable routes of parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needleless injectors and infusion equipment.

In another embodiment, the compounds of the invention may also be administered topically to the skin or mucosa, i. dermal or transdermal. In another embodiment, the compounds of the invention may also be administered intranasally or by inhalation. In another embodiment, the compounds of the invention may be administered rectally or vaginally. In another embodiment, the compounds of the invention may also be administered directly to the eye or ear.

The dosage regimen for the compounds of the invention and/or compositions containing said compounds is determined by taking into account a number of factors including the type, age, body weight, sex and medical condition of the patient; the severity of the condition; route of administration; and the activity of the particular compound used. Therefore, the dosage regimen can vary within very wide limits. In one embodiment, the total daily dose of a compound of the invention is typically from about 0.001 to about 100 mg/kg (i.e., mg of a compound of the invention per kg of body weight) for the treatment of the conditions discussed herein. In another embodiment, the total daily dose of a compound of the invention is from about 0.01 to about 30 mg/kg, and in another embodiment from about 0.03 to about 10 mg/kg, and in another embodiment from about 0.1 to about 3. It is not uncommon that the administration of a compound of the invention must be repeated several times a day (usually no more than 4 times). Multiple dosing per day can usually be used to increase the total daily dose if required.

For oral administration, the compositions may be provided in the form of tablets containing 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 30.0, 50, 0, 75.0, 100, 125, 150, 175, 200, 250 and 500 mg active ingredient for symptomatic dosage adjustment for the patient. The drug usually contains from about 0.01 to about 500 mg of the active ingredient, or, in another embodiment, from about 1 to about 100 mg of the active ingredient. Intravenously administered doses may range from about 0.01 to about 10 mg/kg/min at a constant infusion rate.

Suitable subjects of the invention include mammalian subjects. In one embodiment, suitable subjects are humans. Human subjects can be of any gender and at any stage of development.

Pharmaceutical compositions

In another embodiment, the invention covers pharmaceutical compositions. Such pharmaceutical compositions contain a compound of the invention, present with a pharmaceutically acceptable carrier. Other physiologically active substances may also be present. As used herein, the term pharmaceutically acceptable carrier encompasses any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, which are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of the following: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof, and may have isotonic agents, e.g., sugars, sodium chloride, in the composition. , or polyalcohols such as mannitol or sorbitol. Pharmaceutically acceptable substances such as wetting agents or small amounts of adjuvants such as wetting agents or emulsifiers, preservatives or buffers that increase the shelf life or potency of the antibody or antibody region.

The compositions of this invention may be in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms such as liquid solutions (eg, injectable and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The form depends on the intended route of administration and therapeutic application.

Typical compositions are in the form of injectable or infusion solutions, such as compositions such as those used to passively immunize humans with antibodies, as a rule. One route of administration is parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). In another embodiment, the antibody is administered by intravenous infusion or injection. In yet another embodiment, the antibody is administered by intramuscular or subcutaneous injection.

For oral administration, the solid dosage form may be presented, for example, in discrete units such as hard or soft capsules, pills, cachets, lozenges or tablets, each containing a predetermined amount of at least one compound of the invention. In another embodiment, oral administration may be in the form of a powder or granules. In another embodiment, the oral dosage form is sublingual, such as, for example, a lozenge. In such solid dosage forms, the compounds of formula I are usually combined with one or more excipients. Such capsules or tablets may be controlled release preparations. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents or may be prepared with an enteric coating.

In another embodiment, oral administration may be in a liquid dosage form. Liquid dosage forms for oral administration include, for example, pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art (eg, water). Such compositions may also contain adjuvants such as wetting, emulsifying, suspending, flavoring (eg sweetening) agents and/or flavoring agents.

In another embodiment, the invention covers a parenteral dosage form. Parenteral administration includes, for example, subcutaneous injections, intravenous injections, intraperitoneal injections, intramuscular injections, intrasternal injections, and infusion. Injectable preparations (i.e. sterile injectable aqueous or oily suspensions) can be prepared by those known in the art.

- 17 040816 in this area by methods using suitable dispersing, wetting and/or suspending agents.

In another embodiment, the invention encompasses a topical dosage form. Topical administration includes, for example, transdermal administration, such as via transdermal patches or iontophoresis devices, intraocular administration, or intranasal administration, or inhalation administration. Compositions for topical administration also include, for example, topical gels, sprays, ointments and creams. The topical composition may contain a compound that enhances the absorption or penetration of the active ingredient through the skin or other affected areas. When the compounds of this invention are administered via a transdermal device, then administration will be by using a patch of either the reservoir type or the porous membrane or solid matrix type. Topical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, powders, dressings, foams, films, skin patches, cachets, implants, sponges, fibers, bandages, and microemulsions. Liposomes can also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol, and propylene glycol. Penetration enhancers may be included (see, for example, B. C. Finnin and T. M. Morgan, J. Pharm. Sci., vol. 88, p. 955-958, 1999).

Forms of compositions for topical administration to the eye include, for example, eye drops, wherein the compound of this invention is dissolved or suspended in a suitable carrier. A typical formulation suitable for administration to the eyes and ears may be in the form of drops of a micronized suspension or solution in isotonic, pH-adjusted, sterile saline. Other forms of composition suitable for administration to the eyes and ears include ointments, biodegradable (i.e., absorbable gel sponges, collagen) and non-biodegradable (i.e., silicone) implants, cachets, lenses, and particulate or vesicular systems such as like niosomes or liposomes. A polymer such as cross-linked polyacrylic acid, polyvinyl alcohol, hyaluronic acid, a cellulose polymer such as hydroxypropyl methylcellulose, hydroxyethylcellulose or methylcellulose, or a heteropolysaccharide polymer such as gelan gum may be included in the composition along with a preservative such as benzalkonium chloride. Such compositions can also be delivered by iontophoresis.

For intranasal or inhalation administration, the compounds of the invention are conveniently delivered in the form of a solution or suspension from a pump nebulizer container squeezed or pumped by the patient, or as an aerosol spray from a pressurized container or nebulizer using a suitable propellant. Compositions suitable for intranasal administration are usually administered in the form of a dry powder (either alone or as a mixture, such as a dry mixture with lactose, or as a mixture of particulate components, such as a mixture with phospholipids, such as phosphatidylcholine) from a dry powder inhaler or aerosol spray from a pressurized container, pump, atomizer, atomizer (preferably an atomizer using electrohydrodynamics to create a fine mist) or nebulizer, with or without the use of a suitable propellant such as 1,1,1,2-tetrafluoroethane or 1,1 ,1,2,3,3,3-heptafluoropropane. The powder for intranasal use may contain a bioadhesive agent such as chitosan or cyclodextrin.

In another embodiment, the invention encompasses a rectal dosage form. Such a rectal dosage form may be, for example, a suppository. Cocoa butter is the traditional suppository base, but various other alternatives may be used if appropriate.

Other carrier materials and routes of administration known in the pharmaceutical art may also be used. Pharmaceutical compositions according to the invention can be obtained by any methods well known in pharmacy, such as effective methods of preparation and administration. The foregoing considerations regarding effective compositions and methods of administration are known in the art and are described in standard textbooks. The preparation of drugs is discussed in, for example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania, 1975; Liberman et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Kibbe et al., Eds., Handbook of Pharmaceutical Excipients (3rd Ed.), American Pharmaceutical Association, Washington, 1999.

Joint introduction

The compounds of the invention may be used alone or in combination with other therapeutic agents. The invention provides any uses, methods or compositions as defined herein, wherein a compound of any embodiment of Formula I as described herein, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate of said compound or said salt, is used in combination with one or more than one other therapeutic agent discussed in this document. This combination will include a pharmaceutical composition for the treatment of a disease or condition in which a GLP-1R agonist is indicated, comprising a compound of Formula I, II, III, IV, or V, or a pharmaceutically acceptable salt thereof, as defined in any of the embodiments described herein. document, and one or more

- 18 040816 than one other therapeutic agent discussed in this document.

The introduction of two or more compounds in combination means that all compounds are administered close enough in time that each can produce a biological effect within the same period of time. The presence of one agent may alter the biological effects of the other(s) compound(s). Two or more compounds can be administered simultaneously, in parallel or sequentially. In addition, simultaneous administration can be carried out by mixing the compounds before administration, or by administering the compounds at the same time, but in separate dosage forms, at the same site or at different sites of administration.

The phrases co-administration, co-administration, simultaneous administration and simultaneous administration mean that the compounds are administered in combination.

Also provided are methods of treatment comprising administering the compounds of the present invention in combination with one or more other pharmaceutical agents, wherein the one or more other pharmaceutical agents may be selected from those discussed herein.

The compounds of this invention may be administered with an antidiabetic agent including, but not limited to, a biguanide (eg, metformin), a sulfonylurea (eg, tolbutamide, glibenclamide, gliclazide, chlorpropamide, tolazamide, acetohexamide, glycopyramide, glimepiride, or glipizide), a thiazolidinedione (eg, pioglitazone , rosiglitazone, or lobeglitazone), glitazar (eg, saroglitazar, aleglitazar, muraglitazar, or tesaglitazar), meglitinide (eg, nateglinide, repaglinide), dipeptidyl peptidase 4 (DPP-4) inhibitor (eg, sitagliptin, vildagliptin, saxagliptin, linagliptin, gemigliptin, anagliptin , teneligliptin, alogliptin, trelagliptin, dutogliptin, or omarigliptin), glitazone (eg, pioglitazone, rosiglitazone, balaglitazone, rivoglitazone, or lobeglitazone), sodium-dependent glucose cotransporter 2 (SGLT2) inhibitor (eg, empagliflozin, canagliflozin, dapagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate, remogliflozin etabonate or ertugliflozin), inhibition an SGLTL1 agonist, a GPR40 agonist (FFAR1/FFA1 agonist such as fasiglypham), glucose-dependent insulinotropic peptide (GIP) and its analogs, an alpha-glucosidase inhibitor (such as voglibose, acarbose, or miglitol), or insulin or an insulin analog, including pharmaceutically acceptable salts of the specifically named agents; and pharmaceutically acceptable solvates of said agents and salts.

The compounds of this invention may be administered with an anti-obesity agent, including, but not limited to, a YY peptide or analog thereof, a type 2 neuropeptide Y receptor (NPYR2) agonist, an NPYR1 or NPYR5 antagonist, a cannabinoid type 1 receptor (CB1R) antagonist, a lipase inhibitor (e.g. , orlistat), human proislet peptide (HIP), melanocortin 4 receptor agonist (eg, setmelanotide), melanin-concentrating hormone 1 receptor antagonist, farnesoid X receptor (FXR) agonist (eg, obeticholic acid), zonisamide, phentermine (alone or in combination with topiramate), a norepinephrine/dopamine reuptake inhibitor (eg, bupropion), an opioid receptor antagonist (eg, naltrexone), a combination of a norepinephrine/dopamine reuptake inhibitor and an opioid receptor antagonist (eg, a combination of bupropion and naltrexone), a GDF-analogue 15, sibutramine, a cholecystokinin agonist, amylin and analogues (eg pramlintide), leptin and analogues (eg metrol ptin), serotonergic agent (eg, lorcaserin), methionine aminopeptidase 2 (MetAP2) inhibitor (eg, beloranib or ZGN-1061), phendimetrazine, diethylpropion, benzphetamine, SGLT2 inhibitor (eg, empagliflozin, canagliflozin, dapagliflozin, ipragliflozin, tofogliflozin, sergliflozin etabonate , remogliflozin etabonate or ertugliflozin), SGLTL1 inhibitor, SGLT2/SGLT1 dual inhibitor, fibroblast growth factor receptor (FGFR) modulator, AMP-activated protein kinase (AMPK) activator, biotin, MAS receptor modulator or glucagon receptor agonist (alone or in combination with another agonist GLP-1R, eg, liraglutide, exenatide, dulaglutide, albiglutide, lixisenatide or semaglutide), including pharmaceutically acceptable salts of the specifically named agents and pharmaceutically acceptable solvates of these agents and salts.

The compounds of this invention may be administered with an NASH treatment agent including, but not limited to, PF-05221304, an FXR agonist (e.g., obeticholic acid), a PPAR α/δ agonist (e.g., elafibranor), a synthetic fatty acid-bile acid conjugate (e.g. , aramhol), caspase inhibitor (eg, emrikasan), anti-lysyl oxidase 2 (LOXL2) homologue monoclonal antibody (eg, simtuzumab), galectin 3 inhibitor (eg, GR-MD-02), MAPK5 inhibitor (eg, GS-4997), dual chemokine receptor 2 (CCR2) and CCR5 antagonist (eg, cenicriviroc), fibroblast growth factor 21 (FGF21) agonist (eg, BMS-986036), leukotriene D4 receptor (LTD4) antagonist (eg, tipelukast), niacin analog (eg, ARI 3037MO), ASBT inhibitor (eg, volixibate), acetyl-CoA carboxylase (ACC) inhibitor (eg, NDI 010976), ketohexokinase (KNK) inhibitor, diacylglyceryl acyltransferase 2 (DGAT2) inhibitor, CB1 receptor antagonist, anti-CB1R antibody, or inhibition an apoptosis signal-regulating kinase 1 (ASK1) agent, including pharmaceutically acceptable salts of the specifically named agents and pharmaceutically acceptable solvates of said agents and salts.

Some specific compounds that may be used in combination with

- 19 040816 Indications of the present invention for the treatment of diseases or disorders described herein (for example, NASH) include: 4-(4-(1-isopropyl-7-oxo-1,4,6,7-tetrahydrospiro[indazole -5,4'piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid, which is an example of a selective inhibitor of ACC (acetyl-coenzyme A-carboxylase) and was obtained as a free acid in Example 9 of the patent US No. 8859577, which is the US national phase of International Application PCT/GV2011/054P9, the descriptions of which are incorporated herein by reference in their entirety for all purposes; crystalline forms of 4-(4-(1-isopropyl-7-oxo-1,4,6,7tetrahydrospiro[indazol-5,4'-piperidine]-1'-carbonyl)-6-methoxypyridin-2-yl)benzoic acid , including the anhydrous mono-tris form (form 1) and the mono-tris salt trihydrate (form 2), are described in International Application PCT/IB2018/058966, the disclosure of which is incorporated herein by reference in its entirety for all purposes; (S)-2-(5-((3-ethoxypyridin-2-yl)oxy)pyridin-3-yl)-N(tetrahydrofuran-3-yl)pyrimidine-5-carboxamide, or a pharmaceutically acceptable salt thereof and crystalline solids forms (Form 1 and Form 2), which is an example of a DGAT2 (diacylglycerol-O-acyltransferase 2) inhibitor described in Example 1 of US Patent No. 10071992, the disclosure of which is incorporated herein by reference in its entirety for all purposes; [(1R, 5S, 6R)3-{2-[(2S)-2-methylazetidin-1-yl]-6-(trifluoromethyl)pyrimidin-4-yl}-3-azabicyclo[3.1.0]hex-6yl ]acetic acid, or a pharmaceutically acceptable salt thereof (including its free acid crystalline form), which is an example of a ketohexokinase inhibitor and is described in Example 4 of US Pat. No. 9,809,579, the disclosure of which is incorporated herein by reference in its entirety for all purposes; and the FXR agonist tropifexor, or a pharmaceutically acceptable salt thereof, as described in Example 1-1B of US Pat. No. 9,150,568, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

These agents and compounds of the invention may be combined with pharmaceutically acceptable carriers such as saline, Ringer's solution, dextrose solution, and the like. The specific dosage regimen, i.e. the dose, time of administration, and repeatability will depend on the particular individual and the individual's medical history.

Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and may contain buffers such as phosphate buffer, citrate buffer and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as ammonium octadecyldimethylbenzyl chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and meta-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or Ig (immunoglobulins); hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Liposomes containing these agents and/or compounds of the invention are made by methods known in the art, such as those described in US Pat. Nos. 4,485,045 and 4,544,545. made by reverse phase evaporation with a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through filters with a defined pore size to obtain liposomes of the desired diameter.

These agents and/or compounds of the invention may also be microencapsulated, for example, by coacervation or interfacial polymerization methods, such as hydroxymethylcellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, respectively, in colloidal drug delivery systems (e.g. , albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such methods are disclosed in Remington, The Science and Practice of Pharmacy, 20th Ed., Mack Publishing (2000).

Sustained release formulations may be used. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing a compound of formula I, II, III, IV or V in the form of molded articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl methacrylate) or polyvinyl alcohol, polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-E-glutamate, non-degradable ethylene vinyl acetate, degradable copolymers of lactic acid and glycolic acid, such as the copolymers used in LUPRON DEPOT™ (injectable microspheres consisting of a copolymer of lactic acid and glycolic acid and leuprolide acetate), sucrose acetate isobutyrate, and poly-O-(-)-3-hydroxybutyric acid.

Compositions intended for intravenous administration must be sterile. This is easily accomplished, for example, by filtration through sterile filtration membranes. The compounds of the invention are usually placed in a container having a sterile access port, such as an intravenous solution bag or a vial having a stopper pierced by a hypodermic needle.

Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient is either dissolved in a premixed emulsion formulation or alternatively it can be dissolved in an oil (e.g. soybean oil, sunflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion is formed when mixed with a phospholipid ( e.g. egg phospholipids, soy phospholipids or soy lecithin) and water. It is understood that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, eg 5 to 20%. The fat emulsion may contain fat droplets from 0.1 to 1.0 microns, especially from 0.1 to 0.5 microns, and may have a pH in the range of 5.5-8.0.

Compositions in the form of an emulsion may be emulsions which are prepared by mixing a compound of the invention with Intralipid™ or its components (soybean oil, egg phospholipids, glycerin and water).

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set forth above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic exposure. Compositions in preferably sterile pharmaceutically acceptable solvents can be sprayed using gases. Nebulized solutions can be inhaled directly from a nebulizing device, or the nebulizing device can be attached to a face mask, awning, or intermittent positive pressure breathing machine. Compositions in the form of a solution, suspension or powder can be administered preferably orally or nasally from devices that deliver the composition in an appropriate manner.

Sets

In yet another aspect of the invention, kits are disclosed containing a compound of formula I, II, III, IV or V or a pharmaceutical composition containing a compound of formula I, II, III, IV or V of the invention. The kit may include, in addition to a compound of formula I, II or III according to the invention or a pharmaceutical composition containing it, diagnostic or therapeutic agents. The kit may also include instructions for use in a diagnostic or therapeutic method. In some embodiments, the kit includes a compound of formula I, II, III, IV, or V, or a pharmaceutical composition containing it, and a diagnostic agent. In other embodiments, the kit includes a compound of formula I, II, III, IV or V or a pharmaceutical composition containing it.

In yet another embodiment, the invention encompasses kits that are suitable for use in the implementation of the methods of treatment described in this document. In one embodiment, the kit contains a first dosage form containing one or more compounds of the invention in amounts sufficient to carry out the methods of the invention. In another embodiment, the kit contains a first dosage form containing one or more compounds of the invention in amounts sufficient to carry out the methods of the invention, and a dosage container.

Receipt

Compounds of formula I, II, III, IV or V may be prepared by the general and specific methods described below using the normal general knowledge of a person skilled in the art of organic chemistry. Such common general knowledge can be found in standard reference books such as Comprehensive Organic Chemistry, Ed. Barton and Ollis, Elsevier; Comprehensive Organic Transformations: A Guide to Functional Group Preparations, Larock, John Wiley and Sons; and Compendium of Organic Synthetic Methods, Vol. IXII (published by Wiley-Interscience). Used starting materials are commercially available or can be obtained by routine methods known in this field.

It should be noted that when preparing compounds of formula I, II, III, IV or V, some of the preparations described herein require protection of the terminal functional group (for example, primary amine, secondary amine, carboxyl in precursors of compounds of formula I). The need for such protection will depend on the nature of the terminal functional group and the conditions of the methods of preparation. The need for such protection will be readily determined by one skilled in the art. The use of such protection/deprotection methods is also within the skill of those skilled in the art. A general description of protecting groups and their uses is given in T.W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

For example, some compounds contain a primary amino functional group or a carboxyl functional group, which can interfere with reactions at other sites in the molecule if left unprotected. Accordingly, such functional groups may be protected with an appropriate protecting group, which may be removed in a subsequent step. Suitable protecting groups for amine and carboxylic acid protection include those protecting groups commonly used in peptide synthesis (such as N-tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz) and 9-fluorenylmethyleneoxycarbonyl (Fmoc) for amines and lower alkyl or benzyl esters for carboxylic acids) which are generally not chemically reactive under the reaction conditions described and can usually be removed without chemically changing the other functional group in the compounds of formula I.

The schemes described below are intended to provide a general description of the methodology used in preparing the compounds of the present invention. Some compounds of the present invention may contain a single chiral center and multiple chiral centers with the stereochemical designation (R) or (S). One of skill in the art will appreciate that all synthetic transformations can be carried out in a similar manner whether the substances are enantioenriched or racemic. Moreover, resolution to the desired optically active species can take place at any desired point in the reaction sequence using well known methods such as those described herein and in the chemical literature. For example, intermediates (eg S4, S7, S8, S24, S40 and S41) and final products (eg S25 and S42) can be isolated using chiral chromatographic techniques. Alternatively, chiral salts can be used to isolate enantiomerically enriched intermediates and final compounds.

In the schemes below, the variables X, Y, Z ¹ , Z ² , Z ³ , R, R ¹ , R ² , R ³ , R ⁴ , m, p and q are as described herein for compounds of the formula I, II, III, IV or V unless otherwise noted. In the schemes below, each X ¹ , X ² , X ³ and X ⁴ independently may be a leaving group such as any alkyl or aryl sulfonate (eg mesylate, tosylate or triflate) or halogen or any other a group that can be substituted with an amine or used in a metal-mediated coupling reaction. X ⁴ may also be a protected carboxyl group (ie ester). When the protecting group is identified as Pg ¹ then it may be an alkylamine protecting group such as benzyl, benzhydryl or the like; a carbamate protecting group such as Boc, Cbz or the like; or an amide protecting group such as trifluoroacetamide. When the protecting group is identified as Pg ² then it may be an acid protecting group such as methyl, ethyl, benzyl, t-butyl or the like. When the protecting group is identified as Pg ³ then it may be an alcohol protecting group such as trimethylsilylethoxyethyl; or an acyl group such as acetyl, benzoyl or the like; or a trialkylsilyl group such as trimethylsilyl, t-butyldimethylsilyl, triisopropylsilyl or the like. R ^2a is H or -C1.2 alkyl, where alkyl may have 0-1 OH. R ^4a is C1. ₂ alkyl, C ₀ _ ₂ alkylene-C ₃ _ ₆ cycloalkyl, C ₀ . ₂ alkylene J ⁵ or C1.2α alkylene-R ⁶ , wherein said alkyl, alkylene or cycloalkyl may be independently substituted, when valency permits, with 0-3 F atoms and 0-1 substituent independently selected from C0. ₁ alkylene-OR ^O and -N(R ⁿ ) ₂ .

Substituted piperidine S8, where R ² = H and Y = CH, can be prepared as shown in Scheme 1. Aryl or heteroaryl bromide S1 can be treated with an alkyl lithium, such as butyl lithium or t-butyl lithium, to give an aryl or heteroaryl lithium a compound which can be reacted with an aldehyde S2 to give a diol S3. Other aryl or heteroaryl organometallic reagents, such as, without limitation, Grignard reagents, can also be used in the preparation of S3. The reaction is usually carried out at a temperature of about -70°C. The diol S3 can then be oxidized with NaIO4 to give the acetal S4 (R ² = H). Compound S4 can then be reacted with a substituted boronic acid or borate ester (S5) in the presence of a palladium catalyst and a ligand complex by a Suzuki reaction (Maluenda and Navarro, Molecules, 2015, 20, 7528-7557) to give a compound of general formula S6. Reduction of the olefin to give a compound of general structure S7 can be carried out under a hydrogen atmosphere (15-100 psi H ₂ ) in an alcoholic solvent such as MeOH or EtOH, or alternatively in an aprotic organic solvent such as EtOAc or THF , in the presence of a suitable catalyst such as palladium on carbon, Pd(OH)2 on carbon (Pearlman catalyst), PtO2 (Adams catalyst), or tris(triphenylphosphine)rhodium(I) chloride (Wilkinson catalyst ( Wilkinson). Hydrogen transfer hydrogenation reagents such as ammonium formate or dihydrobenzene or the like can be used using a suitable catalyst. Alternatively, the reduction may be carried out by alternative methods known to those skilled in the art using reagents such as triethylsilane or other silanes under acid or metal catalysis conditions, or metal reducing agents such as magnesium or

- 22 040816 similar. Alternatively, the olefin may be functionalized by methods known to those skilled in the art to introduce R ³ groups. For example, an olefin can be hydroborated to give an alcohol, which can be alkylated or further converted to a nitrile, F, or alkyl group. Removal of Pg ¹ can be carried out by many methods described in the literature, with the receipt of amines S8.

Scheme 1

Scheme 2 shows an alternative preparation of compounds of general structure S4. The reaction of appropriately substituted diols of general structure S9 with aldehydes or ketones of general structure S10a in the presence of an acid such as p-toluenesulfonic acid or pyridinium p-toluenesulfonate in an aprotic organic solvent such as toluene or benzene can provide compounds of general structure S4. The reaction mixture is usually refluxed using a Dean-Stark trap to remove water azeotropically. Diols S9 can also be reacted with cyclic (dashed line present) or acyclic (dashed line absent) acetals or ketals of general structure S10b under acid catalysis conditions. The same applies to cyclic or acyclic thioacetals or thioketals of the general structure S10c when exposed to a mercury salt, mild oxidants or alkylating agents to give S4 compounds. Alternatively, diols of general formula S9 may be reacted with an appropriately substituted alkyne S11 in an aprotic solvent such as toluene in the presence of triruthenium dodecacarbonyl at a temperature of about 100° C. to give compounds of general structure S4 where R ² =CH2R ^2a . In cases where R ² contains an alcohol functional group, such as CH2OH, an alcohol (Pg ³ ) protecting group, such as acetate, can be introduced into compounds of the general structure S10. The protecting group can then be removed in a subsequent step. Intermediate S4 can then be further modified using the methods described for Scheme 1 to give amines of general structure S8.

Scheme 2

As shown in Scheme 3, the conversion of S4 to compounds of general structure S7, where Y = N, can be carried out in such a way as CN Buchwald-Hartwig coupling between compounds of general structure S4 and appropriately substituted and protected piperazine S12 in the presence of a palladium or copper catalyst and ligand complex. These reactions are typically carried out at 0-110° C. in aprotic organic solvents such as, but not limited to, 1,4-dioxane and PhCH ₃ with the addition of a base such as Cs ₂ CO ₃ , LiHMDS or NaOtBu. Removal of Pg ¹ to obtain amines S8, where Y = N, can be carried out in many ways described in the literature.

Scheme 3

Amino compounds S8 prepared by the methods of Schemes 1-3 may be alkylated with protected 2-bromoacetate in the presence of a suitable base such as K ₂ CO ₃ , Et ₃ N, NaH or LiHMDS in a polar aprotic solvent such as as, without limitation, DMF, DMAc, DMSO, or NMP, to give compounds of the general structure S13 (X=N, L=CH ₂ ). Standard ester hydrolysis can be carried out to give S14 acids. If Pg ² is t-butyl, then standard acid deprotection methods such as TFA/DCM, HCl/1,4-dioxane, HCl/EtOAc, or other suitable conditions can be used to produce S14 acids. If Pg ² is methyl or ethyl, then standard base deprotection methods such as aqueous NaOH in methanol or ethanol or other suitable conditions can be used to prepare S14 acids.

Scheme 4

Compounds of general structure S15 (Scheme 5) can be reacted with amines R ⁴ NH2 in the presence of bases such as sodium, potassium or cesium carbonate, bicarbonate, hydroxide, acetate or an organic amine base such as Et3N, DIPEA, DBU, etc. p., in a polar aprotic solvent such as, but not limited to, THF, DMF, DMAc, DMSO, or NMP, or in a protic solvent such as water, MeOH, EtOH, or iPrOH, or a mixture thereof, to give compounds of general structure S16. It should be noted that if the example provides R ⁴ with a separated enantiomeric center, the other enantiomer or its racemic mixture can be obtained by choosing the appropriate starting material. Preferred X ³ substituents include F, Cl and Br, preferred X ⁴ groups include Cl, Br and -CO 2 -Pg ² . The reduction of the nitro group can be carried out by hydrogenation under conditions of 1-6 atm H ₂ with a metal catalyst such as palladium on carbon or Raney nickel, in a protic solvent such as MeOH or EtOH, or in an aprotic solvent such as DMF, THF or EtOAc . Alternatively, the nitro group can be reduced with iron, zinc, SnCl ₂ or other suitable metal in an acid medium such as 1N HCl. HC, AcOH, or in aqueous NH ₄ Cl in THF or methanol to give compounds of general structure S17 (Scheme 5a). Compounds such as S18 can be acylated with acyl halides in the standard manner or with carboxylates using standard amide coupling protocols to give S19 compounds. Reduction to S20 compounds can be carried out under standard conditions with reducing agents such as LAH or BH ₃ -THF or BH3-DMS (Scheme 5b).

Scheme 5

E) r4r4 χ3 z3 _X 4 HN Z^ χ4 HN^Z^X ⁴

1% - 1% - 1% o ₂ n^^ o ₂ n^^h

S15 S16S17 ^b) r ⁴ ^or h ₂ n^^x ⁴ HN^^X ⁴ hn^^x ⁴ j T --- TT ---TT

H ₂ NNR ^z H2N NR ^z H2N NR ^z

S18 S19S20

Diamino compounds S17 and S20 obtained by the methods presented in schemes 5a and 5b, collectively designated as diamine S21 (scheme 6), can be acylated with acids of general structure S14 under standard amide coupling conditions to obtain amides S22, which will exist as a mixture from 100% S22a to 100% S22b. This mixture of amines S22 can be cyclized to give compounds of the general structure S23 in various ways. Amines S22 can be heated with a dehydrating agent such as T ₃ P® or an alkyl alcohol such as n-butanol under microwave conditions (10-60 min at 120-180°C) to give compounds S23. Alternatively, a mixture of S22 compounds can be heated under acidic conditions such as AcOH at 60-100°C, or under basic conditions such as aqueous NaOH or KOH in 1,4-dioxane at 60100°C to give S23. Compounds of general structure S23 (X ⁴ = C, Br, or I) can be converted to esters of structure S24 by palladium-catalyzed carbonylation under 15-100 psi carbon monoxide at 20-100°C using the appropriate alcohol, such as MeOH or EtOH or other alkyl alcohol. The hydrolysis of the S24 ester can be carried out as shown in Scheme 4 to give the S25 acids. For compounds S22, where X ⁴ = CO2-Pg ² , the conversion to ester S24 proceeds under conditions similar to those described previously, except for the use of the alkaline cyclization method, where the compound S25 can be isolated directly from the reaction mixture. For S24 compounds where X ⁴ is CO ₂ tBu, deprotection of the S25 acid can be carried out under the acidic conditions shown in Scheme 4.

- 24 040816

Alternatively, for S24 compounds where Pg ² is C1- _C8 alkyl such as methyl, ethyl, hexyl or octyl, deprotection of the ester protecting group can be accomplished using a range of enzymes including esterases, proteases, peptidases, lipases and glycosidases, which are well known to those skilled in the art. Hydrolysis can also be carried out by treating the ester with an aqueous solution of 1,5,7-triazabicyclo[4.4.0]dec-5-ene at rt.

Scheme 6

^R4

Additionally, diamine S21 can be converted to 2-chloromethylbenzimidazole S26 (Scheme 7) in several ways. Treatment with 2-chloroacetyl chloride or chloroacetic anhydride in an aprotic solvent such as 1,4-dioxane followed by heating at 40-100°C for 2-18 hours can provide the desired benzimidazole S26, where Z ¹ , Z ² and Z ³ are CH. In cases where Z ¹ , Z ² and Z ³ are not all CR ^z , after treatment with 2-chloroacetyl chloride in an aprotic solvent such as 1,4-dioxane for a period of 30 minutes to 4 hours, the solvent is replaced with an acid medium , such as AcOH or TFA, followed by heating at 40-100°C for 2-18 hours to obtain the target compound S26. Diamine S21 may also be treated with chloroacetic anhydride at 0-80°C in an aprotic solvent such as, but not limited to, 1,4-dioxane, THF or MeCN followed by heating for 2-18 hours at 60-100°C to obtain the target compound S26. Additionally, diamine S21 can be treated with 2-chloro-1,1,1-trimethoxyethane in an aprotic solvent such as but not limited to 1,4-dioxane, THF or MeCN, or in a protic solvent such as MeOH or EtOH, in the presence of an acid catalyst, for example pTSA, at 20-100°C. Alternatively, diamines S21 can be subjected to heating at 100-180° C. with 2-hydroxyacetic acid in an aprotic solvent such as, but not limited to, mesitylene to give a hydroxymethyl intermediate. The conversion of the hydroxymethyl group to the chloromethyl compound S26 can be carried out by standard methods, including treatment with SOCl2 in an aprotic solvent. Compounds of general structure S26 may be reacted with compounds S8 in the presence of bases such as sodium, potassium or cesium carbonate, sodium, potassium or cesium bicarbonate, NaH or an organic amine base such as Et3N, DIPEA, DBU, etc., in a polar aprotic solvent such as, but not limited to, THF, MeCN, DMF, DMAc, DMSO, or NMP to give compounds S23 (X ⁴ = C, Br, I) or compounds S24 (X ⁴ = CO2-Pg ² ), which are then used to prepare S25 compounds by the methods presented in Scheme 6.

Scheme 7

R ⁴ N^Z ³ X ⁴ S8 IS21 - 3 ; ₂ --------- S24 - S25

Cl N^ _Z 1 ^Z _X 4 _\u003d co ₂ -Rd ²

S26

S8 S26 --------- S23

X ⁴ \u003d CI, Br, I

Alternatively (Scheme 8), compounds of general structure S26 can be reacted with suitably substituted and protected piperazines S12 in the presence of bases such as sodium, potassium or cesium carbonate, sodium, potassium or cesium bicarbonate, NaH, or an organic amine base. , such as Et ₃ N, DIPEA, DBU, etc., in a polar aprotic solvent such as, without limitation, THF, MeCN, DMF, DMAc, DMSO, or NMP, to obtain compounds S27 (scheme 8). Removal of Pg ¹ can be carried out by many methods described in the literature, with the receipt of amines S28. Conversion to compounds of general structure S23 (X ⁴ = C, Br or I) or S24 (X ⁴ = CO ₂ -Pg ² ) can be carried out in a manner such as CN Buchwald-Hartwig coupling between compounds of general structure S4, and as shown earlier in Scheme 3. Compounds of general structure S23 or S24 can then be used to prepare compounds of structure S25 by the methods presented in Scheme 6.

Scheme 8

^{R4 R4}

S12 -Z ³ __uz S4 ^S26 ζ 1 ^N \ Y ^_ _χ 4 N ^ / VX ⁴ * S23 or S24 ^g (R ³ )q Z ¹ \u003d Z ² (R ³ )q Z ¹ \u003d Z ²

S27 _S28

Compounds of the general structure S25 can also be prepared as shown in Scheme 9. Diol S9 can be protected to form S29. The trimethylsilylethoxymethyl group is a preferred protecting group. Protection of the diol as the appropriate acetal, eg formaldehyde acetal, is also preferred. Compound S29 can then be reacted with a substituted boronic acid or borate ester (S5) and an olefin, followed by reduction by the methods shown in Scheme 1, to give a compound of general formula S31 where Y=CH. Alternatively, compound S29 can be coupled to piperazines of general structure S12 using the methods shown in Scheme 3 to give S31 where Y=N. Compounds of general structure S31 can be deprotected and then coupled to S26 to give compounds of general structure S33 with using the methods shown in Scheme 7. Alternatively, compounds of the general structure S33 can be obtained from S32 by converting S32 to the corresponding N-acetic acid derivative and subsequent condensation with diamines S21, as shown in Scheme 4 and Scheme 6. Deprotection of S33 with using methods known to those skilled in the art, can provide diols of general structure S34, which can then be reacted with alkynes of general structure S11 using the methods presented in Scheme 2 to obtain S23 or S24. Alternatively, compound S34 can be converted to compound S23 or S24 using aldehydes, ketones or their derivatives as shown in Scheme 2. Compounds of general structure S23 or S24 can then be used to prepare compounds of structure S25 by the methods shown in Scheme 6.

Scheme 9

OH Rd ³ O Rd ³ O γΥ ⁹

HO^A^Br Pg ³ O^Y ^Br S5

TJ ---kJ ---kJ ^{( )q} (R)p ^ (R)p ^ (R)p'^ ^x

S9 S29 S30

S11

---------► S23 or S24

Compounds of general structure S24 and general structure S33 where Y=N and XL=cyclopropyl can be prepared as shown in Scheme 10. The protected piperidinone S35 can be homologated to the unsaturated ester S36 using methods well known to those skilled in the art. . For example, Horner-Wadsworth Emmons olefinization of S42 with a phosphonate such as ethyl(diethoxyphosphoryl)acetate that has been deprotonated with a strong base such as lithium, sodium or potassium tert-butoxide can provide S36. The reaction is usually carried out in an aprotic solvent such as THF or DME at a temperature of from about 0 to -50°C. The conversion of S36 to the cyclopropane derivative S37 can be carried out by treatment with a sulfoxonium ylide prepared from trimethylsulfoxonium iodide and a base such as potassium tert-butoxide or sodium hydride. Deprotection of S37 and subsequent combination of the resulting carboxylic acid S38 with S21, where X ⁴ = CO ₂ Pg ² , using the methods presented on

- 26 040816 scheme 6, can provide compounds of General formula S39. Deprotection of S39 and coupling to S4 using the methods of Scheme 3 can give compounds of the general structure S24 where Y=N and X-L is cyclopropyl. Compounds of general structure S24 can then be used to prepare compounds of structure S25 by the methods shown in Scheme 6. Alternatively, S40 can be reacted with S29 using the methods shown in Scheme 3 to give S33 where Y=N and X-L= cyclopropyl. Compounds of general structure S33 can then be used to prepare compounds of structure S25 by the methods presented in Scheme 6 and Scheme 9.

Scheme 10

Alternatively, compounds of general structure S25 where Y=N and XL is cyclopropyl can be prepared as shown in Scheme 11. Removal of Pg ¹ from S37 provides the piperidine derivative S43. The combination of S43 with S4 in a manner similar to the method presented in scheme 3, provides S13, where Y = N, and XL represents cyclopropyl. Deprotection can then provide compounds of the general structure S14, which can then be used to obtain S25, as shown in Scheme 6.

Scheme 11

Examples

The following illustrates the synthesis of non-limiting compounds of the present invention. Additional compounds within the scope of this invention may be prepared using the methods illustrated in these examples, either alone or in combination with methods known in the art.

The experiments were usually carried out in an inert atmosphere (nitrogen or argon), in particular, in cases where oxygen- or moisture-sensitive reagents or intermediates were used. Commercial solvents and reagents were used, as a rule, without further purification. Anhydrous solvents were used where appropriate, typically AcroSeal® products from Acros Organics, Aldrich® Sure/Seal™ from Sigma-Aldrich, or DriSolv® products from EMD Chemicals. In other cases, commercial solvents were passed through columns packed with 4A molecular sieves until QC (quality control) standards for water were achieved: a) <100 ppm. for dichloromethane, toluene, N,N-dimethylformamide and tetrahydrofuran; b) <180 ppm for methanol, ethanol, 1,4-dioxane and diisopropylamine. For very sensitive reactions, the solvents were further treated with sodium metal, calcium hydride, or molecular sieves and distilled just prior to use. Typically, the products were dried under vacuum before being further reacted or subjected to biological testing. Mass spectrometric data were obtained either on liquid chromatography-mass spectrometry (LC-MS) with atmospheric pressure chemical ionization (APCI) or gas chromatography-mass spectrometry (GC-MS) instruments. The symbol ♦ means that a pattern with peaks of chlorine isotopes was observed in the mass spectrum.

Chiral resolution has been used to separate the enantiomers or diastereomers of certain intermediates to obtain compounds of the invention. When the chiral separation was performed, the separated enantiomers were designated as ENT-1 or ENT-2 (or DIAST-1 or DIAST-2) in the order they were eluted. In some embodiments, enantiomers designated as ENT-1 or ENT-2 may be used as starting materials to obtain other enantiomers or diastereomers. In such situations, the resulting enantiomers are designated ENT-X1 and ENT-X2, respectively, according to their starting materials; similarly, the resulting diastereomers are designated DIAST-X1 and DIAST-X2, respectively (or DIAST-according to their starting materials. DIAST-Y and DIAST-Z nomenclature is used similarly in syntheses using a variety of intermediates

- 27 040816 connections.

For compounds with two chiral centers, the stereoisomers for each stereocenter were separated at different times. The designation ENT-1 or ENT-2 (or DIAST-1 or DIAST-2) of an intermediate or exemplary compound refers to the order of elution in the separation performed in this step. It is understood that when stereoisomers at a chiral center are separated in conjunction with two or more centers, the separated enantiomers are each other's diastereomers. By way of example, but not limitation, the compounds of examples 15 and 16 have two chiral centers. The chiral center of the cyclopropyl moiety was separated when intermediate C36 was separated into ENT-1 giving intermediate P17 and ENT-2 giving intermediate P18. P18 was then used in the preparation of C70, which has one stereoisomer enriched in the chiral carbon of cyclopropyl and a mixture of stereoisomers in the carbon of dioxolane. C70 was then separated into DIAST-Y1 at the dioxolane carbon to give intermediate C71 and DIAST-Y2 at the dioxolane carbon to give intermediate C72, where these intermediates are enriched in a single stereoisomer. C71 was then used to prepare the compound of example 15, which was identified by the name 2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6-azaspiro[2.5]oct -1 -yl }-1 -(2-methoxyethyl) -1 H-benzimidazole-6-carboxylic acid, DIAST-X1, trifluoroacetate salt [from P18 through C71]. In these preparations, after the mixture has been subjected to resolution, the chiral center is identified by indicating abs near that center, implying that the resolved enantiomers may not be enantiomerically pure. Typically, the enriched enantiomer at each chiral center is >90% isolated. Preferably, the enriched enantiomer at each center is >98% of the mixture.

In some examples, the optical rotation of an enantiomer was measured using a polarimeter. According to the obtained rotation data (or specific rotation data), the right hand rotation enantiomer was designated as the (+)-enantiomer and the left hand rotation enantiomer was designated as the (-)-enantiomer. Racemic compounds are indicated either by the absence of the stereochemistry depicted or described, or by the presence of (+/-) next to the structure; in this latter case, the indicated stereochemistry is the relative (rather than absolute) configuration of the substituents of the compound.

