Patent Application
20250236627
The technical field relates to certain octahydrofuro[3,4-b]pyrazines, to their use in the treatment of cardiovascular disease and metabolic conditions, for example type 2 diabetes, and to pharmaceutical compositions containing them.
Obesity and type 2 diabetes (T2D) are major and growing health problems worldwide (Lancet, 2014, 9922, 1068-1083). The two diseases are strongly associated with each other, with obesity proceeding development of insulin resistance and T2D. T2D is associated with several comorbidities including cardiovascular disease, renal disease, hypertension, stroke, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) (Lancet, 2005, 9468, 1415-1428).
Incretin hormones including GLP-1 (glucagon-like peptide-1) and GIP (glucose-dependent insulinotropic polypeptide) are gut peptides that are secreted after nutrient intake and stimulate insulin secretion (Diabetes Obes Metab., 2018, 20(Suppl.1), 5-21). GLP-1 secretion from the gut is impaired in obese subjects which may indicate a role in the pathophysiology of obesity (Regulatory Peptides, 2004, 122, 209-217).
GLP-1 is secreted from the L-cells in the lower gut in response to food intake. GLP-1 stimulates insulin secretion from the pancreatic β-cells, in a glucose dependent manner (Diabetologia, 1993, 36, 741-744). GLP-1 also inhibits glucagon secretion, reduces appetite and slows down gastric emptying. The GLP-1 receptor is also present in the heart, kidneys and immune system and activation has been shown to reduce blood pressure, increase natriuresis and decrease inflammation.
GLP-1 is a 37-amino acid peptide, post-translationally processed from pro-glucagon, a 158 amino acid precursor polypeptide (www.uniprot.org, pro-glucagon entry P01275). Several other peptides are also derived from proglucagon and processed in a tissue specific manor, including glucagon and oxyntomodulin. GLP-1 has very short half-life in vivo as it is rapidly degraded by dipeptidyl peptidase-4 (DPP-IV) (Front. Endocrinol. 2019, 10, Article 260, 1-10).
Incretin-based glucose- and body weight-lowering medications include GLP-1 receptor agonists, DPP-IV inhibitors and more recently also combinations of GLP-1 agonists and glucose-dependent insulinotropic polypeptide (GIP) agonists (Peptides, 2020, 125, Article 170202). Traditionally GLP-1 analogues are peptide hormones which have been modified to minimize DPP-IV cleavage and are administered as injectables. The first oral GLP-1 peptide was recently approved but bioavailability is low and the drug needs to be administered in the fasting state, 30 min before nutrient intake which may limit patient compliance (JAMA, 2017, 318(15), 1460-1470). The injectable peptides show increased efficacy over the oral peptides but are limited by the route of administration. Small molecule GLP-1 receptor agonists are in development from several companies and are expected to provide a therapeutic benefit versus peptide based therapies due to early use in the treatment paradigm.
Pharmacological stimulation of GLP-1 receptors has been shown to significantly reduce HbA1c levels, provide long term weight loss and reduce blood pressure. GLP-1 receptor agonists have also been shown to reduce cardiovascular events and prolong life in high-risk patients with T2D and are therefore recommended by the European Association for the Study of Diabetes (EASD) and American Diabetes Association (ADA) in patients with multiple risk factors of cardiovascular disease (CVD) independent of the patients glycemic control (Diabetes Care, 2020, 43, 487-493).
There remains a need for an easily-administered prevention and/or treatment for cardiometabolic and associated diseases.
WO2018/109607 discloses 6-carboxylic acids of benzimidazoles and 4-aza-, 5-aza-, 7-aza- and 4,7-diazabenzimidazoles as GLP-1 receptor agonists, processes to make said compounds, and methods comprising administering said compounds to a mammal in need thereof.
WO2019/239319 and WO2019/239371 disclose 6-carboxylic acids of benzimidazoles and 4-aza-, 5-aza- and 7-aza-benzimidazoles as GLP-1 receptor agonists, processes to make said compounds, and methods comprising administering said compounds to a mammal in need thereof.
WO2020/103815 disclose GLP1 receptor agonist compounds and pharmaceutical compositions thereof, for use in e.g. treating type 2 diabetes mellitus, pre-diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and cardiovascular disease.
WO2020/207474 disclose GLP1 receptor agonist compounds and pharmaceutical compositions thereof, for use in e.g. treating type 2 diabetes mellitus, pre-diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and cardiovascular disease.
WO2020/234726 disclose combinations of GLP-1 receptor agonist compounds and pharmaceutical compositions thereof and an acetyl-CoA carboxylase (ACC) inhibitor or a diacylglycerol acyltransferase (DGAT2) inhibitor, or a ketohexokinase (KHK) inhibitor or farnesoid X receptor (FXR) agonist, for use in e.g. treating type 2 diabetes mellitus, pre-diabetes, obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and related diseases.
WO2020/263695 discloses glucagon-like peptide-1 receptor agonists and therapeutic uses of the compounds to treat type II diabetes mellitus.
WO2021/081207 discloses compounds that bind to and act as agonists or modulators of the glucagon-like peptide-1 receptor (GLP-1R) and act as agonists or modulators of GLP-1R. The disclosure further relates to the use of the compounds for the treatment and/or prevention of diseases and/or conditions by said compounds.
WO2021/018023 discloses compounds for modulating a Glucagon-like peptide-1 (GLP-1) receptor, and a pharmaceutical use thereof.
WO2021/096284 and WO2021/096304 discloses compounds that act as GLP-1 receptor agonists, for use as therapeutic agents for metabolic diseases.
WO2021/112538 discloses compounds which serves as a GLP-1 receptor agonist and may be useful in the prevention or treatment of a disease associated with GLP-1 activity.
WO2021/154796 discloses GLP-1R agonists, and compositions, methods, and kits thereof. Such compounds are generally useful for treating a GLP-1R mediated disease or condition.
WO2021/160127 discloses GLP-1 agonists, pharmaceutical compositions, and methods of use thereof.
WO2021116874 discloses of solid forms of 2-[[4-[(S)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl]piperidin-1-yl]methyl]-1-[[(S)-oxetan-2-yl]methyl]-1H-benzo[d]imidazole-6-carboxylic acid, 1,3-dihydroxy-2-(hydroxymethyl)propan-2-amine salt for pharmaceutical use.
CN113493447A discloses a compound that can be used as a GLP-1 receptor agonist. WO2021197464 discloses fused imidazole derivatives, preparation methods and medical use as a therapeutic agent, especially as GLP-1 receptor agonists.
CN113480534A discloses benzimidazole or azabenzimidazole-6-carboxylate compound that can activate GLP-1R downstream signaling pathway.
WO2021154796 discloses compounds as GLP-1R agonists, and compositions, methods, and kits thereof.
WO2021219019 discloses GLP-1 agonists of formula I, including pharmaceutically acceptable salts and solvates thereof, pharmaceutical compositions, and methods of using the same.
WO2021244645 discloses five-membered heteroaromatic imidazole compounds I and their medical use.
WO2021249492 discloses methyl-substituted benzobisoxazole compound and the use thereof in the preparation of drugs for treating related diseases.
CN113816948A discloses fused imidazole derivatives as GLP-1 receptor agonist in the treatment of diabetes.
WO2021254470 discloses preparation of 6-oxo-3,6-dihydropyridine derivative and a pharmaceutical composition containing the derivative, are used as therapeutic agents, in particular as GLP-1 receptors agonist and in the preparation of drugs for the treatment and/or prevention of diabetes.
WO2022007979 discloses a fused imidazole derivative, a preparation method therefor, a pharmaceutical composition containing the derivative, and the use of same as a therapeutic agent, in particular the use thereof as a GLP-1 receptor agonist.
CN113831337A discloses heterocyclic nitrogen compounds as GLP-1 receptor agonist.
WO2022068772 discloses a kind of benzimidazole derivative, its preparation method and application as GLP-1R agonists.
WO2022042691 discloses GLP-1 agonists, including pharmaceutically acceptable salts and solvates thereof, and pharmaceutical compositions including the same.
WO2022040600 discloses compounds that may be used as a glucagon-like peptide-1 receptors (GLP-1R) agonist.
WO2022028572 discloses GLP-1 agonists, including pharmaceutically acceptable salts and solvates thereof, and pharmaceutical compositions including the same.
WO2022031994 discloses compounds and pharmaceutical compositions thereof, for use in, e.g. treating type 2 diabetes mellitus, pre-diabetes, obesity, non-alc. fatty liver disease, non-alc. steatohepatitis, and cardiovascular disease.
CN114591308A discloses piperazine-imidazole containing GLP-1R receptor agonist compounds and application thereof.
WO2022111624 discloses benzimidazole derivatives that are agonists of a glucagon-like peptide-1 receptor (GLP-1R).
WO2022109182 discloses polyheterocyclic benzimidazole compounds and their preparation and use in the treatment of GLP-1R mediated diseases.
CN114478497A discloses a kind of aryl alkyl acid GLP-1 receptor agonist, its preparation method and application in treatment or prevention of GLP-1-mediated diseases and related diseases.
WO2022078380 discloses compounds that are GLP-1 agonists.
WO2022078407 discloses compounds that are GLP-1 agonists.
WO2022078152 discloses a kind of benzimidazolone compounds, their preparation method and application as GLP-1 receptor agonist.
CN114716423A discloses 5,6-dihydro-1,2,4-triazine compounds as GLP-1 receptor agonist.
CN114634510A discloses imidazolopyridine derivatives, which can be used to prepare drugs for treating GLP-1 receptor agonist mediated diseases.
CN114591296A discloses aromatic heterocyclic derivatives as GLP-1R agonists.
WO2022192430 discloses GLP-1R agonists and compositions, methods, and kits thereof.
WO2022192428 discloses GLP-1R agonists and compositions, methods, and kits thereof.
WO2022184849 discloses GLP-1R agonists, uses and pharmaceutical compositions thereof.
CN114907351A discloses tricyclic GLP-1 receptor agonists.
WO2022165076 discloses substituted benzimidazolecarboxylic acids which are GLP-1 receptor modulator compounds.
CN114805336A discloses fused imidazole compounds that are GLP-1 receptor agonists.
CN114763352A discloses benzimidazole derivatives and its application as GLP-1 receptor agonist.
J. Med. Chem. 2022, 65, 12, 8208-8226 discloses A Small-Molecule Oral Agonist of the Human Glucagon-like Peptide-1 Receptor.
Cell Research 2020, (39), 1140-1142 discloses structural insights into the activation of GLP-1R by a small molecule agonist.
An object is to provide novel GLP-1 receptor modulators useful in therapy. A further object is to provide novel compounds having improved safety profile, e.g with regards to selectivity for the GLP-1 receptor over e.g. phosphodiesterase 3 (PDE3) and/or having improved metabolic stability in the body.
There is provided compounds that are modulators of the glucagon-like peptide-1 (GLP1) receptor, their use as medicaments, pharmaceutical compositions containing them and synthetic routes to their production.
In one embodiment, there is provided a compound of Formula (I).
wherein
The compounds of Formula (I) are modulators of the GLP-1 receptor. Thus, the compounds of Formula (I) can be used as a medicament, in particular for disorders, disease or conditions responsive to modulation of the GLP-1 receptor, and more specifically cardiovascular disease and metabolic conditions.
In another embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt of a compound of Formula (I), wherein the stereochemistry is undefined, e.g. a racemate or a mixture of diastereomers.
In another embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt of a compound of Formula (I), wherein the stereochemistry is defined.
In another embodiment there is provided a pharmaceutical formulation comprising a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt of a compound of Formula (I), and a pharmaceutically acceptable diluent, excipient and/or inert carrier.
In a further embodiment there is provided a pharmaceutical formulation comprising a compound of Formula (I), or a pharmaceutically acceptable salt of a compound of Formula (I), for use in the treatment of a condition where modulation of the GLP-1 receptor would be beneficial.
In a further embodiment there is provided a compound of Formula (I), or a pharmaceutically acceptable salt of a compound of Formula (I), for use in therapy, especially in the treatment of cancer in a mammal, particularly a human.
In a further embodiment there is provided the use of a compound of Formula (I), or a pharmaceutically acceptable salt of a compound of Formula (I), for the manufacture of a medicament for the treatment of cardiovascular disease and metabolic conditions.
According to another aspect there is provided a process for the preparation of compounds of Formula (I), or pharmaceutically acceptable salts of compounds of Formula (I), and the intermediates used in the preparation thereof.
The compounds of Formula (I) described herein have the advantage that they may be more efficacious, be less toxic, be more selective, be more potent, produce fewer side effects, be more easily absorbed, and/or have a better pharmacokinetic profile (e.g. higher oral bioavailability and/or lower clearance), than compounds known in the prior art.
This detailed description and its specific examples, while indicating embodiments, are intended for purposes of illustration only. Therefore, there is no limitation to the illustrative embodiments described in this specification. In addition, it is to be appreciated that various features that are, for clarity reasons, described in the context of separate embodiments, also may be combined to form a single embodiment. Conversely, various features that are, for brevity reasons, described in the context of a single embodiment, also may be combined to form sub-combinations thereof.
Listed below are definitions of various terms used in the specification and claims.
It is to be understood that where in this specification a group is qualified by “defined above” the said group encompasses the first occurring and broadest definition as well as each and all of the other definitions for that group.
In this specification, the term “modulator” is used to describe a compound that exhibit varying receptor agonism, either full agonism, or partial agonism.
It is to be understood that in this specification “C1-4” means a carbon group having 1, 2, 3 or 4 carbon atoms.
It is to be understood that in this specification “C1-2” means a carbon group having 1 or 2 carbon atoms.
In this specification, unless stated otherwise, the term “alkyl” includes both straight and branched chain alkyl groups and may be, but is not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, iso-butyl or tert-butyl.
It is to be understood that in this specification “(5- to 6-membered)heteroaryl” means an aromatic ring with 5 to 6 atoms and containing one or more heteroatoms independently selected from nitrogen, oxygen or sulphur.
It is to be understood that in this specification “(6-membered)heteroaryl” means an aromatic ring with 6 atoms and containing one or more heteroatoms independently selected from nitrogen, oxygen or sulphur.
It is to be understood that in this specification “(6-membered)heteroaryl” means for example pyridine.
It is to be understood that in this specification “(5-membered)heteroaryl” means an aromatic ring with 5 atoms and containing one or more heteroatoms independently selected from nitrogen, oxygen or sulphur.
It is to be understood that in this specification “(4- to 6-membered)heterocycloalkyl” means a partially or completely saturated ring system with 4 to 6 atoms and wherein at least one of the ring carbon atoms is replaced with a heteroatom independently selected from nitrogen, oxygen or sulphur.
It is to be understood that in this specification a “heterocycloalkyl” substituent may be attached via a nitrogen atom having the appropriate valences, or via any ring carbon atom.
It is to be understood that in this specification a “heterocycloalkyl” or “heteroaryl” substituent may be further substituted.
In this specification, unless stated otherwise, the term “pharmaceutically acceptable” is used to characterize a moiety (e.g. a salt, dosage form, or excipient) as being appropriate for use in accordance with sound medical judgment. In general, a pharmaceutically acceptable moiety has one or more benefits that outweigh any deleterious effect that the moiety may have. Deleterious effects may include, for example, excessive toxicity, irritation, allergic response, and other problems and complications.
There is provided compounds of Formula (I) wherein X1, X2, Z1, Z2, Z3, R1-R7, m, n, p and q are as defined in Formula (I).
In one embodiment X1 is N or C.
In a further embodiment X1 is N.
In still a further embodiment X1 is C.
R1 is 0, 1, 2 or 3 substituents independently selected from F, Cl, Br, CN, OCH3, OCFH2, OCF2H, OCF3, CH3, CFH2, CF2H and CF3.
In one embodiment X2 is independently N or C, provided that no more than two atoms in the aromatic ring A are N.
In a further embodiment X2 is C.
In one embodiment Z1 is N or CR3.
In a further embodiment Z1 is N.
In still a further embodiment Z1 is CR3.
R3 is selected from H, F, Cl, N(CH3)2, C1-2alkyl and OC1-2alkyl, wherein said C1-2alkyl is substituted by 0, 1, 2 or 3 F.
In one embodiment Z2 and Z3 are each independently N or CR4, provided that when Z1 or Z3 is N, Z2 is CR4.
In a further embodiment Z1 and Z2 are N.
In still a further embodiment Z1 and Z3 are N.
In still a further embodiment Z2 and Z3 are N.
In still a further embodiment Z1 is N, Z2 and Z3 are CR4.
In still a further embodiment Z2 is N, Z1 and Z3 are CR4.
In still a further embodiment Z3 is N, Z1 and Z2 are CR4.
In still a further embodiment Z1, Z2 and Z3 are CR4.
R4 is independently selected from H, F, Cl, OH, CH3, CFH2, CF2H, CF3, OCH3, OCFH2, OCF2H and OCF3.
In one embodiment R1 is 0, 1, 2 or 3 substituents independently selected from F, Cl, Br, CN, OCH3, OCFH2, OCF2H, OCF3, CH3, CFH2, CF2H and CF3.
In a further embodiment R1 is 0, 1 or 2 substituents independently selected from F, Cl, Br, CN, OCH3, OCFH2, OCF2H, OCF3, CH3, CFH2, CF2H and CF3.
In still a further embodiment R1 is 0, 1, 2 or 3 substituents independently selected from F, Cl, Br, CN, OCH3.
In still a further embodiment R1 is 0, 1 or 2 substituents independently selected from F, Cl, Br, CN, OCH3.
In still a further embodiment R1 is 0, 1 or 2 substituents independently selected from F, Cl and CN.
In still a further embodiment R1 is 0 or 1 substituents selected from F, Cl and CN.
In one embodiment R2 is selected from 0 or 1 F, Cl or CN.
In one embodiment R3 is selected from H, F, Cl, N(CH3)2, C1-2alkyl and OC1-2alkyl, wherein said C1-2alkyl is substituted by 0, 1, 2 or 3 F.
In a further embodiment R3 is selected from H, F, Cl, C1-2alkyl and OC1-2alkyl, wherein said C1-2alkyl is substituted by 0, 1, 2 or 3 F.
In still a further embodiment R3 is selected from H, F, Cl, CH3, CFH2, CF2H, CF3, OCH3, OCFH2, OCF2H and OCF3.
In still a further embodiment R3 is selected from H, F, Cl, CH3 and OCH3.
In one embodiment R4 is independently selected from H, F, Cl, OH, CH3, CFH2, CF2H, CF3, OCH3, OCFH2, OCF2H and OCF3.
In a further embodiment R4 is independently selected from H, F, Cl, OH, CH3 and OCH3.
In still a further embodiment R4 is independently selected from H, F, Cl, CH3 and OCH3.
In still a further embodiment R4 is independently selected from H, F and Cl.
In one embodiment R5 is selected from H, CH3, CFH2, CF2H and CF3; In a further embodiment R5 is selected from H, and CH3.
In still a further embodiment R5 is CH3.
In one embodiment R6 is selected from (4- to 6-membered)heterocycloalkyl, (5- to 6-membered)heteroaryl, CN, C1-4alkyl, O(C1-4alkyl), S(C1-4alkyl), cyclopropyl, cyclobutyl, O(cyclopropyl) or S(cyclopropyl), wherein said C1-4alkyl is substituted by 0 or 1 substituent selected from CN or OCH3, and 0, 1, 2 or 3 F and where said cyclopropyl and cyclobutyl is substituted by 0 or 1 substituent selected from CN, OCH3, OCFH2, OCF2H, OCF3 and CH2CN and 0, 1, 2 or 3 F.
In a further embodiment R6 is selected from C1-4alkyl, O(C1-4alkyl) and S(C1-4alkyl), wherein said C1-4alkyl is substituted by 0 or 1 substituent selected from CN or OCH3, and 0, 1, 2 or 3 F.
In still a further embodiment R6 is selected cyclopropyl, cyclobutyl, O(cyclopropyl) or S(cyclopropyl), said cyclopropyl and cyclobutyl is substituted by 0 or 1 substituent selected from CN, OCH3, OCFH2, OCF2H, OCF3 and CH2CN and 0, 1, 2 or 3 F.
In still a further embodiment R6 is selected from (4- to 6-membered)heterocycloalkyl and (5- to 6-membered)heteroaryl.
In still a further embodiment R6 is selected from (5- to 6-membered)heteroaryl.
In still a further embodiment R6 is selected from (4- to 6-membered)heterocycloalkyl.
In still a further embodiment R6 is oxetan-2-yl.
In one embodiment R7 is independently selected from F, C1-2alkyl and OC1-2alkyl, wherein said C1-2alkyl is substituted by 0, 1, 2 or 3 substituents independently selected from F.
In a further embodiment R7 is independently selected from F, C1-2alkyl and OC1-2alkyl.
In still a further embodiment R7 is independently selected from F, CH3 and OCH3.
In one embodiment m is 0, 1, 2 or 3.
In a further embodiment m is 0, 1, or 2.
In still a further embodiment m is 1 or 2
In still a further embodiment m is 0 or 1.
In still a further embodiment m is 1.
In still a further embodiment m is 0.
In one embodiment n is 0 or 1.
In a further embodiment n is 1.
In still a further embodiment n is 0.
In one embodiment p is 1, 2 or 3.
In a further embodiment p is 1 or 2.
In still a further embodiment p is 1.
In one embodiment q is 0, 1 or 2.
In a further embodiment q is 0 or 1.
In still a further embodiment q is 0.
In one embodiment, there is provided a compound of Formula (Ia),
wherein
In a further embodiment, there is provided a compound of Formula (Ia),
wherein
In still a further embodiment, there is provided a compound of Formula (Ia),
wherein
In one embodiment, there is provided a compound of Formula (Ib),
wherein
In a further embodiment, there is provided a compound of Formula (Ib),
wherein
In still a further embodiment, there is provided a compound of Formula (Ib),
wherein
In one embodiment, there is provided a compound of Formula (Ic),
wherein
In a further embodiment, there is provided a compound of Formula (Ic),
wherein
In still a further embodiment, there is provided a compound of Formula (Ic),
wherein
In one embodiment, there is provided a compound of Formula (Id),
wherein
In a further embodiment, there is provided a compound of Formula (Id),
wherein
In still a further embodiment, there is provided a compound of Formula (Id),
wherein
In one embodiment the compounds of formula (I) are selected from:
It shall be noted that any one of these specific compounds may be disclaimed from any of the herein mentioned embodiments.
In one embodiment there is provided a process for the preparation of compounds of formula (I), or pharmaceutically acceptable salts of compounds of formula (I), and the intermediates used in the preparation thereof.
Another embodiment is a product obtainable by any of the processes or examples disclosed herein.
The compounds of formula (I) and their pharmaceutically acceptable salts are believed to be useful in the prevention or treatment of cardiovascular disease and metabolic conditions, including but not limited to type 2 diabetes (T2D), obesity, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), in a mammal, particularly a human.
For the avoidance of doubt, as used herein, the term “treatment” includes therapeutic and/or prophylactic treatment.
When a compound or salt described herein is administered as therapy for treating a disorder, a “therapeutically effective amount” is an amount sufficient to reduce or completely alleviate symptoms or other detrimental effects of the disorder, cure the disorder, reverse, completely stop, or slow the progress of the disorder or reduce the risk of the disorder getting worse.
The compounds described herein are thus indicated both in the therapeutic and/or prophylactic treatment of these conditions.
The compounds described herein have the advantage that they may be more efficacious, be less toxic, be more selective, be more potent, produce fewer side effects, be more easily absorbed, and/or have a better pharmacokinetic profile (e.g. higher oral bioavailability and/or lower clearance), than compounds known in the prior art.
For the above-mentioned therapeutic indications, the dosage administered will vary with the compound employed, the mode of administration and the treatment desired. However, in general, satisfactory results are obtained when the compounds are administered at a dosage of the solid form of between 1 mg and 2000 mg per day.
The compounds of formula (I), and pharmaceutically acceptable derivatives thereof, may be used on their own, or in the form of appropriate pharmaceutical compositions in which the compound or derivative is in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. Thus, another aspect concerns a pharmaceutical composition comprising a novel compound of formula (I), or a pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. Administration may be by, but is not limited to, enteral (including oral, sublingual or rectal), intranasal, inhalation, intravenous, topical or other parenteral routes. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, Pharmaceuticals—The Science of Dosage Form Designs, M. E. Aulton, Churchill Livingstone, 2nd Ed. 2002. In one embodiment the pharmaceutical composition preferably comprises less than 80% and in another embodiment less than 50% of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
In one embodiment there is provided a compound selected from any one of the compounds of formula (I), or a pharmaceutically acceptable salt of a compound of formula (I), for use in therapy, especially in the prevention or treatment of cardiovascular disease and metabolic conditions, including but not limited to type 2 diabetes (T2D), obesity, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).
These and other embodiments are described in greater detail herein below, where further aspects will be apparent to one skilled in the art from reading this specification.
The compounds of formula (I), or pharmaceutically acceptable salts thereof, may also be administered in conjunction with other compounds used for the treatment of the above conditions.
In another embodiment, there is a combination therapy wherein a compound selected from any one of the compounds of formula (I), or a pharmaceutically acceptable salt thereof, and a second active ingredient are administered concurrently, sequentially or in admixture, for the treatment of one or more of the conditions listed above. Such a combination may be used in combination with one or more further active ingredients.
When used in a combination therapy, it is contemplated that the a compound selected from any one of the compounds of formula (I), or pharmaceutically acceptable salts thereof, and the other active ingredients may be administered in a single composition, completely separate compositions, or a combination thereof. It also is contemplated that the active ingredients may be administered concurrently, simultaneously, sequentially, or separately. The particular composition(s) and dosing frequency(ies) of the combination therapy will depend on a variety of factors, including, for example, the route of administration, the condition being treated, the species of the patient, any potential interactions between the active ingredients when combined into a single composition, any interactions between the active ingredients when they are administered to the animal patient, and various other factors known to physicians (in the context of human patients), veterinarians (in the context of non-human patients), and others skilled in the art.
There is provided a method of treatment of a condition where modulation of GLP-1 receptor is required, which method comprises administration of a therapeutically effective amount of a compound selected from any one of the compounds of formula (I) to a person suffering from, or susceptible to, such a condition.
The compounds of formula (I) will normally be administered via the oral, topical, parenteral, intravenous, intramuscular, subcutaneous or in other injectable ways, buccal, rectal, vaginal, transdermal and/or nasal route and/or via inhalation, in the form of pharmaceutical preparations comprising the active ingredient or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses. Conventional procedures for the selection and preparation of suitable pharmaceutical formulations are described in, for example, Pharmaceuticals—The Science of Dosage Form Designs, M. E. Aulton, Churchill Livingstone, 2nd Ed. 2002.
In one embodiment suitable daily doses of the compounds of formula (I) in therapeutical treatment of humans are about 0.0001-100 mg/kg body weight, in another embodiment about 0.01-10 mg/kg body weight.
The optimum dosage and frequency of administration will depend on the particular condition being treated and its severity; the species of the patient; the age, sex, size and weight, diet, and general physical condition of the particular patient; brain/body weight ratio; other medication the patient may be taking; the route of administration; the formulation; and various other factors known to physicians and others skilled in the art.
According to a further aspect there is thus provided a pharmaceutical formulation comprising a compound selected from any one of the compounds of formula (I), or pharmaceutically acceptable derivatives thereof, in admixture with a pharmaceutically acceptable adjuvant, diluent and/or carrier.