The course of reactions through detectable intermediates was usually followed by LCMS and continued until complete conversion before adding further reagents. With respect to syntheses with reference to procedures in other examples or methods, the reaction conditions (reaction time and temperature) may vary. As a rule, thin layer chromatography or mass spectrometry was performed after the reactions and processing, when it was appropriate. Purifications may vary from experiment to experiment: as a rule, solvents and solvent ratios used for eluents/gradients were chosen to obtain appropriate Rf values or retention times. All starting materials in the preparations and examples are either commercially available or can be prepared by methods known in the art or as described herein.

Getting P1. tert-Butyl 4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidine-1carboxylate (Р1)

Stage 1. Synthesis of 2-bromo-6-[(4-chloro-2-fluorophenyl)(hydroxy)methyl]phenol (С1).

This experiment was carried out in two batches of the same scale, n-butyl lithium (2.5 M solution in hexanes; 32.8 ml, 82.0 mmol) was slowly added to a -70 ° C solution of 1-bromo-4chloro-2-fluorobenzene ( 17.2 g, 82.1 mmol) in diethyl ether (100 ml), keeping the temperature of the reaction mixture below -60°C. After stirring the reaction mixture at -70°C for 20 min, a solution of 3-bromo-2-hydroxybenzaldehyde (5.5 g, 27 mmol) in diethyl ether (100 ml) was slowly added, keeping the temperature of the reaction mixture below -60°C. After stirring for another 1 hour at -70°C, the reaction mixture was quenched by adding an aqueous solution of ammonium chloride (50 ml) at -70°C, and the resulting mixture was diluted with water (100 ml). The two batches were combined at this time point and extracted with ethyl acetate (400 ml). The organic layer was washed with saturated aqueous sodium chloride (200 ml), dried over sodium sulfate, filtered and concentrated in vacuo. Chromatography on silica gel (gradient: 0 to 7% ethyl acetate in petroleum ether) gave C1 as a white solid. Overall yield: 15.7 g, 47.4 mmol, 88%.

1Н NMR (400 MHz, chloroform-d) δ 7.44 (dd, J=8.0, 1.5 Hz, 1H), 7.37 (dd, J=8.1, 8.1 Hz, 1H), 7.15 (br dd, J= 8.5, 2.1 Hz, 1H), 7.12-7.05 (m, 2H), 6.80 (dd, J=7.8, 7.8 Hz, 1H), 6.78 (s, 1H), 6.31 (d, J=4, 8 Hz, 1H), 3.02 (br d, J=4.9 Hz, 1H).

Stage 2. Synthesis of 4-bromo-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxole (С2).

To a solution of C1 (15.7 g, 47.4 mmol) in methanol (450 ml) was added a solution of sodium periodate (25.4 g, 119 mmol) in water (105 ml) and the reaction mixture was stirred at 30°C in for 16 h, after which it was concentrated in vacuo. After diluting the residue with dichloromethane (500 ml), it was washed with water (500 ml). The dichloromethane solution was then dried over sodium sulfate, filtered and concentrated in vacuo. Purification by chromatography on silica gel (eluent: petroleum ether) gave C2 as a white solid. Yield: 10.0 g, 30.3 mmol, 64%. The following 1H NMR data were obtained from an experiment carried out in the same manner, but on a smaller scale.

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.67-7.61 (m, 2H), 7.50 (s, 1H), 7.43 (br dd, J=8, 2 Hz, 1H), 7.09 (dd, J= 8.3, 1.1 Hz, 1H), 7.01 (dd, J=7.9, 1.1 Hz, 1H), 6.86 (dd, J=8.1, 8.1 Hz, 1H).

Step 3. Synthesis of tert-butyl 4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]-3,6dihydropyridine-1(2H)-carboxylate (С3).

Reaction flask containing a suspension of C2 (8.00 g, 24.3 mmol), tert-butyl-4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6- dihydropyridine-1(2H)-carboxylate (9.01 g, 29.1 mmol), sodium carbonate (5.15 g, 48.6 mmol) and [1,H-bis(diphenylphosphino)ferrocene]dichloropalladium(II) [ Pd(dppf)Cl ₂ ; 888 mg, 1.21 mmol] in 1,4-dioxane (80 ml) and water (32 ml), evacuated and filled with nitrogen. This evacuation cycle was repeated twice, and then the reaction mixture was stirred at 90° C. for 16 hours. After removal of the solvent in vacuo, the residue was partitioned between ethyl acetate (200 ml) and water (200 ml). The organic layer was washed with saturated aqueous sodium chloride (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. Chromatography on silica gel (gradient: 0% to 4.3% ethyl acetate in petroleum ether) gave a product which was combined with the material from a similar reaction carried out using C2 (2.00 g, 6.07 mmol), with obtaining C3 in the form of a light yellow resin. Overall yield: 10.3 g, 23.8 mmol, 78%.

1H NMR (400 MHz, chloroform-d) δ 7.53 (dd, J=8.3, 7.8 Hz, 1H), 7.23-7.16 (m, 3H), 6.88-6.83 (m, 2H), 6.81-6.76 (m , 1H), 6.34-6.28 (br m, 1H), 4.10-4.05 (m, 2H), 3.61 (br dd, J=6.5 Hz, 2H), 2.59-2.50 (brm, 2H), 1.48 (s , 9H).

Step 4. Synthesis of tert-butyl 4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidine-1carboxylate (P1).

A solution of C3 (10.3 g, 23.8 mmol) and tris(triphenylphosphine)rhodium(I) chloride (Wilkinson's catalyst; 1.54 g, 1.66 mmol) in methanol (100 ml) was stirred at 50° C. under atmosphere hydrogen (45 psi) for 18 hours. The reaction mixture was then filtered through a pad of diatomaceous earth and the filtrate was concentrated under reduced pressure and subjected to silica gel chromatography (gradient: 0 to 9% ethyl acetate in petroleum ether). The resulting material was combined with that obtained from a similar reaction carried out using C3 (1.67 g, 3.87 mmol) to give P1 as a colorless gum. Overall yield: 10.3 g, 23.7 mmol, 86%. LC-MS m/z 456.1 [M+Na+].

1Н NMR (400 MHz, chloroform-d) δ 7.52 (dd, J=8.5, 7.6 Hz, 1H), 7.23-7.17 (m, 2H), 7.16 (s, 1H), 6.83 (dd, J=7.8 , 7.8 Hz, 1H), 6.78-6.69 (m, 2H), 4.35-4.10 (br m, 2H), 2.89-2.71 (m, 3H), 1.89-1.77 (m, 2H), 1.771.63 ( m, 2H), 1.47 (s, 9H).

Getting P2. tert-Butyl-4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1carboxylate(Р2)one

Pd(dppf)CI ₂

Na ₂ CO ₃ (Ph ₃ P) ₃ RhCI

Stage 1. Synthesis of 4-bromo-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxole (С4).

To a solution of 3-bromobenzene-1,2-diol (330 g, 1.75 mol) in toluene (1.5 L) was added 1-(4-chloro-2fluorophenyl)ethanone (316 g, 1.83 mol) and para -toluenesulfonic acid (6.02 g, 35.0 mmol). The reaction apparatus was equipped with a Dean-Stark trap, and the reaction mixture was heated at 140°C for 60 h, after which the solution was concentrated in vacuo and purified by chromatography on silica gel (eluent:

- 29 040816 petroleum ether); C4 was obtained as a mixture of a yellow oil and a solid. Yield: 158 g, 460 mmol, 26%.

1H NMR (400 MHz, chloroform-d): δ 7.54 (dd, J=8.4, 8.4 Hz, 1H), 7.17-7.10 (m, 2H), 6.95 (dd, J=7.9, 1.4

Hz, 1H), 6.75 (dd, ABX system component, J=7.8, 1.4 Hz, 1H), 6.70 (dd, ABX system component, J=7.9,

7.9 Hz, 1H), 2.11 (d, J=1.1 Hz, 3H).

Step 2. Synthesis of tert-butyl 4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-3,6dihydropyridine-(2Н)-carboxylate (С5) .

tert-Butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)carboxylate (62 g, 200 mmol) and carbonate sodium (100 g, 940 mmol) was added to a solution of C4 (58.0 g, 169 mmol) in 1,4-dioxane (600 ml). After [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (6.0 g, 8.2 mmol) was added, the reaction mixture was heated to 90° C. and stirred for 16 h. Water (500 mL) was then added , and the resulting mixture was extracted with ethyl acetate (2x500 ml). The combined organic layers were washed with saturated aqueous sodium chloride (2x500 ml), dried over sodium sulfate, filtered and concentrated in vacuo. Chromatography on silica gel (gradient: 0 to 9% ethyl acetate in petroleum ether) gave C5 as a yellow oil. Yield: 56.0 g, 126 mmol, 75%.

1H NMR (400 MHz, chloroform-d) δ 7.50 (dd, J=8.2, 8.2 Hz, 1H), 7.17-7.09 (m, 2H), 6.83-6.77 (m, 2H), 6.74 (dd, component ABX systems, J=5.4, 3.6 Hz, 1H), 6.39-6.33 (br m, 1H), 4.14-4.08 (m, 2H), 3.70-3.56 (m, 2H), 2.66-2.45 (m, 2H ), 2.07 (d, J=1.1 Hz, 3H), 1.50 (s, 9H).

Stage 3. Synthesis of tert-butyl 4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine 1-carboxylate (P2).

To a solution of C5 (56.0 g, 126 mmol) in methanol (200 ml) was added tris(triphenylphosphine)rhodium(1) chloride (Wilkinson's catalyst; 8.10 g, 8.75 mmol) and the reaction mixture was heated to 50 °C for 18 hours under a hydrogen atmosphere (45 psi). It was then cooled to 25°C and filtered through diatomaceous earth. The filtrate was concentrated in vacuo and purified twice by silica gel chromatography (first column: gradient: 0 to 9% ethyl acetate in petroleum ether; second column: gradient: 0 to 2% ethyl acetate in petroleum ether) to give P2 as a yellow solid. Yield: 37.0 g, 82.6 mmol, 66%. LC-MS m/z 392.1 [(M-2-methylprop-1-ene)+H]+.

1H NMR (400 MHz, chloroform-d) δ 7.51 (dd, J=8.3, 8.0 Hz, 1H), 7.17-7.09 (m, 2H), 6.77 (dd, ABC system component, J=7.8, 7, 8 Hz, 1H), 6.70 (dd, ABC system component, J=1.1, 1.3 Hz, 1H), 6.66 (dd, ABC system component, J=7.8, 1.3 Hz, 1H), 4.37-4.13 ( br m, 2H), 2.92-2.73 (m, 3H), 2.05 (d, J=1.1 Hz, 3H), 1.901.63 (m, 4H), 1.49 (s,9H).

Getting P3. 4-[(2S)-2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine p-toluenesulfonate salt (Р3)

Stage 1. Isolation of tert-butyl-4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4yl]piperidine-1-carboxylate (С6) and tert- butyl 4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1-carboxylate (C7).

Separation of P2 (75.2 g, 168 mmol) into its constituent enantiomers was performed by SFC [column: Chiral Technologies Chiralpak AD-H, 5 μm; mobile phase: 4:1 carbon dioxide/(2-propanol containing 0.2% 1-aminopropan-2-ol)]. The first eluting compound was designated C6 and the second eluting enantiomer was designated C7. These absolute configurations are established on the basis of the determination carried out by X-ray diffraction analysis of a single crystal of compound C8, which is a derivative of compound C6 (see below).

C6 - Yield: 38.0 g, 84.8 mmol, 50%. Retention time 3.64 min [column: Chiral Technologies Chiralpak AD-H, 4.6x250 mm, 5 µm; mobile phase A: carbon dioxide; mobile phase B: 2-propanol containing 0.2% 1-aminopropan-2-ol; gradient: 5% B over 1.00 min, then 5 to 60% B over 8.00 min; flow rate: 3.0 ml/min; back pressure: 120 bar].

C7 - Yield: 36.8 g, 82.2 mmol, 49%. Retention time 4.19 min (analytical SFC conditions,

- 30 040816 identical to the conditions used for C6).

Step 2. Synthesis of 4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine p-toluenesulfonate salt (P3).

A solution of C7 (1.62 g, 3.62 mmol) in ethyl acetate (36 ml) was treated with p-toluenesulfonic acid monohydrate (791 mg, 4.16 mmol) and heated at 45°C. After 23 hours the reaction mixture was allowed to cool to room temperature and the solid was collected by filtration. This was washed with a mixture of ethyl acetate and heptane (1:1.2x15 ml) to give P3 as a white solid. Yield: 1.37 g, 2.63 mmol, 73%. LC-MS m/z 348.1 ♦ [M+H]+.

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.53 (v br s, 1H), 8.29 (v br s, 1H), 7.65-7.55 (m, 2H), 7.47 (d, J=8.1 Hz, 2H), 7.35 (dd, J=8.4, 2.0 Hz, 1H), 7.11 (d, J=7.8 Hz, 2H), 6.88-6.81 (m, 2H), 6.75-6.68 (m, 1H) , 3.42-3.33 (m, 2H), 3.11-2.93 (m, 3H), 2.29 (s, 3H), 2.03 (s, 3H), 1.98-1.82 (m, 4H).

Conversion of C6 to 4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine methanesulfonate salt (C8) to determine absolute stereochemistry

C6C8 para-Toluenesulfonic acid (377 mg, 2.19 mmol) was added to a solution of C6 (490 mg, 1.09 mmol) in ethyl acetate (5.5 mL) and the reaction mixture was stirred at room temperature overnight. After dilution with additional ethyl acetate, the reaction mixture was washed successively with aqueous sodium bicarbonate, water and saturated aqueous sodium chloride, dried over sodium sulfate, filtered and concentrated in vacuo. Yield: 375 mg, 1.08 mmol, 99%.

¹ H NMR (400 MHz, methanol-d ₄ ) δ 7.59 (dd, J=8.3.8.3 Hz, 1H), 7.27 (dd, J=10.9, 2.0 Hz, 1H), 7.20 (br dd, J=8.4, 2.1 Hz, 1H), 6.81-6.75 (m, 1H), 6.74-6.67 (m, 2H), 3.18-3.09 (m, 2H), 2.88-2.77 (m, 1H), 2.77- 2.67 (m, 2H), 2.02 (d, J=0.7 Hz, 3H), 1.85-1.73 (m, 4H).

A 0.1 M solution of this free base (4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine) in ethyl acetate was prepared and subjected to salt screening. Only the formation of the methanesulfonate salt is described here. A mixture of methanesulfonic acid (25 μl, 39 μmol) and substrate solution (0.1 M; 0.25 ml, 25 μmol) was stirred overnight. Sufficient methanol was then added to dissolve the solid present and ethyl acetate (3 ml) was added. The resulting solution was allowed to slowly evaporate without stirring to give C8 crystals; one of these crystals was used to determine the structure by single crystal X-ray diffraction analysis described below.

X-ray diffraction analysis of single crystal C8.

X-ray diffraction analysis of a single crystal.

Data collection was carried out on a Bruker D8 Quest diffractometer at room temperature. Data collection consisted of omega and fi scans.

Structure was revealed by internal phasing using the SHELX software package in orthorhombic class space group P212121. The structure was then refined by the least squares method in the full matrix approximation. All non-hydrogen atoms were identified and refined using anisotropic substitution parameters.

The formation of the methanesulfonate salt was confirmed by N1 H1X 04 proton transfer.

Hydrogen atoms located on nitrogen and oxygen were determined from the difference Fourier map and refined with limited distances. The remaining hydrogen atoms were placed in the calculated positions and left on the atoms carrying them. The final refinement included isotropic substitution parameters for all hydrogen atoms.

Absolute structure analysis using probabilistic methods (Hooft, 2008) was performed using PLATON (Spek). The results show that the absolute structure was determined correctly; by this method it is calculated that the probability that the structure is accurate is 100%. The Hooft parameter is presented as 0.02 with esd (estimated standard deviation) 0.0012, and the Parson parameter (Parson) is presented as 0.07 with esd 0.009. The absolute configuration of C7 was confirmed as (R).

The asymmetric unit consists of one molecule of the protonated C8 free base and one molecule of the deprotonated methanesulfonic acid. The final R-index was 4.6%. The finite Fourier difference did not reveal any missing or inappropriate electron density.

Relevant information about the crystal, data collection, and refinement is summarized in Table 1. A. Atomic coordinates, bond lengths, bond angles, and substitution parameters are listed in Table 1. B-D.

- 31 040816

Software and sources of information.

SHELXTL, Version 5.1, Bruker AXS, 1997.

PLATON, A. L. Spek, J. Appl. Cryst. 2003, 36, 7-13.

MERCURY, C. F. Macrae, P. R. Edington, P. McCabe, E. Pidcock, G. P. Shields, R.

Taylor, M. Towler, and J. van de Streek, J. Appl. Cryst. 2006, 39, 453-457.

OLEX2, O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, and H. Puschmann, J. Appl. Cryst. 2009, 42, 339-341.

R. W. W. Hooft, L. H. Straver, and A. L. Spek, J. Appl. Cryst. 2008, 41, 96-103.

H. D. Flack, Acta Cryst. 1983, A39, 867-881.

Table A

Crystal data and structure refinement for C8

Empirical formula Molecular weight according to the formula Temperature Wavelength C20H23CIFNO5S 443.90 296(2)K 1.54178 A Crystal system Space group Unit cell parameters orthorhombic P212121 a = 6.5348(5) A a = 90° b = 9.3688(7) A β = 99° c = 35.214(3) A y = 90° Volume 2155.9(3) A ³ Z 4 Density (calculated) Absorption coefficient A(000) 1.368 mg/m ³ 2.823 mm' ¹ 928 Crystal dimensions Theta range for data collection 0.480 x 0.100 x 0.040 mm ³ from 2.509 to 70.483° Index ranges Collected Reflections Independent Reflections -l<=h<=7, -11<=K=8, -42<=/<=42 16311 4035 [R _int = 0.0638] Completion to theta = 67.679° 99.0% Absorption correction refinement method Empirical Full Matrix Least Squares by F ² Data/limitations/parameters F ² goodness of fit Final R indices [Ι>2σ(Ι)] R indices (all data) Absolute Structural 4035/2/271 0.832 RI = 0.0463, wR2 = 0.1227 RI = 0.0507, wR2 = 0.1294 -0.003(18) parameter extinction coefficient Biggest diff, peak and gap 0.0051(6) 0.256 and -0.305 e.A' ³

- 32 040816

Table B

Atomic coordinates (x 104) and equivalent isotropic substitution parameters (A ² x10 ³ ) for C8. U (eq) is defined as one third of the trace of the orthogonalized Ui ^j tensor x y z U(eq)

S(l) 3842(2) 9910(1) 5317(1) 57(1) Cl(l) -1625(2) -718(1) 6588(1) 80(1) 0(1) 6138(4) 3727(3) 6876(1) 53(1) F(l) 639(5) 3071(4) 7503(1) 89(1) 0(2) 3445(4) 5043(3) 7117(1) 57(1) 0(4) 2909(6) 11013(4) 5082(1) 78(1) 0(3) 3708(7) 10299(4) 5708(1) 83(1) N(l) 10461(5) 2909(4) 5493(1) 56(1) C(9) 5652(6) 4826(4) 6629(1) 44(1) C(l) 3361(7) 1662(4) 6697(1) 53(1) C(6) 2957(6) 2523(4) 7012(1) 49(1) C(10) 4075(6) 5613(4) 6776(1) 47(1) C(14) 6628(6) 5138(4) 6294(1) 47(1) 0(5) 5833(7) 9578(4) 5179(1) 96(1) C(15) 8265(6) 4182(4) 6130(1) 49(1) C(5) 1105(7) 2270(5) 7190(1) 59(1) C(16) 7309(6) 3048(5) 5874(1) 54(1) C(2) 1971(7) 670(4) 6567(1) 55(1) C(4) -286(7) 1288(5) 7080(1) 64(1) C(7) 4448(6) 3667(4) 7142(1) 52(1) C(13) 5876(8) 6374(5) 6113(1) 60(1) C(11) 3359(7) 6819(4) 6602(1) 57(1) C(8) 5296(8) 3485(6) 7537(1) 64(1) S(19) 9905(7) 4976(6) 5902(1) 67(1) S(17) 8902(7) 2063(5) 5702(1) 59(1) C(12) 4316(8) 7178(5) 6263(1) 65(1) C(3) 150(7) 497(4) 6756(1) 56(1) S(18) 11476(7) 3977(6) 5738(1) 73(1) C(20) 2328(14) 8399(7) 5260(2) 117(3)

- 33 040816

Table C

Lengths [A] and angles [°] of bonds for C8

S(l)-O(5) 1.423(4) C(4)-C(3) 1.389(6) S(l)-O(3) 1.428(3) C(4)-H(4) 0.9300 S(l)-O(4) 1.458(3) C(7)-C(8) 1.506(6) S(l)-C(20) 1.738(6) C(13)-C(12) 1.373(7) Cl(l)-C(3) 1.729(5) C(13)-H(13) 0.9300 O(l)-C(9) 1.385(4) C(ll)-C(12) 1.388(7) O(l)-C(7) 1.449(4) C(ll)-H(ll) 0.9300 F(l)-C(5) 1.367(4) C(8)-H(8A) 0.9600 O(2)-C(10) 1.376(4) C(8)-H(8B) 0.9600 O(2)-C(7) 1.449(4) C(8)-H(8C) 0.9600 N(l)-C(18) 1.478(6) C(19)-C(18) 1.505(7) N(l)-C(17) 1.486(5) C(19)-H(19A) 0.9700 N(1)-H(1X) 0.99(2) C(19)-H(19B) 0.9700 N(1)-H(1Y) 0.97(2) C(17)-H(17A) 0.9700 C(9)-C(10) 1.369(5) C(17)-H(17B) 0.9700 C(9)-C(14) 1.375(5) C(12)-H(12) 0.9300

C(l)-C(2) 1.378(6) C(18)-H(18A) 0.9700 C(l)-C(6) 1.395(5) C(18)-H(18B) 0.9700 C(l)-H(l) 0.9300 C(20)-H(20A) 0.9600 C(6)-C(5) 1.384(6) C(20)-H(20B) 0.9600 C(6)-C(7) 1.519(6) C(20)-H(20C) 0.9600 C(10)-C(ll) 1.369(5) C(14)-C(13) 1.409(6) O(5)-S(l)-O(3) 116.2(3) C(14)-C(15) 1.509(5) O(5)-S(l)-O(4) 110.1(2) C(15)-C(16) 1.527(5) O(3)-S(l)-O(4) 109.9(2) C(15)-C(19) 1.531(6) O(5)-S(l)-C(20) 107.6(4) C(15)-H(15) 0.9800 O(3)-S(l)-C(20) 106.6(3) C(5)-C(4) 1.351(7) O(4)-S(l)-C(20) 105.9(4) C(16)-C(17) 1.518(6) C(9)-O(l)-C(7) 105.0(3) C(16)-H(16A) 0.9700 C(10)-0(2)-0(7) 105.2(3) C(16)-H(16B) 0.9700 C(18)-N(l)-C(17) 112.3(3) C(2)-C(3) 1.372(6) C(18)-N(1)-H(1X) 107(3) C(2)-H(2) 0.9300 C(17)-N(1)-H(1X) 113(3) C(18)-N(1)-H(1Y) 113(3) C(l)-C(2)-H(2) 120.3 C(17)-N(1)-H(1Y) 103(3) 0(5)-0(4)-0(3) 117.5(4) H(1X)-N(1)-H(1Y) 108(4) C(5)-C(4)-H(4) 121.2 C(10)-C(9)-C(14) 124.1(3) C(3)-C(4)-H(4) 121.2 C(10)-C(9)-O(l) 109.6(3) 0(1)-0(7)-0(2) 105.7(3) C(14)-C(9)-O(l) 126.3(3) 0(1)-0(7)-0(8) 108.7(3) C(2)-C(l)-C(6) 121.9(4) 0(2)-0(7)-0(8) 108.8(3) C(2)-C(l)-H(l) 119.0 0(1)-0(7)-0(6) 108.7(3) C(6)-C(l)-H(l) 119.0 0(2)-0(7)-0(6) 108.6(3) C(5)-C(6)-C(l) 115.3(4) 0(8)-0(7)-0(6) 115.8(3) C(5)-C(6)-C(7) 123.0(3) 0(12)-0(13)-0(14) 122.4(4) C(l)-C(6)-C(7) 121.7(4) C(12)-C(13)-H(13) 118.8 C(9)-C(10)-C(ll) 122.1(4) C(14)-C(13)-H(13) 118.8 C(9)-C(l 0)-0(2) 110.3(3) C(10)-C(ll)-C(12) 115.6(4) C(11)-C(10)-0(2) 127.5(4) 0(10)-0(1l)-H(ll) 122.2

- 34 040816

С(9)-С(14)-С(13) 113.6(4) С(12)-С(11)-Н(11) 122.2 С(9)-С(14)-С(15) 122.1(3) С(7)-С(8)-Н(8А) 109.5 С(13)-С(14)-С(15) 124.2(3) С(7)-С(8)-Н(8В) 109.5 С(14)-С(15)-С(16) 110.4(3) H(8A)-C(8)-H(8V) 109.5 С(14)-С(15)-С(19) 114.1(3) С(7)-С(8)-Н(8С) 109.5 С(16)-С(15)-С(19) 108.4(3) H(8A)-C(8)-H(8C) 109.5 С(14)-С(15)-Н(15) 107.9 H(8V)-C(8)-H(8C) 109.5 С(16)-С(15)-Н(15) 107.9 С(18)-С(19)-С(15) 112.2(4) С(19)-С(15)-Н(15) 107.9 С(18)-С(19)-Н(19А) 109.2 C(4)-C(5)-F(l) 117.2(4) С(15)-С(19)-Н(19А) 109.2 С(4)-С(5)-С(6) 125.0(4) С(18)-С(19)-Н(19В) 109.2 F(l)-C(5)-C(6) 117.9(4) С(15)-С(19)-Н(19В) 109.2 С(17)-С(16)-С(15) 112.2(3) H(19A)-C(19)-H(19B) 107.9 С(17)-С(16)-Н(16А) 109.2 N(l)-C(17)-C(16) 110.1(3) С(15)-С(16)-Н(16А) 109.2 N(1)-C(17)-H(17A) 109.6 С(17)-С(16)-Н(16В) 109.2 С(16)-С(17)-Н(17А) 109.6 С(15)-С(16)-Н(16V) 109.2 N(1)-C(17)-H(17B) 109.6 H(16A)-C(16)- 107.9 С(16)-С(17)-Н(17В) 109.6 H(16V) С(3)-С(2)-С(1) 119.4(4) H(17A)-C(17)-H(17V) 108.2 С(3)-С(2)-Н(2) 120.3 С(13)-С(12)-С(11) 122.1(4) С(1)-С(2)-Н(2) 120.3 С(13)-С(12)-Н(12) 118.9 С(11)-С(12)-Н(12) 118.9 С(2)-С(3)-С(4) 120.8(4) H(18A)-C(18)-H(18V) 108.2 С(2)-С(3)-С1(1) 119.6(3) S(l)-C(20)-H(20A) 109.5 С(4)-С(3)-С1(1) 119.6(3) S(l)-C(20)-H(20B) 109.5 N(l)-C(18)-C(19) 109.9(3) H(20A)-C(20)-H(20V) 109.5 N(1)-C(18)-H(18A) 109.7 S(l)-C(20)-H(20C) 109.5 С(19)-С(18)-Н(18А) 109.7 H(20A)-C(20)-H(20C) 109.5 N(1)-C(18)-H(18B) 109.7 H(20V)-C(20)-H(20C) 109.5 С(19)-С(18)-Н(18V) 109.7

Symmetry transformations used to form equivalent atoms.

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Table D

Anisotropic substitution parameters (A ² x10 ³ ) for C8. The exponent of the anisotropic substitution factor takes the form:

-2π ² [ή ² a* ² U ^u + ... + 2 hka* b* U ¹² ]

and ¹¹ and ²² and ³³ and ²³ and ¹³ and ¹² S(l) 73(1) 48(1) 48(1) -2(1) 7(1) -1(1) С1(1) 81(1) 78(1) 81(1) -8(1) 1(1) -8(1) 0(1) 54(1) 50(1) 56(1) 14(1) Yu(1) 17(1) F(l) 83(2) 103(2) 79(2) -40(2) 38(2) -6(2) 0(2) 66(2) 49(1) 54(1) 2(1) I(1) 18(1) 0(4) 87(2) 84(2) 64(2) 19(2) 17(2) 21(2) 0(3) 122(3) 80(2) 47(1) -3(1) 7(2) -13(2) N(l) 47(2) 73(2) 48(2) 7(2) 3(1) AND 2) C(9) 51(2) 38(2) 44(2) 2(1) -7(1) 2(2) C(1) 63(2) 46(2) 50(2) 5(2) 21(2) 13(2) C(6) 55(2) 47(2) 45(2) 5(1) AND 2) 19(2) C(10) 55(2) 39(2) 46(2) -5(1) -4(2) 6(2) C(14) 54(2) 46(2) 42(2) 0(1) -9(2) -5(2) 0(5) 88(2) 88(3) 113(3) -24(2) 13(2) 21(2) C(15) 47(2) 61(2) 40(2) 3(2) -3(1) -2(2) C(5) 60(2) 62(2) 54(2) -6(2) 19(2) 13(2) C(16) 43(2) 53(2) 65(2) -4(2) 8(2) -6(2) C(2) 72(3) 49(2) 45(2) 2(2) 16(2) 14(2) C(4) 57(2) 68(3) 65(2) -3(2) 23(2) 6(2) C(7) 54(2) 50(2) 51(2) 7(2) 12(2) 16(2) C(13) 81(3) 54(2) 46(2) 9(2) -4(2) 4(2) C(11) 70(3) 46(2) 54(2) -8(2) -14(2) 17(2) C(8) 69(3) 71(3) 51(2) 4(2) 4(2) 15(2) S(19) 54(2) 78(3) 70(3) -13(2) 2(2) -25(2) S(17) 54(2) 57(2) 67(2) -3(2) 8(2) 3(2) C(12) 96(3) 43(2) 56(2) 5(2) -14(2) 13(2) C(3) 64(2) 52(2) 52(2) 4(2) 2(2) 14(2) S(18) 43(2) 103(4) 73(3) 7(3) 3(2) -18(2) C(20) 153(7) 87(4) 110(5) -14(4) -6(5) -57(5)

Getting P3. Di-p-toluoyl-L-tartrate salt of 4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3benzodioxol-4-yl]piperidine di-p-toluoyl-L- tartrate salt (P3, di-para-toluoyl-L-tartrate salt)

A solution of C13, free base (519 mg, 1.49 mmol) and di-p-toluoyl-L-tartaric acid (278 mg, 0.719 mmol) in acetonitrile (7.5 ml) was stirred at 50°C for 1 5 hours The mixture was left to cool to room temperature at 0.2°C per minute. After 15 hours at room temperature, the mixture was heated to 65° C. and charged with acetonitrile (15 ml). The mixture was allowed to cool to room temperature at 0.2° C. per minute. After 15 hours at room temperature, the mixture was heated to 54°C. After 3 hours, the solid was collected by filtration and dried in a vacuum oven at 35°C under nitrogen to give P3, di-p-toluoyl-L-tartrate salt, as a white solid (217 mg, 0.296 mmol, 20%, 82% her).

A solution of P3, di-p-toluoyl-L-tartrate salt, (217 mg, 0.296 mmol, 82% ee) in acetonitrile (8.0 ml) at 50°C was allowed to cool to room temperature at 0.2°C per minute . After 15 hours, the solid was collected by filtration and dried in a vacuum oven at 35°C under nitrogen to give P3, the di-p-toluoyl-L-tartrate salt, as a white solid (190 mg, 0.259 mmol, 88%, 88%

- 36 040816 her). LC-MS m/z 348.1 ♦ [M+H]+.

1H NMR (400 MHz, DMSC-d ₆ ) δ 8.9-8.5 (br s, 2H), 7.79 (d, J=8.1 Hz, 4H), 7.64-7.54 (br, 2H), 7.34 (dd, J =8.4, 2.1 Hz, 1H), 7.26 (d, J=8.0 Hz, 4H), 6.87-6.78 (m, 2H), 6.69 (dd, J=6.7, 2.5 Hz, 1H), 5.58 (s, 2H), 3.373.28 (m, 2H, hypothesized; partially hidden by water peak), 3.05-2.89 (m, 3H), 2.33 (s, 6H), 2.02 (s, 3H), 1.92-1.80 ( m, 4H). Retention times: Peak 1 (4.97 min, minor) and Peak 2 (5.31 min, major) {column: Chiralpak IC-U 3.0x50 mm, 1.6 µm; mobile phase A: carbon dioxide; mobile phase B: 0.1% isopropylamine in methanol; gradient: 10% B over 5.00 min, then 45% B over 0.6 min; flow rate: 1.7 ml per minute; back pressure: 130 bar}.

Getting P4. tert-Butyl 4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine 1-carboxylate (Р4)

Suspension of P2 (2.00 g, 4.46 mmol), zinc cyanide (734 mg, 6.25 mol), zinc (70.1 mg, 1.07 mmol), 1,1'-bis(diphenylphosphino)ferrocene ( dppf; 198 mg, 0.357 mmol) and tris(dibenzylideneacetone)dipalladium(0) (164 mg, 0.179 mmol) in N,N-dimethylacetamide (20 ml) were stirred at 120° C. for 16 h after which it was filtered. The filtrate was mixed with water (50 ml) and extracted with ethyl acetate (3x50 ml). The combined organic layers were then washed successively with water (30 ml) and saturated aqueous sodium chloride (20 ml) and concentrated in vacuo. Chromatography on silica gel (gradient: 0 to 30% ethyl acetate in petroleum ether) gave a solid which was treated with acetonitrile (15 ml) and water (15 ml) and lyophilized. This gave P4 as a light yellow solid. Yield: 1.17 g, 2.67 mmol, 60%. LC-MS m/z 461.3 [M+Na ⁺ ].

¹ H NMR (400 MHz, chloroform-d) 7.71 (dd, J=1.1, 7.6 Hz, 1H), 7.45 (dd, J=8.0, 1.6 Hz, 1H), 7.42 (dd, J=10.0 , 1.5 Hz, 1H), 6.79 (dd, ABC system component, J=1.1, 7.6 Hz, 1H), 6.72 (dd, ABC system component, J=7.8, 1.3 Hz, 1H), 6.68 (dd, ABC system component, J=7.8, 1.3 Hz, 1H), 4.37-4.14 (br m, 2H), 2.912.73 (m, 3H), 2.07 (d, J=1.1 Hz, 3H ), 1.89–1.62 (m, 4H), 1.49 (s, 9H).

Getting P5 and P6. 4-Bromo-2-phenyl-1,3-benzodioxole, ENT-1, (P5) and 4-bromo-2-phenyl-1,3-benzodioxole, ENT-2, (P6)

R5 R6

Stage 1. Synthesis of 2-bromo-6-[hydroxy(phenyl)methyl]phenol (С9).

Phenyllithium (1.9 M solution in 1-butoxybutane; 78.5 ml, 149 mmol) was slowly added to a solution of 3-bromo-2-hydroxybenzaldehyde (10.0 g, 49.7 mmol) cooled to -70°C in tetrahydrofuran (70 ml) at a rate at which the temperature of the reaction mixture was maintained below -60°C. The resulting suspension was stirred at -70°C for 1 h and then left to warm to room temperature overnight, after which it was poured into a 0°C aqueous ammonium chloride solution (30 ml). This mixture was extracted with ethyl acetate (3x30 ml) and the combined organic layers were washed with saturated aqueous sodium chloride (30 ml), dried over sodium sulfate, filtered and concentrated in vacuo. Chromatography on silica gel (gradient: 0 to 5% ethyl acetate in petroleum ether) gave C9 as a yellow solid. Yield: 6.11 g, 21.9 mmol, 44%.

1Н NMR (400 MHz, chloroform-d) δ 7.45-7.28 (m, 6H), 7.22-7.18 (m, 1H), 7.06 (br d, J=7.7 Hz, 1H), 6.77 (dd, J= 7.9, 7.8 Hz, 1H), 6.06 (br s, 1H), 2.89 (br s, 1H).

Stage 2. Synthesis of 4-bromo-2-phenyl-1,3-benzodioxole (C10).

To a solution of C9 (6.11 g, 21.9 mmol) in methanol (370 ml) was added a solution of sodium periodate (11.7 g, 54.7 mmol) in water (175 ml). The reaction mixture was stirred at 30° C. for 40 h after which most of the methanol was removed by concentration in vacuo. The resulting mixture was extracted with dichloromethane (5x100 ml) and the combined organic layers were washed successively with aqueous sodium sulfite (100 ml) and saturated aqueous sodium chloride (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. Chromatography on silica gel (eluent: petroleum ether) gave C10 as a colorless oil. Yield: 4.50 g,

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16.2 mmol, 74%. LC-MS m/z 278.5 (bromine isotope pattern observed) [M+H]+.

'H NMR (400 MHz, chloroform-J) δ 7.62-7.57 (m, 2H), 7.49-7.43 (m, 3H), 7.04 (s, 1H), 7.00 (dd, J=8.0, 1.4 Hz, 1H), 6.79 (dd, ABX system component, J=7.8, 1.4 Hz, 1H), 6.75 (dd, ABX system component, J=7.9, 7.8 Hz, 1H).

Step 3. Isolation of 4-bromo-2-phenyl-1,3-benzodioxole, ENT-1, (P5) and 4-bromo-2-phenyl-1,3-benzodioxole, ENT-2, (P6).

Enantiomers constituting C10 (5.00 g, 18.0 mmol) were separated using SFC [column: Chiral Technologies ChiralCel OD, 10 μm; mobile phase: 3:1 carbon dioxide/(methanol containing 0.1% ammonium hydroxide)]. The first eluting enantiomer was designated ENT-1 (P5) and the second eluting enantiomer was designated ENT-2 (P6); both were obtained as a yellow oil.

P5 Yield: 2.20 g, 7.94 mmol, 44%. LC-MS m/z 277.0 (bromine isotope pattern observed) [M+H]+.

'H NMR (400 MHz, chloroform-d) δ 7.63-7.55 (m, 2H), 7.51-7.42 (m, 3H), 7.04 (s, 1H), 7.00 (dd, J=8.0, 1.3 Hz, 1H), 6.80 (dd, ABX system component, J=7.8, 1.4 Hz, 1H), 6.75 (dd, ABX system component, J=7.9, 7.8 Hz, 1H). Retention time 3.28 min (column: Chiral Technologies ChiralCel OD-H, 4.6x150 mm, 5 µm; mobile phase A: carbon dioxide; mobile phase B: methanol containing 0.05% diethylamine; gradient: 5 to 40 % B in 5.5 min; flow rate: 2.5 ml per minute).

P6 Yield: 2.00 g, 7.22 mmol, 40%. LC-MS m/z 276.9 (bromine isotope pattern observed) [M+H]+.

'H NMR (400 MHz, chloroform-d) δ 7.63-7.55 (m, 2H), 7.50-7.42 (m, 3H), 7.04 (s, 1H), 7.00 (dd, J=8.0, 1.4 Hz, 1H), 6.80 (dd, ABX system component, J=7.8, 1.4 Hz, 1H), 6.75 (dd, ABX system component, J=7.9, 7.9 Hz, 1H). Retention time 3.73 min (analytical conditions identical to those used for P5).

Getting P7. tert-Butyl-4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidine-1carboxylate (Р7)

C11

Stage 1. Synthesis of 2-(4-bromo-2-methyl-1,3-benzodioxol-2-yl)-5-chloropyridine (С11).

A mixture of 5-chloro-2-ethynylpyridine (1.80 g, 13.1 mmol), 3-bromobenzene-1,2-diol (2.47 g, 13.1 mmol) and triruthenium dodecacarbonyl (167 mg, 0.261 mmol) in toluene (25 ml) was degassed for 1 min and then heated at 100° C. for 16 h. The reaction mixture was diluted with ethyl acetate (30 ml) and filtered through a pad of diatomaceous earth. The filtrate was concentrated in vacuo and purified by silica gel chromatography (gradient: 0 to 1% ethyl acetate in petroleum ether) to give C11 as a yellow oil. Yield: 1.73 g, 5.30 mmol, 40%. LC-MS m/z 325.6 (a pattern with peaks of bromine and chlorine isotopes was observed) [M+H]+.

'H NMR (400 MHz, chloroform-J) δ 8.63 (dd, J=2.4, 0.7 Hz, 1H), 7.71 (dd, component of the ABX system, J=8.4, 2.4 Hz, 1H), 7.60 ( dd, ABX system component, J=8.4, 0.7Hz, 1H), 6.97 (dd, J=8.0, 1.4Hz, 1H), 6.76 (dd, ABX system component, J=7.8, 1.4Hz , 1H), 6.72 (dd, component of the ABX system, J=8.0, 7.8 Hz, 1H), 2.10 (s, 3H).

Step 2. Synthesis of tert-butyl 4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]-3,6dihydropyridine-(2Н)-carboxylate (С12) .

[1,H-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (388 mg, 0.530 mmol) was added to a suspension of C11 (1.73 g, 5.30 mmol), tert-butyl-4-(4.4.5 ,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6dihydropyridine-1(2H)-carboxylate (1.64 g, 5.30 mmol) and cesium carbonate (5.18 g, 15, 9 mmol) in 1,4dioxane (35 ml) and water (6 ml). This reaction mixture was stirred at 90°C for 4 h, after which it was diluted with ethyl acetate (30 ml) and water (5 ml). The organic layer was concentrated in vacuo and the residue was subjected to silica gel chromatography (gradient: 0 to 5% ethyl acetate in petroleum ether) to give C12 as a yellow gum. Yield: 1.85 g, 4.31 mmol, 81%. LC-MS m/z 451.04 [M+Na+].

'H NMR (400 MHz, chloroform-d) δ 8.62 (dd, J=2.5, 0.8 Hz, 1H), 7.69 (dd, component of the ABX system, J=8.4, 2.4 Hz, 1H), 7.57 ( dd, component of the ABX system, J=8.4, 0.8 Hz, 1H), 6.84-6.79 (m, 2H), 6.78-6.73 (m, 1H), 6.39-6.33 (br m, 1H), 4.13-4.07 ( m, 2H), 3.68–3.58 (m, 2H), 2.60–2.51 (br m, 2H), 2.07 (s, 3H), 1.49 (s, 9H).

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Step 3. Synthesis of tert-butyl 4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidine 1-carboxylate (P7).