The compound of formula (I) may be present in the pharmaceutical formulation in a concentration from 0.1 to 99.5%, such as from 0.5 to 95%, by weight of the total formulation.
The protection and deprotection of functional groups is described in Protective Groups in Organic Synthesis, 4th Ed, T. W. Greene and P. G. M. Wuts, Wiley-Interscience (2006) and Protecting Groups, 3rd Ed, P. J. Kocienski, Georg Thieme Verlag (2005).
A further embodiment encompasses pharmaceutically acceptable salts of the compounds of formula (I).
A salt of a compound selected from any one of formula (I) may be advantageous due to one or more of its chemical or physical properties, such as stability in differing temperatures and humidities, or a desirable solubility in H2O, oil, or other solvent. In some instances, a salt may be used to aid in the isolation or purification of the compound. In some embodiments (particularly where the salt is intended for administration to an animal, e.g. a human, or is a reagent for use in making a compound or salt intended for administration to an animal), the salt is pharmaceutically acceptable.
The term “pharmaceutically acceptable” is used to characterize a moiety (e.g. a salt, dosage form, or excipient) as being appropriate for use in accordance with sound medical judgment. In general, a pharmaceutically acceptable moiety has one or more benefits that outweigh any deleterious effect that the moiety may have. Deleterious effects may include, for example, excessive toxicity, irritation, allergic response, and other problems and complications.
Where the compound is sufficiently basic, pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid addition salts.
For reviews on suitable salts, see Berge et al., J. Pharm. Sci., 1977, 66, 1-19 or Handbook of Pharmaceutical Salts: Properties, selection and use, P. H. Stahl, P. G. Vermuth, IUPAC, Wiley-VCH, 2002.
Where an acid co-former is a solid at r.t. and there is no or only partial proton transfer between the compound of formula (I) and such an acid co-former, a co-crystal of the co-former and compound of formula (I) may result rather than a salt. All such co-crystal forms of the compound of formula (I) are encompassed herein.
It is also to be understood that certain compounds of formula (I) may exist in solvated form, e.g. hydrates, including solvates of a pharmaceutically acceptable salt of a compound of formula (I).
In a further embodiment, certain compounds of formula (I) may exist as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. Certain compounds of formula (I) may also contain linkages (e.g. carbon-carbon bonds, carbon-nitrogen bonds such as amide bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring bond or double bond. Stereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallization, or the stereoisomers may be made by stereoselective synthesis.
In a further embodiment, the compounds of formula (I) encompass any isotopically-labelled (or “radio-labelled”) derivatives of a compound of formula (I). Such a derivative is a derivative of a compound of formula (I) wherein one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that may be incorporated include 2H (also written as “D” for deuterium).
In a further embodiment, the compounds of formula (I) may be administered in the form of a prodrug which is broken down in the human or animal body to give a compound of the formula (I).
Various forms of prodrugs are known in the art. For examples of prodrug derivatives, see: Nature Reviews Drug Discovery 2008, 7, 255 and references cited therein. Intermediate compounds may also exist in enantiomeric forms and may be used as purified enantiomers, diastereomers, racemates or mixtures.
The following examples are non-limiting examples.
PrepMethod A: The compound was purified by preparative HPLC on a Kromasil C8 column (10 μm, 250×20 mm ID) using a gradient of MeCN in H2O/MeCN/FA (95/5/0.2) as mobile phase.
PrepMethod B: The compound was purified by preparative HPLC on a YMC Triart C18 column (5 μm, 100×20 mm ID) using a gradient of H2O/MeCN/0.1% NH4OH as mobile phase.
PrepMethod C: The compound was purified by preparative HPLC on a XBridge™ C18 column (10 μm, 250×19 mm ID) using a gradient of MeCN in H2O/MeCN/NH3 (95/5/0.2) as mobile phase.
PrepMethod D: The compound was purified by preparative HPLC on a SunFire column (5 μm, 100×19 mm ID) using a gradient of MeCN in H2O as mobile phase.
PrepMethod E: The compound was purified by preparative HPLC on a SunFire column (5 μm, 150×30 mm ID) using a gradient of MeCN in 0.15 M TFA (aq) at pH3 as mobile phase.
PrepMethod F: The compound was purified by preparative HPLC on a SunFire C18 ODB column (5 μm, 150×30 mm ID) using a gradient of MeCN in FA (aq) at pH3 as mobile phase.
PrepMethod G: The compound was purified by preparative HPLC on a Chromatorex C18 SMB100-5T column (5 μm, 100×19 mm ID) using a gradient of MeCN in H2O as mobile phase.
PrepMethod H: The compound was purified by preparative HPLC on a XBridge™ C18 column (10 μm, 250×50 mm ID) using a gradient of MeCN in H2O/MeCN/NH3 (95/5/0.2) as mobile phase.
Relevant fractions were collected, combined and freeze-dried to give the purified compound or relevant fractions were collected, combined and concentrated at reduced pressure, extracted with DCM or EtOAc, and the organic phase was dried either over Na2SO4 or by using a phase-separator, and then concentrated at reduced pressure to give the purified compound.
ChemDraw is optionally using labels in the graphical representation of stereocenters such as ‘&’ and ‘or’ to describe the configuration of the stereochemical centers present in the structure.
In general chemical structures of Examples and Intermediates containing the label ‘&’ at a stereocenter, means the configuration of such Example or Intermediate at that stereocenter is a mixture of both (R) and (S); and a label ‘or’ means the configuration of such Example or Intermediate at that stereocenter is either (S) or (R). Absolute, unspecified, ‘&’, and ‘or’ stereocenters can all be present in a single structure.
In general for structures of Examples and Intermediates where all of the stereocenters are designated as ‘&’, the structure is named with a “rac-” prefix. For structures of Examples and Intermediates where all of the stereocenters are designated as ‘or’, the structure is named with a “rel-” prefix.
In general Examples and Intermediate compounds are named using the descriptors (RS) and (SR) to denote general ‘&’ centers for chemical structures with multiple chiral centers where only some are designated as ‘&’. The descriptors (R*) and (S*) are used to denote the general ‘or’ centers for chemical structures with multiple chiral centers where only some are designated as ‘or’.
In general, for structures of Examples and Intermediates where all stereocenters present are racemic, no flag is designated to the stereocenter(s) and the structure is drawn with a straight bond at each stereocenter.
In general, for structures of Examples and Intermediates where two or more stereocenters are present in a ring and fixed to each other and do not vary independently of each other, e.g. are cis or trans to each other, said stereocenters are drawn with stereobonds representing their internal relationship. Said stereocenters are labelled with an “&1” flag representing a mixture of cis-configuration or a mixture of trans-configuration, or an “or1” flag representing a single cis-isomer or a single trans-isomer with unknown absolute stereochemistry. In general, should the structure of said Example or Intermediate further contain one or more stereocenters that are racemic and not fixed in relation to the former stereocenters, said stereocenter(s) is drawn with a straight bond at said stereocenters.
In general the descriptors (r) and (s) are used to describe the absolute configuration of any pseudoasymmetric centers in the structures of Examples and Intermediates.
In general the label “Isomer 1” corresponds to the first eluted isomer, and “Isomer 2” corresponds to the second eluted isomer, on a given chiral HPLC column and eluent, and are used to distinguish two isomers containing one or more stereocenters with absolute unknown configuration;
K2CO3 (5.43 g, 39.27 mmol) was added to a solution of methyl 3-fluoro-5-methoxy-4-nitrobenzoate (3 g, 13.09 mmol) and (S)-oxetan-2-ylmethanamine (1.14 g, 13.09 mmol) in THF/DMF (5:2, 110 mL) and the reaction mixture was stirred at 90° C. for 16 h. The solvent was removed under reduced pressure, and the residue was suspended in water (250 mL). The aqueous layer was extracted with EtOAc (3×250 mL), and the combined organic layer was dried over Na2SO4, filtered and evaporated at reduced pressure. The crude product was purified by straight phase flash column chromatography on silica (10-20% EtOAc in petroleum ether) to give the title compound (1.8 g, 46%) as a yellow solid; MS (ESI) m/z [M+H]+ 297.1.
A suspension of Pd—C (0.144 g, 1.35 mmol) and methyl (S)-3-methoxy-4-nitro-5-((oxetan-2-ylmethyl)amino)benzoate Intermediate 1 (4.0 g, 13.50 mmol) in THF (100 mL) was stirred under an atmosphere of H2(g) at 2 atm and 15° C. for 3 h. The reaction mixture was filtered through celite, and the filter cake was washed with MeOH (3×300 mL). The filtrate was concentrated under reduced pressure. The crude product was purified by straight phase flash column chromatography on silica (50-70% EtOAc in petroleum ether) to give the title compound (3.0 g, 83%) as a light yellow solid; MS (ESI) m/z [M+H]+ 267.3.
pTsOH (0.119 g, 0.63 mmol) was added to a solution of methyl (S)-4-amino-3-methoxy-5-((oxetan-2-ylmethyl)amino)benzoate Intermediate 2 (333 mg, 1.25 mmol) and 2-chloro-1,1,1-trimethoxyethane (387 mg, 2.50 mmol) in MeCN (10 mL) and the reaction mixture was stirred at 45° C. for 30 min. The reaction mixture was concentrated under reduced pressure and the residue was purified by straight phase flash column chromatography on silica (50-100% EtOAc in heptane) to give the title compound (155 mg, 38%); MS (ESI) m/z [M+H]+ 325.0.
DIPEA (3.73 ml, 21.35 mmol) was added to a solution of (S)-(tetrahydrofuran-2-yl)methanamine (1.08 g, 10.68 mmol) and 5-bromo-1-fluoro-3-methoxy-2-nitrobenzene (2.67 g, 10.68 mmol) in MeCN (20 mL) and the reaction mixture was stirred at 57° C. for 3 h. The reaction mixture was concentrated at reduced pressure and the residue was purified by straight phase flash column chromatography on silica (0-20% EtOAc in heptane) to give the title product (2.39 g, 68%); MS (ESI) m/z [M+H]+ 333.1.
DIPEA (6.07 mL, 34.73 mmol) and HSiCl3 (2.453 ml, 24.31 mmol) was added dropwise at 0° C. to a solution of (S)-5-bromo-3-methoxy-2-nitro-N-((tetrahydrofuran-2-yl)methyl)aniline Intermediate 4 (2.3 g, 6.95 mmol) in MeCN (20 mL). The reaction mixture was stirred at 0° C. for 2 min and then at rt for 30 min. NaHCO3(aq, 10 mL) was added dropwise and the biphasic mixture was stirred at rt for 30 min and then extracted with EtOAc (2×5 mL). The combined organic layer was dried over MgSO4, filtered and evaporated at reduced pressure to give the title compound (2.0 g, 96%); MS (ESI) m/z [M+H]+ 303.1.
DIPEA (8.70 mL, 49.80 mmol) was added to a suspension of (S)-5-bromo-3-methoxy-N1-((tetrahydrofuran-2-yl)methyl)benzene-1,2-diamine Intermediate 5 (1.5 g, 4.98 mmol) and Pd(dppf)Cl2 (292 mg, 0.40 mmol) in MeOH (20 mL) and the reaction mixture was stirred under an atmosphere of CO(g) at 9 atm and 85° C. for 16 h. The reaction mixture was filtered through a pad of celite, and the filter cake was rinsed with MeOH (10 mL). The filtrate was concentrated at reduced pressure and the residue was purified by straight phase flash column chromatography on silica (20-60% EtOAc in heptane) to give the title compound (444 mg, 32%); MS (ESI) m/z [M+H]+ 281.2.
pTSOH (149 mg, 0.78 mmol) was added to a solution of methyl (S)-4-amino-3-methoxy-5-(((tetrahydrofuran-2-yl)methyl)amino)benzoate Intermediate 6 (440 mg, 1.57 mmol) and 2-chloro-1,1,1-trimethoxyethane (485 mg, 3.14 mmol) in MeCN (10 mL) and the reaction mixture was stirred at 50° C. for 30 min. The reaction mixture was diluted with EtOAc (10 mL) and washed with NaHCO3 (2×3 mL). The organic phase was dried over MgSO4, filtered and evaporated at reduced pressure to give the title compound (450 mg, 85%); MS (ESI) m/z [M+H]+ 399.0.
DIPEA (3.45 mL, 19.77 mmol) was added to a solution of (S)-(tetrahydrofuran-2-yl)methanamine (1 g, 9.89 mmol) and 5-bromo-1-chloro-3-fluoro-2-nitrobenzene (2.52 g, 9.89 mmol) in MeCN (20 mL) and the reaction mixture was stirred at 45° C. for 2 h. The reaction mixture was evaporated at reduced pressure and the residue was purified by straight phase flash column chromatography on silica (0-20% EtOAc in heptane) to give the title compound (2.70 g, 81%); MS (ESI) m/z [M+H]+ 332.8.
Fe(s) (3.59 g, 64.37 mmol) was added to a mixture of (S)-5-bromo-3-chloro-2-nitro-N-((tetrahydrofuran-2-yl)methyl)aniline Intermediate 8 (2.7 g, 8.05 mmol) and NH4Cl (3.44 g, 64.37 mmol) in water (5 mL) and MeOH (15 mL), and the reaction mixture was heated at 60° C. for 1 h. The reaction mixture was filtered through a pad of Celite. The precipitate formed in the filtrate was filtered off, rinsed with water, and dried in vacuo, to give the title compound (2.20 g, 89%); MS (ESI) m/z [M+H]+ 305.11.
DIPEA (12.57 mL, 71.99 mmol) was added to a mixture of (S)-5-bromo-3-chloro-N1-((tetrahydrofuran-2-yl)methyl)benzene-1,2-diamine Intermediate 9 (2.2 g, 7.20 mmol), Pd(dppf)Cl2 (421 mg, 0.58 mmol) in MeOH (20 mL) and the reaction mixture was stirred under an atmosphere of CO(g) at 5 atm and 80° C. for 16 h, and then at 9 bar and 80° C. for 72 h. The reaction mixture was filtered through a pad of Celite, and the pad was rinsed with MeOH (10 mL), and the filtrate was concentrated at reduced pressure. The residue was purified by straight phase flash column chromatography on silica (20-60% EtOAc in heptane) to give the title compound (960 mg, 47%); MS (ESI) m/z [M+H]+ 285.0.
pTsOH (160 mg, 0.84 mmol) was added to a solution of methyl (S)-4-amino-3-chloro-5-(((tetrahydrofuran-2-yl)methyl)amino)benzoate Intermediate 10 (480 mg, 1.69 mmol) and 2-chloro-1,1,1-trimethoxyethane (521 mg, 3.37 mmol) in MeCN (10 mL) and the reaction mixture was stirred at 45° C. for 90 min. The reaction mixture was concentrated at reduced pressure and the residue was purified by straight phase flash column chromatography on silica (50-100% EtOAc in heptane) to give the title compound (280 mg, 48%); MS (ESI) m/z [M+H]+ 343.0.
A mixture of methyl 3-fluoro-4-nitrobenzoate (2.34 g, 11.75 mmol), 2-(1H-pyrazol-1-yl)ethan-1-amine (1.30 g, 11.70 mmol) and DIPEA (6 mL, 34.35 mmol) in MeCN (20 mL) was stirred at 45° C. overnight. The reaction mixture was cooled and concentrated, and the residue was purified by flash chromatography on silica (0-50% EtOAC in heptane). Product containing fractions were pooled and concentrated to give the title compound (1.82 g, 54%) as an orange powder; MS (ESI) m/z [M+H]+ 281.17.
A mixture of methyl 3-((2-(1H-pyrazol-1-yl)ethyl)amino)-4-nitrobenzoate Intermediate 12 (1.82 g, 6.27 mmol) and Pd—C (0.133 g, 0.06 mmol) in AcOH (50 mL) was stirred under an atmosphere of H2 (g) (1 bar) at rt for 2 h. The reaction mixture was filtered through a syringe filter, and the filtrate was concentrated. The residue was dissolved in EtOAc (150 mL) and washed with sat NaHCO3 (aq, 50 mL) and water (50 mL). The organic phase was dried over MgSO4, filtered, and concentrated to give the title compound (1.59 g, 97%) as a yellow oil which solidified upon standing; MS (ESI) m/z [M+H]+ 261.18.
DIPEA (6.91 mL, 39.55 mmol) was added to a solution of (S)-(tetrahydrofuran-2-yl)methanamine (2.0 g, 19.8 mmol) and methyl 3-fluoro-4-nitrobenzoate (3.94 g, 19.8 mmol) in MeCN (20 mL), and the reaction mixture was stirred at 35° C. for 18 h. The reaction mixture was evaporated and the residue was purified by flash chromatography on silica (0-40% EtOAc in heptane) to give the title compound (4.34 g, 78%); 1H NMR (500 MHz, CDCl3) 1.65-1.76 (1H, m), 1.94-2.03 (2H, m), 2.11 (1H, td), 3.35-3.43 (1H, m), 3.52 (1H, d), 3.79-3.86 (1H, m), 3.94 (4H, s), 4.22 (1H, qd), 7.24 (1H, dd), 7.59 (1H, d), 8.22 (2H, d).
Pd—C (5%, 0.330 g, 3.10 mmol) was added to a solution of methyl (S)-4-nitro-3-(((tetrahydrofuran-2-yl)methyl)amino)benzoate Intermediate 14 (4.34 g, 15.48 mmol) in MeOH (40 mL) and the reaction mixture was stirred under an atmosphere of H2(g) (1 atm) at rt for 30 min. The catalyst was filtered off and the filtrate was concentrated under reduced pressure to give the title compound (3.70 g, 9%); MS (ESI) m/z [M+H]+ 251.25.
pTsOH (0.281 g, 1.48 mmol) was added to a solution of methyl (S)-4-amino-3-(((tetrahydrofuran-2-yl)methyl)amino)benzoate Intermediate 15 (3.7 g, 14.78 mmol) and 2-chloro-1,1,1-trimethoxyethane (2.74 g, 17.74 mmol) in MeCN (10 mL), and the reaction mixture was stirred at 45° C. for 90 min. The solvent was evaporated and the residue was purified by flash chromatography on silica (50-100% EtOAc in heptane) to give the title compound (4.30 g, 94%); MS (ESI) m/z [M+H]+ 309.0.
DIPEA (8.45 mL, 48.36 mmol) was added to a solution of methyl 3,5-difluoro-4-nitrobenzoate (3.50 g, 16.12 mmol) and (S)-oxetan-2-ylmethanamine (1.40 g, 16.12 mmol) in THF/DMF (125 mL, 5:2), and the reaction mixture was stirred at 20° C. for 4 h. The solvent was removed under reduced pressure, and the residue was suspended in water (300 mL). The aqueous layer was extracted with EtOAc (3×500 mL), and the combined organic layer was dried over Na2SO4, filtered and evaporated. The crude product was purified by straight phase flash chromatography on silica (gradient: 10-20% EtOAc in petroleum ether) to give the title compound (4.50 g, 98%) as a yellow solid; MS (ESI) m/z [M+H]+ 285.0.
A suspension of methyl (S)-3-fluoro-4-nitro-5-((oxetan-2-ylmethyl)amino)benzoate Intermediate 17 (4.2 g, 14.78 mmol) and 10% Pd—C (1.57 g, 1.48 mmol) in THF (150 mL) was stirred under an atmosphere of H2 (g) at 3 atm and 25° C. for 2 h. The reaction mixture was filtered through celite and the filter cake was washed with MeOH (3×100 mL). The filtrate was concentrated under reduced pressure. The crude product was purified by straight phase flash chromatography on silica (gradient: 70-80% EtOAc in petroleum ether), to give the title compound (3.20 g, 85%) as a light red solid; MS (ESI) m/z [M+H]+ 254.95.
pTsOH (0.108 g, 0.57 mmol) was added to a solution of methyl (S)-4-amino-3-fluoro-5-((oxetan-2-ylmethyl)amino)benzoate Intermediate 18 (1.45 g, 5.70 mmol) and 2-chloro-1,1,1-trimethoxyethane (1.06 g, 6.84 mmol) in MeCN (10 mL) and the reaction mixture was stirred at rt for 18 h. The solvent was evaporated at reduced pressure and the crude compound was purified by straight phase flash chromatography on silica (gradient: 50-100% EtOAc in heptane) to give the title compound (1.54 g, 86%); MS (ESI) m/z [M+H]+ 313.26.
Pd2(dba)3 (454 mg, 0.50 mmol) was added to as suspension of 2-(4-bromo-2-methylbenzo[d][1,3]dioxol-2-yl)-5-chloropyridine (WO2020234726) (1.62 g, 4.96 mmol), rac-tert-butyl (4aR,7aS)-hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate (1.36 g, 5.95 mmol), RuPhos (926 mg, 1.98 mmol) and sodium 2-methylpropan-2-olate (1.91 g, 19.84 mmol) in degassed toluene (10 mL). The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at 35° C. for 18 h. The reaction mixture was cooled to rt and filtered through a pad of celite and the filter cake was rinsed with toluene. The filtrate was concentrated at reduced pressure and the residue was purified by straight phase flash column chromatography on silica (0-20% EtOAc in heptane) to give the title product (1.38 g, 59%); MS (ESI) m/z [M+H]+ 474.4.
Pd2(dba)3 (0.800 g, 0.87 mmol) was added to a suspension of 4-bromo-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxole (WO2020234726) (3 g, 8.73 mmol), rac-tert-butyl (4aR,7aS)-hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate (1.9 g, 8.32 mmol), RuPhos (0.815 g, 1.75 mmol) and sodium 2-methylpropan-2-olate (3.36 g, 34.93 mmol) in degassed toluene (10 mL). The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at 37° C. for 16 h. The reaction mixture was cooled to rt and filtered through a pad of Celite. The pad was rinsed with MeOH (10 mL) and the filtrate was evaporated. The residue was purified by straight phase flash column chromatography on silica (0-20% EtOAc in heptane) to give the title compound (4.00 g, 93%); MS (ESI) m/z [M+H]+ 491.3.
A solution of HCl in 1,4-dioxane (4 M, 1.5 mL, 6.00 mmol) was added to a solution of rac-tert-butyl (4aR,7aS)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 21 (441 mg, 0.90 mmol) in 1,4-dioxane (5 mL), and the reaction mixture was stirred at rt overnight. The reaction mixture was concentrated under reduced pressure to give the title compound as an HCl salt (0.417 g, 100%); MS (ESI) m/z [M+H]+ 391.33.
TFA (187 μL, 2.43 mmol) was added to a solution of rac-tert-butyl (4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 20 (230 mg, 0.49 mmol) in DCM (3.0 mL) and the reaction mixture was stirred at rt for 20 min. The reaction mixture was evaporated at reduced pressure to give the TFA-salt of the sub-title compound; MS (ESI) m/z [M+H]+ 374.2.
The crude product from Step a) was dissolved in MeCN (3 mL) and K2CO3 (201 mg, 1.46 mmol) and methyl (S)-2-(chloromethyl)-4-methoxy-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 3 (155 mg, 0.48 mmol) were added, and the reaction mixture was heated at 75° C. for 2 h, and then at 85° C. for 1 h. The reaction mixture was cooled to rt and filtered through a pad of Celite. The filtrate was evaporated under reduced pressure and the residue was purified by straight phase flash column chromatography on silica (30-100% EtOAc in heptane) to give the title compound (170 mg, 53%); MS (ESI) m/z [M+H]+ 662.5.
The diastereomers of methyl 2-(((4aRS,7aSR)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 23 (170 mg, 0.26 mmol) were separated by chiral chromatography on a LUX C3 (OJ) column (250×20 mm, 5 μm), eluted with 12% MeOH/DEA (100/20 mM) in CO2, 125 bar, at a flow rate of 70 mL/min and detected at 220 nm;
The stereoisomers of the first eluted compound mixture (52 mg) were separated by chiral chromatography on a YMC SA (IA) column (250×30 mm, 5 μm), eluted with 25% MeOH/DEA (100/20 mM) in CO2, 130 bar, at a flow rate of 120 mL/min and detected at 220 nm;
TFA (171 μL, 2.22 mmol) was added to a solution of rac-tert-butyl (4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 20 (210 mg, 0.44 mmol) in DCM (3.0 mL), and the reaction mixture was stirred at rt for 30 min. The reaction mixture was evaporated at reduced pressure and co-evaporate with toluene (10 mL) to give the TFA salt of the sub-title compound; MS (ESI) m/z [M+H]+ 374.0.
The crude product from Step a) was dissolved in MeCN (3 mL), and K2CO3 (184 mg, 1.33 mmol) and methyl (S)-2-(chloromethyl)-4-methoxy-1-((tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 7 (150 mg, 0.44 mmol) were added, and the reaction mixture was heated at 60° C. for 18 h. The reaction mixture was cooled to rt and filtered through a pad of Celite. The filtrate was evaporated under reduced pressure and the residue was purified by straight phase flash column chromatography on silica (30-100% EtOAc in heptane, then 4% EtOH in EtOAc) to give the title compound (200 mg, 67%); MS (ESI) m/z [M+H]+ 676.4.
The diastereomers of methyl 2-(((4aRS,7aSR)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 26 (200 mg, 0.30 mmol) were separated by chiral chromatography on a YMC SJ (imob OJ) column (250×30 mm, 5 μm), eluted with 15% EtOH/DEA (100/20 mM) in CO2, 130 bar, at a flow rate of 130 mL/min and detected at 220 nm;
TFA (392 μL, 5.09 mmol) was added to a solution of rac-tert-butyl (4aR,7aS)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 21 (500 mg, 1.02 mmol) in DCM (10 mL) and the reaction mixture was stirred at rt for 30 min. The reaction mixture was evaporated, and the residue was co-evaporated with toluene (10 mL) followed by MeCN (2×10 mL) to give the TFA salt of the subtitle compound; MS (ESI) m/z [M+H]+ 392.9.
The crude product from Step a) was dissolved in MeCN (10 mL) and K2CO3 (422 mg, 3.06 mmol) and methyl (S)-2-(chloromethyl)-4-methoxy-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 3 (397 mg, 1.22 mmol) were added, and the reaction mixture was heated at 70° C. for 18 h. The reaction mixture was cooled to rt and EtOAc (10 mL) was added, and the mixture was washed with NaHCO3 (aq, 2×5 mL). The organic layer was evaporated at reduced pressure and the residue was purified by straight phase flash column chromatography on silica (10-100% EtOAc in heptane). The product containing fractions were collected and evaporated and the crude compound was purified by preparative HPLC, PrepMethod C, (gradient: 30-95%) to give the title compound (265 mg, 38%); MS (ESI) m/z [M+H]+ 679.5.
The diastereoisomers of methyl 2-(((4aRS,7aSR)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1—(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 29 (265 mg) were separated by chiral chromatography on a YMC SA (IA) column (250×30 mm, 5 μm), eluted with 30% EtOH/DEA (100/20 mM) in CO2, 130 bar, at a flow rate of 120 mL/min and detected at 220 nm;
The stereoisomers of the first eluted compound mixture (116 mg) were separated by chiral chromatography on a LUX C3 (OJ) column (250×30 mm, 5 μm), eluted with 17-22% EtOH/DEA (100/20 mM) in CO2, 120 bar, at a flow rate of 100 mL/min and detected at 220 nm;
The stereoisomers of the second eluted compound mixture (123 mg) were separated by chiral chromatography on a LUX C3 (OJ) column (250×30 mm, 5 μm), eluted with 12-17% EtOH/DEA (100/20 mM) in CO2, 120 bar, at a flow rate of 100 mL/min and detected at 220 nm; the second eluted compound was collected and evaporated to yield the title compound Isomer 4, Intermediate 32 (40 mg); MS (ESI) m/z [M+H]+ 679.57.