A solution of C12 (2.61 g, 6.08 mmol) and tris(triphenylphosphine)rhodium(I) chloride (Wilkinson's catalyst; 563 mg, 0.608 mmol) in methanol (100 mL) was degassed under vacuum and then purged with hydrogen;

this evacuation-purge cycle was performed a total of three times. The reaction mixture was then stirred at 60°C in hydrogen (50 psi) for 16 h, after which it was filtered. The filtrate was concentrated in vacuo and the residue was purified by silica gel chromatography (gradient: 0 to 10% ethyl acetate in petroleum ether). The resulting material was combined with the material obtained from a similar hydrogenation of C12 (110 mg, 0.256 mmol) to give P7 as a light yellow gum. Overall yield: 2.05 g, 4.76 mmol, 75%. LC-MS m/z 431.3^ [M+H]+.

¹ H NMR (400 MHz, chloroform-d) δ 8.62 (d, J=2.3 Hz, 1H), 7.69 (dd, component of the ABX system, J=8.4, 2.4 Hz, 1H), 7.57 (d, half AB quartet, J=8.4 Hz, 1H), 6.79 (dd, ABC component, J=7.8, 7.7 Hz, 1H), 6.72 (dd, ABC component, J=7.8, 1.3 Hz , 1H), 6.68 (br d, ABC system component, J=7.9 Hz, 1H), 4.32-4.12 (br m, 2H), 2.91-2.73 (m, 3H), 2.05 (s, 3H), 1.90 -1.62 (m, 4H), 1.48 (s, 9H).

Getting P8 and P9. tert-Butyl 4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4yl]piperidine-1-carboxylate, ENT-1, (P8) and tert-butyl-4 -[2-(5-chloropyridin-2-yl)-2-methyl-1,3benzodioxol-4-yl]piperidine-1-carboxylate, ENT-2, (P9)

Separation of P7 (500 mg, 1.16 mmol) into its constituent enantiomers was performed using SFC {column: Phenomenex Lux Amylose-1, 5 μm; mobile phase: 9:1 carbon dioxide/[2-propanol containing 0.2% (7M ammonia in methanol)]}. The first eluting enantiomer was designated ENT-1 (P8) and the second eluting enantiomer was designated ENT-2 (P9).

P8 Yield: 228 mg, 0.529 mmol, 46%. Retention time 4.00 min {column: Phenomenex Lux Amy lose-1, 4.6x250 mm, 5 µm; mobile phase A: carbon dioxide; mobile phase B: [2-propanol containing 0.2% (7M ammonia in methanol)]; gradient: 5% B over 1.00 min, then 5 to 60% B over 8.00 min; flow rate: 3.0 ml per minute; back pressure: 120 bar}.

P9 Yield: 229 mg, 0.531 mmol, 46%. Retention time 4.50 min (analytical conditions identical to those used for P8).

Getting P10. {4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}acetic acid (Р10)

Step 1. Synthesis of 4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine p-toluenesulfonate salt (C13).

A solution of P2 (5.0 g, 11 mmol) and para-toluenesulfonic acid (4.81 g, 27.9 mmol) in ethyl acetate (100 ml) was stirred at 60°C for 2 h, after which it was concentrated in vacuo with obtaining C13 in the form of a yellow resin. This substance was directly transferred to the next stage. LC-MS m/z 347.9 ♦ [M+H] ⁺ .

Stage 2. Synthesis of ethyl {4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}acetate (С14).

Potassium carbonate (7.71 g, 55.8 mmol) and ethyl bromoacetate (1.86 g, 11.2 mmol) were added to a solution of C13 (from the previous step; <11 mmol) in acetonitrile (150 ml) and this reaction mixture stirred at 55°C for 16 hours. It was then filtered and the filtrate was concentrated in vacuo and purified by silica gel chromatography (gradient: 0 to 30% ethyl acetate in petroleum ether) to give C14 in

- 39 040816 yellow resin. According to ¹ H NMR analysis this material was not quite pure. Yield: 3.57 g, 8.23 mmol, 75% over 2 steps.

¹ H NMR (400 MHz, chloroform-d), C14 peaks only: δ 7.52 (dd, J=8.4, 8.0 Hz, 1H), 7.17-7.07 (m, 2H), 6.77 (dd, component of the ABC system, J=7.8, 7.8 Hz, 1H), 6.72-6.67 (m, 2H), 4.21 (q, J=7.1 Hz, 2H), 3.27 (s, 2H), 3.07 (m, 2H), 2.70 (tt, J=12.1, 3.8 Hz, 1H), 2.35 (ddd, J=11.5, 11.5, 2.7 Hz, 2H), 2.04 (d, J=1.1 Hz, 3H), 2.021.76 (m, 4H), 1.29 (t, J=7.1 Hz, 3H).

Step 3. Synthesis of {4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}acetic acid (P10).

A solution of C14 (3.57 g, 8.23 mmol) and aqueous sodium hydroxide solution (3 M; 13.7 ml, 41.1 mmol) in a mixture of methanol (80 ml) and tetrahydrofuran (40 ml) were stirred at 25°C within 16 hours After removing the solvents in vacuo, the aqueous residue was acidified to pH 7 by adding 1 M hydrochloric acid and then extracted with a mixture of dichloromethane and methanol (10:1, 2x100 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure to give P10 as a yellow solid. Yield: 2.95 g, 7.27 mmol, 88%. LC-MS m/z 406.2^ [M+H]+.

¹ H NMR (400 MHz, methanol^) δ 7.61 (dd, J=8.3, 8.3 Hz, 1H), 7.29 (dd, J=10.9, 2.0 Hz, 1H), 7.22 (ddd, J=8.4 , 2.0, 0.8 Hz, 1H), 6.82 (dd, ABC system component, J=8.3, 7.1 Hz, 1H), 6.78-6.72 (m, 2H), 3.65-3.54 (br m, 2H), 3.51 (s, 2H), 3.04-2.88 (m, 3H), 2.23-2.07 (m, 2H), 2.07-1.93 (m, 2H), 2.04 (d, J=1.1 Hz, 3H).

Getting P11. Methyl 2-(chloromethyl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate (P11)

P11, HCl salt

Stage 1. Synthesis of methyl 3-[(2-methoxyethyl)amino]-4-nitrobenzoate (С15).

To a colorless solution of methyl 3-fluoro-4-nitrobenzoate (50 g, 250 mmol) in tetrahydrofuran (400 ml) was added triethylamine (40.7 g, 402 mmol, 55.8 ml), then 2-methoxyethanamine (30. 2 g, 402 mmol) in tetrahydrofuran (100 ml) dropwise at room temperature. The resulting yellow solution was stirred at 55° C. for 18 hours. The solution was cooled to room temperature and concentrated under reduced pressure to remove tetrahydrofuran. The resulting yellow solid was dissolved in ethyl acetate (800 ml) and washed with saturated aqueous ammonium chloride (250 ml). The aqueous phase was separated and extracted with ethyl acetate (200 ml). The combined organic layers were washed with saturated aqueous sodium chloride (3x250 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure to give C15 (60.2 g, 94%) as a yellow solid.

¹ H NMR (600 MHz, chloroform-d) δ 8.23 (d, 1H), 8.17 (br s, 1H), 7.58 (d, 1H), 7.25 (dd, 1H), 3.95 (s, 3H), 3.69- 3.73 (m, 2H), 3.56 (m, 2H), 3.45 (s, 3H); LC-MS m/z 255.4 [M+H] ⁺ .

Stage 2. Synthesis of methyl 4-amino-3-[(2-methoxyethyl)amino]benzoate (С16).

Pd/C (10 g, 94 mmol) was added to a solution of C15 (30 g, 118 mmol) in methanol (500 ml). The reaction mixture was stirred at room temperature in 15 psi hydrogen for 18 hours. The black suspension was filtered through diatomaceous earth and the filter cake was washed with methanol (500 ml).

The combined filtrates were concentrated in vacuo to give C16 (26.5 g, quantitative) as a brown oil which solidified on standing.

¹ H NMR (400 MHz, chloroform-d) δ 7.48 (dd, 1H), 7.36 (d, 1H), 6.69 (d, 1H), 3.87 (s, 3H), 3.77 (br s, 2H), 3.68 ( t, 2H), 3.41 (s, 3H), 3.32 (t, 2H); LC-MS m/z 224.7 [M+H] ⁺ .

Stage 3. Synthesis of methyl 2-(chloromethyl)-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate (P11).

To a solution of C16 (5.00 g, 22.3 mmol) in tetrahydrofuran (100 mL) was added 2-chloro-1,1,1-trimethoxyethane (3.31 mL, 24.6 mmol), then p-toluenesulfonic acid monohydrate ( 84.8 mg, 0.446 mmol). This reaction mixture was heated at 45°C for 5 h, after which it was concentrated in vacuo; the residual oil was dissolved in ethyl acetate (10 ml) and heated until a solution formed. This solution was stirred slowly while cooling to room temperature overnight. The precipitate was collected by filtration and washed with heptane to give P11 as a gray solid. Yield: 5.73 g, 20.3 mmol, 91%.

¹ H NMR (600 MHz, chloroform-d) δ 8.12 (br s, 1H), 8.01 (br d, J=8.6 Hz, 1H), 7.79 (d, J=8.4 Hz, 1H),

- 40 040816

4.96 (s, 2H), 4.52 (t, J=5.1 Hz, 2H), 3.96 (s, 3H), 3.74 (t, J=5.1 Hz, 2H), 3.28 (s, 3H).

Step 4. Synthesis of methyl 2-(chloromethyl)-1-(2-methoxyethyl)-III-benzimidazole-6-carboxylate hydrochloride salt (P11, HCl salt).

A solution of C16 (5.0 g, 24 mmol) in 1,4-dioxane (100 ml) was heated to 100°C, a solution of chloroacetic anhydride (4.1 g, 24.5 mmol) in 1,4-dioxane (60 ml) through an addition funnel over a period of 10 h, and the reaction mixture was stirred for another 12 h at 100°C. The next day, the reaction mixture was cooled to room temperature, and 1,4-dioxane was removed under reduced pressure. The crude reaction mixture was dissolved in ethyl acetate and washed with saturated aqueous sodium bicarbonate. The ethyl acetate layer was separated, dried over sodium sulfate and filtered. A solution of 4 M hydrogen chloride in 1,4-dioxane (1.1 eq.) was added to the ethyl acetate solution with constant stirring. The hydrochloride salt P11 precipitated as a pale yellow solid. The suspension was stirred for 1 hour and the P11 hydrochloride salt was then collected by filtration to give a yellow solid (6.1 g, 86%).

1H NMR (600 MHz, CD3OD) δ 8.64 (s, 1H), 8.30 (d, 1H), 7.92 (d, 1H), 5.32 (s, 2H), 4.84 (m, 2H), 3.99 (s, 3H) , 3.83 (t, 2Н), 3.31 (s, 3Н). LC-MS m/z 283.2 [M+H]+.

Obtaining carboxylate (P12)

Methyl-1-(2-methoxyethyl)-2-(piperazin-1-ylmethyl)-1 H-benzimidazole-6P12.

Step 1. Synthesis of methyl 2-{[4-(tert-butoxycarbonyl)piperazin-1-yl]methyl}-1-(2-methoxyethyl)1H-benzimidazole-6-carboxylate (C17).

Compound P11 (1.59 g, 5.62 mmol) was added to a 15° C. mixture of tert-butylpiperazine-1-carboxylate (1.00 g, 5.37 mmol) and potassium carbonate (2.97 g, 21. 5 mmol) in acetonitrile (15 ml) and this reaction mixture was stirred at 55°C for 12 h. 1.07 mmol) and the mixture was filtered. After concentrating the filtrate in vacuo, the residue was purified by silica gel chromatography (gradient: 0 to 60% ethyl acetate in petroleum ether) to give C17 as a pale yellow solid. Overall yield: 2.30 g, 5.32 mmol, 83%. LC-MS m/z 433.0 [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 8.12 (d, J=1.5 Hz, 1H), 7.96 (dd, J=8.4, 1.5 Hz, 1H), 7.73 (d, J=8.5 Hz, 1H), 4.58 (t, J=5.4 Hz, 2H), 3.95 (s, 3H), 3.89 (s, 2H), 3.73 (t, J=5.4 Hz, 2H), 3.46-3.37 (br m, 4H), 3.28 (s, 3H), 2.54–2.44 (br m, 4H), 1.45 (s, 9H).

Stage 2. Synthesis of methyl 1-(2-methoxyethyl)-2-(piperazin-1-ylmethyl)-1H-benzimidazole-6carboxylate (P12).

To a solution of C17 (2.30 g, 5.32 mmol) in dichloromethane (80 ml) was added a solution of hydrogen chloride in ethyl acetate (20 ml). This reaction mixture was stirred at 20°C for 2 h, after which it was concentrated in vacuo. The residue was diluted with water (20 ml), adjusted to pH 9-10 with saturated aqueous sodium bicarbonate and extracted with a mixture of ethyl acetate and methanol (10:1, 15x50 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo to give P12 as a pale yellow solid. Yield: 1.68 g, 5.05 mmol, 95%. LC-MS m/z 332.8 [M+H]+.

¹ H NMR (400 MHz, chloroform-d) δ 8.13 (br s, 1H), 7.96 (br d, J=8.5 Hz, 1H), 7.72 (d, J=8.5 Hz, 1H), 4.59 (t, J=5.5 Hz, 2H), 3.95 (s, 3H), 3.86 (s, 2H), 3.75 (t, J=5.5 Hz, 2H), 3.29 (s, 3H), 2.87 ( t, J=4.8 Hz, 4H), 2.50 (br m, 4H).

Getting P13. 6-Bromo-2-(chloromethyl)-1-(2-methoxyethyl)-1H-imidazo[4,5-b]pyridine (Р13)

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Stage 1. Synthesis of 5-bromo-N-(2-methoxyethyl)-2-nitropyridine-3-amine (C18).

A solution of 5-bromo-3-fluoro-2-nitropyridine (400 mg, 1.81 mmol) and 2-methoxyethanamine (408 mg, 5.43 mmol) in tetrahydrofuran (10 ml) was stirred at 25°C for 2 h, after which it was diluted with ethyl acetate (100 ml) and washed with water (50 ml). The organic layer was washed with saturated aqueous sodium chloride (50 ml), dried over magnesium sulfate, filtered and concentrated to give C18 as a yellow solid. Yield: 430 mg, 1.56 mmol, 86%.

Stage 2. Synthesis of 5-bromo-N ³ -(2-methoxyethyl)pyridine-2,3-diamine (C19).

A solution of C18 (430 mg, 1.56 mmol), ammonium chloride (833 mg, 15.6 mmol) and iron powder (870 mg, 15.6 mmol) in a mixture of methanol (10 ml) and water (2 ml) was stirred at 80°C for 30 min. The resulting suspension was poured into water (50 ml) and extracted with ethyl acetate (2x50 ml). The combined organic layers were dried over magnesium sulfate, filtered and concentrated to give C19 as a brown solid. Yield: 350 mg, 1.42 mmol, 91%.

¹ H NMR (400 MHz, chloroform-d) δ 7.63 (d, J=2.1 Hz, 1H), 6.88 (d, J=2.0 Hz, 1H), 4.33-4.19 (br s, 2H), 3.65 (dd, J=5.6, 4.6 Hz, 2H), 3.40 (s, 3H), 3.22 (br t, J=5 Hz, 2H).

Stage 3. Synthesis of 6-bromo-2-(chloromethyl)-1-(2-methoxyethyl)-1H-imidazo[4,5-b]pyridine (P13).

A solution of C19 (400 mg, 1.63 mmol) in 1,4-dioxane (8 mL) was treated with chloroacetyl chloride (0.284 mL, 3.57 mmol) and stirred at room temperature until LC-MS analysis showed complete conversion C19 to an amide intermediate. After removal of 1,4-dioxane in vacuo, the residue was dissolved in trifluoroacetic acid (8 ml) and heated at 80°C for 18 h, after which the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting oil was dissolved in ethyl acetate (50 ml) and neutralized by adding saturated aqueous sodium bicarbonate. The aqueous layer was extracted with ethyl acetate (20 ml) and the combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. Chromatography on silica gel (gradient: 0 to 80% ethyl acetate in heptane) gave P13 as a solid. Yield: 176 mg, 0.578 mmol, 35%. LC-MS m/z 306.1 (a pattern with peaks of bromine and chlorine isotopes was observed) [M+H]+.

1H NMR (600 MHz, chloroform-d) δ 8.58 (br s, 1H), 7.89 (br s, 1H), 4.92 (s, 2H), 4.44 (t, J=5.0 Hz, 2H), 3.71 ( t, J=5.0 Hz, 2H), 3.28 (s, 3H).

Getting P14. Methyl 2-{[4-(2,3-dihydroxyphenyl)piperidin-1-yl]methyl}-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylate (Р14)

- 42 040816

Stage 1. Synthesis of [(3-bromobenzene-1,2-diyl)bis(oxymethanediyloxyethane-2,1diyl)]bis(trimethylsilane) (С20).

This reaction was carried out in two batches of identical scale. Ν,N-Diisopropylethylamine (37.8 ml, 217 mmol) was added dropwise to a solution of 3-bromobenzene-1,2-diol (10.0 g, 52.9 mmol) in tetrahydrofuran (300 ml). After stirring the mixture for 10 min at 20°C, [2(chloromethoxy)ethyl](trimethyl)silane (19.2 mL, 108 mmol) was added dropwise over 5 min and stirring was continued for 16 h at room temperature ( 18°C). N,Ndiisopropylethylamine (27.6 ml, 158 mmol) was added again, then [2(chloromethoxy)ethyl](trimethyl)silane (14.0 ml, 79.1 mmol) was added dropwise at room temperature (18°C). After an additional 2.5 hours at room temperature, the reaction mixture was filtered and the filtrate was concentrated in vacuo. At this point, the crude products from the two batches were combined and purified by silica gel chromatography (gradient: 0 to 7% ethyl acetate in petroleum ether) to give C20 as a colorless oil. According to 1H NMR analysis this material was not quite pure. Overall yield: 22.9 g, 50.9 mmol, 48%.

¹ H NMR (400 MHz, chloroform-d), C20 peaks only: δ 7.19 (dd, J=8.1, 1.5 Hz, 1H), 7.12 (dd, J=8.3, 1.4 Hz, 1H), 6.90 (dd, J=8.2. 8.2 Hz, 1H), 5.26-5.19 (m, 4H), 4.00-3.92 (m, 2H), 3.80-3.73 (m, 2H), 1.00-0.91 (m, 4H) , 0.03 (s, 9H), 0.00 (s, 9H).

Step 2. Synthesis of tert-butyl 4-(2,3-bis{[2-(trimethylsilyl)ethoxy]methoxy}phenyl)-3,6dihydropyridine-1(2H)-carboxylate (С21).

Reaction vessel containing a suspension of C20 (6.11 g, 13.6 mmol), tert-butyl-4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6- dihydropyridine-1(2H)-carboxylate (5.04 g, 16.3 mmol), aqueous sodium carbonate solution (1 M; 40.8 ml, 40.8 mmol) and [1,1'bis(diphenylphosphino)ferrocene] dichloropalladium(II) (497 mg, 0.679 mmol) in 1,4-dioxane (100 mL) was evacuated and filled with nitrogen. This vacuum cycle was repeated twice, and then the reaction mixture was stirred at 85°C for 16 h, after which the reaction mixture was diluted with water (40 ml) and was extracted with ethyl acetate (3x150 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. Purification by chromatography on silica gel (gradient: 0 to 8% methanol in dichloromethane) gave C21 as a yellow oil. Yield: 5.47 g, 9.91 mmol, 73%.

1H NMR (600 MHz, chloroform-d) δ 7.10 (br d, J=8.2 Hz, 1H), 6.98 (dd, J=7.9, 7.9 Hz, 1H), 6.81 (br d, J=1 ,1 Hz, 1H), 5.79 (br s, 1H), 5.23 (s, 2H), 5.07 (s, 2H), 4.03 (br s, 2H), 3.83–3.74 (m, 4H), 3.59 (br s , 2H), 2.52 (br s, 2H), 1.49 (s, 9H), 1.01–0.89 (m, 4H), 0.01 (s, 9H), 0.01 (s, 9H).

Step 3. Synthesis of tert-butyl 4-(2,3-bis{[2-(trimethylsilyl)ethoxy]methoxy}phenyl)piperidine-1carboxylate (С22).

A solution of C21 (12.5 g, 22.6 mmol) in methanol (300 mL) was treated with 10% palladium on carbon (2.94 g, 2.76 mmol) and hydrogenated for 16 hours at 40 psi. inch and 25°C. LC-MS analysis at this point in time showed the conversion to the product: LC-MS m/z 576.0 [M+Na+]. After filtering the reaction mixture and washing the filter cake with methanol (2x100 ml), the combined filtrates were concentrated in vacuo to give C22 as a colorless oil. Yield: 11.2 g, 20.1 mmol, 89%.

1Н NMR (400 MHz, chloroform-d) δ 7.05-6.97 (m, 2H), 6.83 (dd, J=6.9, 2.5 Hz, 1H), 5.22 (s, 2H), 5.13 (s, 2H), 4.38-4.10 (br m, 2H), 3.90-3.82 (m, 2H), 3.81-3.73 (m, 2H), 3.22 (tt, J=12.2, 3.5 Hz, 1H), 2.79 (br dd, J =12.8, 12.8 Hz, 2H), 1.78 (br d, J=13 Hz, 2H), 1.65-1.52 (m, 2H), 1.48 (s, 9H), 1.04-0.91 (m, 4H), 0.03 (s, 9H),

- 43 040816

0.00(s, 9H).

Step 4. Synthesis of 4-(2,3-bis{[2-(trimethylsilyl)ethoxy]methoxy}phenyl)piperidine (С23).

To a room temperature (15°C) solution of C22 (7.23 g, 13.0 mmol) in dichloromethane (90 ml) was added 2,6-dimethylpyridine (2.39 g, 22.3 mmol), then added dropwise trimethylsilyl trifluoromethanesulfonate (3.80 g, 17.1 mmol). The reaction mixture was stirred at 15° C. for 16 h after which 2,6-dimethylpyridine (909 mg, 8.48 mmol) and trimethylsilyl trifluoromethanesulfonate (1.45 g, 6.52 mmol) were further added. After stirring at room temperature (15° C.) for another 5 hours, LC-MS analysis of the reaction mixture showed the presence of the product: LC-MS m/z 454.1 [M+H]+. The reaction mixture was concentrated in vacuo and the residue was washed successively with aqueous ammonium chloride (3x100 ml) and saturated aqueous sodium chloride (100 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure to give C23 as a brown oil (6, 6 g). This substance was directly transferred to the next stage.

Step 5. Synthesis of methyl 2-{[4-(2,3-bis{[2-(trimethylsilyl)ethoxy]methoxy}phenyl)piperidin-1yl]methyl} -1 -(2-methoxyethyl)-1 H-benzimidazole -6-carboxylate (C24).

To a solution of C23 (from the previous step; 6.6 g, <13 mmol) in acetonitrile (150 mL) was added P11 (3.08 g, 10.9 mmol) followed by potassium carbonate (10.1 g, 73.1 mmol) and the reaction mixture was stirred at room temperature (15° C.) for 16 hours. LC-MS analysis at this point in time showed the presence of the product: LC-MS m/z 700.2 [M+H] ⁺ . The reaction mixture was filtered and the filtrate was concentrated in vacuo. Purification by silica gel chromatography (gradient: 34% to 56% ethyl acetate in petroleum ether) gave C24 as a yellow oil. Yield: 5.4 g, 7.7 mmol, 59% over 2 steps.

¹ H NMR (400 MHz, chloroform-d) δ 8.16-8.12 (m, 1H), 7.96 (dd, J=8.5, 1.5 Hz, 1H), 7.73 (d, J=8.5 Hz, 1H) , 7.04-6.96 (m, 2H), 6.86 (dd, J=6.7, 2.6 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.63 (t, J=5.5 Hz , 2Н), 3.95 (s, 3Н), 3.93-3.83 (m, 4H), 3.80-3.72 (m, 4Н), 3.31 (s, 3Н), 3.17-3.06 (m, 1H), 2.99 (br d, J=11.2 Hz, 2H), 2.352.22 (m, 2H), 1.81 (br d, half AB quartet, J=12.6 Hz, 2H), 1.75-1.61 (m, 2H), 1.04-0.91 (m, 4H), 0.05 (s, 9H), -0.01 (s, 9H).

Step 6. Synthesis of methyl 2-{[4-(2,3-dihydroxyphenyl)piperidin-1-yl]methyl}-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylate (P14).

A solution of hydrogen chloride in 1,4-dioxane (4 M; 96 mL, 384 mmol) was added to a room temperature (18°C) solution of C24 (6.40 g, 9.14 mmol) in 1,4-dioxane ( 120 ml). After completion of the addition, the reaction mixture was stirred at room temperature (18°C) for 16 h, combined with the product of a similar reaction carried out using C24 (1.00 g, 1.43 mmol) and concentrated in vacuo. The residue was treated with a mixture of dichloromethane and methanol (20:1, 150 ml) and stirred at room temperature (18°C) for 1 h, after which the solid (4.85 g) was collected by filtration. This material was treated with water (100 ml) and the mixture was adjusted to pH 7-8 by addition of aqueous sodium bicarbonate solution, stirred at room temperature (18°C) for 30 min and filtered. The filter cake was washed with water (2x20 ml), then mixed with methanol (100 ml) and concentrated in vacuo. The resulting material was treated with petroleum ether (100 ml) and stirred at room temperature (18° C.) for 30 minutes. After filtration, the filter cake was mixed with toluene (30 ml) and concentrated in vacuo to give P14 as a gray solid. Overall yield: 2.92 g, 6.64 mmol, 63%. LC-MS m/z 440.1 [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.21 (d, J=1.6 Hz, 1H), 7.81 (dd, J=8.5, 1.6 Hz, 1H), 7.66 (d, J=8, 5 Hz, 1H), 6.64-6.51 (m, 3H), 4.63 (t, J=5.3 Hz, 2H), 3.88 (s, 3H), 3.84 (s, 2H), 3.75 (t, J=5 .3 Hz, 2H), 3.22 (s, 3H), 2.97-2.78 (m, 3H), 2.18 (br dd, J=11, 11 Hz, 2H), 1.75-1.64 (m, 2H), 1.64-1.49 (m, 2H).

Getting P15. Methyl 2-(chloromethyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (Р15)

- 44 040816

szo

This whole sequence of reactions was carried out on a large scale. As a rule, before carrying out the reactions, as well as adding reagents, the reactors were evacuated to -0.08 to -0.05 MPa and then filled with nitrogen to normal pressure. This process was typically repeated 3 times, and then the oxygen content was evaluated to ensure that it was <1.0%. For the processes of extraction and washing of the organic layers, the mixtures were usually stirred for 15-60 minutes and then left to settle for 15-60 minutes, after which the layers were separated.

Stage 1. Synthesis of (2S)-2-[(benzyloxy)methyl]oxetane (С25).

This reaction was carried out in three batches of approximately the same scale. A 2000 L glass lined reactor was charged with 2-methylpropan-2-ol (774.7 kg). Potassium tert-butoxide (157.3 kg, 1402 mol) was added via a solids funnel and the mixture stirred for 30 minutes. Trimethylsulfoxonium iodide (308.2 kg, 1400 mol) was then added in the same manner and the reaction mixture was heated at 55-65°C for 2-3 h, after which (28)-2-[(benzyloxy)methyl]oxirane was added (92.1 kg, 561 mol) at a rate of 5 to 20 kg per hour. After keeping the reaction mixture at 55-65°C for 25 h, it was cooled to 25-35°C and filtered through diatomaceous earth (18.4 kg). The filter cake was washed with tert-butyl methyl ether (3x340 kg) and the combined filtrates were transferred to a 5000 L reactor, treated with purified water (921 kg) and stirred for 15-30 min at 1530°C. The organic layer was then washed twice with a solution of sodium chloride (230.4 kg) in purified water (920.5 kg) and concentrated under reduced pressure (< -0.08 MPa) at <45°C. Nheptane (187 kg) was added and the resulting mixture was concentrated under reduced pressure (< -0.08 MPa) at <45°C. The organic phase was purified by chromatography on silica gel (280 kg) with sodium chloride (18.5 kg) at the top of the column. The crude material was loaded onto the column using n-heptane (513 kg) and then eluted with a mixture of n-heptane (688.7 kg) and ethyl acetate (64.4 kg). Three batches were combined to give C25 as a light yellow oil of 85% purity (189.7 kg, 906 mmol, 54%).

1H NMR (400 MHz, chloroform-d), C25 peaks only: δ 7.40-7.32 (m, 4H), 7.32-7.27 (m, 1H), 4.98 (dddd, J=8.1, 6.7, 4.9, 3.7 Hz , 1H), 4.72-4.55 (m, 4H), 3.67 (dd, ABX system component, J=11.0, 4.9 Hz, 1H), 3.62 (dd, ABX system component, J=11.0, 3.7 Hz, 1H), 2.72-2.53 (m, 2H).

Stage 2. Synthesis of (2S)-oxetan-2-ylmethanol (С26).

10% palladium on carbon (30.7 kg) was added via a hopper to a 10-30° C. solution of 85% pure C25 (from the previous step; 185.3 kg, 884.8 mol) in tetrahydrofuran (1270 kg) into a stainless steel autoclave reactor with a capacity of 3000 l. The hopper was washed with purified water and tetrahydrofuran (143 kg) and the washings were added to the reaction mixture. After purging the contents of the reactor with nitrogen, it was similarly purged with hydrogen, increasing the pressure to 0.3–0.5 MPa and then dropping it to 0.05 MPa. This hydrogen purge was repeated 5 times, after which the hydrogen pressure was increased to 0.3-0.4 MPa. The reaction mixture was then heated to 35-45°C. After 13 hours, during which the hydrogen pressure was maintained at 0.3-0.5 MPa, the mixture was ventilated to 0.05 MPa and purged five times with nitrogen, increasing the pressure to 0.15-0.2 MPa and then dropping to 0.05 MPa. After the mixture was cooled to 10-25°C, it was filtered and the reactor was washed with tetrahydrofuran (2x321 kg). The filter cake was soaked twice with washing liquid and then filtered. Concentration under reduced pressure (<-0.06 MPa) was performed at <40° C. to give C26 (62.2 kg, 706 mol, 80%) in tetrahydrofuran (251 kg).

Stage 3. Synthesis of (2S)-oxetan-2-ylmethyl-4-methylbenzenesulfonate (С27).

4-(Dimethylamino)pyridine (17.5 kg, 143 mol) was added to a 10-25°C solution of C26 (from the previous step; 62.2 kg, 706 mol) in tetrahydrofuran (251 kg) and triethylamine (92. 7 kg, 916 mol) in dichloromethane (1240 kg). After 30 minutes p-toluenesulfonyl chloride (174.8 kg, 916.9 mol) was added in portions at 20-40 minute intervals and the reaction mixture was stirred at 15-25° C. for 16 hours and 20 minutes. Purified water (190 kg) was added and after stirring, the organic layer was washed with an aqueous sodium bicarbonate solution (prepared from 53.8 kg sodium bicarbonate and 622 kg purified water) and then washed with an aqueous ammonium chloride solution (prepared from 230 ammonium chloride and 624 kg purified water). After a final wash with purified water (311 kg), the organic layer was filtered through a steel suction filter preloaded with silica gel (60.2 kg). The filter cake was soaked in dichloromethane (311 kg) for 20 min and then

- 45 040816 filtered. The combined filtrates were concentrated under reduced pressure (<-0.05 MPa) and <40° C. until 330-400 L remained. Then tetrahydrofuran (311 kg) was added at 15-30° C. and the mixture was concentrated in the same manner to a final volume of 330-400 L. The addition of tetrahydrofuran and concentration was repeated again to a volume of 330-400 L to give a light yellow solution of C27 (167.6 kg, 692 mmol, 98%) in tetrahydrofuran (251.8 kg).

1H NMR (400 MHz, chloroform-d), C27 peaks only: δ 7.81 (d, J=8.4 Hz, 2H), 7.34 (d, J=8.1 Hz, 2H), 4.91 (ddt, J= 8.0, 6.7, 3.9 Hz, 1H), 4.62-4.55 (m, 1H), 4.53-4.45 (m, 1H), 4.14 (d, J=3.9 Hz, 2H), 2.75-2.63 (m, 1H), 2.60-2.49 (m, 1H), 2.44 (s, 3H).

Stage 4. Synthesis of (2S)-2-(azidomethyl)oxetane (С28).

K,K-Dimethylformamide (473 kg), sodium azide (34.7 kg, 534 mol) and potassium iodide (5.2 kg, 31 mol) were combined in a 3000 L glass lined reactor at 10-25°C. After adding C27 (83.5 kg, 344.6 mol) in tetrahydrofuran (125.4 kg), the reaction mixture was heated to 55-65°C for 17 hours and 40 minutes, after which it was cooled to 25-35°C, and nitrogen was bubbled from the bottom valve for 15 minutes. Then tert-butyl methyl ether (623 kg) and purified water (840 kg) were added and the resulting aqueous layer was extracted twice with tert-butyl methyl ether (312 kg and 294 kg). The combined organic layers were washed with purified water (2x419 kg) while maintaining the temperature at 10-25°C to give C28 (31.2 kg, 276 mol, 80%) in a solution of the above organic layer (1236.8 kg).

Step 5. Synthesis of 1-[(2S)-oxetan-2-yl]methanamine (C29).

10% palladium on carbon (3.7 kg) was added via a hopper to a 10-30°C solution of C28 [from the previous step; 1264 kg (31.1 kg C28, 275 mol)] in tetrahydrofuran (328 kg) in a 3000 L steel autoclave reactor. The feed funnel was washed with tetrahydrofuran (32 kg) and the washing liquid was added to the reaction mixture. After purging the contents of the reactor with nitrogen, it was similarly purged with hydrogen, increasing the pressure from 0.05 to 0.15 MPa and then ventilating to 0.03-0.04 MPa. This hydrogen purge was repeated 5 times, after which the hydrogen pressure was increased to 0.05-0.07 MPa. The reaction temperature was increased to 25-33°C, and the hydrogen pressure was maintained at 0.05-0.15 MPa for 22 h, replacing hydrogen every 3-5 h. The mixture was then purged five times with nitrogen, increasing the pressure to 0.15-0. .2 MPa and then ventilating to 0.05 MPa. After filtration, tetrahydrofuran (92 kg and 93 kg) was used to wash the reactor and then the filter cake was impregnated. The combined filtrates were concentrated under reduced pressure (<-0.07 MPa) and <45° C. to give C29 (18.0 kg, 207 mol, 75%) in tetrahydrofuran (57.8 kg).

¹ H NMR (400 MHz, DMSO-d6), C29 peaks only: δ 4.62 (ddt, J=7.6, 6.6, 5.1 Hz, 1H), 4.49 (ddd, J=8.6, 7.3, 5.6 Hz, 1H), 4.37 (dt, J=9.1, 5.9 Hz, 1H), 2.69 (d, J=5.1 Hz, 2H), 2.55-2.49 (m, 1H), 2.39 (m, 1H).

Stage 6. Synthesis of methyl 4-nitro-3-{[(2S)-oxetan-2-ylmethyl]amino}benzoate (С30).

Potassium carbonate (58.1 kg, 420 mol) was added to a solution of methyl 3-fluoro-4-nitrobenzoate (54.8 kg, 275 mol) in tetrahydrofuran (148 kg) in a 100 L glass lined reactor and the mixture was stirred in within 10 min. A solution of C29 (29.3 kg, 336 mol) in tetrahydrofuran (212.9 kg) was added and the reaction mixture was stirred at 20-30°C for 12 h, after which ethyl acetate (151 kg) was added and the mixture was filtered through silica gel (29 kg). The filter cake was washed with ethyl acetate (150 kg and 151 kg) and the combined filtrates were concentrated under reduced pressure (<-0.08 MPa) and <45° C. to a volume of 222-281 L. After cooling the mixture to 10-30°C, n-heptane (189 kg) was added, stirring was carried out for 20 min, and the mixture was concentrated under reduced pressure (<-0.08 MPa) and <45°C to a volume of 222 L. Again n-heptane (181 kg) was added to the mixture at a standard rate of 100-300 kg per hour and stirring was continued for 20 minutes. Mixture samples were analyzed until residual tetrahydrofuran was <5% and residual ethyl acetate was 10%-13%. The mixture was heated to 4045°C and stirred for 1 hour, after which it was cooled to 15-25°C at a rate of 5-10°C per hour and then stirred at 15-25°C for 1 hour. After filtration using steel centrifuge, a filter cake was obtained, which was washed with a mixture of ethyl acetate (5.0 kg) and n-heptane (34 kg) and then stirred with tetrahydrofuran (724 kg) at 10-30° C. for 15 minutes. Filtration gave a yellow solid consisting mainly of C30 (57.3 kg, 210 mol, 76%).

1H NMR (400 MHz, DMSO-d6) 8.34 (t, J=5.8 Hz, 1H), 8.14 (d, J=8.9 Hz, 1H), 7.63 (d, J=1.7 Hz, 1H ), 7.13 (dd, J=8.9, 1.8Hz, 1H), 4.99 (dddd, J=7.7, 6.7, 5.3, 4.1Hz, 1H), 4.55 (ddd, J=8.6, 7.3, 5, 8 Hz, 1H), 4.43 (dt, J=9.1, 6.0 Hz, 1H), 3.87 (s, 3H), 3.67-3.61 (m, 2H), 2.67 (dddd, J=11.1, 8.6, 7.7, 6.2 Hz, 1H), 2.57-2.47 (m, 1H).

Step 7. Synthesis of methyl 2-(chloromethyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6carboxylate (P15).

A solution of C30 (from the previous step; 51.8 kg, 190 mol) in tetrahydrofuran (678 kg) in a 3000 L autoclave reactor was treated with 10% palladium on carbon (5.2 kg) at 10-30°C. The loading tube was washed with tetrahydrofuran (46 kg) and the washing liquid was added to the reaction mixture. After purging the contents of the reactor with nitrogen, it was similarly purged with hydrogen, increasing the pressure to 0.1–0.2 MPa and then dropping it to 0.02–0.05 MPa. This hydrogen purge was repeated 5 times, after which the hydrogen pressure was increased to 0.1-0.25 MPa. The reaction mixture was stirred at 20-30°C and

- 46 040816 every 2-3 hours the mixture was purged with nitrogen three times and then purged with hydrogen five times; after each final replacement of hydrogen, the hydrogen pressure was increased to 0.1-0.25 MPa. After 11.25 hours of reaction time, the reaction mixture was vented to normal pressure and purged five times with nitrogen, increasing the pressure to 0.15-0.2 MPa and then dropping to 0.05 MPa. The mixture was then filtered and the filter cake washed twice with tetrahydrofuran (64 kg and 63 kg). The combined washing liquid and filtrate were concentrated under reduced pressure (<-0.08 MPa) and <40° C. to a volume of 128-160 L. Tetrahydrofuran (169 kg) was added and the mixture was again concentrated to a volume of 128-160 L; this process was repeated a total of 4 times to give a solution of the intermediate methyl 4-amino-3-{[(2S)oxetan-2-ylmethyl]amino}benzoate.

To this solution was added tetrahydrofuran (150 kg), followed by 2-chloro-1,1,1-trimethoxyethane (35.1 kg, 227 mol) and p-toluenesulfonic acid monohydrate (1.8 kg, 9.5 mol). After stirring the reaction mixture for 25 minutes, it was heated at 40-45°C for 5 hours, after which it was concentrated under reduced pressure to a volume of 135-181 L. 2-Propanol (142 kg) was added and the mixture was again concentrated to a volume of 135-181 L, after which 2-propanol (36.5 kg) and purified water (90 kg) were added and stirring was continued until a solution was obtained. This solution was filtered using an inline liquid filter and then treated with purified water (447 kg) at a standard speed of 150-400 kg per hour at 20-40°C. After the mixture was cooled to 20-30° C., it was stirred for 2 hours and the solid was collected by filtration using a centrifuge. The filter cake was washed with a solution of 2-propanol (20.5 kg) and purified water (154 kg); after drying, P15 was obtained as a white solid (32.1 kg, 109 mol, 57%).

¹ H NMR (400 MHz, chloroform-d) δ 8.14-8.11 (m, 1H), 8.01 (dd, J=8.5, 1.1 Hz, 1H), 7.79 (br d, J=8.6 Hz, 1H ), 5.26-5.18 (m, 1H), 5.04 (s, 2H), 4.66-4.58 (m, 2H), 4.53 (dd, component of the ABX system, J=15.7, 2.7 Hz, 1H), 4.34 (dt , J=9.1, 6.0 Hz, 1H), 3.96 (s, 3H), 2.82-2.71 (m, 1H), 2.48-2.37 (m, 1H).

Getting R16. Methyl 2-(chloromethyl)-1-methyl-1H-benzimidazole-6-carboxylate (Р16)

Methyl 4-amino-3-(methylamino)benzoate (206 mg, 1.14 mmol) was dissolved in 1,4-dioxane (11.5 ml) and treated with chloroacetyl chloride (109 μl, 1.37 mmol). The mixture was stirred at 100° C. for 3 hours and cooled to room temperature. Triethylamine (0.8 ml, 7 mmol) and heptane (10 ml) were added and filtered. The filtrate was concentrated under reduced pressure and the crude material was purified by silica gel chromatography (eluent: 40% ethyl acetate in heptane) to give 120 mg of P16 (44%).

1Н NMR (400 MHz, chloroform-d) δ 8.14 (s, 1H), 8.01 (d, 1H), 7.78 (d, 1H), 4.87 (s, 2H), 3.97 (s, 3Н), 3.94 (s, 3H); LC-MS m/z 239.1 [M+H]+.

Getting P17 and P18. Methyl 2-(6-azaspiro[2.5]oct-1-yl)-1-(2-methoxyethyl)-1H-benzimidazole-6carboxylate, ENT-1 (P17) and methyl 2-(6-azaspiro[2.5] oct-1-yl)-1-(2-methoxyethyl)-1H-benzimidazole-6carboxylate, ENT-2 (P18)

Step 1. Synthesis of tert-butyl 4-(2-ethoxy-2-oxoethylidene)piperidine-1-carboxylate (C31).

- 47 040816

A solution of potassium tert-butoxide (65.9 g, 587 mmol) in tetrahydrofuran (500 ml) was added to a 0° C. solution of ethyl (diethoxyphosphoryl)acetate (132 g, 589 mmol) in tetrahydrofuran (500 ml) and the resulting the suspension was stirred at 0°C for 1 h, after which it was cooled to 50°C. A solution of tert-butyl 4-oxopiperidine-1-carboxylate (90.0 g, 452 mmol) in tetrahydrofuran (1.5 L) was added dropwise at −50° C. and the reaction mixture was then allowed to warm slowly to 20° C. and then stirred for 16 h at 20°C. After water (1 L) was added, the mixture was concentrated in vacuo to remove the tetrahydrofuran. The aqueous residue was extracted with ethyl acetate (2x800 ml) and the combined organic layers were washed with saturated aqueous sodium chloride (500 ml), dried over sodium sulfate, filtered and concentrated under reduced pressure. The resulting material was washed several times with petroleum ether (200 ml) to give C31 as a white solid. Yield: 95.0 g, 353 mmol, 78%.