TFA (293 μL, 3.80 mmol) was added to a solution of rac-tert-butyl (4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 20 (360 mg, 0.76 mmol) in DCM (3 mL) and the reaction mixture was stirred at rt for 1 h. The reaction mixture was evaporated, and the residue was co-evaporated with toluene (10 mL) followed by MeCN (2×10 mL); MS (ESI) m/z [M+H]+ 374.3.
The crude product from Step a) was dissolved in MeCN (10 mL) and K2CO3 (315 mg, 2.28 mmol) and methyl (S)-4-chloro-2-(chloromethyl)-1-((tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 11 (280 mg, 0.82 mmol) were added, and the reaction mixture was heated at 60° C. for 18 h. The reaction mixture was cooled to rt and the reaction mixture was filtered through Celite. The filtrate was evaporated at reduced pressure and the residue was purified by straight phase flash column chromatography on silica (30-100% EtOAc in heptane, then 4% EtOH in EtOAc). The product containing fractions were collected and evaporated and the crude compound was purified by preparative HPLC, PrepMethod C, (gradient: 50-95%) to give the title compound (280 mg, 54%); MS (ESI) m/z [M+H]+ 680.4.
The diastereoisomers of methyl 4-chloro-2-(((4aRS,7aSR)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 33 (240 mg) were separated by chiral chromatography on a YMC SJ (imob OJ) column (250×30 mm, 5 μm), eluted with 20% EtOH/DEA (100/20 mM) in CO2, 150 bar, at a flow rate of 130 mL/min and detected at 220 nm;
4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 20 (570 mg, 1.2 mmol) in DCM (15 mL), and the reaction mixture was stirred at ambient temperature for 8 h. The reaction mixture was concentrated under reduced pressure to give the sub-title compound as an HCl salt (300 mg, 95%). MS (ESI) m/z [M+H]+ 374.0.
Methyl (S)-2-(chloromethyl)-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate (385 mg, 1.31 mmol), DIPEA (675 mg, 5.22 mmol) and NaI (587 mg, 3.92 mmol) were added to a solution of rac-(4aR,7aS)-1-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride, (Step a) (488 mg, 1.31 mmol) in MeCN (5 mL) and the reaction mixture was stirred at 45° C. overnight. The reaction mixture was diluted with water, and the mixture was extracted with EtOAc. The combined organic layer was washed with sat NaCl (aq), dried over anhydrous Na2SO4, filtered and concentrated to dryness. The residue was purified by preparative HPLC, PrepMethod D, (gradient: 40-50%) to give the title compound (0.167 g, 43%); MS (ESI) m/z [M+H]+ 632.2.
The diastereomers of methyl 2-(((4aRS,7aSR)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 36 (167 mg) were separated by chiral chromatography on a Chiralcel OD-H column (250×20 mm, 5 μm), eluted with a mixture of hexane-IPA-MeOH (80:10:10), at a flow rate of 14 mL/min, followed by chiral chromatography on a Chiralcel OD-H column (250×20 mm, 5 μm), eluted with a mixture of hexane-IPA-MeOH (70:15:15), at a flow rate of 14 mL/min;
Methyl (S)-2-(chloromethyl)-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate (91 mg, 308 μmol) and DIPEA (139 mg, 1.08 mmol) were added to a solution of rac-(4aR,7aS)-1-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 22 (120 mg, 308 μmol) in MeCN (5 mL) and the reaction mixture was stirred at 45° C. overnight. Water was added to the reaction mixture, and the mixture was extracted with EtOAc. The combined organic layer was washed with sat NaCl (aq), dried over anhydrous Na2SO4, filtered and concentrated to dryness. The residue was purified by preparative HPLC, PrepMethod D, (gradient: 10-50%) to give the title compound (0.312 g, 60%); MS (ESI) m/z [M+H]+ 649.0.
The diastereomers of methyl 2-(((4aRS,7aSR)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 41 were separated by chiral chromatography on a Chiralpak IA column (250×4.6 mm, 5 μm) eluted with a mixture of hexane-IPA-MeOH (70:15:15) at a flow rate of 0.6 mL/min;
The stereoisomers of the first eluted compound mixture were separated by chiral chromatography on a Chiralpak IB column (250×4.6 mm, 5 μm) eluted with a mixture of hexane-IPA-MeOH (70:15:15) at a flow rate of 0.6 mL/min;
A solution of 2-chloro-1,1,1-trimethoxyethane (155 μL, 1.15 mmol) in MeCN (1 mL) was added to a solution of methyl 3-((2-(1H-pyrazol-1-yl)ethyl)amino)-4-aminobenzoate Intermediate 13 (284 mg, 1.09 mmol) and pTsOH (21 mg, 0.11 mmol) in MeCN (4 mL) and the reaction mixture was stirred at 50° C. for 2 h.
K2CO3 (498 mg, 3.60 mmol) and a solution of rac-(4aR,7aS)-1-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine dihydrochloride Intermediate 22 (412 mg, 0.89 mmol) in MeCN (3 mL) were added to the reaction mixture of step a and the reaction mixture was stirred at 50° C. overnight. The reaction mixture was concentrated, and the residue was dissolved in EtOAc (100 mL) and washed with water (50 mL) and brine (50 mL). The organic layer was dried over MgSO4, filtered and concentrated. The residue was purified by straight phase flash chromatography on silica (50-100% EtOAc in heptane), to give the title compound (0.360 g, 60%) as a brown oil; MS (ESI) m/z [M+H]+ 673.54.
The diastereomers of rac-methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-2-(((4aR,7aS)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 46 (360 mg) were separated by chiral chromatography on a Lux C4 column (250×50 mm, 5 μm), eluted with 25% EtOH/DEA (100/20 mM) in CO2, 120 bar, at a flow rate of 400 mL/min and detected at 210 nm;
TFA (374 μL, 4.85 mmol) was added to a solution of rac-tert-butyl (4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 20 (460 mg, 0.97 mmol) in DCM (3.0 mL), and the reaction mixture was stirred at rt for 1 h. The reaction mixture was evaporated and the residue was used directly in the next step
The crude product from step a was dissolved in MeCN (3 mL) and K2CO3 (402 mg, 2.91 mmol) and methyl (S)-2-(chloromethyl)-1-((tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 16 (330 mg, 1.07 mmol) were added and the reaction mixture was heated at 60° C. for 18 h. The reaction mixture was allowed to cool to rt, and filtered through a pad of Celite®. The filtrate was concentrated and the residue was purified by flash chromatography on silica (Gradient: 0-100% EtOAc in heptane) to give the title compound (0.440 g, 70%); MS (ESI) m/z [M+H]+ 646.4.
The diastereoisomers of methyl 2-(((4aRS,7aSR)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 50 (440 mg) were separated by chiral chromatography on a YMC SA (IA) column (250×30 mm, 5 μm), eluted with 25% EtOH/DEA (100/20 mM) in CO2, 130 bar, at a flow rate of 150 mL/min and detected at 220 nm;
Methyl (S)-2-(chloromethyl)-4-fluoro-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 19 (337 mg, 1.08 mmol), Cs2CO3 (1.22 g, 3.7 mmol) and NaI (485 mg, 3.24 mmol) were added to a solution of rac-(4aR,7aS)-1-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine (Intermediate 36, Step a) (483 mg, 1.29 mmol) in DMF (7 mL) and the reaction mixture was stirred at 60° C. overnight. Water was added to the reaction mixture and the mixture was extracted with EtOAc. The combined organic layer was washed with sat NaCl (aq), dried over anhydrous Na2SO4, filtered and concentrated to dryness. The residue was purified by preparative HPLC, PrepMethod D, (gradient: 40-65%), to give the title compound (158 mg, 51% yield) as a mixture of isomers; MS (ESI) m/z [M+H]+ 650.2.
The diastereoisomers of methyl 2-(((4aRS,7aSR)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 53 were separated by chiral chromatography on a Chiral ART Cellulose-SC column (250×20 mm, 5 μm), eluted with Hexane/IPA/MeOH (80:10:10), at a flow rate of 12 mL/min;
Cs2CO3 (856 mg, 2.63 mmol) was added to a mixture of 4-bromo-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxole WO2020234726 (361 mg, 1.05 mmol), tert-butyl (4aS,7aR)-hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 163 (200 mg, 0.88 mmol), 2′-(bis(3,5-bis(trifluoromethyl)phenyl)phosphino)-3′,6′-dimethoxy-N2,N2,N6,N6-tetramethyl-[1,1′-biphenyl]-2,6-diamine (14 mg, 0.04 mmol) and methanesulfonato(2-bis(3,5-di(trifluoromethyl)phenylphosphino)-3,6-dimethoxy-2′,6′-bis(dimethylamino)-1,1′-biphenyl)(2′-methylamino-1,1′-biphenyl-2-yl)palladium(II) (50 mg, 0.04 mmol) in 1,4-dioxane (4 mL) at 30° C. under an atmosphere of N2 (g), and the reaction mixture was stirred at 90° C. for 16 h. Another batch was prepared as described above and the reaction mixtures were combined. The combined reaction mixture was diluted with EtOAc (100 mL) and washed sequentially with sat brine (100 mL) and water (100 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The crude product was purified by preparative TLC (petroleum ether:EtOAc, 3:1) to give the title compound (0.890 g, 83%) as a yellow oil which solidified upon standing; MS (ES+) m/z [M+H]+ 491.
tert-Butyl (4aS,7aR)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 58 (870 mg, 1.77 mmol) was dissolved in DCM (30 mL) and TFA (10 mL) at 25° C. and the reaction mixture was stirred at 25° C. for 3 h. The solvent was removed under reduced pressure and the crude product was precipitated from EtOAc. The suspension was filtered and the solids were collected and dried under vacuum to give the title compound as a TFA salt (890 mg, 99%); MS (ESI) m/z [M+H]+ 391.
2-Chloro-1,1,1-trimethoxyethane (0.75 mL, 5.58 mmol) was added to a solution of methyl 3-((2-(1H-pyrazol-1-yl)ethyl)amino)-4-aminobenzoate Intermediate 13 (1.09 g, 4.19 mmol) and pTsOH hydrate (0.086 g, 0.45 mmol) in MeCN (20 mL) and the reaction mixture was stirred at 50° C. for 1.5 h. Additional 2-chloro-1,1,1-trimethoxyethane (0.2 mL, 1.49 mmol) was added and the mixture was heated for 1 h. EtOAc (200 mL) was added and the mixture was washed with sat NaHCO3 (aq, 2×50 mL). The organic layer was dried over MgSO4, filtered, and concentrated to give the title compound (1.27 g, 95%) as a brown solid; MS (ESI) m/z [M+H]+ 319.2.
2-(1H-Pyrazol-1-yl)ethan-1-amine (0.934 g, 8.40 mmol) was added to 5-bromo-1,3-difluoro-2-nitrobenzene (2.0 g, 8.40 mmol) and DIPEA (3.67 mL, 21.01 mmol) in MeCN (25 mL) at 20° C. and the reaction mixture was stirred at 22° C. for 1 h. The reaction mixture was diluted with EtOAc (100 mL) and washed with sat brine (75 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The crude product was purified by preparative TLC (EtOAc:petroleum ether, 5:1), to afford the title compound (2.40 g, 87%) as a yellow solid; MS (ESI) m/z [M+H]+ 329/331.
Zn(s) (3.66 g, 55.91 mmol) was added to N-(2-(1H-pyrazol-1-yl)ethyl)-5-bromo-3-fluoro-2-nitroaniline Intermediate 61 (2.3 g, 6.99 mmol) and NH4Cl (3.74 g, 69.88 mmol) in MeOH (80 mL) and water (20 mL) at 20° C., and the reaction mixture was stirred at 60° C. for 2 h. The reaction mixture was filtered through Celite and the filter cake was washed with MeOH (100 mL). The combined filtrate was concentrated at reduced pressure. The residue was diluted with DCM (100 mL) and washed sequentially with sat brine (100 mL) and water. The organic layer was dried over Na2SO4, filtered and concentrated. The crude product was purified by preparative TLC (MeOH:DCM, 4:1) to give the title product (1.50 g, 72%) as a white solid; MS (ESI) m/z [M+H]+ 299/300.
N1-(2-(1H-Pyrazol-1-yl)ethyl)-5-bromo-3-fluorobenzene-1,2-diamine Intermediate 62 (0.5 g, 1.67 mmol), Pd(dppf)Cl2 (0.122 g, 0.17 mmol) and DIPEA (2.92 mL, 16.71 mmol) in MeOH (150 mL) was stirred under an atmosphere of CO (g) at 60 atm and 120° C. for 12 h. The reaction mixture was concentrated and diluted with EtOAc (150 mL) and washed with sat brine (75 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The crude product was purified by preparative TLC (EtOAc:petroleum ether, 2:1) to give the title compound (0.30 g, 64%) as a yellow solid; MS (ESI) m/z [M+H]+ 279.0.
2-Chloro-1,1,1-trimethoxyethane (0.70 mL, 5.21 mmol) was added to a solution of methyl 3-((2-(1H-pyrazol-1-yl)ethyl)amino)-4-amino-5-fluorobenzoate Intermediate 63 (1.06 g, 3.81 mmol) and pTsOH hydrate (0.086 g, 0.45 mmol) in MeCN (20 mL) and the resulting mixture was stirred at 50° C. for 2 h. EtOAc (100 mL) was added and the mixture was washed with sat NaHCO3 (aq, 2×50 mL). The combined organic layer was dried over MgSO4, filtered, and the solvent was removed at reduced pressure to give the title compound (1.23 g, 96%) as a brown solid; MS (ESI) m/z [M+H]+ 377.0.
The title compound was prepared from 5-bromo-1,3-difluoro-2-nitrobenzene, in 4 steps, in analogy with the description for Intermediate 7 with the exception that in step 3 a solvent mixture of MeOH:DMSO (2:1) was used. The preparation gave the title compound (500 mg, 93%); MS (ESI) m/z [M+H]+ 327.2.
A mixture of methyl 3,5-difluoro-4-nitrobenzoate (2.0 g, 9.21 mmol), (pyridin-3-yl)methanamine (996 mg, 9.21 mmol) and DIPEA (5.95 g, 46.06 mmol) in THF (10 mL) was stirred at 45° C. for 16 h, and then cooled to rt. The mixture was diluted with water and extracted with MTBE. The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated to give the title compound (2.5 g, 89%); MS (ESI) m/z [M+H]+ 306.2.
Methyl 3-fluoro-4-nitro-5-((pyridin-3-yl)methyl)aminobenzoate Intermediate 66 (2.5 g, 8.19 mmol) was dissolved in MeOH (15 mL) and treated with Pd/C (10%, 250 mg). The resulting mixture was hydrogenated under an atmosphere of H2 (g) at ambient pressure and temperature until the reaction was complete. The reaction mixture was filtered and the filtrate was collected and concentrated. The residue was purified by flash chromatography on silica (DCM:MeCN, 1:1) to give the title compound (1.25 g, 53%); 1H NMR (500 MHz, DMSO-d6) δ 8.58 (d, 1H), 8.45 (dd, 1H), 7.74 (dt, 1H), 7.35 (dd, 1H), 6.99 (dd, 1H), 6.83 (d, 1H), 5.64 (t, 1H), 5.43 (s, 2H), 4.36 (d, 2H), 3.69 (d, 3H).
Methyl 4-amino-3-fluoro-5-((pyridin-3-yl)methyl)aminobenzoate Intermediate 67 (600 mg, 2.18 mmol) was dissolved in THF (10 mL). 2-Chloro-1,1,1-trimethoxyethane (404 mg, 2.61 mmol) and pTsOH hydrate (41 mg, 218 μmol) were added, and the reaction mixture was stirred at 50° C. overnight. 4 M HCl in 1,4-dioxane (15 mL) was added and the reaction mixture was filtered. The solids were collected and dried in vacuo to give the HCl salt of the title compound (700 mg, 96%); MS (ESI) m/z [M+H]+ 334.0.
DIPEA (15.44 mL, 88.43 mmol) and 5-bromo-1-chloro-3-fluoro-2-nitrobenzene (7.5 g, 29.48 mmol) were added dropwise to a solution of (S)-oxetan-2-ylmethanamine (2.57 g, 29.48 mmol) in THF (200 mL), and the reaction mixture was stirred at 60° C. for 3 h. The reaction mixture was diluted with EtOAc (300 mL) and washed with sat brine (4×300 mL). The organic layer was dried over Na2SO4, filtered and evaporated. The crude product was purified by flash chromatography on silica (0-50% EtOAc in petroleum ether) to give the title compound (9.00 g, 95%) as a yellow oil; MS (ESI) m/z [M+H]+ 321/323.
Fe(s) (24.66 g, 441.60 mmol) was added to a mixture of (S)-5-bromo-3-chloro-2-nitro-N-(oxetan-2-ylmethyl)aniline Intermediate 69 (14.2 g, 44.16 mmol) and NH4Cl (23.62 g, 441.60 mmol) in MeOH (400 mL) and water (100 mL) at 20° C., and the reaction mixture was stirred at 60° C. for 6 h. The reaction mixture was filtered and the precipitate was washed with MeOH (4×100 mL). The filtrate was concentrated under reduced pressure and the crude product was diluted with EtOAc (500 mL). The organic layer was washed sequentially with water (500 mL) and sat brine (500 mL), dried over Na2SO4, filtered and evaporated. The crude product was purified by flash chromatography on silica (30-50% EtOAc in petroleum ether) to give the title compound (12.0 g, 93%) as a white solid; MS (ESI) m/z [M+H]+ 292/291.
A mixture of (S)-5-bromo-3-chloro-N1-(oxetan-2-ylmethyl)benzene-1,2-diamine Intermediate 70 (1.5 g, 5.14 mmol), Pd(dppf)Cl2·DCM (0.38 g, 0.51 mmol) and DIPEA (8.99 mL, 51.45 mmol) in MeOH (300 mL) was stirred under an atmosphere of CO (g) at 60 atm and 120° C. for 30 h. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (20-25% EtOAc in petroleum ether) to give the title compound (1.0 g, 72%) as a white solid; MS (ESI) m/z [M+H]+ 271.
pTsOH (0.357 g, 1.88 mmol) was added to a solution of methyl (S)-4-amino-3-chloro-5-((oxetan-2-ylmethyl)amino)benzoate Intermediate 71 (5.08 g, 18.77 mmol) and 2-chloro-1,1,1-trimethoxyethane (3.77 g, 24.40 mmol) in MeCN (20 mL) and the reaction mixture was stirred at 50° C. for 30 min. 2-Chloro-1,1,1-trimethoxyethane (1.16 g, 7.51 mmol) was added and the reaction mixture was stirred at 50° C. for 20 min. The reaction mixture was diluted with EtOAc (10 mL) and washed with NaHCO3 (aq, 2×3 mL). The organic layer was dried over MgSO4, filtered and concentrated at reduced pressure. The crude compound was purified by flash chromatography on silica (50-100% EtOAc in heptane) to give the title compound (5.30 g, 86%); MS (ESI) m/z [M+H]+ 329.1.
A mixture of methyl 3,5-difluoro-4-nitrobenzoate (3.0 g, 13.82 mmol), 2-(2-methyl-1H-imidazol-1-yl)ethan-1-amine (1.73 g, 13.82 mmol) and DIPEA (8.93 g, 69.08 mmol) in THE (15 mL) was stirred at 45° C. for 16 h, and then cooled to rt. The mixture was diluted with water and extracted with MTBE. The combined organic layer was washed with brine, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated under reduced pressure to give the title compound (3.5 g, 57%); MS (ESI) m/z [M+H]+ 323.0.
Methyl 3-fluoro-5-(2-(2-methyl-1H-imidazol-1-yl)ethyl)amino-4-nitrobenzoate Intermediate 73 (3.5 g, 10.9 mmol) was dissolved in MeOH (15 mL) and treated with Pd/C (10%, 350 mg). The reaction mixture was hydrogenated under an atmosphere of H2 (g) at ambient pressure and temperature until the reaction was complete. The reaction mixture was filtered, and the filtrate was collected and concentrated. The residue was purified by flash chromatography on silica (0-95% MeOH in MeCN) to give the title compound (1.4 g, 44%); 1H NMR (400 MHz, DMSO-d6) δ 7.19 (t, 1H), 7.08-6.97 (m, 1H), 6.89 (d, 1H), 6.81 (d, 1H), 5.39 (s, 2H), 5.29 (d, 1H), 4.14-4.04 (m, 2H), 3.75 (d, 3H), 3.42 (q, 2H), 2.27 (d, 3H).
Methyl 4-amino-3-fluoro-5-(2-(2-methyl-1H-imidazol-1-yl)ethyl)aminobenzoate Intermediate 74 (1.4 g, 4.79 mmol) was dissolved in THF (80 mL). 2,2,2-Triethoxyethan-1-ol (2.56 g, 14.37 mmol) and pTsOH hydrate (91 mg, 479 μmol) were added, and the reaction mixture was stirred at 50° C. overnight. The mixture was poured into an excessive amount of water and extracted with EtOAc (×3). The combined organic layer was washed with brine and concentrated under vacuum to give the title compound (1.5 g, 94%); MS (ESI) m/z [M+H]+ 333.2.
Thionyl chloride (2.68 g, 22.56 mmol) was added dropwise to a solution of methyl 4-fluoro-2-(hydroxymethyl)-1-(2-(2-methyl-1H-imidazol-1-yl)ethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 75 (1.5 g, 4.51 mmol) in DCM (15 mL) and the reaction mixture was stirred at 50° C. for 2 h. The reaction mixture was concentrated under vacuum, and the residue was suspended in dry Et2O (250 mL) and filtered, to give the dihydrochloride of the title compound (1.0 g, 63%); MS (ESI) m/z [M+H]+ 351.0.
A solution of 2-(2-methyl-1H-imidazol-1-yl)ethan-1-amine dihydrochloride (2.63 g, 13.28 mmol) in dry DMF (50 mL) was slowly added to a mixture of methyl 3-chloro-5-fluoro-4-nitrobenzoate (3.1 g, 13.28 mmol) and DIPEA (6.01 g, 46.49 mmol) in dry DMF (15 mL), and the reaction mixture was stirred at 60° C. for 16 h. The reaction mixture was poured into water (150 mL) and extracted with DCM (3×60 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give the title compound (3.46 g, 77% yield) as an orange oil; MS (ESI) m/z [M+H]+ 339.2.
Pt/C (10%, 0.6 g) was added to a solution of methyl 3-chloro-5-(2-(2-methyl-1H-imidazol-1-yl)ethyl)amino-4-nitrobenzoate Intermediate 77 (3.46 g, 10.2 mmol) in MeOH (100 mL), and the reaction mixture was stirred under an atmosphere of H2 (g) at 1 atm and rt for 5 days. The reaction mixture was filtered through a pad of Celite, and the filtrate was concentrated in vacuo. The residue was purified by chromatography on silica (10% MeOH in CHCl3) to give the title compound (3 g, 96%) as yellow solid; 1H NMR (400 MHz, CDCl3) δ 7.58 (d, 1H), 7.24 (s, 2H), 6.87 (s, 1H), 6.82-6.73 (m, 1H), 4.33 (s, 2H), 4.08 (t, 2H), 3.85 (s, 3H), 3.69 (s, 1H), 3.53 (s, 2H), 2.29 (s, 2H).
2,2,2-Triethoxyethan-1-ol (1.85 g, 10.36 mmol) and pTsOH (59 mg, 345 μmol) were added to a solution of methyl 4-amino-3-chloro-5-(2-(2-methyl-1H-imidazol-1-yl)ethyl)aminobenzoate Intermediate 78 (1.07 g, 3.45 mmol) in MeCN (7 mL) and the reaction mixture was stirred at 60° C. for 16 h. The reaction mixture was allowed to cool to rt and then concentrated in vacuo. The residue was diluted with water (50 mL), and the pH was adjusted to 7 with sat Na2CO3 (aq). The mixture was extracted with EtOAc (3×50 mL) and the combined organic layer was washed with brine (50 mL) and water (50 mL), dried over Na2SO4, filtered, and concentrated in vacuo to give the crude title compound (1.14 g); MS (ESI) m/z [M+H]+ 349.2.
A catalytic amount of DMF followed by thionyl chloride (3.89 g, 32.68 mmol) were added dropwise to a vigorously stirred solution of methyl 4-chloro-2-(hydroxymethyl)-1-(2-(2-methyl-1H-imidazol-1-yl)ethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 79 (1.14 g, 3.27 mmol) in DCM (15 mL). The reaction mixture was stirred at 40° C. for 16 h. The reaction mixture was concentrated in vacuo to give the HCl salt of the title compound (0.95 g, 79%) as a yellow solid; MS (ESI) m/z [M+H]+ 367.0.
A mixture of methyl 3-chloro-5-fluoro-4-nitrobenzoate (4.2 g, 17.97 mmol), (1,3-oxazol-4-yl)methanamine hydrochloride (2.42 g, 17.97 mmol) and DIPEA (5.81 g, 44.94 mmol) in DMF (20 mL) was heated to 50° C. for 16 h. The reaction mixture was poured into water (100 mL) and filtered to give the crude title compound (4.45 g) as dark yellow solid; MS (ESI) m/z [M+H]+ 312.0.
Wet Pt/C (10%, 0.8 g) was added to a yellow suspension of methyl 3-chloro-4-nitro-5-((oxazol-4-ylmethyl)amino)benzoate Intermediate 81 (4.45 g, 14.94 mmol) in MeOH (100 mL), and the reaction mixture was stirred under an atmosphere of H2 (g) at 1 atm and 20° C. for 16 h. The reaction mixture was filtered, and the filter cake was washed with MeOH (50 mL). The filtrate was concentrated under reduced pressure and the crude product was purified by flash chromatography on silica (0-99% MTBE in hexane) to give the title compound (2.13 g, 68%); 1H NMR (500 MHz, CDCl3) δ 7.90 (d, 1H), 7.69-7.54 (m, 2H), 7.30 (q, 1H), 4.51-4.00 (m, 5H), 3.85 (s, 3H).
2-Chloro-1,1,1-trimethoxyethane (1.36 g, 8.77 mmol) and pTsOH (137 mg, 797 μmol) were added to a stirred solution of methyl 4-amino-3-chloro-5-((oxazol-4-ylmethyl)amino)benzoate Intermediate 82 (2.13 g, 7.97 mmol) in MeCN (30 mL) and the reaction mixture was heated at 60° C. for 18 h. The reaction mixture was evaporated under reduced pressure, and the residue was extracted with EtOAc (3×20 mL). The combined organic layer was washed with NaHCO3, dried over Na2SO4, filtered and evaporated. The crude product was purified by column chromatography on silica (CHCl3:EtOAc, 60:40) to give the title compound (1.1 g, 43%); 1H NMR (400 MHz, CDCl3) δ 8.10-8.04 (m, 1H), 8.03-7.96 (m, 1H), 7.82 (s, 1H), 7.65 (t, 1H), 5.45 (s, 2H), 5.07 (s, 2H), 4.00-3.84 (m, 3H).