¹ H NMR (400 MHz, chloroform-d) δ 5.71 (s, 1H), 4.16 (q, J=7.2 Hz, 2H), 3.55-3.43 (m, 4H), 2.94 (br t, J=5 .5 Hz, 2H), 2.28 (br t, J=5.5 Hz, 2H), 1.47 (s, 9H), 1.28 (t, J=7.0 Hz, 3H).

Stage 2. Synthesis of 6-tert-butyl-1-ethyl-6-azaspiro [2.5]octane-1,6-dicarboxylate (С32).

To a solution of trimethylsulfoxonium iodide (140 g, 636 mmol) in dimethyl sulfoxide (800 ml) was added potassium tert-butoxide (71.2 g, 634 mmol) in one portion at 20°C. After the reaction mixture was stirred at 20° C. for 1.5 h, a solution of C31 (95.0 g, 353 mmol) in dimethyl sulfoxide (800 ml) was added dropwise, and stirring was continued at 20° C. for 16 h. aqueous sodium chloride solution (2.0 l). The resulting mixture was neutralized by adding ammonium chloride and extracted with ethyl acetate (3.0 L). The combined organic layers were washed successively with water (2x1.0 L) and saturated aqueous sodium chloride (2.0 L), dried over sodium sulfate, filtered and concentrated in vacuo. Purification by chromatography on silica gel (eluent: 10:1 petroleum ether/ethyl acetate) gave C32 as a yellow oil. 1H NMR analysis indicated that a more recent aliphatic was present. Yield: 80 g, 280 mmol, 79%.

1H NMR (400 MHz, chloroform-d), C32 peaks only: δ 4.19-4.09 (m, 2H), 3.55-3.39 (m, 3H), 3.27 (ddd, J=13.0, 7.0, 4.5 Hz, 1H ), 1.76-1.64 (m, 2H), 1.56 (dd, J=8.0, 5.5 Hz, 1H, inferred; partially obscured by water peak), 1.47 (s, 9H), 1.47-1.37 (m, 2H), 1.27 (t, J=7.0 Hz, 3H), 1.17 (dd, J=5.0, 5.0 Hz, 1H), 0.93 (dd, J=8.0, 4.5 Hz, 1H).

Stage 3. Synthesis of 6-(tert-butoxycarbonyl)-6-azaspiro[2.5]octane-1-carboxylic acid (С33).

To a mixture of C32 (80 g, 280 mmol) in tetrahydrofuran (500 ml) and water (500 ml) was added lithium hydroxide monohydrate (37.4 g, 891 mmol) in one portion. This reaction mixture was stirred at 25°C for 16 h, after which it was diluted with water (600 ml) and washed with ethyl acetate (3x300 ml). The organic layers were discarded and the aqueous layer was acidified to pH 3-4 with 6 M hydrochloric acid. The resulting mixture was extracted with ethyl acetate (3x600 ml) and the combined organic layers were dried over sodium sulfate, filtered and concentrated in vacuo. Trituration of the residue with petroleum ether (300 ml) gave C33 as a white solid. Yield: 42.0 g, 164 mmol, 59%. LC-MS m/z 278.2 [M+Na+].

1H NMR (400 MHz, DMSO-d ₆ ) δ 12.15-12.03 (br s, 1H), 3.43-3.25 (m, 3H, hypothetical; partially obscured by water peak), 3.23-3.12 (m, 1H), 1.64-1.50 (m, 2H), 1.52 (dd, J=7.5, 5.5 Hz, 1H), 1.39 (s, 9H), 1.39-1.28 (m, 2H), 0.96-0.88 (m, 2H).

Step 4. Synthesis of tert-butyl 1-({4-(methoxycarbonyl)-2-[(2-methoxyethyl)amino]phenyl}carbamoyl)-6-azaspiro[2.5]octane-6-carboxylate (С34).

A solution of C33 (570 mg, 2.23 mmol), C16 (500 mg, 2.23 mmol) and O-(1-azabenzotriazol-1-yl)-N,N,N',N'tetramethyluronium hexafluorophosphate (HATU; 1 .27 g, 3.34 mmol) in N,N-dimethylformamide (10 ml) was stirred at 30°C for 30 min, after which triethylamine (902 mg, 8.91 mmol) was added and stirring was continued at 30°C within 16 hours the Reaction mixture was then poured into water (60 ml) and was extracted with ethyl acetate (3x50 ml). The combined organic layers were washed with saturated aqueous sodium chloride (3x50 ml), dried over sodium sulfate, filtered and concentrated in vacuo. Chromatography on silica gel (eluent: 1:1 petroleum ether/ethyl acetate) gave C34 as a brown oil, which was taken directly to the next step.

Step 5. Synthesis of methyl 2-[6-(tert-butoxycarbonyl)-6-azaspiro[2.5]oct-1-yl]-1-(2-methoxyethyl)1 H-benzimidazole-6-carboxylate (С35).

A solution of C34 (from the previous step, <2.23 mmol) in acetic acid (15 ml) was stirred at 50° C. for 16 h after which it was concentrated in vacuo to give C35 as a brown oil. This material was directly used in the next step. LC-MS m/z 444.1 [M+H] ⁺ .

Step 6. Synthesis of methyl 2-(6-azaspiro[2.5]oct-1-yl)-1-(2-methoxyethyl)-1H-benzimidazole-6carboxylate (С36).

Trifluoroacetic acid (5 ml) was added to a solution of C35 (from the previous step; <2.23 mmol) in dichloromethane (10 ml) and the reaction mixture was stirred at 25° C. for 2 h. After the solvents were removed in vacuo, the residue was made basic by adding saturated aqueous carbonate solution

- 48 040816 potassium (40 ml) and was extracted with a mixture of dichloromethane and methanol (10:1, 3x40 ml). The combined organic layers were dried over magnesium sulfate, filtered, concentrated in vacuo and chromatographed on silica gel (eluent: 10:1:0.1 dichloromethane/methanol/concentrated ammonium hydroxide) to give C36 as a yellow solid. Yield: 640 mg, 1.86 mmol, 83% over three steps.

LC-MS m/z 344.1 [M+H]+.

Step 7. Isolation of methyl 2-(6-azaspiro[2.5]oct-1-yl)-1-(2-methoxyethyl)-1H-benzimidazole-6carboxylate ENT-1 (P17) and methyl 2-(6-azaspiro [2.5]oct-1-yl)-1-(2-methoxyethyl)-1H-benzimidazole-6carboxylate ENT-2 (P18).

Separation of C36 (630 mg, 1.83 mmol) into its constituent enantiomers was performed using SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 55:45 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting peak was designated ENT-1 (P17) and the second eluting enantiomer was designated ENT-2 (P18); both were isolated as a pale yellow solid.

P17 Yield: 300 mg, 0.874 mmol, 48%. LC-MS m/z 344.1 [M+H]+. Retention time: 5.10 min (column: Chiral Technologies Chiralpak AD-3, 4.6x150 mm, 3 µm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5 to 40% B for 5.5 minutes, then hold at 40% B for 3.0 minutes; flow rate: 2.5 ml per minute).

P18 Yield: 240 mg, 0.699 mmol, 38%. LC-MS m/z 344.1 [M+H]+. Retention time: 7.35 min (analytical conditions identical to those used for P17).

Getting R19. Methyl 4-amino-3-{[(1-ethyl-1H-imidazol-5-yl)methyl]amino}benzoate (P19) o

C37 R19

Stage 1. Synthesis of methyl 3-{[(1-ethyl-1H-imidazol-5-yl)methyl]amino}-4-nitrobenzoate (С37).

Triethylamine (3.65 ml, 26.2 mmol) was added to a solution of methyl 3-fluoro-4-nitrobenzoate (1.00 g, 5.02 mmol) and 1-(1-ethyl-III-imidazol-5-yl )methanamine dihydrochloride salt (1.00 g, 5.05 mmol) in a mixture of tetrahydrofuran (12 ml) and methanol (8 ml). The reaction mixture was stirred at 60° C. for 40 h after which it was concentrated in vacuo and purified by silica gel chromatography (gradient: 0 to 2% methanol in dichloromethane) to give C37 as an orange solid. Yield: 1.27 g, 4.17 mmol, 83%.

¹ H NMR (400 MHz, chloroform-d) δ 8.24 (d, J=8.8 Hz, 1H), 7.98-7.91 (m, 1H), 7.68 (d, J=1.7 Hz, 1H), 7.57 (br s, 1H), 7.33 (dd, J=8.8, 1.7 Hz, 1H), 7.11 (br s, 1H), 4.53 (d, J=4.9 Hz, 2H), 3.99 (q, J =7.3 Hz, 2H), 3.95 (s, 3H), 1.47 (t, J=7.3 Hz, 3H).

Stage 2. Synthesis of methyl 4-amino-3-{[(1-ethyl-1H-imidazol-5-yl)methyl]amino}benzoate (P19).

A mixture of wet palladium on carbon (144 mg) and C37 (412 mg, 1.35 mmol) in methanol (13 mL) was stirred under balloon hydrogen for 16 hours at 25°C. The reaction mixture was then filtered through a pad of diatomaceous earth and the filtrate was concentrated in vacuo to give P19 as a gray solid. Yield: 340 mg, 1.24 mmol, 92%.

1H NMR (400 MHz, methanol-d ₄ ) δ 7.66 (br s, 1H), 7.38-7.29 (m, 2H), 6.97 (br s, 1H), 6.67 (d, J=7.9 Hz, 1H) , 4.35 (s, 2H), 4.11 (q, J=7.3 Hz, 2H), 3.81 (s, 3H), 1.44 (t, J=7.3 Hz, 3H).

Getting P20. Methyl 4-amino-3-(methylamino)benzoate (Р20)

D1 P20

Stage 1. Synthesis of methyl 3-(methylamino)-4-nitrobenzoate (D1).

To a solution of methyl 3-fluoro-4-nitrobenzoate (5.10 g, 25.6 mmol) in tetrahydrofuran (60 ml) was added methylamine (38.4 ml, 76.8 mmol, 2 M in tetrahydrofuran) dropwise over 10 min. Immediately after the addition, the pale yellow solution turned into a rich orange and was stirred for 2 hours at room temperature. The reaction mixture was then diluted with diethyl ether (100 ml) and the organic layer was washed successively with water (50 ml) and saturated aqueous sodium chloride (50 ml). The organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give 5.26 g of methyl 3-(methylamino)-4-nitrobenzoate (98%) as a deep orange solid. LC-MS m/z 211.1 [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 8.22 (d, J=8.9 Hz, 1H), 8.00 (br s, 1H), 7.56 (d, J=1.7 Hz, 1H), 7.25 (dd , J=8.9, 1.7 Hz, 1H, hypothesized; partially obscured by the solvent peak), 3.95 (s, 3H), 3.09 (d, J=5.1 Hz, 3H).

Stage 2. Synthesis of methyl 4-amino-3-(methylamino)benzoate (P20).

- 49 040816

A solution of D1 (5.26 g, 25.0 mmol) in ethanol (150 mL) was added to a 500 mL Parr® vessel preloaded with 10% palladium on carbon (50% water; 1 g ). The mixture was shaken under a 50 psi hydrogen atmosphere for 1 hour at room temperature, after which it was filtered and the filter cake was washed with ethanol (100 ml). The filtrate was concentrated under reduced pressure to give 4.38 g of P20 (97%) as an off-white solid. LC-MS m/z 181.1 [M+H]+.

¹ H NMR (400 MHz, chloroform-d) δ 7.46 (dd, J=8.0, 1.9 Hz, 1H), 7.34 (d, J=1.8 Hz, 1H), 6.68 (d, J=8, 0 Hz, 1H), 3.87 (s, 3H), 3.72 (br s, 2H), 3.21 (br s, 1H), 2.91 (s, 3H).

Getting P21 and P22. 5-Bromo-N ³ -methylpyridine-2,3-diamine (Р21) and 5-Bromo-N ³ ,6-dimethylpyridine2,3-diamine (Р22) n n n ₃ u ii η n ₃ b Tj d η ₂ ν ^v^ h ₂ n^n^ch ₃

P21 P22

Intermediate P21 was synthesized according to the procedure described in the literature (Choi, J. Y. et al. J. Med. Chem. 2012, 55, 852-870). Intermediate P22 was synthesized using the same method.

Getting R23. Methyl 2-(chloromethyl)-[(1-methyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6carboxylate (P23)

D4 P23

Stage 1. Synthesis of methyl 3-{[(1-methyl-1H-imidazol-5-yl)methyl]amino}-4-nitrobenzoate (D2).

To a colorless solution of methyl 3-fluoro-4-nitrobenzoate (1.0 g, 5.0 mmol) in N,N-dimethylformamide (10 mL) was slowly added 1-(1-methyl-1H-imidazol-5-yl) methanamine (670 mg, 6.0 mmol) and triethylamine (762 mg, 7.53 mmol). This reaction mixture was stirred at 60°C for 16 h, after which it was poured into water (30 ml) and extracted with dichloromethane (3x30 ml). The combined organic layers were dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (eluent: 20% methanol in dichloromethane). The resulting yellow solid was triturated with 30:1 petroleum ether/ethyl acetate to give D2 (1.2 g, 82%) as a yellow solid. LC-MS m/z 290.9 [M+H] ⁺ .

1H NMR (400 MHz, chloroform-d) δ 8.25 (d, J=8.9 Hz, 1H), 7.98-7.92 (m, 1H), 7.70 (d, J=1.7 Hz, 1H), 7.49 ( s, 1H), 7.34 (dd, J=8.9, 1.7 Hz, 1H), 7.12 (s, 1H), 4.54 (d, J=5.0 Hz, 2H), 3.96 (s, 3H), 3.67 (s, 3H).

Stage 2. Synthesis of methyl 4-amino-3-{[(1-methyl-1H-imidazol-5-yl)methyl]amino}benzoate (D3).

To a suspension of D2 (5.46 g, 18.8 mmol) in methanol (160 ml) was added wet 10% palladium on carbon (1 g). This mixture was stirred in a hydrogen atmosphere of 1 atm for 36 h at 20°C. The reaction mixture was filtered and the filter cake was washed with methanol (200 ml). The filtrate was concentrated under reduced pressure to give D3 (4.8 g, 98%) as a brown solid. LCMS m/z 260.9 [M+H] ⁺ .

1H NMR (400 MHz, DMSC-d6) δ 7.56 (s, 1H), 7.18 (br d, J=8.1 Hz, 1H), 7.12 (brs, 1H), 6.87 (s, 1H), 6.55 (d , J=8.2 Hz, 1H), 5.50 (s, 2H), 4.84 (t, J=5.2 Hz, 1H), 4.23 (d, J=5.0 Hz, 2H), 3.73 (s, 3Н), 3.63 (s, 3Н).

Step 3. Synthesis of methyl 2-(hydroxymethyl)-1-[(1-methyl-1H-imidαzol-5-yl)methyl]-1H-benzimidazole-6-carboxylate (D4).

A mixture of D3 (780 mg, 3.00 mmol) and 2-hydroxyacetic acid (342 mg, 4.49 mmol) in 1,3,5trimethylbenzene (8 mL) was stirred at 140°C for 14 h and at 25°C for over 48 hours. The clear yellow solution was decanted to give a brown residue, which was dissolved in methanol (50 ml) and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (eluent: 20% methanol in dichloromethane) to give D4 (318 mg, 35%) as a yellow foam. LC-MS m/z 300.9 [M+H] ⁺ .

1H NMR (400 MHz, DMSC-d6) δ 8.13-8.11 (m, 1H), 7.83 (dd, J=8.4, 1.6 Hz, 1H), 7.71 (d, J=8.5 Hz, 1H), 7.59 (s, 1H), 6.58 (s, 1H), 5.69 (s, 2H), 4.75 (s, 2H), 3.84 (s, 3H), 3.53 (s, 3H).

Stage 4. Synthesis of methyl-2-(chloromethyl)-1-[(1-methyl-1H-imidazol-5-yl)methyl]-1H-benzimidazol-6

- 50 040816 carboxylate (P23).

To a suspension of D4 (500 mg, 1.66 mmol) in dichloromethane (10 ml) and N,N-dimethylformamide (3 ml) was added thionyl chloride (990 mg, 0.60 ml, 8.32 mmol) dropwise at room temperature. This reaction mixture was stirred at room temperature for 1 hour, then concentrated under reduced pressure. The resulting brown residue was triturated with dichloromethane (10 ml). The solid was collected by filtration and washed with dichloromethane (5 mL) to give P23 (431 mg, 73%) as an off-white solid. LC-MS m/z 318.9^ [M+H]+.

¹ H NMR (400 MHz, DMSO-d6) δ 9.17 (s, 1H), 8.31 (s, 1H), 7.93 (br d, J=8.5 Hz, 1H), 7.82 (d, J=8.5 Hz, 1H), 7.11 (s, 1H), 5.92 (s, 2H), 5.13 (s, 2H), 3.87 (s, 3H), 3.87 (s, 3H).

Getting P24. 5-Chloro-2-(chloromethyl)-3-methyl-3H-imidazo[4,5-b]pyridine (Р24)

Stage 1. Synthesis of 6-chloro-N-methyl-3-nitropyridine-2-amine (D5).

A solution of methylamine _in tetrahydrofuran ( _2.0 M ; 622 ml, 1.24 mol) dropwise at 0°C with a syringe. After the addition was complete, the reaction mixture was stirred at 18° C. for 6 hours. The mixture was filtered and the filtrate was concentrated under reduced pressure to give a yellow solid. This crude material was purified by silica gel chromatography (gradient: 0 to 5% ethyl acetate in petroleum ether) to give D5 (158 g, 81% yield) as a yellow solid.

1H NMR (400 MHz, DMSO-d6) δ 8.72 (br s, 1H), 8.41 (d, J=8.6 Hz, 1H), 6.76 (d, J=8.6 Hz, 1H), 3.00 (d , J=4.8 Hz, 3H).

Stage 2. Synthesis of 6-χλορ-Ν -methylpyridine-2,3-diamine (D6).

To a mixture of D5 (15.8 g, 84.2 mmol) in acetic acid (100 ml) was added iron powder (15.4 g, 276 mmol). This reaction mixture was stirred at 80°C for 3 h, after which it was cooled to room temperature and filtered. The filter cake was washed with ethyl acetate (2x100). The combined organic layers were concentrated under reduced pressure and the crude material was purified by silica gel chromatography (eluent: 1:1 ethyl acetate/petroleum ether) to give D6 (8.40 g, 63% yield) as a brown solid.

1H NMR (400 MHz, chloroform-d) δ 6.79 (d, J=1.1 Hz, 1H), 6.49 (d, J=7.7 Hz, 1H), 3.00 (s, 3H).

Stage 3. Synthesis of 5-chloro-2-(chloromethyl)-3-methyl-3H-imidazo[4,5-b]pyridine (P24).

To a solution of D6 (50.0 g, 317 mmol) in 1,4-dioxane (1.2 L) was added chloroacetyl chloride (55.5 ml, 698 mmol) and the reaction mixture was stirred at 15°C for 50 min. It was then concentrated under reduced pressure to give a brown solid which was taken up in trifluoroacetic acid (1.2 L) and stirred at 80° C. for 60 h. The mixture was concentrated under reduced pressure to give a brown oil which was diluted with ethyl acetate (1 L ) and neutralized by adding saturated aqueous sodium bicarbonate. When the evolution of carbon dioxide subsided, the layers were separated and the aqueous layer was extracted with ethyl acetate (200 ml). The organic extracts were combined, dried over sodium sulfate, filtered and concentrated under reduced pressure. The crude material was purified by silica gel chromatography (gradient: 10% to 25% ethyl acetate in petroleum ether) to give P24 (61.0 g, 79% yield) as a yellow solid. LC-MS m/z 215.7 (dichloroisotope pattern observed) [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) δ 8.13 (d, J=8.3 Hz, 1H), 7.37 (d, J=8.4 Hz, 1H), 5.11 (s, 2H), 3.84 (s , 3H).

Examples 1 and 2.

2-({4-[2-(4-Chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1-yl}methyl)-1-(2-methoxyethyl)1H-benzimidazol-6 -carboxylic acid ENT-X1 trifluoroacetate salt (1) [from C39]; and 2-({4-[2-(4-chloro2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1-yl}methyl)-1-(2-methoxyethyl)-1 H-benzimidazol- 6carboxylic acid ENT-X2 trifluoroacetate salt (2) [from C40]

- 51 040816

Stage 1. Synthesis of methyl-2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1yl}methyl)-1 -(2-methoxyethyl)- 1 H-benzimidazole-6-carboxylate (C3 8).

This experiment was carried out in two batches of identical scale. Reaction vessel containing a mixture of C2 (500 mg, 1.52 mmol), P12 (530 mg, 1.59 mmol), [2',6'-bis(propan-2-yloxy)biphenyl-2yl](dicyclohexyl)phosphane (Ruphos; 142 mg, 0.304 mmol), tris(dibenzylideneacetone)dipalladium(0) (139 mg, 0.152 mmol) and cesium carbonate (1.48 g, 4.54 mmol) in toluene (15 ml) were evacuated and filled with nitrogen. This evacuation cycle was repeated twice, after which the reaction mixture was stirred at

100°C for 16 h, combined with the second batch and filtered. The filtrate was concentrated and the residue was subjected to silica gel chromatography (gradient: 0 to 60% ethyl acetate in petroleum ether), then preparative thin layer chromatography (eluent: 1:1 petroleum ether/ethyl acetate) to give C38 as a pale yellow solid. Overall yield: 600 mg, 1.03 mmol, 34%. LC-MS m/z 581.0 ♦ [M+H]+.

Step 2. Isolation of methyl-2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1yl}methyl)-1-(2-methoxyethyl)- 1H-benzimidazole-6-carboxylate ENT-1 (C39) and methyl-2-({4-[2-(4-chloro-2fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1 -yl}methyl )-1 -(2-methoxyethyl)-1 H-benzimidazole-6carboxylate ENT-2 (C40).

Separation of C38 (780 mg, 1.34 mmol) into its constituent enantiomers was performed using SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 3:2 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The enantiomer that eluted first, designated ENT1 (C39), was obtained as a white solid. Yield: 282 mg, 0.485 mmol, 36%. LC-MS m/z 581.0^ [M+H]+. Retention time 1.90 min (column: Chiral Technologies Chiralpak AD-3, 4.6x50 mm, 3 µm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5% B in for 0.20 min, then 5 to 40% B over 1.4 min, then hold at 40% B for 1.05 min; flow rate: 4.0 ml per minute).

The second eluting enantiomer, designated ENT-2 (C40), was subjected to a second purification using SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 3:2 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. This gave C40 as a pale brown solid. Yield: 280 mg, 0.482 mmol, 36%. LC-MS m/z 581.0^ [M+H]+. Retention time 2.18 min (analytical conditions identical to those used for C39).

Stage 3. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1-yl}methyl)-1(2-methoxyethyl)-1H -benzimidazole-6-carboxylic acid ENT-X1 trifluoroacetate salt (1) [from C39].

An aqueous solution of lithium hydroxide (2 M; 0.30 ml, 0.60 mmol) was added to a solution of C39 (70 mg, 0.12 mmol) in a mixture of methanol (3 ml) and tetrahydrofuran (3 ml). After the reaction mixture was stirred at 25° C. for 16 h, an aqueous solution of lithium hydroxide (2 M; 0.30 ml, 0.60 mmol) was added again, and

- 52 040816 stirring was continued for another 20 hours the Reaction mixture was then brought to pH 7 by adding 1 M hydrochloric acid and then concentrated in vacuo to remove methanol and tetrahydrofuran. The residue was adjusted to pH 5-6 with trifluoroacetic acid and then purified by reverse phase HPLC (column: Agela Durashell C18, 5 µm; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: 30 to 60 % B) to give 1 as a white solid. Yield: 40.5 mg, 59.5 µmol, 50%. LC-MS m/z 567.0^ [M+H]+.

1H NMR (400 MHz, methanol-d ₄ ) δ 8.37 (br s, 1H), 8.07 (dd, J=8.5, 1.5 Hz, 1H), 7.79 (d, J=8.6 Hz, 1H), 7.59 (dd, J=8.0, 8.0Hz, 1H), 7.34 (dd, J=10.2, 2.0Hz, 1H), 7.30 (br dd, J=8.3, 2.0Hz, 1H), 7.22 (s, 1H), 6.87 (dd, J=8.1, 8.1 Hz, 1H), 6.63 (brd, J=8 Hz, 1H), 6.60 (br d, J=8 Hz, 1H), 4.70 (s , 2H), 4.65 (t, J=4.8 Hz, 2H), 3.75 (t, J=4.8 Hz, 2H), 3.59-3.42 (m, 8H), 3.29 (s, 3H).

Step 4. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperazin-1-yl}methyl)-1(2-methoxyethyl)-1H -benzimidαzol-6-carboxylic acid ENT-X2 trifluoroacetate salt (2) [from C40].

An aqueous solution of lithium hydroxide (2 M; 0.30 ml, 0.60 mmol) was added to a solution of C40 (69 mg, 0.12 mmol) in a mixture of methanol (3 ml) and tetrahydrofuran (3 ml). After stirring the reaction mixture at 25° C. for 16 h, an aqueous solution of lithium hydroxide (2 M; 0.30 ml, 0.60 mmol) was again added and stirring was continued for another 20 h. The reaction mixture was adjusted to pH 7 by adding 1 M hydrochloric acid and then concentrated in vacuo to remove methanol and tetrahydrofuran. The residue was adjusted to pH 5-6 with trifluoroacetic acid and then purified by reverse phase HPLC (column: Agela Durashell C18, 5 µm; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: from 30 to 60% B) to give 2 as a white solid. Yield: 22.9 mg, 33.6 µmol, 28%. LC-MS m/z 567.0^ [M+H] ⁺ .

'and NMR (400 MHz, methanol) δ 8.40-8.35 (m, 1H), 8.07 (dd, J=8.6, 1.5 Hz, 1H), 7.79 (d, J=8.6 Hz, 1H), 7.59 (dd, J=8.0, 8.0 Hz, 1H), 7.35 (dd, J=10.2, 2.0 Hz, 1H), 7.31 (br dd, J=8, 2 Hz, 1H), 7.22 (s, 1H), 6.87 (dd, J=8.3, 8.0 Hz, 1H), 6.63 (br d, J=8 Hz, 1H), 6.60 (br d, J=8 Hz, 1H), 4.68 (s, 2H ), 4.65 (t, J=4.9 Hz, 2H), 3.76 (t, J=4.8 Hz, 2H), 3.57-3.40 (m, 8H), 3.29 (s, 3H).

Example 3

2-({4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(2methoxyethyl)-1H- imidazo[4,5-b]pyridine-6-carboxylic acid trifluoroacetate salt (3)

Step 1. Synthesis of 4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine (C13, free base).

To a solution of P2 (300 mg, 0.670 mmol) in ethyl acetate (3.5 ml) was added para-toluenesulfonic acid monohydrate (318 mg, 1.67 mmol). The reaction mixture was stirred at 60° C. for 1 h, after which it was made basic by adding saturated aqueous potassium carbonate (20 ml) and extracted with a mixture of dichloromethane and methanol (10:1, 3x50 ml). The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo to give the C13 free base as a brown solid. Yield: 230 mg, 0.661 mmol, 99%.

Step 2. Synthesis of 6-bromo-2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1 - (2-methoxyethyl)-1H-imidazo[4,5-b]pyridine (C41).

A suspension of C13, free base (130 mg, 0.374 mmol), P13 (130 mg, 0.427 mmol) and potassium carbonate (172 mg, 1.24 mmol) in acetonitrile (2 ml) was stirred at 50°C for 16 h. the mixture was then purified by preparative thin layer chromatography (eluent: ethyl acetate) to give C41 as a brown oil. Yield: 114 mg, 0.185 mmol, 49%. LC-MS m/z 617.1 (a pattern was observed with peaks of bromine and chlorine isotopes) [M+H]+.

Stage 3. Synthesis of methyl-2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-(2 -methoxyethyl)-1H-imidazo[4,5-b]pyridine-6-carboxylate (C42).

A solution of C41 (114 mg, 0.185 mmol), 1,3-bis(diphenylphosphino)propane (15.3 mg, 37.1 µmol), palladium(II) acetate (8.3 mg, 37 µmol) and triethylamine (187 mg , 1.85 mmol) in a mixture of methanol (5 ml) and N,Ndimethylformamide (1 ml) was stirred at 80°C in an atmosphere of carbon monoxide (50 psi) for 16 hours. After diluting the reaction mixture with ethyl acetate (50 ml) it was washed with saturated aqueous sodium chloride solution (2x50 ml), dried over magnesium sulfate, filtered and concentrated under reduced pressure. Purification by preparative thin layer chromatography (eluent: ethyl acetate) gave C42 as a colorless oil. Yield: 60.0 mg, 0.101 mmol, 55%. LC-MS m/z 617.2 (a pattern was observed with peaks of chlorine isotopes [M+Na ⁺ ]).

Step 4. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-(2-methoxyethyl )-1H-imidazo[4,5-b]pyridine-6-carboxylic acid trifluoroacetate salt (3).

To a solution of C42 (60.0 mg, 0.101 mmol) in methanol (2.0 mL) was added an aqueous solution of sodium hydroxide (3 M; 1.0 mL, 3.0 mmol) and the reaction mixture was stirred at 20°C for 2 hours. It was then brought to pH 7 with 1 M hydrochloric acid and extracted with a mixture of dichloromethane and methanol (10:1, 3x30 ml). The combined organic layers were dried over magnesium sulfate, filtered, concentrated in vacuo and purified using reverse phase HPLC (column: Boston Green ODS, 5 μm; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: 10% to 95% B) to give 3 as a white solid. Yield: 29.6 mg, 42.6 µmol, 42%. LC-MS m/z 581.0^ [M+H] ⁺ .

¹ H NMR (400 MHz, methanol-d4) δ 9.13 (d, J=1.9 Hz, 1H), 8.74 (d, J=1.9 Hz, 1H), 7.63 (dd, J=8.3, 8, 3 Hz, 1H), 7.30 (dd, J=10.9, 2.0 Hz, 1H), 7.24 (ddd, J=8.4, 2.0, 0.7 Hz, 1H), 6.89-6.84 (m, 1H), 6.82 -6.77 (m, 2H), 4.98-4.89 (m, 2H, hypothesized; most of it is hidden by the water peak), 4.64 (t, J=4.8 Hz, 2H), 4.043.92 (br m, 2H), 3.75 (dd, J=5.4, 4.2 Hz, 2H), 3.51-3.39 (m, 2H), 3.31 (s, 3H), 3.19-3.06 (m, 1H), 2.41-2.24 (m, 2H), 2.24 -2.12 (m, 2H), 2.06 (d,J=1.0Hz, 3H).

Examples 4 and 5.

2-({4-[(2R)-2-(4-Chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-ammonium carboxylate (4) and 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)1,3-benzodioxol-4-yl]piperidine -1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6ammonium carboxylate (5)

Stage 1. Synthesis of 4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidine trifluoroacetate salt (C43).

Trifluoroacetic acid (1.3 ml) was added to a solution of P1 (300 mg, 0.691 mmol) in dichloromethane (5 ml). This reaction mixture was stirred at 29°C for 2 h, after which it was concentrated in vacuo to give C43 as a brown oil, which was used directly in the next step.

Step 2. Synthesis of methyl 2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[( 2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C44) and methyl-2-({4-[(2S)-2-(4chloro-2-fluorophenyl)-1,3-benzodioxol- 4-yl]piperidin-1-yl}methyl)-1-[(28)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylate (C45).

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To a solution of C43 (from the previous step, <0.691 mmol) in acetonitrile (10 mL) was added P15 (204 mg, 0.692 mmol) followed by potassium carbonate (956 mg, 6.92 mmol). This reaction mixture was stirred at 29°C for 16 h, after which it was filtered; the filtrate was concentrated in vacuo to give a residue which was purified by preparative thin layer chromatography (eluent: 2:1 petroleum ether/ethyl acetate) to give a mixture of diastereomeric products as a yellow gum (178 mg). Separation into two products was performed by the SFC method [column: Chiral Technologies ChiralCel OD, 5 μm; mobile phase: 55:45 carbon dioxide/(methanol containing 0.1% ammonium hydroxide)]. The diastereomer that eluted first and was obtained as a yellow oil was designated C44. Yield: 44.3 mg, 74.8 µmol, 11% over 2 steps. LC-MS m/z 592.1 ♦ [M+H]+. Retention time 4.26 min (column: Chiral Technologies ChiralCel OD-3, 4.6 x 100 mm, 3 µm; mobile phase A: carbon dioxide; mobile phase B: methanol containing 0.05% diethylamine; gradient: 5 to 40 % B for 4.5 min, then hold at 40% B for 2.5 min; flow rate: 2.8 ml per minute).

The second eluted diastereomer was subjected to a second SFC purification [column: Chiral Technologies ChiralCel OD, 5 μm; mobile phase: 3:2 carbon dioxide/(methanol containing 0.1% ammonium hydroxide)] to give the second eluting diastereomer as a colorless oil, which is designated C45. Yield: 38 mg, 64 µmol, 9% over 2 steps. LC-MS m/z 592.1^ [M+H] ⁺ . Retention time 4.41 min (analytical conditions identical to those used for C44).

The indicated absolute stereochemistry for dioxolane is assigned by correlating activity 5 with sample 5, the free acid synthesized from intermediate C48; the absolute stereochemistry of this intermediate was determined by X-ray diffraction analysis of a single crystal (see below) of C49, the hemisulphate salt of C48.

Step 3. Synthesis of 2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S) -oxetan-2-ylmethyl]-1H-benzimidazole-6-ammonium carboxylate (4).

An aqueous solution of lithium hydroxide (2 M; 0.80 ml, 1.6 mmol) was added to a solution of C44 (44.3 mg, 74.8 µmol) in a mixture of methanol (1 ml) and tetrahydrofuran (1 ml), and this reaction the mixture was stirred at 26° C. for 3 hours. It was then adjusted to pH 7 by adding trifluoroacetic acid and the resulting mixture was concentrated in vacuo and purified by reverse phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.05 % ammonium hydroxide in water, mobile phase B: acetonitrile, gradient: 30 to 50% B) to give 4 as a white solid. Yield: 26.6 mg, 44.7 µmol, 60%. LCMS m/z 578.0^ [M+H] ⁺ .

¹ H NMR (400 MHz, methanol-^) δ 8.31 (d, J=1.4 Hz, 1H), 7.96 (dd, J=8.5, 1.6 Hz, 1H), 7.66 (d, J=8, 5 Hz, 1H), 7.57 (dd, J=8.0, 8.0 Hz, 1H), 7.34 (dd, J=10.1, 2.0 Hz, 1H), 7.29 (br dd, J=8.3, 2.0 Hz, 1H), 7.20 (s, 1H), 6.86-6.79 (m, 1H), 6.77 (br dd, ABC system component, J=7.9, 1.3 Hz, 1H), 6.73 (dd, ABC system component, J=7.5, 1.4 Hz, 1H), 5.29-5.18 (m, 1H), 4.9-4.78 (m, 1H, hypothesized; partially obscured by water peak), 4.68 (dd, J=15.3, 2.7 Hz, 1H), 4.54 (td, J=8.0, 5.9 Hz, 1H), 4.44 (dt, J=9.2, 5.9 Hz, 1H), 4.02 (AB quartet, J _AB = 13.9 Hz, Av _ab = 49.0 Hz, 2H), 3.18-3.08 (m, 1H), 3.05-2.96 (m, 1H), 2.81-2.68 (m, 2H), 2.56-2.45 (m, 1H), 2.45-2.30 (m , 2H), 2.03-1.88 (m, 2H), 1.88-1.79 (m, 2H).

Step 4. Synthesis of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S) -oxetan-2-ylmethyl]-1H-benzimidazole-6-ammonium carboxylate (5).

An aqueous solution of lithium hydroxide (2 M; 0.80 ml, 1.6 mmol) was added to a solution of C45 (38 mg, 64 µmol) in a mixture of methanol (1 ml) and tetrahydrofuran (1 ml) and the reaction mixture was stirred at 24 °C for 2.5 hours. It was then adjusted to pH 7 with 1 M hydrochloric acid and the resulting mixture was concentrated in vacuo and purified by reverse phase HPLC (column: Agela Durashell C18, 5 µm; mobile phase A: 0.05 % ammonium hydroxide in water, mobile phase B: acetonitrile, gradient: 29% to 49% B) to give 5 as a white solid. Yield: 27.9 mg, 46.9 µmol, 73%. LC-MS m/z 577.9^ [M+H] ⁺ .

1H NMR (400 MHz, methanol-d ₄ ) δ 8.32 (d, J=1.4 Hz, 1H), 7.96 (dd, J=8.5, 1.5 Hz, 1H), 7.66 (d, J=8, 5 Hz, 1H), 7.56 (dd, J=8.0, 8.0 Hz, 1H), 7.34 (dd, J=10.2, 2.0 Hz, 1H), 7.29 (br dd, J=8.3, 2.0 Hz, 1H), 7.20 (s, 1H), 6.85-6.80 (m, 1H), 6.77 (dd, ABC system component, J=8.0, 1.3 Hz, 1H), 6.73 (dd, ABC system component, J =7.5, 1.4 Hz, 1H), 5.30-5.20 (m, 1H), 4.9-4.79 (m, 1H), hypothetical; partially hidden by water peak), 4.68 (dd, J=15.4, 2.7 Hz, 1H), 4.62-4.54 (m, 1H), 4.44 (dt, J=9.2, 5.9 Hz, 1H), 4.02 (AB quartet, J _AB = 13.9 Hz, Av _ab = 44.6 Hz, 2Н), 3.18-3.09 (m, 1H), 3.06-2.97 (m, 1H), 2.80-2.67 (m, 2H), 2.55- 2.30 (m, 3H), 2.02-1.78 (m, 4H).

Alternative synthesis of the compound of Example 5, free acid 2-({4-[(2S)-2-(4-Chloro-2fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1 - [(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-carboxylic acid (5, free acid)

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5" free acid

Step 1. Isolation of tert-butyl 4-[(2R)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4yl]piperidine-1-carboxylate (С46) and tert-butyl 4-[ (2S)-2-(4-Chloro-2-fluorophenyl)-1,3-benzodioxol-4yl]piperidine-1-carboxylate (C47).

Separation of P1 (10 g, 23 mmol) into its constituent enantiomers was performed using reverse phase HPLC [column: Phenomenex Lux Amylose-1, 5 μm; mobile phase: 9:1 carbon dioxide/(2-propanol containing 0.2% 1-aminopropan-2-ol)]. The first eluting enantiomer was designated C46 and the second eluting enantiomer was designated C47; both were obtained as a colorless oil. The absolute stereochemistry indicated for C46 and C47 was assigned from the results of X-ray diffraction analysis of the C49 single crystal, which was synthesized from C47 (see below).

C46 Yield: 4.47 g, 10.3 mmol, 45%. Retention time: 3.98 min [column: Phenomenex Lux Amylose-1, 4.6 x 250 mm, 5 µm; mobile phase A: carbon dioxide; mobile phase B: 2-propanol containing 0.2% 1-aminopropan-2-ol; gradient: 5% B over 1.00 min, then 5 to 60% B over 8.00 min; flow rate: 3.0 ml per minute; back pressure: 120 bar].

C47 Yield: 4.49 g, 10.3 mmol, 45%. Retention time: 4.32 min (SFC analytical conditions identical to those used for C46).

Stage 2. Synthesis of 4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidine (С48).

Para-toluenesulfonic acid monohydrate (566 mg, 2.98 mmol) was added to a solution of C47 (1.12 g, 2.58 mmol) in ethyl acetate (26 mL). After heating the reaction mixture at 45°C for 16 h, it was concentrated in vacuo, dissolved in ethyl acetate and washed with saturated aqueous sodium bicarbonate. The aqueous layers were extracted with ethyl acetate and the combined organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, filtered and concentrated under reduced pressure to give C48 as a foamy white solid (947 mg), LC-MS m/z 334.0 ^ [M+H]+. A portion of this material, which still contained some p-toluenesulfonic acid, was used in the C50 synthesis below.

The second portion of foamy white solid (440 mg) was dissolved in ethyl acetate (25 ml) and washed with saturated aqueous sodium bicarbonate (2x15 ml). The organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo to give C48 (350 mg) as a colorless oil which no longer contained p-toluenesulfonic acid. Corrected yield: 350 mg, 1.05 mmol, 88%.

'H NMR (400 MHz, chloroform-d) δ 7.53 (dd, J=8.4, 7.8 Hz, 1H), 7.22-7.13 (m, 3H), 6.87-6.80 (m, 1H), 6.79-6.71 ( m, 2H), 3.23-3.14 (m, 2H), 2.86-2.69 (m, 3H), 1.90-1.68 (m, 4H).

Step 3. Synthesis of 4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidine hemisulfate salt (C49).

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A 0.1M solution of C48 (colorless oil from the step above) was prepared in ethyl acetate and salt shielded. Only the formation of the sulfate salt is described here. A mixture of sulfuric acid (25 µmol) and substrate solution (0.1 M, 250 µl, 25 µmol) was heated to 45°C for 1 h, left to cool to room temperature and stirred for 15 h. The resulting suspension was treated with methanol (approx. 150 µl) until a solution is formed. This solution was allowed to slowly evaporate overnight until approximately 50 µl of solvent remained. One of the resulting crystals was analyzed by single crystal X-ray diffraction analysis, establishing the absolute stereochemistry as shown.

X-ray diffraction analysis of single crystal C49.

X-ray diffraction analysis of a single crystal.

Data collection was carried out on a Bruker D8 Venture diffractometer at room temperature. Data collection consisted of omega and fi scans.

The structure was revealed by stepwise use of the SHELX software package in the space group of the triclinic class P1. The structure was then refined by the least squares method in the full matrix approximation. All non-hydrogen atoms were identified and refined using anisotropic substitution parameters.

Hydrogen atoms located on nitrogen and oxygen were determined from the difference Fourier map and refined with limited distances. The remaining hydrogen atoms were placed in the calculated positions and left on the atoms carrying them. The final refinement included isotropic substitution parameters for all hydrogen atoms.

The asymmetric unit consists of two molecules of protonated C48, one molecule of doubly deprotonated sulfuric acid, and one molecule of full-time water. Thus, the structure is a hemisulfate salt and a hemihydrate. The chlorofluorophenyl ring is random and modeled with 60/40 occupancy, with the ring rotated in two positions.