A mixture of methyl 3,5-difluoro-4-nitrobenzoate (1.5 g, 6.91 mmol), (1,3-oxazol-4-yl)methanamine hydrochloride (930 mg, 6.91 mmol) and DIPEA (2.68 g, 20.73 mmol, 3.61 mL) in THF (10 mL) was stirred at 45° C. for 2 h, and then cooled to rt. The mixture was diluted with water and extracted with MTBE (3×20 mL). The combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo to give the title compound (2.3 g, 79%); MS (ESI) m/z [M+H]+ 297.2.
Methyl 3-fluoro-4-nitro-5-((oxazol-4-ylmethyl)amino)benzoate Intermediate 84 (2.3 g, 5.45 mmol) was dissolved in dry THF (10 mL) and treated with Pd/C (10%, 0.2 g). The reaction mixture was hydrogenated under an atmosphere of H2 (g) at 1 atm and ambient temperature until the reaction was complete. The reaction mixture was filtered and the filtrate was concentrated in vacuo. The residue was purified by flash chromatography on silica (0-40% MeOH in CHCl3) to give the title compound (1.5 g, 88%); MS (ESI) m/z [M+H]+ 266.0.
2-Chloro-1,1,1-trimethoxyethane (1.05 g, 6.79 mmol) and pTsOH hydrate (54 mg, 283 μmol) was added to a solution of methyl 4-amino-3-fluoro-5-((oxazol-4-ylmethyl)amino)benzoate Intermediate 85 (1.5 g, 4.81 mmol) in THF (50 mL) and the reaction mixture was stirred at 50° C. overnight. The mixture was poured into water and extracted with EtOAc (3×10 mL). The combined organic layer was washed with brine and concentrated under vacuum to give the title compound (1.6 g, 60%); MS (ESI) m/z [M+H]+ 324.2.
A mixture of methyl 3,5-difluoro-4-nitrobenzoate (5.0 g, 23.03 mmol), 2-(1-(aminomethyl)cyclopropyl)acetonitrile hydrochloride (3.38 g, 23.03 mmol) and DIPEA (12.0 mL, 69.08 mmol) in THF (20 mL) was stirred at 60° C. for 16 h. The reaction mixture was cooled to rt and diluted with water. The aqueous phase was extracted with MTBE (3×50 mL), and the combined organic layer was washed with brine, dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated in vacuo to give the title compound (5.1 g, 54%); MS (ESI) m/z [M+H]+ 308.0.
Pd/C (10%, 0.5 g) was added to a solution of methyl 3-(((1-(cyanomethyl)cyclopropyl)methyl)amino)-5-fluoro-4-nitrobenzoate Intermediate 87 (5.1 g, 12.45 mmol) in dry MeOH (20 mL) and the reaction mixture was stirred under an atmosphere of H2 (g) at 1 atm and at ambient temperature until the reaction was complete. The reaction mixture was filtered and concentrated in vacuo. The residue was purified by flash chromatography on silica (0-99% of MTBE in hexane) to give the title compound (2.8 g, 77%); MS (ESI) m/z [M+H]+ 278.2.
2-Chloro-1,1,1-trimethoxyethane (1.34 mL, 9.92 mmol) and pTsOH (155 mg, 902 μmol) were added to a solution of methyl 4-amino-3-(((1-(cyanomethyl)cyclopropyl)methyl)amino)-5-fluorobenzoate Intermediate 88 (2.5 g, 9.02 mmol) in THF (50 mL) and the reaction mixture was stirred at 50° C. overnight. The reaction mixture was poured into water and extracted with EtOAc (3×10 mL). The combined organic layer was washed with brine and concentrated under vacuum to give the title compound (2.25 g, 67%); MS (ESI) m/z [M+H]+ 336.2.
The title compound was prepared in three steps as described for Intermediate 89 from methyl 3,5-difluoro-4-nitrobenzoate and 1-(aminomethyl)cyclopropane-1-carbonitrile hydrochloride to give the title compound (2.35 g, 69%); MS (ESI) m/z [M+H]+ 322.0.
1-(Aminomethyl)cyclopropane-1-carbonitrile hydrochloride (1.76 g, 13.24 mmol) and DIPEA (5.13 g, 39.72 mmol) was slowly added to a colorless solution of methyl 3-chloro-5-fluoro-4-nitrobenzoate (3.09 g, 13.24 mmol) in DMF (70 mL) and the reaction mixture was stirred at 60° C. for 16 h. The reaction mixture was poured into water (250 mL) and extracted with DCM (3×80 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude title compound (3.6 g); MS (ESI) m/z [M+H]+ 310.0.
Wet Pt/C (10%, 0.12 g) was added to a yellow suspension of crude methyl 3-chloro-5-(((1-cyanocyclopropyl)methyl)amino)-4-nitrobenzoate Intermediate 91 (3.6 g) in MeOH (180 mL) and the reaction mixture was stirred under an atmosphere of H2(g) at 1 atm and at 20° C. for 36 h. The reaction mixture was filtered and the filter cake was washed with MeOH (100 mL). The filtrate was concentrated under reduced pressure and the crude residue was purified by flash chromatography on silica (0-99% MTBE in hexane) to give the title compound (0.68 g, 21%); MS (ESI) m/z [M+H]+ 280.2.
2-Chloro-1,1,1-trimethoxyethane (327 mg, 2.11 mmol) and pTsOH (33 mg, 192 μmol) were added to a stirred solution of methyl 4-amino-3-chloro-5-(((1-cyanocyclopropyl)methyl)amino)benzoate Intermediate 92 (538 mg, 1.92 mmol) in MeCN (50 mL) and the reaction mixture was heated at 60° C. for 2 h. The reaction mixture was cooled to rt and evaporated under reduced pressure. The residue was diluted with EtOAc (70 mL), and washed with NaHCO3 (30 mL) and water (30 mL). The organic layer was dried over anhydrous Na2SO4 and concentrated to give the title compound (0.6 g, 87%); MS (ESI) m/z [M+H]+ 280.2.
A mixture of methyl 3-chloro-5-fluoro-4-nitrobenzoate (5.05 g, 21.62 mmol), 2-(1-(aminomethyl)cyclopropyl)acetonitrile hydrochloride (3.17 g, 21.62 mmol) and DIPEA (8.38 g, 64.87 mmol) in THF (200 mL) was heated at 50° C. for 14 h. The reaction mixture was evaporated under reduced pressure, and the residue was diluted with EtOAc (150 mL). The organic layer was washed with water (2×50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give the crude title compound (5.73 g); MS (ESI) m/z [M+H]+ 324.0.
Wet Pt/C (10%, 0.2 g) was added to a suspension of methyl 3-chloro-5-(((1-(cyanomethyl)cyclopropyl)methyl)amino)-4-nitrobenzoate Intermediate 94 (5.73 g, 17.7 mmol) in MeOH (200 mL) and the reaction mixture was stirred under an atmosphere of H2 (g) at 1 atm and at 20° C. for 64 h. The reaction mixture was filtered, and the catalyst carefully washed with MeOH (100 mL). The filtrate was concentrated under reduced pressure. The crude compound was purified by flash chromatography on silica (0-99% MTBE in hexane) to give the title compound (3.7 g, 71%); 1H NMR (500 MHz, CDCl3) δ 7.66 (s, 1H), 7.34 (s, 1H), 3.87 (s, 3H), 3.18 (s, 2H), 2.61 (s, 2H), 1.58 (s, 3H), 0.76 (s, 4H).
2-Chloro-1,1,1-trimethoxyethane (435 mg, 2.81 mmol) and pTsOH (44 mg, 256 μmol) were added to a stirred solution of methyl 4-amino-3-chloro-5-(((1-(cyanomethyl)cyclopropyl)methyl)amino)benzoate Intermediate 95 (751 mg, 2.56 mmol) in MeCN (100 mL) and the reaction mixture was heated at 60° C. for 2 h. The reaction mixture was cooled to rt and then evaporated under reduced pressure. The residue was diluted with EtOAc (70 mL) and the mixture was washed with NaHCO3 (30 mL) and water (30 mL). The organic layer was dried over Na2SO4 and evaporated at reduced pressure to give the crude title compound (0.72 g); MS (ESI) m/z [M+H]+ 352.0.
A mixture of methyl 3-fluoro-4-nitrobenzoate (3.44 g, 17.28 mmol), 2-[1-(aminomethyl)cyclopropyl]acetonitrile hydrochloride (2.53 g, 17.28 mmol) and DIPEA (6.7 g, 51.85 mmol) in MeCN (50 mL) was stirred at 50° C. for 12 h. The reaction mixture was cooled to rt, then diluted with water and extracted with DCM (3×100 mL). The combined organic layer was washed with brine (100 mL), dried over anhydrous Na2SO4, and filtered. The filtrate was concentrated at reduced pressure to give the title compound (5.0 g, 80%); MS (ESI) m/z [M+H]+ 290.0.
Methyl 3-(((1-(cyanomethyl)cyclopropyl)methyl)amino)-4-nitrobenzoate Intermediate 97 (5.02 g, 17.35 mmol) was dissolved in dry MeOH (50 mL) and treated with Pt/C (1% wt, 0.2 g, 5.21 mmol). The reaction mixture was hydrogenated under an atmosphere of H2 (g) at 1 atm and at ambient temperature until the reaction was complete. The reaction mixture was filtered and the filtrate was concentrated in vacuo to give the title compound (4.5 g, 60%); MS (ESI) m/z [M+H]+ 260.2.
2-Chloro-1,1,1-trimethoxyethane (1.07 g, 6.92 mmol) and pTsOH (54.19 mg, 315 μmol) was added to a solution of methyl 4-amino-3-(((1-(cyanomethyl)cyclopropyl)methyl)amino)benzoate Intermediate 98 (1.63 g, 6.29 mmol) in MeCN (20 mL) and the reaction mixture was stirred at 60° C. overnight. The reaction mixture was poured into water and extracted with DCM (3×15 mL). The combined organic layer was washed with brine and concentrated in vacuo to give the title compound (1.8 g, 89%); MS (ESI) m/z [M+H]+ 318.0.
The title compound was prepared in three steps from methyl 3-fluoro-4-nitrobenzoate and 1-(aminomethyl)cyclopropane-1-carbonitrile hydrochloride as described for Intermediate 99 to give the title compound (700 mg, 80%); MS (ESI) m/z [M+H]+ 304.2.
A mixture of 5-bromo-1-chloro-3-fluoro-2-nitrobenzene (2.03 g, 7.98 mmol), 2-(1H-pyrazol-1-yl)ethan-1-amine (0.89 g, 8.01 mmol), and DIPEA (2.79 mL, 15.96 mmol) in MeCN (5 mL) was stirred at 45° C. overnight. The reaction mixture was cooled to rt and concentrated at reduced pressure. The crude product was purified by flash chromatography on silica (0-50% EtOAC in heptane) to give the title compound (2.55 g, 92%) as a yellow powder; MS (ESI) m/z [M+H]+ 344.9.
A solution of trichlorosilane (1.721 mL, 17.06 mmol) in MeCN (5 mL) was added dropwise over 15 min and under an atmosphere of N2(g) to an ice-cold solution of N-(2-(1H-pyrazol-1-yl)ethyl)-5-bromo-3-chloro-2-nitroaniline Intermediate 101 (1.67 g, 4.83 mmol) and DIPEA (4.5 mL, 25.76 mmol) in MeCN (30 mL). The cooling bath was removed and the reaction mixture was stirred at rt overnight. Sat NaHCO3 (aq, 30 mL) was added dropwise to the reaction mixture and it was then diluted with water and extracted with EtOAc (4×50 mL). The combined organic layer was dried over MgSO4, filtered and concentrated and the crude residue was purified by preparative HPLC, PrepMethod H (gradient 30-70%) to give the title compound (0.750 g, 49%) as a dark oil; MS (ESI) m/z [M+H]+ 315.20.
Pd(dppf)Cl2 (152 mg, 0.21 mmol) was added to a mixture of N1-(2-(1H-pyrazol-1-yl)ethyl)-5-bromo-3-chlorobenzene-1,2-diamine Intermediate 102 (0.75 g, 2.38 mmol) and DIPEA (4.15 mL, 23.76 mmol) in MeOH:DMSO (4:1, 5 mL) and the reaction mixture was stirred under an atmosphere of CO (g) (9 atm) at 85° C. for 16 h. The reaction mixture was filtered through a pad of celite and the pad was rinsed with MeOH until the washings were colourless. The filtrate was concentrated and the residue was purified by preparative HPLC, PrepMethod H (gradient:20-60%) to give the title compound (0.372 g, 53%) as a light-brown solid; MS (ESI) m/z [M+H]+ 295.1.
2-Chloro-1,1,1-trimethoxyethane (0.247 mL, 1.84 mmol) was added to a solution of methyl 3-((2-(1H-pyrazol-1-yl)ethyl)amino)-4-amino-5-chlorobenzoate Intermediate 103 (0.361 g, 1.22 mmol) and pTsOH hydrate (0.025 g, 0.13 mmol) in MeCN (6 mL) and the reaction mixture was stirred at 50° C. for 2 h, then at rt for 2 days. 2-Chloro-1,1,1-trimethoxyethane (0.100 mL, 0.74 mmol) was added and the reaction mixture was heated at 50° C. for 2 h. EtOAc (50 mL) was added to the reaction mixture and the mixture was washed with sat NaHCO3 (aq, 2×30 mL). The organic layer was dried over MgSO4, filtered, and concentrated at reduced pressure to give the title compound (0.402 g, 93%) as a light-brown solid; MS (ESI) m/z [M+H]+ 353.2.