Absolute structure analysis using probabilistic methods (Hooft, 2008) was performed using PLATON (Spek). The results show that the absolute structure was determined correctly; by this method it is calculated that the probability that the structure is accurate is 100%. The Hooft parameter is presented as 0.061 with an esd (estimated standard deviation) of 0.004 and the Parson parameter is presented as 0.063 with an esd of 0.0050.07.

The final R-index was 3.1%. The finite Fourier difference revealed no missing or inappropriate electron density.

Relevant crystal, data collection, and refinement information is summarized in Table 1. E. Atomic coordinates, bond lengths, bond angles, and substitution parameters are listed in Tables F-H.

Software and sources of information.

SHELXTL, Version 5.1, Bruker AXS, 1997.

PLATON, A. L. Spek, J. Appl. Cryst. 2003, 36, 7-13.

MERCURY, C. F. Macrae, P. R. Edington, P. McCabe, E. Pidcock, G. P. Shields, R.

Taylor, M. Towler, and J. van de Streek, J. Appl. Cryst. 2006, 39, 453-457.

OLEX2, O. V. Dolomanov, L. J. Bourhis, R. J. Gildea, J. A. K. Howard, and H.

Puschmann, J. Appl. Cryst. 2009, 42, 339-341.

R. W. W. Hooft, L. H. Straver, and A. L. Spek, J. Appl. Cryst. 2008, 41, 96-103.

H. D. Flack, Acta Cryst. 1983, A39, 867-881.

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Table E

Crystallographic data and structure refinement for С49

Empirical Formula C36H38CI2F2N2O9S Molecular weight according to the formula 783.64 Temperature 296(2)K Wavelength 1.54178 A Crystal system Triclinic space group P1 Unit cell dimensions a = 5.9095(2) A a = 86.5910(10)° b = 6.1712(2) Κβ = 89.3680(10)° c = 25.6096(8) A y = 75.7680(10)° Volume 903.68(5)A ³ Z 1 Density (calculated) 1.440 mg/ ^m3 Absorption coefficient 2.743 mm' ¹ F(000) 408 Crystal dimensions 0.380 x 0.120 x 0.080 mm ³ Theta range for data collection 3.458 to 72.096° Index ranges ~7<=h<=7, ~7<=h<=7, -31 <=/<=31 Collected Reflections 24619 Independent Reflections 6399 \R _mt = 0.0323]] Fullness to Theta = 67.679° 96.6% Absorption Correction empirical refinement method Full-matrix method of least squares in F ² Data/Restrictions/Parameters 6399/9/495 Goodness-of-fit criterion for F ² 1.014 Finite R indices \Ι>2σ(Ι)] RI = 0.0305, wR2 = 0.0805 R indices (all data) RI = 0.0310, wR2 = 0.0810 Absolute Structure Parameter 0.058(4) extinction coefficient η/a (no data) Biggest diff, peak and gap 0.167 and -0.184 e. A' ³

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Table F

Atomic coordinates (x 104) and equivalent parameters of isotropic substitution (A ² x10 ³ ) for C49.

U (equiv.) is defined as one third of the trace of an orthogonalized U ^j tensor.

1 1 ^x 1 ^{1 z1} U(eq) S(l) 8968(1) 2512(1) 4774(1) 33(1) С1(1) 2534(3) 7001(5) 9863(1) 161(1) F(l) 9192(9) 7761(7) 8721(2) 95(1) C(1) 7533(7) 6719(7) 8821(1) 72(1) C(2) 6041(9) 7355(8) 9230(2) 92(1) C(3) 4428(8) 6206(10) 9350(2) 93(2) C(4) 4276(8) 4392(9) 9082(2) 86(1) C(5) 5801(7) 3784(7) 8678(1) 69(1) C(6) 7444(6) 4930(5) 8533(1) 56(1) S1(G) 2534(3) 7001(5) 9863(1) 161(1) F(l') 6045(13) 1811(12) 8450(3) 95(1) S(G) 5801(7) 3784(7) 8678(1) 69(1) C(2') 4276(8) 4392(9) 9082(2) 86(1) С(З') 4428(8) 6206(10) 9350(2) 93(2) C(4') 6041(9) 7355(8) 9230(2) 92(1) С(5') 7533(7) 6719(7) 8821(1) 72(1) C(b') 7444(6) 4930(5) 8533(1) 56(1) С1(2) -2047(5) 12265(3) 154(1) 157(1) F(2) -2662(7) 5436(7) 1220(2) 92(1) S(19) -1591(6) 7059(7) 1154(1) 68(1) C(20) -2327(8) 8653(9) 752(2) 88(1) S(21) -1157(9) 10260(8) 665(2) 88(1) C(22) 728(9) 10361(7) 964(2) 80(1) C(23) 1431(6) 8731(6) 1364(1) 65(1) C(24) 274(5) 7058(5) 1472(1) 54(1) C1(2') -2047(5) 12265(3) 154(1) 157(1) F(2') 3433(15) 8441(16) 1630(4) 92(1)

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C(19') 1431(6) 8731(6) 1364(1) 65(1) C(20') 728(9) 10361(7) 964(2) 80(1) S(2G) -1157(9) 10260(8) 665(2) 88(1) C(22') -2327(8) 8653(9) 752(2) 88(1) C(23') -1591(6) 7059(7) 1154(1) 68(1) C(24') 274(5) 7058(5) 1472(1) 54(1) N(l) 4370(3) 2950(4) 5713(1) 41(1) N(2) 4133(4) 8236(3) 4386(1) 42(1) 0(1) 10923(4) 2331(5) 8233(1) 77(1) 0(2) 7874(4) 3730(4) 7651(1) 64(1) 0(3) 1766(4) 6201(4) 2352(1) 64(1) 0(4) 2966(5) 3591(4) 1729(1) 75(1) 0(5) 9024(3) 2305(3) 4214(1) 50(1) 0(6) 7650(4) 989(3) 5024(1) 63(1) 0(7) 11358(3) 1934(4) 4982(1) 64(1) 0(8) 7789(3) 4827(3) 4909(1) 46(1) O(1W) 10276(4) 6879(4) 5537(1) 54(1) C(7) 9086(6) 4293(6) 8090(1) 63(1) C(8) 9234(4) 1745(5) 7490(1) 44(1) C(9) 11056(5) 930(6) 7834(1) 54(1) C(10) 12654(5) -1059(6) 7768(1) 62(1) C(ll) 12316(5) -2213(6) 7338(1) 58(1) C(12) 10459(4) -1405(5) 6994(1) 47(1) C(13) 8826(4) 623(4) 7066(1) 38(1) C(14) 6762(4) 1637(4) 6711(1) 37(1) 0(15) 7243(4) 3516(4) 6343(1) 42(1) 0(16) 5126(4) 4639(4) 6009(1) 44(1) 0(17) 3883(5) 1105(5) 6056(1) 50(1) 0(18) 5997(4) -38(4) 6386(1) 41(1) 0(25) 996(6) 5296(6) 1900(1) 60(1) C(26) 3848(5) 4738(4) 2505(1) 45(1) C(27) 4542(6) 3183(5) 2133(1) 52(1) C(28) 6579(6) 1567(5) 2178(1) 56(1) C(29) 7932(6) 1577(5) 2620(1) 56(1) C(30) 7236(5) 3123(5) 2992(1) 51(1) 0(31) 5126(5) 4786(4) 2944(1) 42(1) C(32) 4261(4) 6474(4) 3352(1) 39(1) C(33) 6145(5) 7543(5) 3544(1) 51(1) C(34) 5139(5) 9272(4) 3932(1) 50(1) C(35) 2313(5) 7116(5) 4227(1) 49(1) C(36) 3263(4) 5420(4) 3826(1) 42(1)

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Table G

Bond lengths [A] and angles [°] for C49

S(l)-O(5) 1.4463(18) S(l)-O(7) 1.4668(19) S(l)-O(6) 1.475(2) S(l)-O(8) 1.4863(18) Cl(l)-C(3) 1.731(4) F(l)-C(l) 1.314(6) C(l)-C(6) 1.375(5) C(l)-C(2) 1.374(6) C(2)-C(3) 1.343(8) C(2)-H(2) 0.9300 C(3)-C(4) 1.369(8) C(4)-C(5) 1.373(6) C(4)-H(4) 0.9300 C(5)-C(6) 1.370(5) C(5)-H(5) 0.9300 C(6)-C(7) 1.493(5) С1(Г)-С(3') 1.731(4) F(l')-C(l') 1.357(8) C(l')-C(6') 1.370(5) S(G)-S(2') 1.373(6) C(2')-C(3') 1.369(8)

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C(2')-H(2') 0.9300 С(3')-С(4') 1.343(8) С(4')-С(5') 1.374(6) С(4')-Н(4') 0.9300 С(5')-С(6') 1.375(5) С(5')-Н(5') 0.9300 С(6')-С(7) 1.493(5) С1(2)-С(21) 1.739(4) F(2)-C(19) 1.312(5) С(19)-С(24) 1.378(5) С(19)-С(20) 1.378(6) С(20)-С(21) 1.348(7) С(20)-Н(20) 0.9300 С(21)-С(22) 1.375(7) С(22)-С(23) 1.384(6) С(22)-Н(22) 0.9300 С(23)-С(24) 1.385(5) С(23)-Н(23) 0.9300 С(24)-С(25) 1.485(5) С1(2')-С(2Г) 1.739(4) F(2')-C(19') 1.340(9) С(19')-С(20') 1.384(6) С(19')-С(24') 1.385(5) С(20')-С(2Г) 1.375(7) С(20')-Н(20') 0.9300 С(2Г)-С(22') 1.348(7) С(22')-С(23') 1.378(6) С(22')-Н(22') 0.9300 С(23')-С(24') 1.378(5) С(23')-Н(23') 0.9300 С(24')-С(25) 1.485(5) N(l)-C(17) 1.480(4) N(l)-C(16) 1.480(3)

- 62 040816

N(1)-H(1X) 0.95(2) N(1)-H(1Y) 0.97(2) N(2)-C(34) 1.483(4) N(2)-C(35) 1.487(4) N(2)-H(2X) 0.96(2) N(2)-H(2Y) 0.99(2) O(l)-C(9) 1.368(4) O(l)-C(7) 1.445(4) O(2)-C(8) 1.373(3) O(2)-C(7) 1.443(3) O(3)-C(26) 1.380(3) O(3)-C(25) 1.440(3) O(4)-C(27) 1.369(4) O(4)-C(25) 1.447(4) O(1W)-H(1WX) 0.93(2) O(1W)-H(1WY) 0.94(2) C(7)-H(7) 0.9800 C(8)-C(9) 1.374(4) C(8)-C(13) 1.376(4) C(9)-C(10) 1.370(5) C(10)-C(ll) 1.387(5) C(10)-H(10) 0.9300 C(ll)-C(12) 1.390(4) C(ll)-H(ll) 0.9300 C(12)-C(13) 1,400(4) C(12)-H(12) 0.9300 C(13)-C(14) 1.514(3) C(14)-C(18) 1.518(3) C(14)-C(15) 1.528(3) C(14)-H(14) 0.9800 C(15)-C(16) 1.518(3) C(15)-H(15A) 0.9700 C(15)-H(15B) 0.9700

- 63 040816

C(16)-H(16A) 0.9700 С(16)-Н(16V) 0.9700 С(17)-С(18) 1.513(4) С(17)-Н(17А) 0.9700 С(17)-Н(17В) 0.9700 С(18)-Н(18А) 0.9700 S(18)-H(18V) 0.9700 С(25)-Н(25) 0.9800 С(26)-С(31) 1.367(4) С(26)-С(27) 1.379(3) С(27)-С(28) 1.363(4) С(28)-С(29) 1.394(5) S(28)-H(28) 0.9300 С(29)-С(30) 1.376(4) S(29)-H(29) 0.9300 С(30)-С(31) 1.408(4) С(ЗО)-Н(ЗО) 0.9300 С(31)-С(32) 1.514(3) С(32)-С(33) 1.527(4) С(32)-С(36) 1.524(3) С(32)-Н(32) 0.9800 С(33)-С(34) 1.510(4) C(33)-H(33A) 0.9700 C(33)-H(33B) 0.9700 С(34)-Н(34А) 0.9700 С(34)-Н(34В) 0.9700 С(35)-С(36) 1.515(3) С(35)-Н(35А) 0.9700 С(35)-Н(35V) 0.9700 С(36)-Н(36А) 0.9700 С(36)-Н(36В) 0.9700 O(5)-S(l)-O(7) 109.68(13) O(5)-S(l)-O(6) 109.65(13)

- 64 040816

O(7)-S( 1)-0(6) 109.45(15) O(5)-S(l)-O(8) 111.22(11) O(7)-S( 1)-0(8) 109.11(11) O(6)-S( 1)-0(8) 107.69(11) F(l)-C(l)-C(6) 118.6(4) F(l)-C(l)-C(2) 119.1(4) C(6)-C( 1)-0(2) 122.1(4) C(3)-C(2)-C(l) 118.9(4) C(3)-C(2)-H(2) 120.5 C(l)-C(2)-H(2) 120.5 C(2)-C(3)-C(4) 121.6(4) C(2)-C(3)-C1(1) 119.3(4) C(4)-C(3)-C1(1) 119.1(5) C(3)-C(4)-C(5) 118.2(5) C(3)-C(4)-H(4) 120.9 C(5)-C(4)-H(4) 120.9 C(6)-C(5)-C(4) 122.4(4) C(6)-C(5)-H(5) 118.8 C(4)-C(5)-H(5) 118.8 C(5)-C(6)-C(l) 116.7(3) C(5)-C(6)-C(7) 122.7(3) C(l)-C(6)-C(7) 120.6(3) F(l')-C(r)-C(6') 114.7(4) F(l')-C(l')-C(2') 122.1(5) С(6')-С(Г)-С(2') 122.4(4) С(3')-С(2')-С(Г) 118.2(5) C(3')-C(2')-H(2') 120.9 C(G)-C(2')-H(2') 120.9 C(4')-C(3')-C(2') 121.6(4) C(4')-C(3')-C1(G) 119.3(4) C(2')-C(3')-C1(G) 119.1(5) C(3')-C(4')-C(5') 118.9(4) C(3')-C(4')-H(4') 120.5

- 65 040816

C(5')-C(4')-H(4') 120.5 С(6')-С(5')-С(4') 122.1(4) C(6')-C(5')-H(5') 118.9 С(4')-С(5')-Н(5') 118.9 C(G)-C(6')-C(5') 116.7(3) C(G)-C(6')-C(7) 122.7(3) С(5')-С(6')-С(7) 120.6(3) F(2)-C(19)-C(24) 119.3(4) F(2)-C(19)-C(20) 118.1(4) С(24)-С(19)-С(20) 122.5(4) С(21)-С(20)-С(19) 118.4(4) С(21)-С(20)-Н(20) 120.8 С(19)-С(20)-Н(20) 120.8 С(20)-С(21)-С(22) 122.4(4) С(20)-С(21)-С1(2) 118.9(4) С(22)-С(21)-С1(2) 118.7(4) С(21)-С(22)-С(23) 117.8(4) С(21)-С(22)-Н(22) 121.1 С(23)-С(22)-Н(22) 121.1 С(22)-С(23)-С(24) 122.0(4) С(22)-С(23)-Н(23) 119.0 С(24)-С(23)-Н(23) 119.0 С(19)-С(24)-С(23) 116.8(3) С(19)-С(24)-С(25) 120.3(3) С(23)-С(24)-С(25) 122.9(3) F(2')-C(19')-C(20') 123.5(5) F(2')-C(19')-C(24') 113.9(5) С(20')-С(19')-С(24') 122.0(4) S(2G)-S(20')-S(19') 117.8(4) С(21')-С(20')-Н(20') 121.1 С(19')-С(20')-Н(20') 121.1 С(22')-С(2Г)-С(20') 122.4(4) С(22')-С(2Г)-С1(2') 118.9(4)

- 66 040816

С(20')-С(2Г)-С1(2') 118.7(4) С(2Г)-С(22')-С(23') 118.4(4) С(2Г)-С(22')-Н(22') 120.8 C(23')-C(22')-H(22') 120.8 С(24')-С(23')-С(22') 122.5(4) С(24')-С(23')-Н(23') 118.7 С(22')-С(23')-Н(23') 118.7 С(23')-С(24')-С(19') 116.8(3) С(23')-С(24')-С(25) 120.3(3) С(19')-С(24')-С(25) 122.9(3) C(17)-N(l)-C(16) 112.6(2) C(17)-N(1)-H(1X) 110.7(19) C(16)-N(1)-H(1X) 108(2) C(17)-N(1)-H(1Y) 108(2) C(16)-N(1)-H(1Y) 112.4(19) H(1X)-N(1)-H(1Y) 105(3) C(34)-N(2)-C(35) 112.2(2) C(34)-N(2)-H(2X) 109.7(19) C(35)-N(2)-H(2X) 109.7(19) C(34)-N(2)-H(2Y) 107.7(19) C(35)-N(2)-H(2Y) 110.8(19) H(2X)-N(2)-H(2Y) 107(3) C(9)-O(l)-C(7) 106.0(2) C(8)-O(2)-C(7) 105.9(2) C(26)-O(3)-C(25) 105.9(2) C(27)-O(4)-C(25) 105.7(2) H( 1 WX)-O( 1 W)-H( 1W Y) 105(4) O(2)-C(7)-O(l) 106.5(3) O(2)-C(7)-C(6) 110.4(3) O(l)-C(7)-C(6) 111.2(3) O(2)-C(7)-C(6') 110.4(3) O(l)-C(7)-C(6') 111.2(3) O(2)-C(7)-H(7) 109.6

- 67 040816

O(l)-C(7)-H(7) 109.6 С(6)-С(7)-Н(7) 109.6 С(9)-С(8)-О(2) 110.0(2) С(9)-С(8)-С(13) 123.4(2) O(2)-C(8)-C(13) 126.6(2) O(1)-C(9)-C(Yu) 128.1(3) O(1)-C(9)-C(8) 110.1(3) С(10)-С(9)-С(8) 121.7(3) С(9)-С(10)-С(11) 116.3(3) С(9)-С(10)-Н(10) 121.8 С(11)-С(10)-Н(10) 121.8 С(10)-С(11)-С(12) 122.0(3) С(10)-С(11)-Н(11) 119.0 С(12)-С(11)-Н(11) 119.0 С(11)-С(12)-С(13) 121.3(3) С(11)-С(12)-Н(12) 119.4 С(13)-С(12)-Н(12) 119.4 С(8)-С(13)-С(12) 115.3(2) С(8)-С(13)-С(14) 119.8(2) С(12)-С(13)-С(14) 124.9(2) С(13)-С(14)-С(18) 114.2(2) С(13)-С(14)-С(15) 111.38(19) С(18)-С(14)-С(15) 108.70(19) С(13)-С(14)-Н(14) 107.4 С(18)-С(14)-Н(14) 107.4 С(15)-С(14)-Н(14) 107.4 С(16)-С(15)-С(14) 111.7(2) С(16)-С(15)-Н(15А) 109.3 С(14)-С(15)-Н(15А) 109.3 С(16)-С(15)-Н(15V) 109.3 С(14)-С(15)-Н(15V) 109.3 H(15A)-C(15)-H(15V) 107.9 N(l)-C(16)-C(15) 109.9(2)

- 68 040816

N(1)-C(16)-H(16A) 109.7 C(15)-C(16)-H(16A) 109.7 N(1)-C(16)-H(16B) 109.7 C(15)-C(16)-H(16B) 109.7 H(16A)-C(16)-H(16B) 108.2 N(l)-C(17)-C(18) 110.94(19) N(1)-C(17)-H(17A) 109.5 C(18)-C(17)-H(17A) 109.5 N(1)-C(17)-H(17B) 109.5 C(18)-C(17)-H(17B) 109.5 H(17A)-C(17)-H(17B) 108.0 C(17)-C(18)-C(14) 110.6(2) C(17)-C(18)-H(18A) 109.5 C(14)-C(18)-H(18A) 109.5 C(17)-C(18)-H(18B) 109.5 C(14)-C(18)-H(18B) 109.5 H(18A)-C(18)-H(18B) 108.1 O(3)-C(25)-O(4) 106.6(2) O(3)-C(25)-C(24') 111.0(3) O(4)-C(25)-C(24') 109.4(3) O(3)-C(25)-C(24) 111.0(3) O(4)-C(25)-C(24) 109.4(3) O(3)-C(25)-H(25) 109.9 O(4)-C(25)-H(25) 109.9 C(24)-C(25)-H(25) 109.9 C(31)-C(26)-C(27) 123.2(3) C(31)-C(26)-O(3) 127.3(2) C(27)-C(26)-O(3) 109.5(2) C(28)-C(27)-O(4) 127.7(2) C(28)-C(27)-C(26) 121.9(3) O(4)-C(27)-C(26) 110.3(2) C(27)-C(28)-C(29) 116.3(2) C(27)-C(28)-H(28) 121.9

- 69 040816

С(29)-С(28)-Н(28) 121.9 С(30)-С(29)-С(28) 121.8(3) С(30)-С(29)-Н(29) 119.1 С(28)-С(29)-Н(29) 119.1 С(29)-С(30)-С(31) 121.7(3) С(29)-С(30)-Н(30) 119.2 С(31)-С(30)-Н(30) 119.2 С(26)-С(31)-С(30) 115.1(2) С(26)-С(31)-С(32) 121.5(2) С(30)-С(31)-С(32) 123.4(2) С(31)-С(32)-С(33) 113.3(2) С(31)-С(32)-С(36) 111.48(19) С(33)-С(32)-С(36) 108.02(19) С(31)-С(32)-Н(32) 107.9 С(33)-С(32)-Н(32) 107.9 С(36)-С(32)-Н(32) 107.9 С(34)-С(33)-С(32) 110.5(2) C(34)-C(33)-H(33A) 109.6 C(32)-C(33)-H(33A) 109.6 C(34)-C(33)-H(33B) 109.6 C(32)-C(33)-H(33B) 109.6 H(33A)-C(33)-H(33B) 108.1 N(2)-C(34)-C(33) 110.6(2) N(2)-C(34)-H(34A) 109.5 С(33)-С(34)-Н(34А) 109.5 N(2)-C(34)-H(34B) 109.5 С(33)-С(34)-Н(34V) 109.5 H(34A)-C(34)-H(34V) 108.1 N(2)-C(35)-C(36) 110.71(19) N(2)-C(35)-H(35A) 109.5 С(36)-С(35)-Н(35А) 109.5 N(2)-C(35)-H(35B) 109.5 С(36)-С(35)-Н(35V) H(35A)-C(35)-H(35V) 109.5 108.1 С(35)-С(36)-С(32) 111.9(2) С(35)-С(36)-Н(36А) 109.2 С(32)-С(36)-Н(36А) 109.2 С(35)-С(36)-Н(36В) 109.2 С(32)-С(36)-Н(36В) 109.2 H(36A)-C(36)-H(36V) 107.9

Symmetry transformations used to create equivalent atoms.

- 70 040816

Table H

Anisotropic substitution parameters (A ² x10 ³ ) for C49.

The exponent of the anisotropic substitution factor takes the form: ^{-2π a} * U + ... + 2hka*b*U ].

and ¹¹ U ²² and ³³ and ²³ and ¹³ and ¹² S(l) 32(1) 32(1) 32(1) -3(1) -2(1) -1(1) Cl(l) 107(1) 258(2) 90(1) -63(1) 19(1) 19(1) F(l) 1I(2) 91(2) 98(2) -30(2) 6(2) -46(2) C(l) 81(2) 71(2) 60(2) -20(2) -16(2) -6(2) C(2) 100(3) 92(3) 74(3) -42(2) -16(2) 7(2) C(3) 70(2) 134(4) 53(2) -27(2) -7(2) 19(3) C(4) 71(2) 116(3) 67(2) 0(2) -1(2) -16(2) C(5) 75(2) 70(2) 59(2) -AND 2) -7(2) -U(2) C(6) 65(2) 54(2) 42(1) -8(1) -18(1) -1(1) S1(G) 107(1) 258(2) 90(1) -63(1) 19(1) 19(1) F(l') 1I(2) 91(2) 98(2) -30(2) 6(2) -46(2) C(l') 75(2) 70(2) 59(2) -AND 2) -7(2) -U(2) C(2') 71(2) 116(3) 67(2) 0(2) -1(2) -16(2) С(З') 70(2) 134(4) 53(2) -27(2) -7(2) 19(3) C(4') 100(3) 92(3) 74(3) -42(2) -16(2) 7(2) С(5') 81(2) 71(2) 60(2) -20(2) -16(2) -6(2) C(b') 65(2) 54(2) 42(1) -8(1) -18(1) -1(1) С1(2) 243(2) 1Yu(1) 80(1) 12(1) -39(1) 26(1) F(2) 88(2) 106(2) 93(2) -12(2) -22(2) -44(2)

- 71 040816

S(19) 62(2) 77(2) 62(2) -26(2) -12(2) -5(2) C(20) 85(3) 98(3) 66(2) -20(2) -31(2) Yu(2) S(21) 117(3) 74(3) 51(2) -AND 2) -U(2) 18(2) C(22) 104(3) 70(2) 60(2) -9(2) 8(2) -8(2) C(23) 58(2) 73(2) 60(2) -13(2) -3(1) -6(2) C(24) 50(2) 60(2) 47(2) -23(1) -4(1) -2(1) C1(2') 243(2) 1Yu(1) 80(1) 12(1) -39(1) 26(1) F(2') 88(2) 106(2) 93(2) -12(2) -22(2) -44(2) С(19') 58(2) 73(2) 60(2) -13(2) -3(1) -6(2) С(20') 104(3) 70(2) 60(2) -9(2) 8(2) -8(2) S(2G) 117(3) 74(3) 51(2) -AND 2) -U(2) 18(2) С(22') 85(3) 98(3) 66(2) -20(2) -31(2) Yu(2) С(23') 62(2) 77(2) 62(2) -26(2) -12(2) -5(2) С(24') 50(2) 60(2) 47(2) -23(1) -4(1) -2(1) N(l) 30(1) 59(1) 32(1) -3(1) -4(1) -7(1) N(2) 49(1) 38(1) 37(1) -u(1) -5(1) 0(1) 0(1) 58(1) 107(2) 55(1) -23(1) -26(1) 6(1) 0(2) 64(1) 66(1) 50(1) -21(1) -23(1) 12(1) 0(3) 66(1) 62(1) 52(1) -27(1) -19(1) I(1) 0(4) 92(2) 64(1) 56(1) -32(1) -20(1) Yu(1) 0(5) 62(1) 51(1) 34(1) -5(1) -2(1) -9(1) 0(6) 76(1) 43(1) 70(1) -4(1) 32(1) -14(1) 0(7) 45(1) 68(1) 69(1) -29(1) -22(1) 13(1) 0(8) 45(1) 35(1) 53(1) -9(1) -4(1) 2(1) O(1W) 56(1) 50(1) 51(1) -3(1) -12(1) 1(1) C(7) 68(2) 73(2) 45(2) -12(1) -14(1) -12(2) C(8) 38(1) 51(1) 36(1) -4(1) -3(1) 0(1) C(9) 42(1) 76(2) 39(1) -1(1) -9(1) -4(1) C(10) 38(1) 87(2) 48(2) Yu(1) -8(1) 6(1) C(11) 45(1) 60(2) 55(2) 9(1) 2(1) 13(1) C(12) 41(1) 46(1) 47(1) 0(1) 3(1) 0(1) C(13) 34(1) 43(1) 34(1) 2(1) -1(1) -4(1) C(14) 30(1) 44(1) 31(1) -4(1) -1(1) 0(1) C(15) 41(1) 38(1) 45(1) 0(1) -12(1) -7(1) C(16) 44(1) 43(1) 39(1) -3(1) -6(1) 4(1) S(17) 39(1) 73(2) 42(1) -1(1) -3(1) -23(1) S(18) 41(1) 46(1) 39(1) -4(1) 2(1) -14(1) C(25) 65(2) 62(2) 51(2) -22(1) -9(1) -8(1) C(26) 55(1) 37(1) 37(1) -8(1) 1(1) -2(1) C(27) 72(2) 41(1) 39(1) -9(1) -2(1) -6(1) C(28) 79(2) 39(1) 43(1) -yu(1) I(1) 1(1) C(29) 62(2) 45(2) 48(2) -2(1) 7(1) 8(1) C(30) 58(2) 45(2) 42(1) -1(1) -1(1) 1(1) C(31) 54(1) 34(1) 34(1) -4(1) 2(1) -4(1) C(32) 50(1) 30(1) 33(1) -4(1) -6(1) 0(1) C(33) 63(2) 45(1) 54(2) -9(1) 17(1) -28(1) C(34) 59(2) 38(1) 58(2) -9(1) -1(1) -22(1) C(35) 46(1) 46(1) 56(2) -17(1) 16(1) -u(1) C(36) 39(1) 36(1) 53(1) -15(1) 12(1) -13(1)

Step 4. Synthesis of methyl 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[( 2S)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylate (C50).

Solution C48 (500 mg foamy white solid from step above, corrected

- 72 040816 p-toluenesulfonic acid: 1.25 mmol) in acetonitrile (6 ml) was treated with N,N-diisopropylethylamine (0.68 ml, 3.9 mmol) and stirred for 5 min at 45°C. After P15 (319 mg, 1.08 mmol) was added, stirring at 45°C was continued for 7.25 h, after which the reaction mixture was diluted with water (6 ml) and acetonitrile (2 ml) at 45°C. The resulting heterogeneous mixture was allowed to cool to room temperature and stirred for 72 hours. Water (5 ml) was added again and after an additional 30 minutes of stirring, the solid was collected by filtration and washed with a mixture of acetonitrile and water (15:85.3x5 ml) to give C50 in as a white solid with a slight pink tint. Yield: 605 mg, 1.02 mmol, 82%. LC-MS m/z 592.0^ [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 8.17 (d, J=1.6 Hz, 1H), 7.96 (dd, J=8.5, 1.5 Hz, 1H), 7.73 (d, J=8.4 Hz, 1H), 7.51 (dd, J=8.0, 8.0 Hz, 1H), 7.19 (br s, 1H), 7.18-7.14 (m, 2H), 6.85-6.79 (m, 1H), 6.76-6.71 (m, 2H), 5.26-5.18 (m, 1H), 4.73 (dd, ABX system component, J=15.3, 5.9 Hz, 1H), 4.67 (dd, ABX system component, J=15.3, 3.5 Hz, 1H), 4.63-4.55 (m, 1H), 4.38 (ddd, J=9.1, 6.0, 5.9 Hz, 1H), 3.94 (s, 5H), 3.03-2.89 (m, 2H), 2.77- 2.65 (m, 2H), 2.51-2.39 (m, 1H), 2.34-2.20 (m, 2H), 1.91-1.76 (m, 4H).

Step 5. Synthesis of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S) -oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid (5, free acid).

A suspension of C50 (595 mg, 1.00 mmol) in methanol (10 ml) was heated to 45° C. and treated with aqueous sodium hydroxide solution (1 M; 2.01 ml, 2.01 mmol). After 21 h at 45°C the reaction mixture was allowed to cool to room temperature, then it was treated with an aqueous solution of citric acid (1 M, 1 ml), which brought the pH to 5-6. Water (10 ml) was added and the mixture was stirred for 1 h, after which the solid was collected by filtration. It was washed with a mixture of methanol and water (1:10, 3x5 ml) to obtain a solid (433 mg). A portion of this material (300 mg) was stirred with a mixture of heptane and ethyl acetate (1:3, 5 ml) at 40°C for 1 h. After cooling to room temperature with stirring, the solid was collected by filtration and washed with a mixture of heptane and ethyl acetate (3: 1, 3x3 ml) to give 5, the free acid, as a white solid. Yield: 260 mg, 0.450 mmol, corresponding to 65% for the entire reaction. LC-MS m/z 578.0^ [M+H] ⁺ .

1H NMR (400 MHz, DMSO-d ₆ ) δ 12.75 (v br s, 1H), 8.26 (br s, 1H), 7.79 (dd, J=8.4, 1.6 Hz, 1H), 7.667.56 (m , 3H), 7.40 (dd, J=8.3, 2.0 Hz, 1H), 7.35 (s, 1H), 6.87-6.75 (m, 3H), 5.13-5.03 (m, 1H), 4.76 (dd, component ABX system, J=15.3, 7.2 Hz, 1H), 4.62 (dd, ABX system component, J=15.2, 2.8 Hz, 1H), 4.46-4.38 (m, 1H), 4.34 (ddd, J= 9.0, 5.9, 5.8 Hz, 1H), 3.84 (AB quartet, J _AB = 13.5 Hz, Av _ab = 67.7 Hz, 2H), 3.00 (br d, J=11.2 Hz, 1H) , 2.84 br (d, J=11.3 Hz, 1H), 2.71-2.56 (m, 2H), 2.45-2.34 (m, 1H), 2.28-2.08 (m, 2H), 1.84-1.65 (m, 4H ).

This substance was determined to have the same absolute configuration as the compound of example 5 above by comparing its biological activity with that of both 4 and 5: in assay 2, this sample of compound 5, the free acid, showed an EC ₅₀ 25 nM (geometric mean of 3 repetitions). The activity in assay 2 for the ammonium salt of the compound of example 4 and the compound of example 5 was >20,000 nM (2 repeats) and 20 nM (geometric average of 3 repeats), respectively.

Synthesis of the compound of Example 5,1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt 4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2ylmethyl]-1H-benzimidazole-6-carboxylate 1,3-hydroxy-2-(hydroxymethyl)propan-2-aminium (5 ,1,3dihydroxy-2-(hydroxymethyl)propane-2-aminium salt) he he ⁴⁼⁷ he

5, free acid

5,1,3-dihydroxy-2(hydroxymethyl)propane-2-aminium salt

A mixture of 5, free acid, (0.50 g, 0.86 mmol) in tetrahydrofuran (4 ml) was treated with an aqueous solution of 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris, 1.0 M; 0.5 ml, 1.0 mmol). After 20 h the mixture was concentrated in vacuo with ethanol (2x6 ml). The mixture was treated with ethanol (4 ml). After stirring for 48 h, the solid was collected by filtration, washed with ethanol (2x10 ml) and dried under vacuum to give 5,1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt, as a white solid. Yield: 410 mg, 0.586 mmol, 68%.

1H NMR (600 MHz, DMSO-d ₆ ), characteristic peaks: δ 8.19 (s, 1H), 7.78 (br d, J=8.4 Hz, 1H), 7.62-7.58 (m, 2H), 7.55 (d , J=8.3 Hz, 1H), 7.40 (dd, J=8.4, 2.0 Hz, 1H), 7.35 (s, 1H), 6.85-6.80 (m, 2H), 6.79 (dd, J=6.9 , 2.4 Hz, 1H), 5.11-5.05 (m, 1H), 4.73 (dd, J=15.2, 7.2 Hz, 1H), 4.60 (dd, J=15.3, 2.9 Hz, 1H), 4.454.39 (m, 1H), 4.34 (ddd, J=9.0, 6.0, 5.8 Hz, 1H), 3.91 (d, J=13.5 Hz, 1H), 3.74 (d, J=13.5 Hz, 1H), 2.99 (br d, J=11.1 Hz, 1H), 2.85 (br d, J=11.3 Hz, 1H), 2.68-2.59 (m, 2H), 2.44-2.37 (m, 1H), 2.25-2.18 (m, 1H), 2.17-2.10 (m, 1H), 1.80-1.69 (m, 4H). So pl. = 168-178°C.

- 73 040816

Examples 6 and 7.

2-({4-[(2K)-2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1[(2S )-oxetan-2-ylmethyl]-1H-benzimidazole-6-ammonium carboxylate (6) and 2-({4-[(28)-2-(4-chloro-2fluorophenyl)-2-methyl-1,3- benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1Hbenzimidazole-6-ammonium carboxylate (7)

Step 1. Synthesis of 4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidine p-toluenesulfonate salt (C13).

A solution of P2 (150 mg, 0.335 mmol) and p-toluenesulfonic acid monohydrate (159 mg, 0.836 mmol) in ethyl acetate (2.0 mL) was stirred at 60°C for 3.5 h. The reaction mixture was concentrated in vacuo to give C13 as a brown oil, which was directly used in the next step. LC-MS m/z 348.1 ♦ [M+H]+.

Step 2. Synthesis of methyl 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[( 2S)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-carboxylate (C51).

To a suspension of C13 (from the previous step; <0.335 mmol) and potassium carbonate (232 mg, 1.68 mmol) in acetonitrile (5.0 ml) was added P15 (99.1 mg, 0.336 mmol). The reaction mixture was stirred at 60° C. for 10 h after which it was filtered and the filtrate was concentrated in vacuo. After combining the residue (390 mg) with the material from a similar reaction carried out using C13 (<0.11 mmol), it was diluted with water (20 ml) and extracted with a mixture of dichloromethane and methanol (10:1, 3x30 ml). The combined organic layers were dried over sodium sulfate, filtered, concentrated in vacuo and subjected to preparative thin layer chromatography (eluent: 1:1 dichloromethane/methanol) to give C51, a mixture of diastereomers, as a colorless oil. Overall yield: 80.6 mg, 0.133 mmol, 30% over 2 steps. LC-MS m/z 606.2^ [M+H] ⁺ .

Step 3. Isolation of methyl-2-({4-[(2K)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)- 1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C52) and methyl 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2- methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C53).

Separation of C51 (180 mg, 0.297 mmol) into its constituent diastereomers was performed by repeated SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 65:35 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The diastereomer that eluted first was designated C52. Yield: 61.2 mg, 0.101 mmol, 34%. LC-MS m/z 627.9^ [M+Na ⁺ ]. Retention time: 5.03 min (column: Chiral Technologies Chiralpak AD-3, 4.6x150 mm, 3 µm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5 to 40% B for 5.5 minutes, then hold at 40% B for 3.0 minutes; flow rate: 2.5 ml per minute).

The second eluting diastereomer was designated C53. As a result of the analysis, it turned out that this substance was contaminated with the corresponding ethyl ester. It was carried to the hydrolysis step (to create 7) as this mixture. Yield: 40.0 mg, 66.0 µmol, 22%. LC-MS m/z 606.0^ [M+H] ⁺ . Retention time: 5.19 min (analytical conditions identical to those used

- 74 040816 for C52).

The indicated absolute stereochemistry for dioxolane was assigned by correlating the activity of 7 with sample 7, the free acid synthesized from intermediate P3 (see below Alternative Synthesis of Example 7, Free Acid). The absolute stereochemistry of P3 was established by X-ray diffraction analysis of a single crystal of compound C8 (see above).

Step 4. Synthesis of 2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1 - [(2S)-oxetan-2-ylmethyl]-1 H-benzimidazole-6-ammonium carboxylate (6).

An aqueous solution of lithium hydroxide (2 M; 0.990 ml, 1.98 mmol) was added to a solution of C52 (60 mg, 99 µmol) in a mixture of methanol (1.0 ml) and tetrahydrofuran (1.0 ml) and the reaction mixture was stirred at 20° C. for 16 h. Trifluoroacetic acid was added until the pH of the reaction mixture reached 7, after which the mixture was concentrated in vacuo and the residue was purified using reverse phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.05% ammonium hydroxide in water mobile phase B: acetonitrile gradient: 29% to 49% B) to give 6 as a white solid. Yield: 14.4 mg, 23.6 µmol, 24%. LC-MS m/z 592.0^ [M+H]+.

1H NMR (400 MHz, methanol-d ₄ ), characteristic peaks: δ 8.35 (d, J=1.3 Hz, 1H), 7.97 (dd, J=8.5, 1.5 Hz, 1H), 7.67 (d, J=8.5Hz, 1H), 7.58(dd, J=8.3, 8.3Hz, 1H), 7.28(dd, J=10.9, 2.0Hz, 1H), 7.21(br dd, J=8.4 , 1.9 Hz, 1H), 6.81-6.75 (m, 1H), 6.74-6.68 (m, 2H), 5.33-5.25 (m, 1H), 4.72 (dd, J=15.4, 2.7 Hz, 1H ), 4.49 (dt, J=9.1, 6.0 Hz, 1H), 4.03 (AB quartet, Jab = 13.9 Hz, Δυλβ = 47.8 Hz, 2Н), 3.14 (br d, J=11 Hz, 1H), 3.02 (br d, J=11.5 Hz, 1H), 2.88-2.78 (m, 1H), 2.77-2.68 (m, 1H), 2.60-2.50 (m, 1H), 2.47-2.32 (m , 2H), 2.03 (d, J=1.1 Hz, 3H), 2.01-1.87 (m, 2H), 1.87-1.78 (brm, 2H).

Step 5. Synthesis of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1- [(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-ammonium carboxylate (7).

An aqueous solution of lithium hydroxide (2 M; 0.642 ml, 1.28 mmol) was added to a solution of C53 (38.9 mg, 64.2 µmol) in a mixture of methanol (1.0 ml) and tetrahydrofuran (1.0 ml). After stirring the reaction mixture at 20° C. for 16 h, it was adjusted to pH 7 with trifluoroacetic acid, concentrated in vacuo and purified using reverse phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.05% ammonium hydroxide in water, mobile phase B: acetonitrile, gradient: 0 to 80% B) to give 7 as a white solid. Yield: 25.1 mg, 41.2 µmol, 64%. LC-MS m/z 591.9^ [M+H] ⁺ .

1H NMR (400 MHz, methanol-d ₄ ), characteristic peaks: δ 8.34 (d, J=1.5 Hz, 1H), 7.98 (dd, J=8.5, 1.6 Hz, 1H), 7.68 (d, J=8.5Hz, 1H), 7.58(dd, J=8.3, 8.3Hz, 1H), 7.28(dd, J=10.9, 2.0Hz, 1H), 7.20(br dd, J=8.4 , 1.9 Hz, 1H), 6.81-6.74 (m, 1H), 6.74-6.67 (m, 2H), 5.33-5.23 (m, 1H), 4.73 (dd, J=15.4, 2.7 Hz, 1H ), 4.68-4.61 (m, 1H), 4.48 (dt, J=9.1, 5.9 Hz, 1H), 4.05 (AB quartet, Jab = 13.9 Hz, Avab = 44.1 Hz, 2H), 3.15 (br d, J=11.7 Hz, 1H), 3.03 (br d, J=11.6 Hz, 1H), 2.87-2.69 (m, 2H), 2.60-2.49 (m, 1H), 2.48-2.33 (m, 2H), 2.03 (br s, 3H), 2.01–1.77 (m, 4H).

Alternative Synthesis of Example 7 Compound, 2-({4-[(2S)-2-(4-chloro-2fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl free acid )-1-[(2 S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid (7, free acid)

7" free acid

Stage 1. Synthesis of methyl-2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)- 1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C53).

Ν,N-Diisopropylethylamine (15.1 ml, 86.9 mmol) was added to a mixture of P3 (8.22 g, 15.8 mmol) in acetonitrile (185 ml). After stirring for 5 min, P15 (4.57 g, 15.5 mmol) was added and the reaction mixture was heated at 45°C. After 4 h the reaction mixture was concentrated in vacuo to half of its original volume, and the resulting mixture was diluted with water (100 ml) and was extracted with ethyl acetate (2x100 ml). The combined organic layers were washed with water (50 ml), dried over magnesium sulfate, filtered and concentrated in vacuo. Chromatography on silica gel (gradient: 50% to 100% ethyl acetate in heptane) gave C53 as a white solid. Yield: 8.4 g, 13.9 mmol, 88%. LC-MS m/z 606.1 ♦ [M+H] ⁺ .

- 75 040816

1H NMR (600 MHz, DMSO-d ₆ ) δ 8.30 (s, 1H), 7.82 (br d, J=8.4 Hz, 1H), 7.67 (d, J=8.4 Hz, 1H), 7.58- 7.53 (m, 2H), 7.33 (dd, J=8.4, 2.1 Hz, 1H), 6.80-6.76 (m, 2H), 6.76-6.72 (m, 1H), 5.14-5.07 (m, 1H), 4.81 (dd, J=15.2, 7.2 Hz, 1H), 4.67 (dd, J=15.3, 2.8 Hz, 1H), 4.51-4.44 (m, 1H), 4.37 (ddd, J=8.9, 5.9 , 5.9 Hz, 1H), 3.97 (d, J=13.6 Hz, 1H), 3.87 (s, 3H), 3.78 (d, J=13.5 Hz, 1H), 3.02 (br d, J =11.1 Hz, 1H), 2.86 (br d, J=11.3 Hz, 1H), 2.74-2.60 (m, 2H), 2.48-2.41 (m, 1H), 2.29-2.22 (m, 1H) , 2.21-2.14 (m, 1H), 2.02 (s, 3H), 1.83-1.73 (m, 2H), 1.73-1.64 (m, 2H).

Step 2. Synthesis of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1- [(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid (7, free acid).

A mixture of C53 (8.40 g, 14.0 mmol) in methanol (135 ml) was heated to 45°C and treated with aqueous sodium hydroxide solution (1 M; 27.7 ml, 27.7 mmol). After 20 hours the reaction mixture was concentrated in vacuo to half of its original volume. The resulting mixture was diluted with water (100 ml) and an aqueous solution of citric acid (1 M, 15 ml) was used to adjust the pH to 5-6. The resulting solid was filtered off, washed with water (2x15 ml) and transferred to a separating funnel as a solution in ethyl acetate (50 ml); this way the residual water was removed. The organic layer was dried over magnesium sulfate, filtered, combined with four batches previously prepared in a similar manner (the amount of C53 used in these reactions was 987 mg, 1.63 mmol; 1.15 g, 1.90 mmol; 8.57 g, 14.1 mmol; and 12.6 g, 20.8 mmol) and concentrated in vacuo. The resulting sticky solid was treated with 10% ethyl acetate in heptane (500 ml). After 4 h, the solid was collected by filtration and washed with 10% ethyl acetate in heptane (2x25 ml) to give the 7 free acid as a white solid. Yield 29.4 g, 0.527 mmol, 74% for combined reactions. LC-MS 592.2 ♦ [M+H]+.

1H NMR (600 MHz, DMSO-d ₆ ): δ 12.74 (br s, 1H), 8.28 (s, 1H), 7.80 (br d, J=8.4 Hz, 1H), 7.64 (d, J=8 .4 Hz, 1H), 7.59-7.52 (m, 2H), 7.33 (dd, J=8.4, 2.1 Hz, 1H), 6.81-6.76 (m, 2H), 6.76-6.72 (m, 1H), 5.14-5.07 (m, 1H), 4.79 (dd, J=15.3, 7.3 Hz, 1H), 4.65 (dd, J=15.2, 2.8 Hz, 1H), 4.51-4.45 (m, 1H), 4.38 (ddd, J=9.0, 5.9, 5.9 Hz, 1H), 3.96 (br d, J=13.6 Hz, 1H), 3.78 (br d, J=13.5 Hz, 1H), 3.02 ( br d, J=11.1 Hz, 1H), 2.86 (br d, J=11.1 Hz, 1H), 2.74-2.60 (m, 2H), 2.48-2.41 (m, 1H), 2.29-2.21 ( m, 1H), 2.21-2.14 (m, 1H), 2.02 (s, 3H), 1.83-1.74 (m, 2H), 1.74-1.64 (m, 2H).

This substance was determined to have the same absolute configuration as the above compound of example 7 by comparing its biological activity with that of both 6 and 7: in assay 2, this sample of compound 7, a free acid, showed an EC ₅₀ of 4.3 nM (geometric mean of 3 repetitions). The activity in assay 2 for the ammonium salt of the compound of example 6 and the compound of example 7 was 2400 nM (geometric average of 5 repetitions) and 2.9 nM (geometric average of 8 repetitions), respectively.

Synthesis of 7S-1. Synthesis of the compound of Example 7,1,3-dihydroxy-2-(hydroxymethyl)propan-2-aminium salt 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1 ,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazol-6-carboxylate 1,3-ihydroxy-2-(hydroxymethyl)propane -2-aminium (7,1,3-dihydroxy-2-(hydroxymethyl) propane-2-aminium salt)

7,1,3-dihydroxy-2(hydroxymethyl)propane-2-aminium salt

A mixture of 7, the free acid, (2.00 g, 3.38 mmol) in tetrahydrofuran (16 ml) was treated with an aqueous solution of 2-amino-2-(hydroxymethyl)propane-1,3-diol (Tris, 1.0 M; 3.55 ml, 3.55 mmol). After 18 h the reaction mixture was concentrated in vacuo and treated with ethanol (30 ml). After stirring this mixture for 23 h, the solid was collected by filtration and washed with ethyl acetate (2x10 ml) to give 7,1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt, as a white solid. Yield: 1.41 g, 1.98 mmol, 59%. LC-MS m/z 592.3^ [M+H] ⁺ .

1H NMR (600 MHz, DMSO-d ₆ ), characteristic peaks: δ 8.20 (s, 1H), 7.79 (d, J=8.4 Hz, 1H), 7.597.52 (m, 3H), 7.33 (br d , J=8.5 Hz, 1H), 6.81-6.72 (m, 3H), 5.14-5.07 (m, 1H), 4.76 (dd, J=15.2, 7.2 Hz, 1H), 4.63 (br d, J=15.4Hz, 1H), 4.50-4.44(m, 1H), 4.37(ddd, J=8.9, 5.9, 5.9Hz, 1H), 3.94(d, J=13.4Hz, 1H) , 3.76 (d, J=13.4 Hz, 1H), 3.01 (br d, J=11.1 Hz, 1H), 2.86 (br d, J=11.2 Hz, 1H), 2.73-2.60 (m , 2H), 2.5-2.41 (m, 1H), 2.27-2.20 (m, 1H), 2.20-2.13 (m, 1H), 2.02 (s, 3H), 1.83-1.64 (m, 4H). So pl. = 175-180°C.

Synthesis of 7S-2. Alternative Synthesis of Example Compound 7,1,3-dihydroxy-2(hydroxymethyl)propane-2-aminium salt. A 3.3 M solution of 2-amino-2-(hydroxymethyl)-1,3-propanediol (1.0 eq., 1.93 L) in water was added to a solution of 7, free acid, (3.74 kg) in isopropanol (20 l) at 65°C. Additional isopropanol (19 L) was added, then methanol (19 L) was added while maintaining the temperature at 65°C. The mixture was slowly cooled to 45°C over 2 h, then held at 45°C for

- 76 040816 at least 12 hours. The mixture was then cooled to 5°C for 3 hours, then kept at 5°C for at least 3 hours. The mixture was then filtered and the solid was collected and washed with ethyl acetate (2x10 ml) to obtain 7,1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt, as a white solid (yield: 3.64 kg, 80.9%). Received LC-MS and 1H NMR data, which are essentially the same as in the synthesis of 7S-1 presented above.

Preparation of X-ray powder diffraction (XRD) data for Form I of the compound of Example 7, 1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt.

The white solid Tris salt of the compound of Example 7 (from Synthesis 7S-1 and Synthesis 7S-2) was subjected to XRD analysis and found to be a crystalline solid (which is designated Form I of this anhydrous crystalline form). Powder X-ray diffraction analysis was performed using a Bruker AXS D8 Endeavor diffractometer equipped with a Cu-radiation source. The divergence slit was set to 15 mm continuous illumination. The diffracted radiation was detected by a PSD-Lynx Eye detector with the PSD (Position Sensitive Detector) detector window set to 2.99 degrees. The voltage and current on the X-ray tube were set to 40 kV and 40 mA, respectively. Data were collected at Cu wavelength (CuKa = 1.5418 λ) in a theta-theta goniometer from 3.0 to 40.0 degrees 2-theta in 0.01 degree steps and 1.0 s time steps. The anti-scatter shield was set to 1.5 mm. Samples were rotated to collect data. Samples were prepared by placing them in a low background silicone holder and rotating to collect data.

Data was collected using DIFFRAC Plus software and analysis was performed using EVA diffract plus software. The XRD data file was not processed prior to the search for peaks. Using the peak search algorithm in the EVA software, peaks selected with a threshold value of 1 were used to create peak pre-assignments. To ensure reliability, adjustments were made manually; The output of the automated assignments was checked visually and the peak positions were corrected for the maximum peak magnitude. As a rule, peaks with a relative intensity of >3% were chosen. Typically, peaks that were not resolved or did not match the noise were not selected. The typical error associated with the position of the peak in the XRD pattern specified in the USP is up to +/- 0.2° 2-theta (USP-941). One diffraction pattern was observed stably and is shown in FIG. 1. The list of diffraction peaks, expressed in degrees 2θ, and relative intensities with a relative intensity of >3.0% XRD from the sample obtained as a result of the synthesis of 7S-2, is presented above in table. x1.

Table X1

Angle (2-theta) 3.7 7.3 8.1 8.5 Yu,1 13.6 14.7 15.2 15.5 15.9 16.9 17.4 17.7 17.9 18.9

Relative intensity (%) 74.3 83.3 12.5 6.5 6.6 3.5 49.8 7.9 28.7 18.3 60.8 26.3 I,4 13.5 75.4

- 77 040816

19.7 18.7 20.2 100.0 20.9 24.8 21.5 14.8 22.2 31.7 22.9 10.1 23.5 34.6 23.7 8.2 24.4 6.5 24.9 8.7 25.2 6.4 25.9 14.7 26.4 48.6 26.7 12.5 27.5 15.8 27.9 6.1 28.3 10.5 29.5 15.5 29.8 12.6 30.2 12.1 30.9 3.4 31.7 16.4 33.3 17.2 34.0 14.9 35.8 4.8 37.5 3.2 38.6 5.3

Examples 8 and 9.

2-({4-[2-(4-Cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X1 (8) [from C56]; and 2-({4-[2-(4-cyano2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)-oxetan- 2-ylmethyl]-N-benzimidazole-6-carboxylic acid, DIAST-X2 (9) [from C57]

NC

CH ₃

C56 C57

Step 1. Synthesis of 3-fluoro-4-[2-methyl-4-(piperidin-4-yl)-1,3-benzodioxol-2-yl]benzonitrile p-toluenesulfonate salt (C54).

To a solution of P4 (161 mg, 0.367 mmol) in ethyl acetate (8 ml) was added para-toluenesulfonic acid (158 mg, 0.919 mmol) and the reaction mixture was stirred at 65°C for 16 hours. C54 was obtained in vacuo as a dark yellow gum. This substance was directly transferred to the next stage.

Step 2. Synthesis of methyl 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[( 2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate (C55).

To a solution of C54 (from the previous step; <0.367 mmol) in acetonitrile (3.7 mL) was added potassium carbonate (219 mg, 1.58 mmol) followed by P15 (115 mg, 0.390 mmol). This reaction mixture was stirred at 50°C for 20 h, after which it was diluted with ethyl acetate (10 ml) and filtered. The filter cake was washed with ethyl acetate (3x10 ml) and the combined filtrates were concentrated in vacuo. Chromatography on silica gel (gradient: 0 to 100% ethyl acetate in petroleum ether) gave C55 as a dark yellow oil. Yield: 191.0 mg, 0.320 mmol, 87% over 2 steps. LC-MS m/z 619.1 [M+Na+].

Step 3. Isolation of methyl 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[( 2S)-oxetan-2-ylmethyl] -1 H-benzimidazole-6-carboxylate ENT -1 (C56) and methyl-2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl- 1,3-Benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate ENT-2 (C57).

Separation of C55 (191 mg, 0.320 mmol) into its constituent dioxolane stereoisomers was performed by SFC [column: Chiral Technologies ChiralCel OD, 5 μm; mobile phase: 3:2 carbon dioxide/2-propanol]. The first eluted isomer, which was obtained as a white gum, was designated ENT-1 (C56). Yield: 114 mg; this substance contained residual ethanol. LC-MS m/z 597.1 [M+H] ⁺ . Retention time 4.40 min (column: Chiral Technologies ChiralCel OD-3, 4.6x100 mm, 3 µm; mobile phase A: carbon dioxide; mobile phase B: 2-propanol containing 0.05% diethylamine; gradient: from 5 to 40% B in 4.5 min, then hold at 40% B for 2.5 min; flow rate: 2.8 ml per minute).

The second eluting isomer was again purified using SFC [column: Chiral Technologies ChiralCel OD, 5 μm; mobile phase: 55:45 carbon dioxide/(2-propanol containing 0.1% ammonium hydroxide)] to give a colorless gum which was designated ENT-2 (C57). Yield: 50 mg, 83.8 µmol, 26%. LC-MS m/z 597.1 [M+H] ⁺ . Retention time 4.74 min (analytical conditions identical to those used for C56).

Step 4. Synthesis of 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S) -oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X1 (8) [from C56].

A solution of C56 (114 mg, 0.191 mmol) in acetonitrile (10 ml) was treated with an aqueous solution of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.97 M, 394 µl, 0.382 mmol) and this reaction mixture was stirred at room temperature for 23 hours. An aqueous solution of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (0 .97 M, 394 μl, 0.382 mmol) and stirring was continued for 6 h, after which the pH was carefully adjusted to 7-8 by adding 1 M hydrochloric acid. After removal of volatiles in vacuo, purification was performed using reverse phase HPLC (column: Agela Durashell C18, 5 µm; mobile phase A: 0.05% ammonium hydroxide in water; mobile phase B: acetonitrile; gradient: 30 to 50% C) to give 8 as a white solid. Yield: 22.2 mg, 38.1 µmol, 20%. LC-MS m/z 583.3 [M+H] ⁺ .

¹ H NMR (400 MHz, methanol^): δ 8.19 (d, J=1.4 Hz, 1H), 7.94 (dd, J=8.4, 1.5 Hz, 1H), 7.77 (dd, J=7.7, 7.7Hz, 1H), 7.64(dd, J=10.6, 1.6Hz, 1H), 7.58(d, J=8.4Hz, 1H), 7.57(dd, J=8.0, 1.5Hz , 1H), 6.81-6.75 (m, 1H), 6.75-6.68 (m, 2H), 5.34-5.25 (m, 1H), 4.73 (dd, J=15.3, 3.0 Hz, 1H), 4.67-4.59 (m, 1H), 4.49 (dt, J=9.2, 6.0 Hz, 1H), 3.96 (AB quartet, J _AB = 13.7 Hz, Av _ab = 41.2 Hz, 2H), 3.06 (br d , J=11 Hz, 1H), 2.95 (br d, J=11 Hz, 1H), 2.87-2.76 (m, 1H), 2.71 (tt, J=12.0, 3.9 Hz, 1H), 2.61-2.50 (m, 1H), 2.36-2.21 (m, 2H), 2.06 (s, 3H), 1.951.72 (m, 4H).

Step 5. Synthesis of 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S) -oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2 (9) [from C57].

A solution of C57 (50 mg, 84 µmol) in acetonitrile (10 ml) was treated with an aqueous solution of 1,3,4,6,7,8hexahydro-2H-pyrimido[1,2-a]pyrimidine (0.97 M; 173 µl, 0.168 mmol). This reaction mixture was stirred at room temperature (about 25° C.) for 16 hours after which time an additional amount of an aqueous solution of 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine was added. (0.97 M; 173 μl, 0.168 mmol) and stirring was continued at 25° C. for 29 hours. The reaction mixture was then carefully adjusted to pH 7-8 with 1 M hydrochloric acid. The resulting mixture was concentrated in vacuo and subjected to reverse phase HPLC (column: Xtimate™ C18, 5 µm; mobile phase A: 0.05% ammonium hydroxide in water; mobile phase B: acetonitrile; gradient: 27 to 67% B) c obtaining 9 as a white solid. Yield: 18.0 mg, 30.9 µmol, 37%. LC-MS m/z 583.3 [M+H] ⁺ .

1H NMR (400 MHz, methanol-d ₄ ) δ 8.36-8.33 (m, 1H), 7.97 (dd, J=8.5, 1.5 Hz, 1H), 7.78 (dd, J=7.7, 7.7 Hz, 1H), 7.70-7.63 (m, 2H), 7.57 (dd, J=8.0, 1.5 Hz, 1H), 6.83-6.76 (m, 1H), 6.76-6.71 (m, 2H), 5.34-5.25 ( m, 1H), 4.95-4.85 (m, 1H, hypothetical; partially obscured by water peak), 4.73 (dd, ABX system component, J=15.3, 2.7 Hz, 1H), 4.68-4.60 (m, 1H), 4.50 (dt, J=9.2, 6.0 Hz, 1H), 4.02 (AB quartet, J _AB = 13.8 Hz, Av _ab =

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48.2 Hz, 2H), 3.13 (br d, J=11 Hz, 1H), 3.01 (br d, J=11.5 Hz, 1H), 2.89-2.78 (m, 1H), 2.78-2.68 (m , 1H), 2.602.50 (m, 1H), 2.45–2.30 (m, 2H), 2.07 (br s, 3H), 2.00–1.86 (m, 2H), 1.83 (m, 2H).

Example 10

2-({4-[2-(5-Chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2 (10) [from P9] but J ³ ci

H ₃

P9

C58

Stage 1. Synthesis of 5-chloro-2-[2-methyl-4-(piperidin-4-yl)-1,3-benzodioxol-2-yl]pyridine, ENT-X2, p-toluenesulfonate salt (C58) [from P9].

A solution of P9 (228 mg, 0.529 mmol) in ethyl acetate (2.7 ml) was treated with p-toluenesulfonic acid monohydrate (116 mg, 0.610 mmol) and the reaction mixture was heated at 50° C. for 16 h. It was then stirred at room temperature in overnight, after which the precipitate was collected by filtration and washed with a mixture of ethyl acetate and heptane (1:1, 2x20 ml) to obtain C58 as a white solid. Yield: 227 mg, 0.451 mmol, 85%. LC-MS m/z 331.0^ [M+H]+.

1H NMR (400 MHz, DMSO-d6): δ 8.73 (d, J=2.4 Hz, 1H), 8.61-8.46 (br m, 1H), 8.35-8.18 (br m, 1H), 8.02 (dd, J=8.5, 2.5Hz, 1H), 7.64 (d, J=8.5Hz, 1H), 7.47 (d, J=7.8, 2H), 7.11 (d, J=7.8Hz, 2H) , 6.89-6.81 (m, 2H), 6.72 (pentet, J=4.0 Hz, 1H), 3.45-3.27 (m, 2H, hypothesized; partially obscured by water peak), 3.10-2.91 (m, 3H), 2.28 (s, 3Н), 2.02 (s, 3Н), 1.97–1.80 (m, 4Н).

Stage 2. Synthesis of methyl-2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[( 2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate, DIAST-Y2 (C59) [from P9].

N,N-Diisopropylethylamine (0.234 ml, 1.34 mmol) was added to a solution of C58 (225 mg, 0.447 mmol) in acetonitrile (2.2 ml). After stirring this mixture for 5 min at 45°C, P15 (120 mg, 0.407 mmol) was added and stirring was continued at 45°C for 16 h, after which P15 (11 mg, 37 µmol) was added again. After stirring for a further 3 h, the reaction mixture was treated with water (2.5 ml) and left to cool to room temperature. More water (5 ml) was added and the resulting suspension was stirred for 2 h, after which the solid was collected by filtration and washed with a mixture of acetonitrile and water (15:85, 3 x 5 ml) to give C59 as an off-white solid (252 mg). This material contained some N,N-diisopropylethylamine as determined by 1H NMR analysis and was carried directly to the next step. LC-MS m/z 589.1 ♦ [M+H] ⁺ .

¹ H NMR (400 MHz, chloroform-d) 8.61 (d, J=2.3 Hz, 1H), 8.18 (d, J=1.5 Hz, 1H), 7.96 (dd, J=8.5, 1.5 Hz, 1H), 7.74 (d, J=8.5 Hz, 1H), 7.67 (dd, ABX system component, J=8.4, 2.4 Hz, 1H), 7.59-7.51 (m, 1H), 6.826. 75 (m, 1H), 6.74-6.66 (m, 2H), 5.28-5.19 (m, 1H), 4.75 (dd, ABX component, J=15.3, 6.0 Hz, 1H), 4.68 (dd, component ABX system, J=15.3, 3.4 Hz, 1H), 4.67-4.58 (m, 1H), 4.41 (ddd, J=9.1, 5.9, 5.9 Hz, 1H), 3.95 (s, 2H), 3.95 (s, 3Н), 3.07-2.89 (m, 2H), 2.81-2.69 (m, 2H), 2.53-2.41 (m, 1H), 2.37-2.22 (m, 2H), 2.05 (s, 3Н), 1.93 -1.74 (m, 4H).

Stage 3. Synthesis of 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(2S) -oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2 (10) [from P9].

A suspension of C59 (from the previous step; 250 mg, <0.407 mmol) in methanol (2 ml) was heated to 40°C after which aqueous sodium hydroxide solution (1 M; 0.81 ml, 0.81 mmol) was added. After 17 hours the reaction mixture was allowed to cool to room temperature and the pH was adjusted to 5-6 with 1M aqueous citric acid. The resulting mixture was diluted with water (2 ml), stirred for 2 h and was extracted with ethyl acetate (3x5 ml). The combined organic layers were washed with saturated aqueous sodium chloride (5 ml), dried over sodium sulfate, filtered and concentrated in vacuo to give a foamy solid. This material was taken up in a mixture of ethyl acetate and heptane (1:1.4 ml), heated to 50°C and then left to cool with stirring overnight.

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After filtration, 10 was obtained as a white solid. Yield: 179 mg, 0.311 mmol, 76% over 2 steps. LC-MS m/z 575.1 ♦ [M+H]+.

¹ H NMR (400 MHz, DMSO-d6) δ 12.73 (br s, 1H), 8.71 (d, J=2.5 Hz, 1H), 8.27 (d, J=1.5 Hz, 1H), 8.00 ( dd, J=8.5, 2.5Hz, 1H), 7.80 (dd, J=8.4, 1.6Hz, 1H), 7.64 (d, J=8.4Hz, 1H), 7.60 (d, J= 8.5 Hz, 1H), 6.83-6.72 (m, 3H), 5.14-5.06 (m, 1H), 4.77 (dd, ABX system component, J=15.2, 7.2 Hz, 1H), 4.63 (dd, component of the ABX system, J=15.2, 2.8 Hz, 1H), 4.50-4.42 (m, 1H), 4.37 (ddd, J=9.0, 5.9, 5.9 Hz, 1H), 3.85 (AB quartet, JAB = 13.6 Hz, ΔνAB = 71.5 Hz, 2H), 3.01 (br d, J=11.2 Hz, 1H), 2.85 (br d, J=11.2 Hz, 1H), 2.74-2.57 (m , 2H), 2.472.38 (m, 1H), 2.29–2.10 (m, 2H), 2.01 (s, 3H), 1.81–1.64 (m, 4H).

Synthesis of 10S-1. Synthesis of the compound of Example 10,1,3-dihydroxy-2-(hydroxymethyl)propane-2aminium salt 4-yl]piperidin-1yl}methyl)-1-[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate of 1,3-dihydroxy-2(hydroxymethyl)propan-2-aminium, DIAST- X2 (10,1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt) [from P9].

10,1,3-dihydroxy-2(hydroxymethyl)propane-2-aminium salt

A mixture of 10 (1.54 g, 2.68 mmol) in tetrahydrofuran (10 ml) was treated with an aqueous solution of 2-amino2-(hydroxymethyl)propane-1,3-diol (Tris, 1.0 M; 2.81 ml, 2 .81 mmol). After 24 h the reaction mixture was concentrated in vacuo with ethanol (2x50 ml). The residue was treated with ethanol (15 ml). After stirring for 20 h, the solid was collected by filtration and washed with cold ethanol (5 ml) to give 10,1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt, as a white solid. Yield: 1.41 g, 2.03 mmol, 76%. LC-MS m/z 575.3^ [M+H] ⁺ .

1H NMR (600 MHz, DMSO-d ₆ ) δ 8.71 (d, J=2.5 Hz, 1H), 8.21 (br s, 1H), 8.00 (dd, J=8.5, 2.5 Hz, 1H), 7.79 (br d, J=8.4 Hz, 1H), 7.60 (d, J=8.5 Hz, 1H), 7.57 (d, J=8.4 Hz, 1H), 6.82-6.73 (m, 3H ), 5.13-5.07 (m, 1H), 4.74 (dd, J=15.3, 7.2 Hz, 1H), 4.61 (dd, J=15.3, 2.9 Hz, 1H), 4.49-4.43 (m, 1H ), 4.37 (ddd, J=9.0, 5.9, 5.9 Hz, 1H), 3.93 (d, J=13.6 Hz, 1H), 3.75 (d, J=13.5 Hz, 1H), 3.01 ( br d, J=11.3 Hz, 1H), 2.86 (br d, J=11.4 Hz, 1H), 2.732.59 (m, 2H), 2.48-2.37 (m, 1H), 2.27-2.20 ( m, 1H), 2.19-2.12 (m, 1H), 2.01 (s, 3H), 1.82-1.66 (m, 4H). So pl. = 184-190°C.

Synthesis of 10S-2. Alternative synthesis of the compound of Example 10,1,3-dihydroxy-2(hydroxymethyl)propane-2-aminium salt.

A mixture of 10 (8.80 g, 15.3 mmol) in 2-methyltetrahydrofuran (90 ml) was concentrated in vacuo on a rotary evaporator in a water bath at 37°C to reduce the total volume to about 54 ml. Isopropanol (90 ml) was added to the mixture and then the resulting mixture was again concentrated to a volume of about 54 ml. Isopropanol (135 ml) was added to the mixture, then aqueous tris-amine (3M; 5.0 ml, 0.98 eq.) was added. The resulting mixture/solution was stirred at ambient temperature and a solid precipitated out within about 15 minutes.

The mixture was then stirred at ambient temperature for an additional 5 hours. The resulting mixture/slurry was cooled to 0°C and the cooled suspension was stirred for about another 2 hours. The suspension was filtered and washed with cold isopropanol (3x15 ml). The collected solid was allowed to air dry on a collecting funnel for about 90 minutes and then transferred to a vacuum oven to dry overnight. After about 16 hours at a vacuum of 50°C/23 mmHg. Art. (with a light flush of nitrogen) 8.66 g of 10,1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt was obtained as a white solid; 99.8% area by UPLC (Ultra Performance Liquid Chromatography) (yield: 12.5 mmol, 81%). LC-MS and 1H NMR data were obtained which are essentially the same as those given above in the 10S-1 synthesis.

Preparation of X-Ray Powder Diffraction (XRD) Data for Example 10 Form A of 1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium Salt (also known as Example 10 Anhydrous Tris Salt Form A)

The white solid Tris salt of the compound of Example 10 (from Synthesis 10S-1 and Synthesis 10S-2) was subjected to XRD analysis and found to be a crystalline compound (which is designated Form A). Powder X-ray diffraction analysis was performed using a Bruker AXS D8 Endeavor diffractometer equipped with a Cu-radiation source. The divergence slit was set to 15 mm continuous illumination. The diffracted radiation was detected by a PSD-Lynx Eye detector with the PSD (Position Sensitive Detector) detector window set to 2.99 degrees. The voltage and current on the x-ray tube were set

- 81 040816 for 40 kV and 40 mA respectively. Data was collected at Cu wavelength (CuKa = 1.5418 λ) in a theta-theta goniometer from 3.0 to 40.0 degrees 2-theta in 0.01 degree steps and 1.0 second time steps. The anti-scatter shield was set to 1.5 mm. Samples were rotated to collect data. Samples were prepared by placing them in a low background silicone holder and rotating to collect data. Data was collected using DIFFRAC Plus software and analysis was performed using EVA diffract plus software. The XRD data file was not processed prior to the search for peaks. Using the peak search algorithm in the EVA software, peaks selected with a threshold value of 1 were used to create peak pre-assignments. To ensure reliability, adjustments were made manually; The output of the automated assignments was checked visually and the peak positions were corrected for the maximum peak magnitude. As a rule, peaks with a relative intensity of >3% were selected. Typically, peaks that were not resolved or did not match the noise were not selected. The typical error associated with the position of the peak in the XRD pattern specified in the USP is up to +/- 0.2° 2-theta (USP-941). One diffraction pattern was observed stably and is shown in FIG. 1. The list of diffraction peaks, expressed in degrees 2θ, and relative intensities with a relative intensity of >3.0% XRD from the sample obtained as a result of the synthesis of 7S-2, is presented in table. X2.

Example 11.

1-(2-Methoxyethyl)-2-({4-[2-methyl-2-(pyridin-3-yl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)1H-benzimidazole -6-carboxylic acid formate salt (11)

- 82 040816

All of this synthesis sequence was carried out in a library format.

Step 1. Synthesis of methyl 1-(2-methoxyethyl)-2-({4-[2-methyl-2-(pyridin-3-yl)-1,3-benzodioxol-4yl]piperidin-1-yl}methyl )-1 H-benzimidazole-6-carboxylate (С60).

A mixture of P14 (44 mg, 100 µmol) and 3-ethynylpyridine (21 mg, 200 µmol) in toluene (800 µl) was treated with sodium bicarbonate (100 µmol) followed by triruthenium dodecacarbonyl (6 mg, 9 µmol). The reaction vessel was then closed and shaken at 120° C. for 16 hours. After removal of the solvent using a Speedvac® concentrator, compound C60 was obtained, which was directly transferred to the next stage.

Stage 2. Synthesis of 1-(2-methoxyethyl)-2-({4-[2-methyl-2-(pyridin-3-yl)-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)- 1H-benzimidazole-6-carboxylic acid formate salt (11).

An aqueous solution of sodium hydroxide (1.0 M; 200 μl, 200 μmol) was added to a solution of C60 (from the previous step, <100 μmol) in a mixture of methanol (400 μl) and tetrahydrofuran (400 μl). The reaction vessel was closed and shaken at 80°C for 16 h, after which the reaction mixture was evaporated using a Speedvac® concentrator and purified using reverse phase HPLC (column: Agela Durashell C18, 5 μm; mobile phase A: 0.225% formic acid in water mobile phase B: acetonitrile gradient: 12 to 52% B) to give 11. Yield: 2.2 mg, 4.2 µmol, 4% over 2 steps. LCMS m/z 529 [M+H]+. Retention time: 2.47 min (column: Waters XBridge C18, 2.1x50 mm, 5 µm; mobile phase A: 0.0375% trifluoroacetic acid in water; mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; gradient: 1 to 5% B in 0.6 min; 5 to 100% B in 3.4 min; flow rate: 0.8 ml per minute).

Example 12.

2-({4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[2(dimethylamino)ethyl ]-1H-benzimidazole-6-carboxylic acid (12)

C64 12

All of this synthesis sequence was carried out in a library format.

Stage 1. Synthesis of methyl 3-{[2-(dimethylamino)ethyl]amino}-4-nitrobenzoate (С61).

Methyl 3-fluoro-4-nitrobenzoate (0.2 M solution in N,N-dimethylformamide; 1 ml, 200 µmol) was treated with N,N-dimethylethane-1,2-diamine (18 mg, 200 µmol) and N, N-diisopropylethylamine (78 mg, 600 µmol). The reaction vessel was then closed and shaken at 50°C for 16 h, after which the reaction mixture was evaporated using a Speedvac® concentrator to give C61. This substance was directly transferred to the next stage.

Stage 2. Synthesis of methyl 4-amino-3-{[2-(dimethylamino)ethyl]amino}benzoate (С62).

Zinc dust was activated with dilute hydrochloric acid. Methanol (2 ml) was added to C61 (from the previous step, <200 µmol), then an aqueous solution of calcium chloride (1.0 M; 200 µl,

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200 µmol) and activated zinc dust (130 mg, 2.0 mmol). The reaction vessel was closed and shaken at 70°C for 16 h, after which the reaction mixture was filtered. The filtrate was concentrated using a Speedvac® concentrator and the residue was taken up in water (2 ml) and then extracted with ethyl acetate (2x3 ml). The combined organic layers were evaporated using a Speedvac® concentrator to give compound C62 (estimated at 150 µmol) which was used directly in the next step.

Step 3. Synthesis of methyl 4-[({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}acetyl)amino]-3 -{[2-(dimethylamino)ethyl]amino}benzoate (C63).

Compound P10 (41 mg, 100 µmol) was added to C62 (from the previous step, approximately 150 µmol) and this mixture was treated with a solution of 2-hydroxypyridine N,N-dimethylacetamide 1-oxide and 1[3-(dimethylamino)propyl]-3 -ethylcarbodiimide hydrochloride (0.1 M each; 1 ml, 100 µmol each). N,N-diisopropylethylamine (39 mg, 300 μmol) was then added and the reaction vessel was sealed and shaken at 50° C. for 16 h. The reaction mixture was then concentrated using a Speedvac® concentrator and purified by preparative thin layer chromatography to give compound C63, which directly transferred to the next stage.

Stage 4. Synthesis of methyl-2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1 -yl}methyl)-1 - [2 -(dimethylamino)ethyl]-1 H-benzimidazole-6-carboxylate (C64).

A mixture of acetic acid (500 μl) and C63 (from the previous step, <100 μmol) was shaken in a closed vessel at 150°C for 2 h, after which the reaction mixture was evaporated using a Speedvac® concentrator. The resulting compound C64 was directly transferred to the next step.

Step 5. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1 -[2-( dimethylamino)ethyl] -1 H-benzimidazole-6-carboxylic acid (12).

A solution of C64 (from the previous step, <100 µmol) in ethanol (500 µl) was treated with an aqueous solution of lithium hydroxide (2.0 M; 500 µl, 1 mmol) and the reaction mixture was shaken at 50°C for 2 h in a sealed closed vessel. After adjusting the pH of the mixture to 7 with 1.0 M hydrochloric acid, the resulting mixture was concentrated using a Speedvac® concentrator and then purified by reverse phase HPLC [column: Agela Durashell C18, 5 μm; mobile phase A: ammonium hydroxide in water (pH 10); mobile phase B: acetonitrile; gradient: 25 to 65% B] to give 12. Yield: 7.0 mg, 12 µmol, 12% over 3 steps. LC-MS m/z 593 [M+H]+. Retention time: 2.45 min (column: Waters XBridge C18, 2.1 x 50 mm, 5 µm; mobile phase A: 0.0375% trifluoroacetic acid in water; mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; gradient: 10 to 100% B in 4.0 min; flow rate: 0.8 ml per minute).

Example 13

2-({4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-3-(1,3oxazol-2 -ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylic acid (13)

Stage 1. Synthesis of methyl 6-[(1,3-oxazol-2-ylmethyl)amino]-5-nitropyridine-2-carboxylate (C65).

Triethylamine (532 mg, 5.26 mmol) was added to a suspension of 1-(1,3-oxazol-2yl)methanamine hydrochloride salt (236 mg, 1.75 mmol) and methyl 6-chloro-5-nitropyridine-2-carboxylate (386 mg, 1.78 mmol) in tetrahydrofuran (5 ml). After stirring the reaction mixture at 25° C. for 14 h, it was poured into water (30 ml) and extracted with dichloromethane (2x50 ml). The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo. After chromatography on silica gel

- 84 040816 (gradient: 0 to 5% methanol in dichloromethane) gave C65 as a yellow solid. Yield: 310 mg, 1.11 mmol, 63%. LC-MS m/z 278.7 [M+H]+.

¹ H NMR (400 MHz, chloroform-d) δ 8.69-8.61 (br m, 1H), 8.58 (d, J=8.4 Hz, 1H), 7.65 (d, J=0.8 Hz, 1H), 7.46 (d, J=8.4Hz, 1H), 7.11 (d, J=1.0Hz, 1H), 5.07 (d, J=5.3Hz, 2H), 3.97 (s, 3H).

The rest of the synthesis sequence was carried out in library format.

Stage 2. Synthesis of methyl 5-amino-6-[(1,3-oxazol-2-ylmethyl)amino]pyridine-2-carboxylate (C66).

An aqueous solution of ammonium chloride (5.0 M; 400 µl, 2.0 mmol), then activated zinc (131 mg, 2.0 mmol) was added to a solution of C65 (56 mg, 200 µmol) in methanol (2.0 ml) . The reaction vessel was then closed and shaken at 30°C for 16 h, after which the reaction mixture was filtered. The filtrate was concentrated using a Speedvac® concentrator, then mixed with water (1.0 ml) and extracted with dichloromethane (3x1.0 ml). The combined organic layers were evaporated using a Speedvac® concentrator to give compound C66, which was taken directly to the next step.

Step 3. Synthesis of methyl 5-[({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}acetyl)amino]-6 -[(1,3-oxazol-2-ylmethyl)amino]pyridine-2-carboxylate (C67).

A mixture of P10 (81 mg, 200 µmol) and C66 (from the previous step, <200 µmol) was mixed with N,N-dimethylacetamide and then treated with N,N-diisopropylethylamine (100 µl, 600 µmol). A solution containing 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (0.24 M) and 2-hydroxypyridine 1 oxide (0.1 M) in N,N-dimethylacetamide (1.0 ml containing 240 µmol 1-[3(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride and 100 µmol 2hydroxypyridine 1-oxide), and the reaction vessel was closed and shaken at 50° C. for 16 hours. The volatiles were then removed using a Speedvac® concentrator, and the residue was subjected to preparative thin layer chromatography to give compound C67, which was directly carried to the next step.

Stage 4. Synthesis of methyl-2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidinyl}methyl)-3-(1,3 -oxazol-2-ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylate (C68).

A mixture of acetic acid (1.0 ml) and C67 (from the previous step, <200 μmol) was shaken at 150°C for 2 h, after which the reaction mixture was evaporated using a Speedvac® concentrator. The resulting compound C68 was directly used in the next step.

Step 5. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-3-(1,3 -oxazol-2-ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylic acid (13).

An aqueous solution of lithium hydroxide (2.0 M; 1.0 ml, 2.0 mmol) was added to a mixture of C68 (from the previous step, <200 μmol) in tetrahydrofuran (1.0 ml). After addition of methanol (500 μl), the reaction vessel was closed and shaken at 50° C. for 16 hours. After removal of volatiles using a Speedvac® concentrator, dimethyl sulfoxide (1.0 ml) was added and the pH was adjusted to 7-8 with concentrated hydrochloric acid. The resulting mixture was purified using reverse phase HPLC [column: Agela Durashell C18, 5 μm; mobile phase A: ammonium hydroxide in water (pH 10); mobile phase B: acetonitrile; gradient: 24 to 64% B] to give 13. Yield: 3.9 mg, 6.5 µmol, 3% over 4 steps. LC-MS m/z 604 [M+H] ⁺ . Retention time: 3.14 min (column: Waters XBridge C18, 2.1 x 50 mm, 5 µm; mobile phase A: 0.0375% trifluoroacetic acid in water; mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; gradient: 1 to 5% B in 0.6 min; 5 to 100% B in 3.4 min; flow rate: 0.8 ml per minute).

Example 14

2-({4-[(2S)-2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1methyl-1H- benzimidazole-b-carboxylic acid (14)

SNA

O-CH3

CH ₃ CH;

CH ₃ N

Stage 1. Synthesis of methyl-2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4yl]piperidin-1 -yl}methyl)- 1-methyl-1 H-benzimidazole-6-carboxylate (C69).

N,N-Diisopropylethylamine (683 μl, 3.92 mmol) was added to a mixture of P3 (680 mg, 1.31 mmol) in acetonitrile (5.2 ml). This mixture was stirred for 5 min at 45°C after which P16 (319 mg, 1.34 mmol) was added. Stirring was continued at 45° C. for 2.75 h and then water (6 ml) was added,

- 85 040816 after which the reaction mixture was left to cool to room temperature and with stirring for 30 minutes. The solid was collected by filtration and washed with a mixture of acetonitrile and water (1:4, 3x5 ml) to give C69 as a white solid. Yield: 635 mg, 1.15 mmol, 88%. LCMS m/z 550.1 ♦ [M+H]+.

1H NMR (400 MHz, chloroform-d) δ 8.15-8.12 (m, 1H), 7.97 (dd, J=8.5, 1.6 Hz, 1H), 7.74 (d, J=8.5 Hz, 1H), 7.50 (dd, J=8.2, 8.2 Hz, 1H), 7.16-7.07 (m, 2H), 6.79-6.73 (m, 1H), 6.72-6.65 (m, 2H), 3.98 (s, 3H), 3.96 (s, 3H), 3.88 (s, 2H), 3.04-2.93 (m, 2H), 2.76-2.66 (m, 1H), 2.37-2.25 (m, 2H), 2.04 (br s, 3H), 1.89 -1.78 (m, 4H).

Step 2. Synthesis of 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1- methyl-1H-benzimidazole-6-carboxylic acid (14).

A mixture of C69 (600 mg, 1.09 mmol) in methanol (11 ml) was heated to 45°C and then treated with aqueous sodium hydroxide solution (1 M; 2.2 ml, 2.2 mmol). After 24 hours, the reaction mixture was adjusted to pH 56 with aqueous citric acid (1 M; 1.1 ml) and then diluted with water (10 ml). The resulting mixture was allowed to cool to room temperature and with stirring for 1 h, after which the precipitated solid was collected by filtration and washed with a mixture of methanol and water (1:4; 3x5 ml). This gave 14 as a white solid. Yield: 535 mg, 0.998 mmol, 92%. LC-MS m/z 536.1 ♦ [M+H] ⁺ .

1H NMR (400 MHz, DMSC-d6) δ 8.16 (d, J=1.5 Hz, 1H), 7.81 (dd, J=8.4, 1.6 Hz, 1H), 7.64 (d, J=8.4 Hz, 1H), 7.59-7.52 (m, 2H), 7.33 (dd, J=8.3, 2.1 Hz, 1H), 6.81-6.70 (m, 3H), 3.94 (s, 3H), 3.84 (s, 2H), 3.01-2.91 (m, 2H), 2.70-2.59 (m, 1h), 2.28-2.16 (m, 2H), 2.02 (s, 3H), 1.73 (m, 4H).

Examples 15 and 16 2-{6-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6azaspiro[2.5]oct-1-yl}-1 -(2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid, DIAST-X1, trifluoroacetate salt (15) [from P18 through C71] and 2-{6-[2-(4-chloro-2-fluorophenyl)- 2-methyl-1,3-benzodioxol-4-yl]-6azaspiro[2.5]oct-1-yl}-1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid, DIAST-X2, trifluoroacetate salt ( 16) [from P18 through C72] ^N3 %

С4 ΒΙΝΑΡ θ ⁷ θ ( ^mixture of diastereomers)

С71 (from Р18) С72 (from Р18)

Stage 1. Synthesis of methyl-2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6azaspiro[2.5]oct-1-yl}- 1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate (C70) [from P18].

Mixture of P18 (240 mg, 0.699 mmol), C4 (275 mg, 0.800 mmol), cesium carbonate (455 mg, 1.40 mmol), tris(dibenzylideneacetone)dipalladium(0) (40.0 mg, 43.7 µmol) and 1,1'-binaphthalene-2,2'diylbis(diphenylphosphane) (BINAP; 52.2 mg, 83.8 µmol) in toluene (5 ml) was degassed with nitrogen for 5 min and then stirred at 90°C for 16 h. The reaction mixture was filtered and the filtrate was concentrated in vacuo. Preparative thin layer chromatography (eluent: 1:1 petroleum ether/ethyl acetate) gave C70, a mixture of diastereomers, as a yellow oil. Yield: 165 mg, 0.272 mmol, 39%. LC-MS m/z 628.14 [M+Na ⁺ ].

Step 2. Isolation of methyl-2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6azaspiro[2.5]oct-1-yl}- 1-(2-methoxyethyl)-1H-benzimidazole-6-carboxylate, DIAST-Y1 (C71) [from P18], and methyl-2-{6-[2-(4-chloro-2-fluorophenyl)-2- methyl 1,3-benzodioxol-4-yl]-6-azaspiro[2.5]oct-1-yl}-1-(2methoxyethyl)-1H-benzimidazole-6-carboxylate, DIAST-Y2 (C72) [from P18] .

- 86 040816

Separation of stereoisomers by dioxolane in C70 (165 mg, 0.272 mmol) was performed using SFC

[column: Chiral Technologies Chiralpak AD, 10 µm; mobile phase: 65:35 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting isomer was designated DIAST-Y1 (C71) and the second eluting isomer was designated DIAST-Y2 (C72); both were isolated as a white solid.

C71 Yield: 55.0 mg, 90.7 µmol, 33%. LC-MS m/z 605.9^ [M+H]+. Retention time 4.47 min (column: Chiral Technologies Chiralpak AD-3, 4.6x100 mm, 3 µm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5 to 40 % B for 4.5 min, then hold at 40% B for 2.5 min; flow rate: 2.8 ml per minute).

C72 Yield: 58.0 mg, 95.7 µmol, 35%. LC-MS m/z 628.0^ [M+Na ⁺ ]. Retention time 4.88 min (analytical conditions identical to those used for C71).

Stage 3. Synthesis of 2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6-azaspiro[2.5]oct1-yl}-1- (2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid, DIAST-X1, trifluoroacetate salt (15) [from P18 through C71].

To a solution of C71 (55.0 mg, 90.7 µmol) in a mixture of methanol (2.0 ml) and tetrahydrofuran (1.0 ml) was added an aqueous solution of sodium hydroxide (3 M; 1.0 ml, 3.0 mmol) . After the reaction mixture was stirred at 20° C. for 2 h, the pH was adjusted to 7 with 1 M hydrochloric acid and the resulting mixture was extracted with a mixture of dichloromethane and methanol (10:1, 3x30 ml). The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo. Reverse phase HPLC (column: Boston Green ODS, 5 µm; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: 10 to 95% B) gave 15 as a white solid . Yield: 35.8 mg, 50.7 µmol, 56%.

LC-MS m/z 592.3 ♦ [M+H] ⁺ .

¹ H NMR (400 MHz, methanol^) δ 8.46 (s, 1H), 8.21 (d, J=8.6 Hz, 1H), 7.78 (d, J=8.6 Hz, 1H), 7.54 (dd, J=8.3, 8.3 Hz, 1H), 7.16-7.08 (m, 2H), 6.76 (dd, J=8.2, 8.1 Hz, 1H), 6.55-6.47 (m, 2H), 4.9-4.70 ( m, 2H, hypothesized; partially hidden by water peak), 3.82 (t, J=4.9 Hz, 2H), 3.66-3.56 (m, 1H), 3.50-3.41 (m, 1H), 3.19-3.09 (m, 1H), 3.15 (s, 3H), 3.08-2.99 (m, 1H), 2.63-2.57 (m, 1H), 2.27-2.17 (m, 1H), 2.01 (s, 3H), 1.761.66 (m, 2H), 1.62-1.50 (m, 2H), 1.35-1.26 (m, 1H).

Stage 4. Synthesis of 2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6-azaspiro[2.5]oct1-yl}-1- (2-methoxyethyl)-1H-benzimidazole-6-carboxylic acid, DIAST-X2, trifluoroacetate salt (16) [from P18 through C72].

To a solution of C72 (58.0 mg, 95.7 µmol) in a mixture of methanol (2.0 ml) and tetrahydrofuran (1.0 ml) was added an aqueous solution of sodium hydroxide (3 M; 1.0 ml, 3.0 mmol) . After stirring the reaction mixture at 20° C. for 2 h, the pH was adjusted to 7 with 1 M hydrochloric acid and the resulting mixture was extracted with a mixture of dichloromethane and methanol (10:1, 3x30 ml). The combined organic layers were dried over magnesium sulfate, filtered and concentrated in vacuo. Reverse phase HPLC (column: Boston Green ODS, 5 µm; mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: acetonitrile; gradient: 35 to 95% B) gave 16 as a white solid . Yield: 33.4 mg, 47.3 µmol, 49%. LC-MS m/z 592.2^ [M+H] ⁺ .

1H NMR (400 MHz, methanol-d4) δ 8.53-8.50 (m, 1H), 8.25 (dd, J=8.6, 1.4 Hz, 1H), 7.80 (br d, J=8.6 Hz, 1H) , 7.57 (dd, J=8.4, 8.2Hz, 1H), 7.25 (dd, J=10.8, 2.0Hz, 1H), 7.19 (br dd, J=8.4, 2.1Hz, 1H), 6.77 (dd, J=8.2, 8.1 Hz, 1H), 6.55-6.50 (m, 2H), 4.9-4.72 (m, 2H, hypothesized; partially obscured by water peak), 3.933.80 (m, 2H), 3.68-3.58 (m, 1H), 3.41-3.3 (m, 1H, hypothesized; partially obscured by solvent peak), 3.25 (s, 3H), 3.22-3.12 (m, 1H), 3.07-2.97 (m, 1H), 2.67 (dd, J=8.3, 5.8 Hz, 1H), 2.28-2.17 (m, 1H), 2.01 (d, J=1.0 Hz, 3H), 1.86-1.71 (m, 2H), 1.69- 1.56 (m, 2H), 1.36-1.26 (m, 1H).

Examples 17 and 18.

2-({4-[2-(4-Chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl1H- imidazol-5-yl)methyl]-1H-benzimidazole-6-ammonium carboxylate, ENT-1 (17), and 2-({4-[2-(4-chloro-2fluorophenyl)-2-methyl-1,3 -benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl-1H-imidazol-5yl)methyl]-1H-benzimidazole-6-ammonium carboxylate, ENT-2 (18)

- 87 040816

CH ₃ CH ₃

Step 1. Synthesis of methyl 4-[({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}acetyl)amino]-3 -{[(1-ethyl-1H-imidazol-5-yl)methyl]amino}benzoate (C73).

O-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (566 mg, 1.49 mmol) was added to a mixture of P19 (340 mg, 1.24 mmol) in N,N -dimethylformamide (10 ml) and this mixture was stirred at 25° C. for 10 minutes. Then a solution of P10 (503 mg, 1.24 mmol) and N,N-diisopropylethylamine (615 μl, 3.53 mmol) in N,N-dimethylformamide (7.7 ml) was added and the reaction mixture was stirred at 25°C for 16 h, after which it was poured into water (10 ml) and extracted with ethyl acetate (3x50 ml). The combined organic layers were washed successively with aqueous ammonium chloride (3x20 ml) and saturated aqueous sodium chloride (2x20 ml), dried over sodium sulfate, filtered and concentrated in vacuo. Purification by silica gel chromatography (gradient: 0 to 5% methanol in ethyl acetate) gave C73 as a pale brown gum. Yield: 316 mg, 0.477 mmol, 38%. LC-MS m/z 662.2^ [M+H]+.

Step 2. Synthesis of methyl 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin1-yl}methyl)-1-[( 1 -ethyl-1 H-imidazol-5 -yl)methyl] -1 H-benzimidazole-6-carboxylate (C74).

A solution of C73 (316 mg, 0.477 mmol) in acetic acid (14 mL) was stirred at 55°C for 16 h. The solvent was removed in high vacuum and the residue was purified by preparative thin layer chromatography (eluent: 10:1 dichloromethane/methanol) to give C74 as a colorless oil. Yield: 200 mg, 0.310 mmol, 65%. LC-MS m/z 644.3^ [M+H] ⁺ .

Step 3. Synthesis of 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1yl}methyl)-1-[(1- ethyl 1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-ammonium carboxylate, ENT-1 (17), and 2-({4-[2-(4-chloro-2-fluorophenyl)-2 -methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl-1Himidazol-5-yl)methyl]-1H-benzimidazole-6-ammonium carboxylate, ENT- 2 (18).

A mixture of C74 (150 mg, 0.233 mmol) and an aqueous solution of sodium hydroxide (2 M; 233 μl, 0.466 mmol) in a mixture of methanol (3 ml) and tetrahydrofuran (3 ml) was stirred at 45 °C for 16 h. After adjusting the pH reaction mixture to 7 with 1 M hydrochloric acid, it was concentrated in vacuo to give a mixture of 17 and 18. These enantiomers were separated by SFC [column: Chiral Technologies ChiralCel OD, 10 μm; mobile phase: 1:1 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluting enantiomer was designated ENT-1 (17) and the second eluting enantiomer was designated ENT-2 (18); both were isolated as a white solid.

Yield: 45.0 mg, 69.5 µmol, 30%. LC-MS m/z 630.3^ [M+H] ⁺ .

¹ H NMR (400 MHz, methanol-d ₄ ) δ 8.15 (br s, 1H), 8.00 (br d, J=8.4 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J=8 .2 Hz, 1H), 7.56 (dd, J=8.3, 8.3 Hz, 1H), 7.28 (dd, J=10.9, 2.0 Hz, 1H), 7.21 (dd, J=8.3, 2.1 Hz, 1H), 6.77 (dd, ABC system component, J=8.0, 7.7 Hz, 1H), 6.69 (dd, ABC system component, J=7.8, 1.2 Hz, 1H), 6.67-6.60 (m , 2Н), 5.82 (s, 2Н), 4.12 (q, J=7.2 Hz, 2Н), 3.89 (AV quartet, Jab = 14.3 Hz, Avab = 6.9 Hz, 2Н), 3.00-2.90 (m, 2H), 2.74-2.64 (m, 1H), 2.32-2.21 (m, 2H), 2.02 (s, 3H), 1.82-1.61 (m, 4H), 1.29 (t, J=7.3 Hz , 3H).

Retention time 5.66 min (column: Chiral Technologies Chiralpak AD-3, 4.6x150 mm, 3 µm; mobile phase A: carbon dioxide; mobile phase B: methanol containing 0.05% diethylamine; gradient: 5 to 40 % B for 5.5 min, then hold at 40% B for 3.0 min; flow rate: 2.5 ml per minute).

Yield: 32.8 mg, 50.7 µmol, 22%. LC-MS m/z 630.3^ [M+H] ⁺ .

¹ H NMR (400 MHz, methanol-d4) δ 8.15 (s, 1H), 8.00 (d, J=8.5 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J=8.5 Hz , 1H), 7.56 (dd, J=8.3, 8.3Hz, 1H), 7.28 (dd, J=10.9, 2.0Hz, 1H), 7.21 (dd, J=8.3, 2.0Hz, 1H ), 6.77 (dd, compo-

- 88 040816 ABC system component, J=7.8, 7.8 Hz, 1H), 6.69 (dd, ABC system component, J=7.9, 1.2 Hz, 1H), 6.67-6.60 (m,

2H), 5.82 (s, 2H), 4.12 (q, J=7.3 Hz, 2H), 3.89 (AB quartet, Jab = 14.1 Hz, Avab = 7.4 Hz, 2H), 3.01-2.90 ( m,

2H), 2.74-2.63 (m, 1H), 2.31-2.21 (m, 2H), 2.02 (s, 3H), 1.82-1.60 (m, 4H), 1.29 (t, J=7.3 Hz, 3H) . Retention time 5.34 min (analytical SFC conditions identical to those used for 17).

The compounds listed in the table. 1 were obtained using techniques similar to those described in the examples identified in table. 2, using the corresponding(their) intermediate(s) compound(s)identified(s) in the table. 2. Compounds were purified using the methods discussed in this description. The final compounds could be isolated as neutral compounds or as acid or base salts.

Table 1

Structure and name according to IUPAC for compounds of examples 19-102

Etc. No. Structure Name according to IUPAC 19 ^h o s ^>0 p ^ν CF ₃ COOH 2-({4-[2-(4-Chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperazin-1 -yl} methyl)-1-(2methoxyethyl)-1/7-benzimid azole -6carboxylic acid trifluoroacetate salt 20 b-x CF3COOH ENT-X2 2-({4-[2-(4-Chloro-2-fluorophenyl)-1,3benzodioxol-4-yl]piperidin-1 yl } methyl)-1-(2-methoxyethyl)-1-Abenzimidazole-6-carboxylic acid, ENT-X2, trifluoroacetate salt [from C77; note 1 in Table 2] 21 0- ^F S CF3COOH ENT-X1 2-({4-[2-(4-Chloro-2-fluorophenyl)-1,3benzodioxol-4-yl]piperide in-1 yl}methyl)-1-(2-methoxyethyl)-Sbenzimidazole-6-carboxylic acid , ENT-X1, trifluoroacetate salt, [from C76; note 1 in Table 2] 22 ^х o О S ау-о 9 ^Ν Ό% _η CF3COOH ENT-X1 1 -(2-methoxyethyl)-2- {[4-(2-phenyl-1,3benzodioxol-4-yl)piperazin-1 - yl] methyl } - 1/7-azol-6carboxylic acid benzimide, ENT-X1, trifluoroacetate salt [from P5] 23 o s ^abs )~O '“'OH • CF3COOH ENT-X2 1 -(2-methoxyethyl)-2- {[4-(2-phenyl-1,3benzodioxol-4-yl)piperazin-1 - yl] methyl } - III-benzimid azol-6carboxylic acid, ENT-X2, trifluoroacetate salt [from P6] 24 ^h o ^ci rr s XvGVw Хд \=/нн • CF3C00H 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4- yl] piperidin-1 -yl } methyl)-1 -(2methoxyethyl)- 1H-benzimid azol-6carboxylic acid trifluoroacetate salt 25 ^c 'XKl l X ^abs/O • CF3COOH DIAST-Z2 2-{6-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzode ioxol-4-yl]-6azaspiro[2.5]oct-1-yl}-1-(2methoxyethyl)- 1R -azol-6carboxylic acid benzimide, DIAST-Z2, trifluoroacetate salt [from P17 through C79; note 2 in Table 2]

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26 CI^^F > xxs abs/O f^ZAabs ' • CF3COOH DIAST-Z1 2-{6-[2-(4-Chloro-2-fluorophenyl)-2methyl-1,3-benzode ioxol-4-yl]-6azaspiro[2.5] oct-1-yl } -1 -(2methoxyethyl)- 1 /7-azol-6-carboxylic acid benzimide, DIAST-Z1, trifluoroacetate salt [from P17 through C78; note 2 in Table 2] 27 NC \ t> ^F s ΛΟ ί^Ν·%<4 ^O U<A> LDu/ OH • CF3COOH 2-({4-[2-(4-cyano-2-fluorophenyl)-1,3benzodioxol-4-yl]piperidin-1 yl } methyl)-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylic acid trifluoroacetate salt 28 9^3 ⁴ o & S AOTKf 1 J ^^OH HCOOH 1 - (2-methoxyethyl) -2- [(4- { 2-methyl-2- [3 (trifluoromethyl) phenyl] -1,3- ioxol-4-yl benzode }piperidin-1 yl)methyl]-III-benzimidazole-6carboxylic acid formate salt 29 ^x o TT ^= ⁷ OH HCOOH 2-({4-[2-(4-ethylphenyl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1 yl}methyl)-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylic acid salt thirty f ⁴ O ύι s •HCOOH 2-({4-[2-(3-Fluoro-4-methoxyphenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-1-(2methoxyethyl)- 1H-benzimid azol-6carboxylic acid formate salt 31 XrX °y\A> rL/y^ ⁰ 1(^1 '^(DH •HCOOH formate salt

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32 °yVL ν-MT ⁵ LJ he nsoon 1 -(2-methoxyethyl)-2-({4-[2-(4methoxyphenyl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1 yl}methyl)- 1/7-benzimid azol-6carboxylic acid formate salt 33 ^x o ^CI IX S TU \=/οη •nsoon formate salt 34 ΟγΟ y jb 9 o ζ 2-({4-[2-(4-cyanophenyl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1 yl } methyl)-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylic acid 35 ^h o ϊ Τ \=/ he nsoon 2-({4-[2-(2-fluoro-4-methoxyphenyl)-2- methyl-1,3-benzodioxol-4- yl] piperidin-1 -yl } methyl)-1 -(2- methoxyethyl)- 1/7-benzimid azole-6carboxylic acid formate salt 36 tu 9=/^ nsoon 1 -(2-methoxyethyl)-2-( { 4- [2-methyl-2-(6methylpyridin-2-yl)-1,3- benzodioxol-4-yl]piperidine-1 - yl } methyl)- 1/7-benzimid azol-6carboxylic acid formate salt 37 । ^h ? gg° T2G° ⁿ 1 -(2-methoxyethyl)-2-({4-[2-(2methoxyphenyl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1 yl } methyl)- 1H-benzimid azol-6carboxylic acid

- 91 040816

38 ^h o ^f Ol S chips nsoon 2-({4-[2-(4-fluorophenyl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1 yl } methyl)-1-(2-methoxyethyl)- 1Hbenzimidazole-6-carboxylic acid formate salt 39 0 + %hop>n° UX \ ₌ /\ _he •ncoon formate salt 40 'XxL' UJ OH •UNSD 1-(2-methoxyethyl)-2-[(4-{2-methyl-2-[4- (trifluoromethyl)phenyl] -1,3- ioxol-4-yl benzode }piperidin-1 yl)methyl]-1H-benzimidazole-6carboxylic acid formate salt 41 1 ΑχθΊ%4> |Q| \=/he 2-({4-[2-(3,4-difluorophenyl)-2-methyl1,3-benzodioxol-4-yl]piperidin-1 yl } methyl)-1-(2-methoxyethyl)-1Hbenzimidazole-6-carboxylic acid acid 42 A. '° nsoon 1 -(2-methoxyethyl)-2-( { 4- [2-methyl-2-(6methylpyridin-3 -yl)-1,3- benzodioxol-4-yl]piperidin-1 yl } methyl)- 1//-benzimid azol-6carboxylic acid formate salt 43 0 Clx^^F < Yli' P ϊ T OH 1 - { 2- [acetyl (methyl) amino] ethyl } -2 ({4- [2- (4-chloro-2-fluorophenyl) -2-methyl- 1,3-benzodioxol-4-yl]piperidin-1 yl} methyl)- 1H-benzimid azol-6carboxylic acid

- 92 040816

44 0 Ац ΟΆ o ^o chAA>nAv< VJ he 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [2(morpholin-4-yl)ethyl ]-\Hbenzimidazole-6-carboxylic acid 45 SK F A GT ^N / X^J ^^^ΟΗ 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl} methyl)-1 (pyridin-2-ylmethyl)- III- benzimidazole-6-carboxylic acid 46 ΟγΟ°?A e V U 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [2-(2oxo-1,3 - oxazolide in-3-yl)ethyl]-1Hbenzimidazole-6-carboxylic acid 47 ^X N^ °Ao X Ti / Aq Frequency response о °hDA> n/W XJj A=/on 2-({4-[2-(4-Chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1-[2(dimethylsulfamoyl)ethyl]-1Hbenzimidazole -6-carboxylic acid 48 ΟγΟ A X l O 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1 - [2-(2oxopyrrolidin-1-yl) ethyl]-\Nbenzimidazole-6-carboxylic acid 49 <An ^C 'A Ao o °hAAA Νγ [TJ \=/ he 1 -[2-(acetylamino)ethyl] -2-({4-[2-(4chloro-2-fluorophenyl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1 yl}methyl)- W- benzimide azole-6carboxylic acid

- 93 040816

50 P Ν ^c 'XIi / Wushu [GD \=/he 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4- yl]piperidin-1 -yl } methyl)-1 - [2-( 1nimidazol-1 -yl)ethyl]- \H- benzimidazole-6-carboxylic acid 51 <Τ^ ^Ν >Λ /° oSHDE n-Ch Uch yj \=/ he 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1 - [(1-ethyl1R-imidazol-2- yl)methyl]-1-I-benzimidazole-6-carboxylic acid 52 Y Η ^C 'YY ^F ^7-0 θ °y\A> N-Uuch Hjr OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [2(methylamino)-2-oxoethyl] -\Nbenzimidazole-6-carboxylic acid 53 a CI^^F <4-o ,Ο °\A/M mu Y4 \=/ he 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4- yl]piperidin-1 -yl } methyl)-1 - [2-(\Hpyrazol-1 -yl)ethyl]- 1H-benzimidazole6-carboxylic acid 54 0^0 ^v t J~' 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4- yl]piperidin-1 -yl } methyl)-1 - [3 -(\H1,2,4-triazol-1 -yl)propyl] -\Hbenzimidazole-6-carboxylic acid 55 ΟγΟ .u u h o 2-({4-[2-(4-Chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [2-(1-methyl-1R-imidazole -4-yl)ethyl]- 1-Abenzimidazole-6-carboxylic acid

- 94 040816

56 ^CI X4 lljr he 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl} methyl)-1 (tetrahydrofuran-3-yl methyl)- 1Hbenzimidazol- 6-carboxylic acid 57 οίγγΡ d~°l P [ΪΤ \=/ OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [(1 methyl- 1H-1,2 ,4-triazol-5-yl)methyl] 1H-benzimidazole-6-carboxylic acid 58 ^c w OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(1,3oxazol-4-ylmethyl)- 1H-benzimidazole6-carboxylic acid 59 y ₀ θ'τΐι s X^J \= ^/Z oh 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [3(d-imethylamino)-3-oxopropyl ]-\Hbenzimidazole-6-carboxylic acid 60 A '5 3~^ ^r 0^0 I 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [2-( 1 methyl-1/7 -1,2,3-triazol-4-yl)ethyl]1H-benzimidazole-6-carboxylic acid 61 ^with| r+| \Ύ \=/ he 2-({4-[2-(4-Chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1(tetrahydrofuran-3-yl)-1Hbenzimidazol-6 -carboxylic acid

- 95 040816

62 ΟγΟ C ABOUT 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [2-(2methyl-1H-imidazol- 1-yl)ethyl]-1 Yabenzimidazole-6-carboxylic acid 63 rfS 0^0 I 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [(1 methyl-1R-1,2 ,3-triazol-4-yl)methyl]1H-benzimidazole-6-carboxylic acid 64 J ar O 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-[(27?)tetrahydrofuran-2-ylmethyl ]-1Abenzimidazole-6-carboxylic acid 65 o A. €>° Ф 0^0 HZ 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl} methyl)-1 (pyridin-3-ylmethyl)- SH- benzimidazole-6-carboxylic acid 66 OyO A a O 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [2(dimethylamino)-2-oxoethyl] -\Nbenzimidazole-6-carboxylic acid 67 °γδ J o 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [2(pyrrolidin-1-yl)ethyl ] - \Hbenzimidazole-6-carboxylic acid

- 96 040816

68 0 Μ Vz- ⁿ 'NH ei^^F \=< Y Τι 7 °y\A> OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl} methyl)-1 - {[3 (methoxymethyl)-III-pyrazole -5yl] methyl } -1 R-benzymide azol-6carboxylic acid 69 OyO & O 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(1,3oxazol-5-ylmethyl)- 1H-benzimidazole6-carboxylic acid 70 Cl F XL Ά ΛνΟΊΟ-uch ⁰ CT 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4- yl]piperidin-1 -yl } methyl)-1 - {[4-(2methoxyethyl)-4R-1,2,4-triazol-3 yl]methyl} - 1R-benzimid azole-6carboxylic acid 71 0N T^r V=/ OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - { 2[methyl(methylsulfonyl)amino]ethyl } 1H-benzimidazole-6-carboxylic acid 72 rr, Y ^0H tjj ^^^OH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [(1 hydroxycyclobutyl)methyl]-1Abenzimidazol- 6-carboxylic acid

- 97 040816

73 Cly^F XVWv TT \=/oh • CF3COOH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1-(1//pyrazol-4-ylmethyl) - 1H- benzimidazole-6-carboxylic acid trifluoroacetate salt 74 ⁿ Cnh T ,° °xAAJ N-# UCH Ϊ Y \=/o _H CF3COOH 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl} methyl)-1 - [2-(\nimidazol-2-yl )ethyl]- \H- benzimidazole-6-carboxylic acid trifluoroacetate salt 75 OyO Λ o O 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl} methyl)-1-(2hydroxyethyl)- 1//-benzimid azole -6carboxylic acid 76 ΟγΟ A o 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl} methyl)-1 - [(1-ethyl177-1,2, 3-triazol-5-yl)methyl]-\Hbenzimidazole-6-carboxylic acid 77 ΟγΟ ..U and 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [(1 methyl-1//-imidazole -4-yl)methyl]-\Hbenzimidazole-6-carboxylic acid 78 J Cr g O 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [(4methyl-4R-1,2, 4-triazol-3-yl)methyl]1H-benzimidazole-6-carboxylic

- 98 040816 acid

2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [(25)tetrahydrofuran-2-ylmethyl] -1-Abenzimidazole-6-carboxylic acid 2-({4-[2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1-(1, 3oxazol-2-ylmethyl)-1H-benzimidazole6-carboxylic acid -3 -(1,3oxazol-5-ylmethyl)-3//-imidazo[4,5/>]pyridine-5-carboxylic acid 1 -(2-methoxyethyl)-2- {[4-(2-methyl- 2phenyl-1,3-benzodioxol-4yl)piperidin-1-yl]methyl }-\Hbenzimidazole-6-carboxylic acid formate salt

2-({4-[2-(2-Chloro-4-methoxyphenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(2methoxyethyl)-III-benzimid azole-6carboxylic acid formate salt 1-(2-methoxyethyl)-2-({4-[2-methyl-2-(4methylphenyl)-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-\Hbenzimidazole-6- carboxylic acid formate salt

- 99 040816

85 OTs ΓΟΆ ρ |ί Τ ^= ⁷ he HCOOH formate salt 86 ^χ ο ry XT ^= ⁷ he HCOOH 2-({4-[2-(2-chlorophenyl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1 yl}methyl)-1-(2-methoxyethyl)-1-benzimidazole-6-carboxylic acid formate salt 87 θΝ ^h o ώι s QWQXyy UD ^= ⁷ he •HCOOH formate salt 88 ^x o N-# UD' [ϊ Ύ ^= ⁷ OH • HCOOH 1 -(2-methoxyethyl)-2-({4-[2-methyl-2-(2methylphenyl)-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1Hbenzimidazole-6-carboxylic acid formate salt 89 ^x o p । S OU GP * N R ° uUU ^ Uch O ^ \u003d ⁷ he • CF ₃ COOH ENT-X2 1 - (2-methoxyethyl) -2- ( { 4- [2-methyl-2 (pyridin-2-yl) -1,3 -benzodioxol-4yl] piperidin-1 -yl } methyl) - 1H benzimidazole-6-carboxylic acid, ENT-X2, trifluoroacetate salt [from C81; note 7 in Table 2] 90 ^h o Qi S CF3COOH ENT-X1 1 - (2-methoxyethyl) -2- ( { 4- [2-methyl-2 (pyridin-2-yl) -1,3 -benzodioxol-4yl] piperidin-1 -yl } methyl) - 1H benzimidazole-6-carboxylic acid, ENT-X1, trifluoroacetate salt [from C80; note 7 in Table 2]

- 100 040816

91 ^x o αγγ \ AGA. ρ I Τ 0' νη ₄ ⁺ ΕΝΤ-Χ1 2-({4-[2-(5-chloropyridin-2-yl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl} methyl)-1-(2methoxyethyl)-1/7-benzimid azole -6 ammonium carboxylate, ENT-X1 [from P8] 92 ⁴ ο ^CI OG S UtOUuX Tsu \=/ 0- nh ₄ ⁺ ENT-X2 2-({4-[2-(5-chloropyridin-2-yl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl} methyl)-1-(2methoxyethyl)-1H-benzimid azole-6carboxylate ammonium, ENT-X2 [from P9] 93 ^Nc n, X T'N'%rj' ^N K _n θγΑγΟ Zug O'NH ₄ ⁺ ENT-X1 2-({4-[2-(5-cyanopyridin-2-yl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 -(2methoxyethyl)- 1//-benzimid azole -6 ammonium carboxylate, ENT-X1 [from P8] 94 ^nc Oq '° 9 T ^x N ^x %f ^N v °yyy> N-Yyy <Y ONH ₄ ⁺ ENT-X2 2-({4-[2-(5-cyanopyridin-2-yl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1-(2methoxyethyl)-1R-benzimid azole-6carboxylate ammonium, ENT-X2 [from P9] 95 ^CI Y^N oX Lif I 7 %аА> IK/ TU '“'OH ·nsoon DIAST-X1 2-({4-[2-(5-chloropyridin-2-yl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1 - [(26)oxetan-2-ylmethyl] -1H-benzimidazole6-carboxylic acid, DIAST-X1 [from P8] 96 °Όί ”7 D.I.AST-1 2-({4-[2-(4-chloro-2-fluorophenyl)-7fluoro-2-methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-1 - [(26)oxetan- 2-ylmethyl]-1H-benzimidazole 6-carboxylic acid, DIAST-1 [note 10 in Table 2]

- 101 040816

97 oL T Τι 7 h. ^F DI AST-2 2-({4-[2-(4-chloro-2-fluorophenyl)-7fluoro-2-methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-1 - [(25)oxetan- 2-ylmethyl]-1 R-benz and midazol6-carboxylic acid, DIAST-2 [note 10 in Table 2] 98 ^NC tXi [G^G \=/o- NH ₄ ⁺ ENT-X2 2-({4-[2-(4-cyano-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1-(1,3oxazol-2-ylmethyl)- 1H-benzimidazole6-ammonium carboxylate, ENT-X2 [from C83; note 12 in Table 2] 99 W C YwTv O' nh ₄ ⁺ ENT-X1 2-({4-[2-(4-cyano-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1 -yl } methyl)-1-(1,3oxazol-2-ylmethyl)- 1H-benzimidazole6-ammonium carboxylate, ENT-X1 [from C82; note 12 in Table 2] 100 ^CI \^Y ^F oY TI] ch ₃ 7 • 1/2 citric acid 2-({4-[(25)-2-(4-chloro-2-fluorophenyl)-2methyl-1,3-benzodioxol-4yl]piperidin-1-yl } methyl)-7-fluoro-1 [(25 )-oxetan-2-ylmethyl]-1-Abenzimidazole-6-carboxylic acid hemicitrate salt [from RH] 101 oX L ί ^H 7 °γ\Α> ν Λ7 OH •1/2 citric acid 2-({4-[(25)-2-(4-chloro-2-fluorophenyl)1,3-benzodioxol-4-yl]piperidin-1 yl } methyl)-7-fluoro-1 -[(25) -oxetan-2ylmethyl]-1H-benzimidazole-6carboxylic acid hemicitrate salt [from C48] 102 ^N s% ¥T \=/oh CF3C00H 2-({4-[2-(hydroxymethyl)-2-phenyl-1,3-benzodioxol-4-yl]piperidin-1 yl } methyl)-1-(2-methoxyethyl)-\H-benzimidazole-6-carboxylic acid trifluoroacetate salt

- 102 040816

table 2

Method of preparation and physico-chemical data for the compounds of examples 19-102

Etc. No. Way 'H NMR (400 MHz, methanol-^) δ; mass spectrum, registered ion m/z [M+H] ⁺ or HPLC retention time; mass spectrum m/z [M+H] ⁺ (unless otherwise indicated) 19 Examples 15 and 16; C4, P12 8.39 (br s, 1H), 8.08 (d, J = 8.5 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.60 (dd, J = 8.3, 8.3 Hz, 1H ), 7.28 (dd, J = 10.9, 2.1 Hz, 1H), 7.22 (dd, J = 8.4, 2.0 Hz, 1H), 6.83 (dd, J = 8.1, 8.1 Hz, 1H), 6.60 (d, J = 7.8 Hz, 1H), 6.55 (d, J = 8.4 Hz, 1H), 4.73 (s, 2H), 4.66 (t, J = 4.9 Hz, 2H), 3.77 (t, J = 4.8 Hz, 2Н), 3.59–3.43 (t, 8Н), 3.30 (s, ZN ^LL ), 2.05 (s, ZN); 581.0 20 Examples 4 and 5 ¹ ; C43, R11 8.34-8.31 (m, 1H), 8.03 (dd, 7=8.5.1.5 Hz, 1H), 7.78 (d, J = 8.6 Hz, 1H), 7.60 (dd, J = 8.0, 8.0 Hz, 1H), 7.36 (dd, J = 10.2, 1.9 Hz, 1H), 7.31 (dd, J = 8.4, 1.8 Hz, 1H), 7.25 (s, 1H), 6.90 (dd, system component ABC, J = 8.9, 6.6 Hz, 1H), 6.86-6.80 (br, 2H), 4.79 (s, 2H), 4.60 (br t, J = 4.8 Hz, 2H), 3.95-3.85 (m , 2H), 3.74 (dd, J = 5.3, 4.2 Hz, 2H), 3.44-3.33 (m, 2H), 3.28 (s, 3H), 3.15-3.05 (m, 1H), 2.37-2.12 (m , 4H); 566.04 21 Examples 4 and 5 ¹ ; C43, R11 8.32 (dd, J= 1.6, 0.7 Hz, 1H), 8.03 (dd, J= 8.5, 1.5 Hz, 1H), 7.78 (dd, J= 8.5, 0.7 Hz, 1H), 7.60 ( dd, J= 8.1, 7.9 Hz, 1H), 7.36 (dd, J= 10.2, 2.0 Hz, 1H), 7.31 (brdd,7=8.3, 1.8 Hz, 1H), 7.25 (s, 1H), 6.90 (dd, ABC system component, J= 8.8, 6.7 Hz, 1H), 6.87-6.80 (m, 2H), 4.79 (s, 2H), 4.60 (t, J= 4.8 Hz, 2H), 3.90 (br d, J= 12.3 Hz, 2H), 3.74 (dd, J= 5.3, 4.2 Hz, 2H), 3.38 (br dd, J= 12.6, 12.5 Hz, 2H) , 3.28 (s, ZN), 3.10 (tt, J= 11.9, 4.0 Hz, 1H), 2.37-2.11 (m, 4H); 566.04 22 Examples 1 and 2; R12, R5 8.37 (d, J= 1.5 Hz, 1H), 8.07 (dd, J= 8.5, 1.5 Hz, 1H), 7.79 (d, J= 8.6 Hz, 1H), 7.60-7.54 (m, 2H), 7.50-7.42 (m, 3H), 6.98 (s, 1H), 6.86 (dd, 7=8.1, 8.1 Hz, 1H), 6.61 (dd,7=7.9, 0.9 Hz, 1H) , 6.59 (dd, 7= 8.4, 0.9Hz, 1H), 4.73 (s, 2H), 4.64 (t, 7= 4.8Hz, 2H), 3.75 (dd, 7= 5.4, 4.3Hz , 2H), 3.61-3.44 (m, 8H), 3.28 (s, 3H); 515.1 23 Examples 1 and 2; R12, R6 8.37 (br s, 1H), 8.07 (dd, 7= 8.6, 1.5 Hz, 1H), 7.79 (d, 7= 8.5 Hz, 1H), 7.61-7.54 (m, 2H), 7.51-7.42 (m, 3H), 6.98 (s, 1H), 6.86 (dd, 7= 8.2, 8.1 Hz, 1H), 6.61 (br d, 7= 8 Hz, 1H), 6.59 (br d, 7= 8 .5 Hz, 1H), 4.69 (s, 2H), 4.64 (t, 7= 4.9 Hz, 2H), 3.75 (t, 7= 4.9 Hz, 2H), 3.59-3.43 (m, 8H) , 3.29(s, 3H); 515.1 24 Examples 4 and 5; C13, P11 8.33 (dd, 7= 1.5, 0.6Hz, 1H), 8.03 (dd, 7= 8.5, 1.5Hz, 1H), 7.79 (dd, 7= 8.5, 0.5Hz, 1H), 7.62 ( dd, 7= 8.4, 8.3Hz, 1H), 7.29 (dd, 7= 10.9, 2.0Hz, 1H), 7.22 (ddd, 7= 8.4, 2.0, 0.7Hz, 1H), 6.88- 6.82 (m, 1H), 6.82-6.76 (m, 2H), 4.83 (s, 2H), 4.63 (t, 7= 4.8 Hz, 2H), 3.98-3.88 (m, 2H), 3.75 (dd, 7= 5.3, 4.2 Hz, 2H), 3.47-3.36 (m, 2H), 3.31 (s, ZN ^LL ), 3.10 (tt, 7= 12.0, 4.1 Hz, 1H), 2.36-2.10 (m , 4H), 2.05 (d, 7= 1.0 Hz, ZN); 580.14 25 Examples 15 and 16 ² ; C4, R17 8.51 (dd, 7= 1.5, 0.7Hz, 1H), 8.25 (dd, 7= 8.6, 1.4Hz, 1H), 7.79 (dd, 7= 8.6, 0.7Hz, 1H), 7.57 ( dd, 7= 8.3, 8.3Hz, 1H), 7.25 (dd, 7= 10.8, 2.0Hz, 1H), 7.19 (ddd, 7= 8.4, 2.0, 0.7Hz, 1H), 6.80- 6.73 (m, 1H), 6.55-6.50 (m, 2H), 4.9-4.73 (m, 2H ^L ), 3.92-3.81 (m, 2H), 3.66-3.58 (m, 1H), 3.41-3.3 (m, 1Н ^LL ), 3.25 (s, 3H), 3.20-3.12 (m, 1H), 3.05-2.97 (m, 1H), 2.70-2.63 (m, 1H), 2.27-2.17 (m, 1H), 2.01 (d ,7= 1.0 Hz, ZN), 1.84-1.71 (m, 2H), 1.67-1.58 (m, 2H), 1.31 (br d,7= 13 Hz, 1H); 592.34

- 103 040816

26 Examples 15 and 16 ² ; C4, R17 8.53-8.50 (m, 1H), 8.26 (dd, J= 8.6, 1.4 Hz, 1H), 7.81 (d, J= 8.6 Hz, 1H), 7.55 (dd, 7=8.3, 8.2 Hz, 1H), 7.16-7.08 (m, 2H), 6.77 (dd, J= 8.3, 7.9 Hz, 1H), 6.52 (br d, J= 8.3 Hz, 1H), 6.51 (br d, J= 7.7 Hz, 1H), 4.9-4.74 (m, 2H ^L ), 3.83 (t, J= 4.8 Hz, 2H), 3.68-3.60 (m, 1H), 3.54-3.46 (m, 1H ), 3.18-3.09 (m, 1H), 3.14 (s, 3H), 3.09-3.01 (m, 1H), 2.69-2.62 (m, 1H), 2.31-2.21 (m, 1H), 2.01 (br s, 3H), 1.78-1.69 (m, 2H), 1.63-1.52 (m, 2H), 1.33-1.25 (m, 1H); 592.34 27 Examples 4 and 5 ³ ; P11 8.32 (br s, 1H), 8.02 (dd, J= 8.5, 1.5 Hz, 1H), 7.82-7.76 (m, 2H), 7.73 (br d, J= 10.0 Hz, 1H), 7.67 ( br d, J= 8.0 Hz, 1H), 7.35 (s, 1H), 6.96-6.89 (m, 1H), 6.88-6.83 (m, 2H), 4.76 (s, 2H), 4.61 (t, J = 4.8 Hz, 2H), 3.87 (br d, J= 12.3 Hz, 2H), 3.74 (t, J= 4.8 Hz, 2H), 3.39-3.3 (m, 2H ^LL ), 3.29 ( s, 3H), 3.15-3.05 (m, 1H), 2.35-2.10 (m, 4H); 557.1 28 Example 11; R14 3.08 min ⁴ ; 596 29 Example 11; P14 3.12 min ⁴ ; 556 thirty Example 11; R14 2.90 min ⁴ ; 576 31 Example 11; R14 2.92 min ⁴ ; 546 32 Example 11; P14 2.88 min ⁴ ; 558 33 Example 11; P14 3.04 min ⁴ ; 562 34 Example 11; P14 2.99 min ⁵ ; 553 35 Example 11; P14 2.92 min ⁴ ; 576 36 Example 11; P14 2.81 min ⁵ ; 543 37 Example 11; P14 2.90 min ⁴ ; 558 38 Example 11; P14 2.91 min ⁴ ; 546 39 Example 11; P14 2.89 min ⁴ ; 558 40 Example 11; P14 3.11 min ⁴ ; 596 41 Example 11; P14 2.97 min ⁴ ; 564 42 Example 11; R14 2.40 min ⁵ ; 543 43 Example 12; P10 2.70 min ⁴ ; 621 44 Example 12; P10 2.49 min ⁴ ; 635

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45 Example 12; PIO 2.79 min ⁴ ; 613 46 Example 12; PIO 2.71 min ⁴ ; 635 47 Example 12; PIO 2.85 min ⁴ ; 657 48 Example 12; PIO 2.71 min ⁴ ; 633 49 Example 12; PIO 2.66 min ⁴ ; 607 50 Example 12; PIO 2.43 min ⁴ ; 616 51 Example 12; PIO 2.74 min ⁴ ; 630 52 Example 12; PIO 2.73 min ⁴ ; 593 53 Example 12; PIO 2.79 min ⁴ ; 616 54 Example 12; PIO 2.67 min ⁴ ; 631 55 Example 12; PIO 2.44 min ⁴ ; 630 56 Example 12; PIO 2.77 min ⁴ ; 606 57 Example 12; PIO 2.72 min ⁴ ; 617 58 Example 12; PIO 2.78 min ⁴ ; 603 59 Example 12; PIO 2.82 min ⁴ ; 621 60 Example 12; PIO 2.74 min ⁴ ; 631 61 Example 12; PIO 2.76 min ⁴ ; 592 62 Example 12; PIO 2.45 min ⁴ ; 630 63 Example 12; PIO 2.78 min ⁴ ; 617 64 Example 12; PIO 2.84 min ⁴ ; 606 65 Example 12; PIO 2.56 min ⁴ ; 613 66 Example 12; PIO 2.75 min ⁴ ; 607 67 Example 12; PIO 2.48 min ⁴ ; 619 68 Example 12; PIO 2.75 min ⁴ ; 646 69 Example 12; PIO 2.73 min ⁴ ; 603 70 Example 12; PIO 2.86 min ⁵ ; 661 71 Example 12; PIO 2.77 min ⁴ ; 657 72 Example 12; PIO 2.79 min ⁴ ; 606 73 Example 12; PIO 2.70 min ⁴ ; 602 74 Example 12; PIO 2.45 min ⁴ ; 616 75 Example 13; PIO 2.92 min ⁴ ; 566

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76 Example 13; PIO 2.99 min ⁴ ; 631 77 Example 13; PIO 2.94 min ⁴ ; 616 78 Example 13; PIO 3.08 min ⁵ ; 617 79 Example 13; PIO 3.09 min ⁴ ; 606 80 Example 13; PIO 3.02 min ⁴ ; 603 81 Example 13; PIO 3.10 min ⁵ ; 604 82 Example 11; P14 2.87 min ⁴ ; 528 83 Example 11; P14 3.00 min ⁴ ; 592 84 Example 11; R14 2.99 min ⁴ ; 542 85 Example 11; P14 2.98 min ⁴ ; 542 86 Example 11; P14 2.97 min ⁴ ; 562 87 Example 11; P14 2.97 min ⁵ ; 553 88 Example 11; P14 2.90 min ⁴ ; 542 89 Examples 4 and 5 ^{6 7} ; P11 8.63 (ddd, J= 4.9, 1.8, 0.9 Hz, 1H), 8.34 (dd, J= 1.6, 0.7 Hz, 1H), 8.03 (dd, J= 8.5, 1.5 Hz, 1H), 7.90 (ddd, J= 7.8, 7.8, 1.7 Hz, 1H), 7.80 (dd, 7=8.5, 0.7 Hz, 1H), 7.74 (ddd, 7= 7.9, 1.1, 1.0 Hz, 1H ), 7.45 (ddd, 7= 7.6, 4.9, 1.2 Hz, 1H), 6.88-6.83 (br, 1H), 6.83-6.76 (m, 2H), 4.83 (s, 2H), 4.63 (t, 7 = 4.8 Hz, 2H), 3.99-3.88 (m, 2H), 3.75 (dd, 7= 5.3, 4.2 Hz, 2H), 3.45-3.34 (br, 2H), 3.31 (s, 3H), 3.15-3.03 (m, 1H), 2.41-2.20 (m, 2H), 2.192.08 (m, 2H), 2.05 (s, 3H); 529.3 90 Examples 4 and 5 ^{6 7} ; P11 8.63 (ddd, 7= 4.9, 1.8, 0.9 Hz, 1H), 8.34 (dd, 7= 1.6, 0.7 Hz, 1H), 8.04 (dd, 7= 8.5, 1.5 Hz, 1H), 7.90 (ddd, 7= 7.8, 7.8, 1.7 Hz, 1H), 7.80 (dd, 7=8.5, 0.7 Hz, 1H), 7.73 (ddd, 7= 8.0, 1.1, 1.0 Hz, 1H ), 7.45 (ddd, 7= 7.6, 4.9, 1.2 Hz, 1H), 6.88-6.83 (br, 1H), 6.83-6.75 (m, 2H), 4.83 (s, 2H), 4.63 (t, 7 = 4.9 Hz, 2H), 3.98-3.88 (m, 2H), 3.75 (t, 7= 4.8 Hz, 2H), 3.44-3.34 (m, 2H), 3.32 (s, ZN ^LL ), 3.15 -3.03 (m, 1H), 2.40-2.19 (m, 2H), 2.18-2.08 (m, 2H), 2.05 (s, 3H); 529.3 91 Examples 6 and 7; R8, R11 8.59 (d, 7= 2.4 Hz, 1H), 8.26 (d, 7= 1.4 Hz, 1H), 7.96 (dd, 7 = 8.5, 1.5 Hz, 1H), 7.87 (dd, 7= 8.5, 2.5 Hz, 1H), 7.68-7.61 (w, 2H), 6.83-6.75 (m, 1H), 6.75-6.67 (t, 2H), 4.67 (t, 7= 5.2 Hz,

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2H), 4.00 (s, 2H), 3.82 (t, J= 5.1 Hz, 2H), 3.29 (s, 3H), 3.133.05 (m, 2H), 2.81-2.70 (m, 1H), 2.45 -2.34 (m, 2H), 2.01 (s, 3H), 1.98-1.77 (m, 4H); 563.34 92 Examples 6 and 7; R9, R11 8.59 (d, J= 2.3 Hz, 1H), 8.26 (d, J= 1.4 Hz, 1H), 7.96 (dd, J= 8.5, 1.5 Hz, 1H), 7.87 (dd, J= 8.5, 2.5 Hz, 1H), 7.68-7.62 (br, 2H), 6.82-6.76 (m, 1H), 6.74-6.68 (m, 2H), 4.67 (t, J= 5.2 Hz, 2H) , 4.00 (s, 2Н), 3.82 (t, J= 5.1 Hz, 2Н), 3.29 (s, ZN), 3.133.04 (m, 2Н), 2.76 (tt, J= 11.8, 4 Hz, 1Н ), 2.45-2.34 (m, 2H), 2.01 (s, 3H), 1.97-1.78 (m, 4H); 563.34 93 Examples 8 and 9 ⁸ ; R8, R11 8.97 (dd, 7=2.1, 0.9 Hz, 1H), 8.27-8.25 (m, 1H), 8.21 (dd, J= 8.2, 2.1 Hz, 1H), 7.96 (dd, J= 8.5, 1.5 Hz, 1H), 7.81 (dd, J= 8.3, 0.9 Hz, 1H), 7.64 (d, J= 8, 6 Hz, 1H), 6.83-6.77 (m, 1H), 6.76-6.68 (m, 2H), 4.68 (t, J= 5.2 Hz, 2H), 3.95 (s, 2H), 3.83 (t, J = 5.2 Hz, 2H), 3.30 (s, 3H), 3.08-2.99 (m, 2H), 2.79-2.69 (t, 1H), 2.39-2.28 (t, 2H), 2.04 (s, 3H), 1.96-1.76 (t, 4H); 554.4 94 Examples 8 and 9 ⁸ ; R9, R11 8.97 (dd, J= 2.2, 0.9 Hz, 1H), 8.26 (br s, 1H), 8.21 (dd, J= 8.2, 2.1 Hz, 1H), 7.96 (dd, J= 8.4, 1, 4 Hz, 1H), 7.81 (dd, J= 8.2, 0.9 Hz, 1H), 7.64 (br d, J= 8.5 Hz, 1H), 6.83-6.77 (m, 1H), 6.766.69 ( m, 2H), 4.68 (t, J= 5.3 Hz, 2H), 3.95 (s, 2H), 3.83 (t, J= 5.2 Hz, 2H), 3.30 (s, 3H), 3.08-2.99 (m, 2H), 2.79-2.69 (m, 1H), 2.39-2.28 (m, 2H), 2.04 (s, 3H), 1.96-1.76 (m, 4H); 554.4 95 Example 10; R8, R15 8.61 (dd, 7=2.5, 0.7Hz, 1H), 8.41 (s, 1H), 8.33 (dd,7 = 1.6, 0.7Hz, 1H), 7.97 (dd, 7= 8.5, 1.5 Hz, 1H), 7.88 (dd, 7= 8.5, 2.5 Hz, 1H), 7.66 (dd, 7 = 8.5, 0.6 Hz, 1H), 7.65 (dd, 7= 8.5, 0.7 Hz, 1H), 6.82-6.77 (m, 1H), 6.76-6.69 (m, 2H), 5.32-5.24 (m, 1H), 4.9-4.83 (m, 1H ^L ), 4.71 (dd, 7= 15.4, 2, 6 Hz, 1H), 4.65-4.58 (m, 1H), 4.48 (ddd, 7= 9.2, 6.0, 5.9 Hz, 1H), 4.03 (AB quartet, 7ab = 13.9 Hz, Avab = 49.7 Hz, 2H), 3.18-3.11 (m, 1H), 3.06-2.98 (m, 1H), 2.87-2.69 (m, 2H), 2.60-2.49 (m, 1H), 2.46-2.31 (m, 2H), 2.02 (s, 3H), 1.98-1.79 (m, 4H); 574.94 96 Examples 6 and 7 ⁹ ,10; p ₁₅ 7.01 min ¹¹ ; 610.54

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97 Examples 6 and 7 ⁹ .1°; p ₁₅ 7.89 min ¹¹ ; 610.54 98 C54 ¹² 8.25-8.23 (m, 1H), 8.00 (dd, J= 8.5, 1.5 Hz, 1H), 7.89 (d, J= 0.9 Hz, 1H), 7.76 (dd, J= 7.9, 7.6 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.64 (dd, J = 10.6, 1.5 Hz, 1H), 7.57 (dd, J = 8.0, 1.5 Hz, 1H) , 7.14 (d, J = 0.9 Hz, 1H), 6.78 (dd, ABC system component, J = 7.9, 7.8 Hz, 1H), 6.70 (dd, ABC system component, J = 7.8, 1.2 Hz, 1H), 6.66 (br d, component of the ABC system, J = 7.9 Hz, 1H), 5.94 (AB quartet, Dav = 17.2 Hz, Avab = 6.5 Hz, 2H), 3.96 (s, 2H), 3.02-2.92 (m, 2H), 2.74-2.63 (m, 1H), 2.31-2.21 (m, 2H), 2.05 (br s, 3H), 1.80-1.58 (m, 4H); 594.3 99 C54 ¹² 8.23 (d, J= 1.4 Hz, 1H), 8.00 (dd, J= 8.5, 1.5 Hz, 1H), 7.89 (d, J= 0.9 Hz, 1H), 7.76 (dd, J= 7.9, 7.6 Hz, 1H), 7.69 (d, J = 8.5 Hz, 1H), 7.64 (dd, J = 10.6, 1.5 Hz, 1H), 7.57 (dd, J = 8.1, 1, 5 Hz, 1H), 7.14 (d, J = 0.9 Hz, 1H), 6.78 (dd, ABC system component, J = 7.8, 7.8 Hz, 1H), 6.70 (dd, ABC system component, J = 7.8, 1.2 Hz, 1H), 6.66 (br d, ABC system component, J = 7.9 Hz, 1H), 5.94 (AB quartet, Dav = 17.1 Hz, Avab = 6.6 Hz, 2H) , 3.96 (s, 2H), 3.01-2.92 (m, 2H), 2.74-2.63 (m, 1H), 2.302.20 (m, 2H), 2.05 (br s, 3H), 1.80-1.58 (m, 4H ); 594.3 100 Example 7, free acid ¹³ ; RZ, S29 characteristic peaks: 7.80 (dd, D= 8.5, 6.6 Hz, 1H), 7.59 (dd, D= 8.3, 8.3 Hz, 1H), 7.51 (d, D= 8.6 Hz, 1H), 7.28 (dd, D = 10.9, 2.0 Hz, 1H), 7.21 (brddV=8.4, 2.0 Hz, 1H), 6.83-6.77 (t, 1H), 6.76-6.71 (t, 2H), 5.32-5.23 (t, 1H), 4.99 (dd, D= 15.5, 7.1 Hz, 1H), 4.79 (dd, D= 15.6, 2.8 Hz, 1H), 4.72-4.63 (t, 1H), 4.47 (ddd , D= 9.1, 6.0, 6.0 Hz, 1H), 4.31 (AB quartet, Jab = 14.4 Hz, Avab = 33.3 Hz, 2H), 3.40 (br d, D = 11.9 Hz, 1H ), 2.92-2.65 (br, 4H), 2.82 (AB quartet, Jab = 15.5 Hz, Avab = 37.5 Hz, 2H), 2.612.49 (m, 1H), 2.13-1.87 (t, 4H) , 2.04 (s, ZN); 610.04 101 Example 5, free acid ¹³ ; C48, characteristic peaks: 7.79 (dd, J = 8.5, 6.6 Hz, 1H), 7.57 (dd, D = 8.0, 8.0 Hz, 1H), 7.49 (d, D = 8.5 Hz, 1H), 7.35 (dd, D = 10.2, 1.9 Hz, 1H), 7.30 (br d, D = 8.4 Hz, 1H), 7.22 (s, 1H), 6.88- C29 6.82 (m, 1H), 6.82-6.74 (m, 2H), 5.30-5.21 (m, 1H), 4.95 (dd, D= 15.4, 7.1 Hz, 1H), 4.77(brd, D= 15.1 Hz, 1H), 4.67-4.59 (m, 1H), 4.44 (ddd, D= 9.1, 5.9, 5.9 Hz, 1H), 4.28 (AV quartet, Dav = 14.4 Hz, Avab = 31.7 Hz , 2Н), 3.37 (br d, J = 12.3 Hz, 1Η ^ΛΛ ), 2.922.61 (m, 4H), 2.82 (АВ quartet, Dav = 15.6 Hz, Avab = 37.1 Hz, 2Н) , 2.58-2.47 (m, 1H), 2.12-1.89 (m, 4H); 596.14 102 Examples 4 and 5 ¹⁴ ; P11 8.34 (dd, D= 1.6, 0.7 Hz, 1H), 8.04 (4phD=8.5, 1.5 Hz, 1H), 7.80 (dd, J = 8.5, 0.7 Hz, 1H), 7.66-7.60 ( m, 2H), 7.46-7.36 (t, 3H), 6.84-6.76 (t, 2H), 6.74 (dd, D= 7.2, 2.0 Hz, 1H), 4.84 (s, 2H), 4.63 (t, D= 4.7 Hz, 2H), 4.01-3.91 (t, 4H), 3.76 (dd, D= 5.3, 4.2 Hz, 2H), 3.47-3.37 (t, 2H), 3.32 (s, ZN) , 3.19-3.08 (t, 1H), 2.412.26 (t, 2H), 2.26-2.13 (t, 2H); 544.2

^l calculated area, the peak is partially hidden by the water peak ^ll calculated area, the peak is partially hidden by the solvent peak ♦the pattern with peaks of chlorine isotopes was observed

1. Racemic methyl ester [methyl-2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4yl]piperidin-1-yl}methyl)-1-(2-methoxyethyl )-1H-benzimidazole-6-carboxylate] was separated into its constituent enantiomers by SFC [column: Chiral Technologies ChiralCel OD-H, 5 μm; mobile phase: 7:3 carbon dioxide/(2-propanol containing 0.1% ammonium hydroxide)]. The first eluting enantiomer, ENT-1 (C76), was used in the synthesis of Example 21, and the second eluting enantiomer, ENT-2 (C77), was converted to Example 20. C76 retention time: 5.72 min (column: Chiral Technologies Chiralpak OD-3, 4.6x150 mm, 3 µm, mobile phase A: carbon dioxide, mobile phase B: 2-propanol containing 0.05% diethylamine, gradient: 5 to 40% B over 5.5 min, then hold at 40% B for 3.0 min; flow rate: 2.5 ml per minute). C77 retention time: 6.01 min (analytical SFC conditions identical to those used for C76).

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2. Methyl ether (2-{6-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]-6azaspiro[2.5]oct-1-yl} -1-(2-methoxyethyl)-III-benzimidazole-6-carboxylate) resulting from the coupling of C4 and P17 was separated into its constituent components by dioxolane by the SFC method [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 65:35 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first isomer that eluted, DIAST-1 (C78), was converted to Example 26; according to the results of the study of 1H NMR data, this substance was an enantiomer of the compound of example 15. The second eluting isomer, DIAST-2 (C79), was used in the synthesis of the compound of example 25; 1H NMR examination showed this to be the enantiomer of Example 16. C78 retention time: 3.60 min (column: Chiral Technologies Chiralpak AD-3, 4.6 x 100 mm, 3 µm; mobile phase A: carbon dioxide; mobile phase B: ethanol containing 0.05% diethylamine; gradient: 5 to 40% B over 4.5 minutes, then hold at 40% B for 2.5 minutes; flow rate: 2.8 ml per minute). C79 retention time: 3.82 min (analytical SFC conditions identical to those used for C78).

3. 4-(4-Bromo-1,3-benzodioxol-2-yl)-3-fluorobenzonitrile was obtained by treating 3-fluoro-4formylbenzonitrile and 3-bromobenzene-1,2-diol with p-toluenesulfonic acid in toluene to remove water using the Dean-Stark apparatus. This substance was then reacted with [1(tert-butoxycarbonyl)piperidin-4-yl](iodo)zinc in the presence of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) and copper(T) iodide followed by cleavage ester using p-toluenesulfonic acid to give the desired 3-fluoro-4[4-(piperidin-4-yl)-1,3-benzodioxol-2-yl]benzonitrile.

4. Conditions for analytical HPLC: column: Waters XBridge C18, 2.1 x 50 mm, 5 µm; mobile phase A: 0.0375% trifluoroacetic acid in water; mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; gradient: 10 to 100% B in 4.0 min; flow rate: 0.8 ml per minute.

5. Conditions for analytical HPLC: column: Waters XBridge C18, 2.1 x 50 mm, 5 µm; mobile phase A: 0.0375% trifluoroacetic acid in water; mobile phase B: 0.01875% trifluoroacetic acid in acetonitrile; gradient: 1 to 5% B in 0.6 min; 5 to 100% B in 3.4 min; flow rate: 0.8 ml per minute.

6. tert-Butyl-4-[2-methyl-2-(pyridin-2-yl)-1,3-benzodioxol-4-yl]-3,6-dihydropyridine-1 (2H) carboxylate was synthesized from 3- bromobenzene-1,2-diol and 2-ethynylpyridine using the procedure described for the synthesis of C12 in Preparation P7. Hydrogenation over palladium on carbon followed by treatment with hydrogen chloride in ethyl acetate gave the desired 2-[2-methyl-4(piperidin-4-yl)-1,3-benzodioxol-2-yl]pyridine hydrochloride salt.

7. Racemic methyl ester [methyl-1-(2-methoxyethyl)-2-({4-[2-methyl-2-(pyridin-2-yl)-1,3benzodioxol-4-yl]piperidin-1-yl }methyl)-1H-benzimidazole-6-carboxylate] was separated into its constituent enantiomers by SFC [column: Chiral Technologies Chiralpak AD, 10 μm; mobile phase: 65:35 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluted enantiomer of ENT-1 (C80) was used in the synthesis of Example 90, and the second eluted enantiomer of ENT-2 (C81) was converted to Example 89. C80 retention time: 4.11 min (column: Chiral Technologies Chiralpak AD-3 , 4.6x100 mm, 3 µm, mobile phase A: carbon dioxide, mobile phase B: ethanol containing 0.05% diethylamine, gradient: 5 to 40% B in 4.5 min, then holding at 40% B in for 2.5 min; flow rate: 2.8 ml per minute). C81 retention time: 4.62 min (analytical SFC conditions identical to those used for C80).

8. The conversion of P8 and P9 to the corresponding cyano-substituted derivatives was carried out using the method described for the synthesis of P4 from P2 in Preparation of P4.

9. Treatment of 1-(4-chloro-2-fluorophenyl)ethanone with trimethyl orthoformate and p-toluenesulfonic acid gave 4-chloro-1-(1,1-dimethoxyethyl)-2-fluorobenzene, which was reacted with 3-bromo-6 -fluorobenzene-1,2-diol in the presence of p-toluenesulfonic acid to give 4-bromo-2-(4-chloro-2-fluorophenyl)-7-fluoro-2-methyl-1,3-benzodioxole. This material was converted to the desired tert-butyl 4-[2-(4-chloro-2-fluorophenyl)-7-fluoro-2-methyl-1,3-benzodioxol-4yl]piperidine-1-carboxylate using the method described in Preparation of P1 for the synthesis of P1 from C2.

10. Dioxolane stereoisomer separation in 96 and 97 was performed using SFC [column: Chiral Technologies Chiralpak IG, 5 µm; mobile phase: 3:1 carbon dioxide/(2-propanol containing 0.2% ammonium hydroxide)]. The first eluting isomer was designated DIAST-1 (96) and the second eluting isomer was designated DIAST-2 (97).

11. Conditions for analytical SFC: column: Chiral Technologies Chiralpak IG, 4.6x100 mm, 5 µm; mobile phase: 7:3 carbon dioxide/(2-propanol containing 0.2% ammonium hydroxide); flow rate: 1.5 ml per minute; back pressure: 150 bar.

12. tert-Butyl-2-(chloromethyl)-1-(1,3-oxazol-2-ylmethyl)-1H-benzimidazole-6-carboxylate was synthesized from tert-butyl-3-fluoro-4-nitrobenzoate and 1- (1,3-oxazol-2-yl)methanamine using the method described for the synthesis of P11. The subsequent reaction with C54 was carried out using triethylamine to give tert-butyl-2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1

- 109 040816 yl}methyl)-1-(1,3-oxazol-2-ylmethyl)-1H-benzimidazole-6-carboxylate, which was separated into its constituent enantiomers using SFC [column: Chiral Technologies ChiralCel OD-H, 5 µm; mobile phase: 55:45 carbon dioxide/(ethanol containing 0.1% ammonium hydroxide)]. The first eluted enantiomer ENT-1 (C82) was used in the synthesis of 99 and the second eluted enantiomer ENT-2 (C83) was converted to 98. C82 retention time: 1.47 min (column: Chiral Technologies Chiralpak OD-3, 4.6 x 50 mm , 3 µm; mobile phase A: carbon dioxide; mobile phase B: methanol containing 0.05% diethylamine; gradient: 5% B in 0.2 min, then 5 to 40% B in 1.4 min, then holding at 40% B for 1.05 min; flow rate: 4 ml per minute). C83 retention time: 1.85 min (analytical SFC conditions identical to those used for C82).

13. As a result of the reaction of 1-bromo-2,3-difluoro-4-nitrobenzene with copper (T) cyanide in 1-methylpyrrolidin-2-one at an elevated temperature, 2,3-difluoro-4-nitrobenzonitrile was obtained, which was treated with thionyl chloride and methanol to give methyl 2,3-difluoro-4-nitrobenzoate. This material was converted using C29 to the desired methyl 2-(chloromethyl)-7-fluoro-1[(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylate by the method described in Preparation P11 for the synthesis of P11 from methyl 3-fluoro-4-nitrobenzoate.

14. The desired [2-phenyl-4-(piperidin-4-yl)-1,3-benzodioxol-2-yl]methanol was synthesized from 2-oxo-2-phenylethyl acetate in a manner analogous to that described for the synthesis of C13.

CHO GLP-1R Clone H6 - analysis 1.

GLP-1R-mediated agonist activity was determined by performing a functional assay on cells using a cAMP (cyclic adenosine monophosphate) assay kit (cAMP HI Range Assay Kit; CisBio catalog number 62AM6PEJ) using the HTRF (Time-Resolved Homogeneous Fluorescence) method, which allows measure cAMP levels in the cell. This method is a competitive immunoassay between native cAMP produced by cells and exogenous cAMP labeled with d2 dye. The binding of the indicator is visualized by a crypt-labeled monoclonal anti-cAMP antibody. The specific signal (ie, energy transfer) is inversely proportional to the cAMP concentration in either the standard or experimental sample.

The human GLP-1R coding sequence (NCBI reference sequence NP_002053.3, including the naturally occurring Gly168Ser variant) was subcloned into pcDNA3 (Invitrogen) and a cell line stably expressing the receptor was isolated (designated Clone H6). Saturation binding assays (filtration assay method) using ¹²⁵ I-GLP-1 ₇ _ ₃₆ (Perkin Elmer) showed that plasma membranes derived from this cell line express GLP-1R at high density (K _d : 0.4 nM , B _max : 1900 fmol/mg protein).

Cells were removed from cryopreservation, resuspended in 40 ml Dulbecco's phosphate buffered saline (DPBS - Lonza Cat # 17-512Q) and centrifuged at 800xg for 5 min at 22°C. The cell debris was then resuspended in 10 ml growth medium (DMEM/F12 mix 1:1 with HEPES, L-Gln, 500 ml (DMEM/F12 Lonza cat. no. 12-719F), 10% heat-inactivated fetal bovine serum (Gibco cat. no. 16140-071), 5 ml 100X Pen-Strep (Gibco part number 15140-122), 5 ml 100X L-glutamine (Gibco part number 25030-081) and 500 µg/ml geneticin (G418) (Invitrogen part number 10131035) 1 ml sample of cell suspension in growth medium was read on a Becton Dickinson ViCell to determine cell viability and count cells per ml. dosed into a white 384-well, tissue culture-treated assay plate (Corning 3570) The assay plate was then incubated for 48 hours at 37° C. in a humid environment in 5% carbon dioxide.

Varying concentrations of each compound to be tested (in DMSO) were diluted in HBSS (Hank's Balanced Salt Solution) assay buffer with calcium/magnesium (Lonza/BioWhittaker catalog number 10-527F)/0.1% BSA (bovine serum albumin ) (Sigma Aldrich catalog number A7409-1L)/20 mM HEPES (Lonza/BioWhittaker catalog number 17-737E) containing 100 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma catalog number I5879). The final concentration of DMSO is 1%.

After 48 hours, the growth medium was removed from the wells of the plate, and the cells were treated with 20 μl of compound serially diluted in assay buffer for 30 minutes at 37°C in a humid environment in 5% carbon dioxide. After a 30 min incubation, 10 µl of d2 labeled cAMP and 10 µl of anti-cAMP antibody (both diluted 1:20 in cell lysis buffer as described by the manufacturer in the assay protocol) were added to each well of the assay plate. The plates were then incubated at room temperature and after 60 min HTRF signal changes were read on an Envision 2104 multichannel plate reader using an excitation wavelength of 330 nm and an emission wavelength of 615 and 665 nm. Primary data were converted to nM cAMP by interpolation from a standard cAMP curve (as described by the manufacturer in the assay protocol) and percentage effect was determined relative to the saturation concentration of the GLP-17.36 full agonist (1 μM) contained in each plate. 110 040816 EC ₅₀ determinations were made from agonist dose-response curves analyzed by a curve fitting program using a 4-parameter logarithmic dose-response equation.

CHO GLP-1R Clone C6 - analysis 2.

GLP-1R-mediated agonist activity was determined by performing a functional assay on cells using a cAMP kit (cAMP HI Range Assay Kit; CisBio catalog number 62AM6PEJ) by the HTRF (Time-Resolved Homogeneous Fluorescence) method, which measures cAMP levels in cell. This method is a competitive immunoassay between native cAMP produced by cells and exogenous cAMP labeled with d2 dye. Indicator binding is visualized with a cryptate-labeled monoclonal anti-cAMP antibody. The specific signal (ie, energy transfer) is inversely proportional to the cAMP concentration in either the standard or experimental sample.

The human GLP-1R coding sequence (NCBI reference sequence NP_002053.3, including the naturally occurring Leu260Phe variant) was subcloned into pcDNA5-FRTTO and a clonal CHO cell line stably expressing the low density receptor was isolated using the Flp-In™ system T-Rex™ as described by the manufacturer (ThermoFisher). Saturation binding assays (filtration assay technique) using ¹²⁵ I-GLP-1 (Perkin Elmer) showed that plasma membranes derived from this cell line (designated Clone C6) expressed GLP-1R at low density (Kd: 0.3 nM, B _max : 240 fmol/mg protein) compared to Clone H6 cell line.

Cells were removed from cryopreservation, resuspended in 40 ml of Dulbecco's phosphate buffered saline (DPBS - Lonza catalog number 17-512Q) and centrifuged at 800 xg for 5 min at 22°C. DPBS was aspirated and cell debris resuspended in 10 ml of complete growth medium (DMEM:F12 1:1 mix with HEPES, L-Gln, 500 ml (DMEM/F12 Lonza cat. no. 12-719F), 10% heat-inactivated fetal bovine serum (Gibco part number 16140-071), 5 ml 100X Pen-Strep (Gibco part number 15140-122), 5 ml 100X L-glutamine (Gibco part number 25030-081), 700 mcg/ml hygromycin (Invitrogen part number 10687010) and 15 µg/ml of blasticidin (Gibco catalog number R21001) A 1 ml sample of cell suspension in growth medium was read on a Becton Dickinson ViCell to determine cell viability and count cells per ml. 1600 live cells/well using the Matrix Combi Multidrop reagent dispenser and cells were dosed into a white 384-well tissue cultured assay plate (Corning 3570) The assay plate was then incubated for 48 h at 37° C in a humid environment (95% O2, 5% CO2).

Varying concentrations of each test compound (in DMSO) were diluted in assay buffer (HBSS with calcium/magnesium (Lonza/BioWhittaker cat. no. 10527F)/0.1% BSA (Sigma Aldrich cat. no. A7409-1L)/ 20 mM HEPES (Lonza/BioWhittaker catalog number 17-737E) containing 100 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma catalog number 15879) The final concentration of DMSO is 1%.

After 48 hours, the growth medium was removed from the wells of the plate, and the cells were treated with 20 μl serially diluted in assay buffer compound for 30 min at 37°C in a humid environment (95% O ₂ , 5% CO ₂ ). After a 30 min incubation, 10 µl of d2 labeled cAMP and 10 µl of anti-cAMP antibody (both diluted 1:20 in cell lysis buffer as described by the manufacturer in the assay protocol) were added to each well of the assay plate. The plates were then incubated at room temperature and after 60 min HTRF signal changes were read on an Envision 2104 multichannel plate reader using an excitation wavelength of 330 nm and an emission wavelength of 615 and 665 nm. Primary data were converted to nM cAMP by interpolation from a standard cAMP curve (as described by the manufacturer in the assay protocol) and percentage effect was determined relative to the saturation concentration of the GLP-1 full agonist (1 μM) contained in each plate. EC ₅₀ determinations were made from agonist dose-response curves analyzed by a curve alignment program using a 4-parameter logarithmic dose-response equation.

In table. 3 shows the assay data to two (2) significant figures as geometric mean (EC50) and arithmetic mean (Emax) per specified number of repeats (Amount). An empty cell means that there is no data for this example or Emax was not calculated.

- 111 040816

Table 3

Biological activity of the compounds of examples 1-102

Etc. No. Assay 1 EU ₅₀ (nM) Analysis 1 Yeshah (%) Analysis 1 Quantity Assay 2 EC ₅₀ (nM) Analysis 2 Emax (%) Analysis 2 Quantity 1 880 99 3 >20000 1 2* 6.6 81 5 260 100 4 3 1.3 94 3 45 120 3 4 1600 87 3 >20000 1 5** 1.3 89 6 23 97 7 6 140 89 7 2400 89 5 ** 0.26 98 3 3.1 93 12 g*** 0.30 92 6 3.6 91 6 2*** 73 88 9 1600 90 4 0.96 99 5 17 96 8 AND 290 78 3 12 29 83 3 690 92 3 13 4.5 95 3 38 110 3 14 7 95 6 79 85 5 15 >18000 100 3 >20000 1 16 7.7 90 3 120 64 3 17 0.079 97 3 1.1 96 4

18 210 97 3 1000 87 3 19 1.2 87 3 25 100 3 20 17 85 3 270 100 3 21 >20000 1 >20000 1 22 >20000 1 23 680 76 3 24 1.4 82 3 49 110 3 25 >20000 1 >20000 1 26 >20000 1 >20000 1 27 61 98 3 1000 100 3 28 480 87 3 29 5.3 87 4 150 93 3 thirty 45 86 4 1100 77 4 31 190 88 3 1900 65 3 32 18 86 3 450 87 3 33 2.6 85 3 100 86 3 34 7.8 98 3 110 88 3 35 6.6 86 3 170 89 3 36 760 85 3 37 81 100 3 1000 83 3 38 10 87 3 240 73 3 39 200 83 3 40 14 88 3 130 73 3 41 91 78 3 2000 74 2 42 120 93 3 1700 83 3 43 3.5 88 4 65 86 3 44 160 78 4 45 9.9 81 3 220 79 3 46 5.2 95 4 57 96 3 47 42 75 3 1400 76 4 48 14 81 3 280 73 3

- 112 040816

49 230 93 3 50 12 87 4 140 92 4 51 19 80 3 280 81 3 52 32 85 3 570 80 3 53 3.1 87 3 52 84 4 54 18 82 3 160 64 3 55 74 81 3 1100 50 3 56 1.2 87 4 And 81 3 57 15 86 3 500 98 3 58 4 98 3 23 88 4 59 74 85 3 680 53 3 60 15 82 3 240 60 3 61 10 79 3 240 85 3 62 2.2 94 3 82 95 3 63 5.2 91 3 66 96 3 64 9.2 94 3 91 80 3 65 1.2 99 3 AND 99 6 66 51 82 3 850 74 3 67 710 83 3 68 73 89 3 1200 94 3 69 10 100 3 8.3 98 3 70 2.8 100 4 97 100 4 71 6.8 80 4 74 80 3 72 14 76 3 310 80 3 73 1.7 98 3 10 100 3 74 460 90 3 75 65 82 3 1000 71 3 76 0.77 93 3 7.6 100 3 77 53 89 3 1700 92 3 78 4.5 89 4 78 100 3 79 1.4 85 3 21 85 3

- 113 040816

80 1.1 87 3 6.9 96 4 81 29 BY 3 54 BY 3 82 47 83 3 1000 83 3 83 3.4 85 4 44 88 4 84 9.1 93 3 100 86 3 85 230 80 3 86 24 91 h 410 100 3 87 570 89 3 88 17 86 3 360 91 3 89 130 85 3 2900 87 3 90 >20000 1 91 14000 100 3 >20000 1 92 4.2 90 5 72 83 3 93 >6500 84 5 >20000 1 94 12 89 5 360 87 3 220 77 3 >13000 5 96 1.1 85 3 AND 93 4 97 14 86 3 140 93 4 98 50 97 3 440 95 3 99 2.8 99 4 5.4 91 2 100 7.6 99 1 101 19 74 1 102 600 86 4

* Tested as ammonium salt and trifluoroacetate salt ** Tested as ammonium salt and 1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium (Tris) salt and free acid ** Tested as ammonium salt and free acid ** ^Tested as formate salt and free acid

All patents, patent applications and sources of information referred to in this specification are incorporated herein by reference in their entirety.

Claims (14)

CLAIM 1. Compound of formula I or a pharmaceutically acceptable salt thereof, wherein R is F; p is 0 or 1; ring A is phenyl or pyridinyl; m is 0, 1, 2, or 3; each R ¹ is independently selected from halogen, -CN, -O _1-3 alkyl, or -OC _1-3 alkyl, where alkyl O _1-3 alkyl and OC _1-3 alkyl are substituted with 0-3 F atoms; R ² is H or -C _1-3 alkyl, where alkyl is substituted with 0-1 OH; X-L is N-CH2 or cyclopropyl; Y is CH or N; R ⁴ is -C1-3 alkyl, -C0-3 alkylene-C3-6 cycloalkyl, -C0-3 alkylene-R ⁵ or -C1-3 alkylene-R ⁶ , wherein said alkyl may be substituted, when valence permits, with 0-3 substituents, independently selected from 0-3 F atoms and 0-1 substituent selected from -C0-1 alkylene-OR ^O , -SO2-N(R ⁿ ) ₂ , -C(O)N(R ⁿ )2, -N(C =O)(R ⁿ ) and -N(R ⁿ )2; R ⁵ is a 4-6 membered heterocycloalkyl selected from oxetanyl, tetrahydrofuranyl, morpholinyl, 1,3-oxazolidinyl and pyrrolidinyl, wherein said heterocycloalkyl may be substituted, when valency permits, with 0-2 substituents independently selected from: 0-1 oxo (=O); - 114 040816 R ⁶ is a 5-6 membered heteroaryl selected from oxazolyl, imidazolyl, pyridinyl, pyrazolyl and triazolyl, wherein said heteroaryl may be substituted, when valency permits, with 0-2 substituents independently selected from: 0-2 -C _1-3 alkyl, where the alkyl may be substituted, when valency permits, with 0-3 substituents independently selected from: 0-1 -ORO; each RO is independently H or —C _1-3 alkyl; each R ⁿ is independently H or —C _1-3 alkyl; each of Z ¹ , Z ² and Z ³ is -CR ^Z ; and each R ^Z is independently H, F, Cl, or —CH ₃ ; or 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-{2[methyl(methylsulfonyl )amino]ethyl}-1H-benzimidazole-6-carboxylic acid or a pharmaceutically acceptable salt thereof. 2. The compound according to claim 1, which is a compound of formula II II or a pharmaceutically acceptable salt thereof, wherein R is F; p is 0 or 1; ring A is phenyl or pyridinyl; m is 0, 1 or 2; each R ¹ is independently selected from halogen, -CN, -C _1-3 alkyl, or -OC _1-3 alkyl, where C _1-3 alkyl and OC _1-3 alkyl are substituted with 0-3 F atoms; R ² is H or CH ₃ ; X-L is N-CH2 or cyclopropyl; Y is CH or N; Z ³ is -CR ^Z ; And R ^Z is H or -CH ₃ . 3. The compound according to claim 1 or 2, which is a compound of formula III III or a pharmaceutically acceptable salt thereof, wherein ring A is phenyl or pyridinyl; m is 0, 1 or 2; each R ¹ is independently selected from F, Cl or -CN; R ² is H or CH ₃ ; And Y is CH or N. 4. The compound according to any one of claims 1-3, where R ⁴ is -CH2-R ⁵ , where R ⁵ is a 4-5 membered heterocycloalkyl selected from oxetanyl, tetrahydrofuranyl, 1,3-oxazolidinyl and pyrrolidinyl, where said heterocycloalkyl may be substituted, when valency permits, with 0-2 substituents independently selected from: 0-1 oxo (=O); or a pharmaceutically acceptable salt thereof. 5. The compound according to any one of claims 1-3, where R ⁴ is -CH2-R ⁶ , where R ⁶ is a 5-membered heteroaryl selected from oxazolyl, imidazolyl, pyrazolyl and triazolyl, where said heteroaryl may be substituted, when valency permits, 0-2 substituents independently selected from: 0-1 -CH3, -CH2CH3 or -CH2CH2OCH3; or a pharmaceutically acceptable salt thereof. 6. A compound according to any one of claims 1 to 5, wherein R ² is CH ₃ , or a pharmaceutically acceptable salt thereof. 7. The compound according to claim 1, which is 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1-[(2S)-oxetan-2ylmethyl] - 1 H-benzimidazole-6-carboxylic acid; - 115 040816 2-({4-[2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro-1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]nineridin-1-yl}methyl)-1-[(2S)-oxetane2 -ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro-1-[( 2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)- 1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-3-(1,3oxazol-2 -ylmethyl)-3H-imidazo[4,5-b]pyridine-5-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl1 H -imidazol-5-yl)methyl]-1 H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3oxazol-4 -ylmethyl)-1 H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(pyridin3-ylmethyl)- III-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3oxazol-5 -ylmethyl)-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(1-ethyl1H- 1,2,3-triazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3oxazol-2 -ylmethyl)-1 H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-chloro-2-fluorophenyl)-7-fluoro-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1[(2S )-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-(1,3oxazol-2 -ylmethyl)-1 H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-7-fluoro1- [(2S)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1[(2S )-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2S)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 1ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2R)-2-(4-cyano-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1 [(2S )-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid; 2-({4-[(2R)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, or 2-({4-[(2R)-2-(4-chloro-2-fluorophenyl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 1ethyl-1H-imidazol-5-yl)methyl]-1H-benzimidazole-6-carboxylic acid, or a pharmaceutically acceptable salt thereof. 8. The compound according to claim 1, which is or a pharmaceutically acceptable salt thereof. 9. The compound according to claim 1, which is 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2S)oxetan- 2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, 2-({4-[(2S)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[( 2S)oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, or - 116 040816 2-({4-[(2K)-2-(5-chloropyridin-2-yl)-2-methyl-1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[ (2S)oxetan-2-ylmethyl]-1 H-benzimidazol-6-carboxylic acid; or a pharmaceutically acceptable salt thereof, wherein the salt is a tris salt. 10. The compound according to claim 1 which is 2-({4-[2-(5-chloropyridin-2-yl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1-yl}methyl) -1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST-X2: or a pharmaceutically acceptable salt thereof. I. The compound according to claim 1, which is a crystalline form (Form I) of anhydrous 1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt 2-({4-[(28)-2-(4 -chloro-2-fluorophenyl)-2-methyl1,3-benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(2 8)-oxetan-2-ylmethyl]-1 H-benzimidazole-6carboxylic an acid having an x-ray powder diffraction pattern (CuKa radiation) containing at least two characteristic peaks selected from peaks at 3.7±0.2°; 7.3±0.2°; 8.5+0.2°; 10.1+0.2°; 14.7+0.2° and 16.9+0.2° in units of 20. 12. The compound according to claim 1, which is a crystalline form (Form A) of anhydrous 1,3-dihydroxy-2-(hydroxymethyl)propane-2-aminium salt of 2-({4-[2-(5-chloropyridine-2 -yl)-2-methyl-1,3benzodioxol-4-yl]piperidin-1-yl}methyl)-1-[(28)-oxetan-2-ylmethyl]-1H-benzimidazole-6-carboxylic acid, DIAST- X2 having an x-ray powder diffraction (CuKa radiation) pattern containing at least two characteristic peaks selected from peaks at 7.7+0.2°; 15.2±0.2°; 15.7±0.2° and 17.6±0.2° in units of 20. 13. A pharmaceutical composition containing (1) a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, and (2) a pharmaceutically acceptable excipient. 14. Use of a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, for the treatment of a disease or disorder selected from the group consisting of T2DM (type 2 diabetes mellitus), hyperglycemia, insulin resistance, hepatic insulin resistance, intolerance to glucose, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, obesity, dyslipidemia, hyperinsulinemia, NAFLD (non-alcoholic fatty liver disease), NASH (non-alcoholic steatohepatitis), fibrosis, cirrhosis and hepatocellular carcinoma. 15. The use of a compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, for the treatment of a disease or condition for which a GLP-1R (glucagon-like peptide-1 receptor) agonist is indicated.