A mixture of rac-(4aR,7aS)-1-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine dihydrochloride Intermediate 36 Step a) (0.625 g, 1.26 mmol), methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-2-(chloromethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 60 (0.407 g, 1.28 mmol), and K2CO3 (0.76 g, 5.50 mmol) in MeCN:H2O (20:1, 10.5 mL) was stirred at 70° C. for 24 h. The reaction mixture was concentrated and the residue was dissolved in EtOAc (100 mL). The organic layer was washed with water (2×50 mL), dried over MgSO4, and concentrated. The crude product was purified by flash chromatography on silica (EtOAc) to give the title compound (0.665 g, 80%) as a light-brown oil; MS (ESI) m/z [M+H]+ 656.4.
A mixture of rac-(4aR,7aS)-1-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine dihydrochloride Intermediate 36 Step a) (0.402 g, 0.81 mmol), methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-2-(chloromethyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate Intermediate 64 (0.275 g, 0.82 mmol), and K2CO3 (0.552 g, 3.99 mmol) in a MeCN:H2O (20:1, 6.3 mL) was stirred at 70° C. for 2 days. The reaction mixture was concentrated, and the residue was dissolved in EtOAc (100 mL). The organic layer was washed with water (2×50 mL), dried over MgSO4, and concentrated. The crude product was purified by flash chromatography on silica (EtOAc) to give the title compound (0.425 g, 78%) as a solid foam; MS (ESI) m/z [M+H]+ 674.62.
TFA (279 μL, 3.62 mmol) was added to a solution of rac-tert-butyl (4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 20 (343 mg, 0.72 mmol) in DCM (3.0 mL) and the reaction mixture was stirred at rt for 30 min. The reaction mixture was evaporated at reduced pressure and co-evaporated with toluene to give the TFA salt of the sub-title compound; MS (ESI) m/z [M+H]+ 374.3. The crude title compound was used in the next step.
rac-(4aR,7aS)-1-(2-(5-Chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine trifluoroacetate Intermediate 107 Step a) was dissolved in MeCN (3 mL) and K2CO3 (300 mg, 2.17 mmol) and methyl (S)-2-(chloromethyl)-4-fluoro-1-((tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 65 (284 mg, 0.87 mmol) were added and the reaction mixture was heated at 60° C. for 18 h, then at 70° C. for 24 h. The reaction mixture was cooled to rt and filtered through Celite. The filtrate was collected, evaporated at reduced pressure and the crude residue was purified by flash chromatography on silica (EtOAc:heptane, 30:70, then 30-100% EtOAc in heptane) to give the title compound (0.443 g, 92% in two steps); MS (ESI) m/z [M+H]+ 664.4.
Methyl 2-(chloromethyl)-4-fluoro-1-(pyridin-3-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate dihydrochloride Intermediate 68 (429 mg, 1.05 mmol) was added to a suspension of rac-(4aR,7aS)-1-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 22 (412 mg, 1.05 mmol), DIPEA (817 mg, 6.32 mmol) and NaI (632 mg, 4.22 mmol) in DMF (25 mL) and the reaction mixture was stirred at 60° C. for 18 h. The reaction mixture was concentrated in vacuo, then diluted with water (20 mL) and extracted with DCM. The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by preparative HPLC, PrepMethod D (gradient:10-50%) to give the title compound (83 mg, 11%); MS (ESI) m/z [M+H]+ 688.2.
DIPEA (620 mg, 4.8 mmol) was added to a suspension of rac-(4aR,7aS)-1-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 36 Step a) (449 mg, 1.2 mmol), methyl (S)-4-chloro-2-(chloromethyl)-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 72 (395 mg, 1.2 mmol) and NaI (540 mg, 3.6 mmol) in MeCN (5 mL) at 20° C., and the reaction mixture was heated at 60° C. overnight. The reaction mixture was concentrated under reduced pressure and the residue was suspended in EtOAc (10 mL). The organic layer was washed with sat NaHCO3 (aq, 20 mL), brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by preparative HPLC, PrepMethod D (gradient 40-50%) to give the title compound (123 mg, 15%); MS (ESI) m/z [M+H]+ 666.2.
Cs2CO3 (1.5 g, 4.61 mmol) was added to a suspension of rac-(4aR,7aS)-1-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 22 (515 mg, 1.32 mmol), methyl (S)-4-chloro-2-(chloromethyl)-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 72 (433 mg, 1.32 mmol) and NaI (395 mg, 2.63 mmol) in MeCN (5 mL) at 20° C. and the reaction mixture was heated at 60° C. overnight. The reaction mixture was concentrated under reduced pressure and the residue was suspended in EtOAc (10 mL). The organic layer was washed with sat NaHCO3 (aq, 20 mL), brine (20 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by preparative HPLC, PrepMethod D (gradient: 40-65%) to give the title compound (158 mg, 17%); MS (ESI) m/z [M+H]+ 683.3.
Methyl (S)-2-(chloromethyl)-4-fluoro-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 19 (375 mg, 1.20 mmol) was added to a suspension of rac-(4aR,7aS)-1-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 22 (469 mg, 1.2 mmol), Cs2CO3 (1.36 g, 4.2 mmol) and NaI (360 mg, 2.4 mmol) in DMF (6 mL), and the reaction mixture was stirred at 60° C. for 18 h. The reaction mixture was concentrated in vacuo, the residue was diluted with water (20 mL) and the obtained mixture was extracted with EtOAc (3×30 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D (gradient: 0-50%) to give the title compound (114 mg, 17%); MS (ESI) m/z [M+H]+ 667.2.
Methyl 2-(chloromethyl)-4-fluoro-1-(2-(2-methyl-1H-imidazol-1-yl)ethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 76 (300 mg, 709 μmol) was added to a suspension of rac-(4aR,7aS)-1-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 22 (277 mg, 709 μmol), DIPEA (550 mg, 4.25 mmol), and NaI (425 mg, 2.84 mmol) in DMF (20 mL) and the reaction mixture was stirred at 60° C. for 18 h. The reaction mixture was concentrated in vacuo, diluted with water (20 mL) and extracted with DCM (3×8 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D (gradient: 30-80%) to give the title compound (136 mg, 27%); MS (ESI) m/z [M+H]+ 705.0.
Methyl 4-chloro-2-(chloromethyl)-1-(2-(2-methyl-1H-imidazol-1-yl)ethyl)-1H-benzo[d]imidazole-6-carboxylate dihydrochloride Intermediate 80 (263 mg, 717 μmol.) was added to a solution of rac-(4aR,7aS)-1-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 22 (280 mg, 717 μmol), DIPEA (556 mg, 4.3 mmol), and NaI (430 mg, 2.87 mmol) in DMF (20 mL) and the reaction mixture was stirred at 60° C. for 18 h. The reaction mixture was concentrated in vacuo, diluted with water (20 mL), and extracted with DCM (3×10 mL). The combined organic layer was washed with brine and water, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D (gradient 10-50%) to give the title compound (76 mg, 15%); MS (ESI) m/z [M+H]+ 721.2.
Methyl 4-chloro-2-(chloromethyl)-1-(oxazol-4-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 83 (248 mg, 759 μmol) was added to a suspension rac-(4aR,7aS)-1-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 22 (297 mg, 759 μmol), DIPEA (343 mg, 2.66 mmol), and NaI (455 mg, 3.04 mmol) in DMF (20 mL) and the reaction mixture was stirred at 60° C. for 18 h. The reaction mixture was concentrated in vacuo, then diluted with water (20 mL) and extracted with DCM (3×20 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D (gradient: 10-50%) to give the title compound (316 mg, 56%); MS (ESI) m/z [M+H]+ 694.0.
Methyl 2-(chloromethyl)-4-fluoro-1-(oxazol-4-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 86 (252 mg, 779 μmol) was added to a suspension of rac-(4aR,7aS)-1-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 22 (304 mg, 779 μmol), DIPEA (352 mg, 2.73 mmol) and NaI (467 mg, 3.12 mmol) in DMF (20 mL) and the reaction mixture was stirred at 60° C. for 18 h. The reaction mixture was concentrated in vacuo, then diluted with water (20 mL) and extracted with DCM (3×10 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D (gradient: 10-50%) to give the title compound (108 mg, 20%); MS (ESI) m/z [M+H]+ 678.2.
Methyl 2-(chloromethyl)-1-((1-(cyanomethyl)cyclopropyl)methyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate Intermediate 89 (0.246 g, 0.73 mmol) was added to a suspension of rac-(4aR,7aS)-1-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 36 Step a) (0.3 g, 0.73 mmol) and DIPEA (0.473 g, 4.10 mmol) in DMF (5 mL) and the reaction mixture was stirred at 50° C. for 16 h. The reaction mixture was concentrated in vacuo, then diluted with water (10 mL) and extracted with EtOAc (3×5 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D (gradient: 10-50%) to give the title compound (252 mg, 50%); MS (ESI) m/z [M+H]+ 673.0.
rac-(4aR,7aS)-1-(2-(5-Chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 36 Step a) (0.3 g, 0.8 mmol) was added to a suspension of methyl 2-(chloromethyl)-1-((1-cyanocyclopropyl)methyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate Intermediate 90 (0.258 g, 0.8 mmol) and DIPEA (0.519 g, 4 mmol) in MeCN (10 mL) and the reaction mixture was stirred at 60° C. for 18 h. The reaction mixture was concentrated in vacuo, diluted with water (70 mL) and extracted with DCM (2×150 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D (gradient: 10-50%) to give the title compound (220 mg, 43%); MS (ESI) m/z [M+H]+ 659.0.
rac-(4aR,7aS)-1-(2-(5-Chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 36 Step a) (476 mg, 1.27 mmol) was added to a suspension of methyl 4-chloro-2-(chloromethyl)-1-((1-cyanocyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 93 (431 mg, 1.2 mmol), DIPEA (988 mg, 7.64 mmol) and NaI (764 mg, 5.1 mmol) in MeCN (150 mL) and the reaction mixture was stirred at 60° C. for 18 h. The reaction mixture was concentrated in vacuo, the residue was diluted with water (50 mL) and the mixture was extracted with DCM (2×85 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D, (gradient: 40-95%) to give the title compound (84 mg, 10%); MS (ESI) m/z [M+H]+ 677.2.
rac-(4aR,7aS)-1-(2-(5-Chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 36 Step a) (792 mg, 2.12 mmol) was added to a suspension of crude methyl 4-chloro-2-(chloromethyl)-1-((1-(cyanomethyl)cyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 96 (746 mg), DIPEA (1.64 g, 12.71 mmol) and NaI (1.27 g, 8.47 mmol) in MeCN (250 mL) and the reaction mixture was stirred at 60° C. for 18 h. The reaction mixture was concentrated in vacuo, diluted with water (70 mL) and extracted with DCM (2×150 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D (gradient 20-45%) to give the title compound (76 mg, 4%); MS (ESI) m/z [M+H]+ 689.2.
Methyl 2-(chloromethyl)-1-((1-(cyanomethyl)cyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 99 (503 mg, 1.58 mmol) was added to a suspension of rac-(4aR,7aS)-1-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 36 Step a) (650 mg, 1.58 mmol), DIPEA (819 mg, 6.34 mmol) and NaI (1.19 g, 7.92 mmol) in MeCN (10 mL) and the reaction mixture was stirred at 60° C. for 12 h. The reaction mixture was concentrated in vacuo, then diluted with water (10 mL) and extracted with DCM (3×15 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D (gradient: 10-50%) to give the title compound (500 mg, 50%); MS (ESI) m/z [M+H]+ 655.2.
A mixture of methyl 2-(chloromethyl)-1-((1-cyanocyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 100 (492 mg, 1.62 mmol), rac-(4aR,7aS)-1-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine hydrochloride Intermediate 36 Step a) (665 mg, 1.62 mmol), DIPEA (838 mg, 6.48 mmol) and NaI (1.21 g, 8.1 mmol) in MeCN (10 mL) was stirred at 60° C. for 12 h. The reaction mixture was concentrated in vacuo, then diluted with water (5 mL) and extracted with DCM (3×10 mL). The combined organic layer was dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by preparative HPLC, PrepMethod D (gradient. 40-50%) to give the title compound (400 mg, 40%); MS (ESI) m/z [M+H]+ 641.0.
A mixture of rac-(4aR,7aS)-1-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine dihydrochloride Intermediate 36 Step a) (0.55 g, 1.11 mmol), methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-4-chloro-2-(chloromethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 104 (0.39 g, 1.10 mmol), and K2CO3 (0.836 g, 6.05 mmol) in MeCN:H2O (20:1, 10.5 mL) was stirred at 70° C. overnight and then concentrated at reduced pressure. The residue was dissolved in EtOAc (100 mL), and the organic layer was washed with water (2×50 mL), dried over MgSO4, and concentrated. The crude product was purified by flash chromatography on silica (EtOAc) to give the title compound (0.642 g, 84%) as a solid off-white foam; MS (ESI) m/z [M+H]+ 690.4.
K2CO3 (1.204 g, 8.72 mmol) was added to (4aR,7aS)-1-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine trifluoro acetate Intermediate 59 (880 mg, 1.74 mmol) and methyl (S)-2-(chloromethyl)-4-methoxy-1-((tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 7 (600 mg, 1.77 mmol) in MeCN (30 mL) and the reaction mixture was stirred at 25° C. for 8 h. The reaction mixture was filtered through Celite and the filter cake was washed with MeCN (3×50 mL). The filtrate was collected and concentrated at reduced pressure. The crude product was purified by flash chromatography on a C18 column (0-100% MeCN in water) to give the title compound (1.02 g, 85%) as a white solid; MS (ESI) m/z [M+H]+ 679.
The stereoisomers of rac-methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-2-(((4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 105 (828 mg, 1.26 mmol) were separated by chiral chromatography on a Lux C4 column (250×30 mm, 5 μm), eluted with 40% (EtOH, 20 mM DEA) in CO2, 120 bar, at a flow rate of 120 mL/min, and detected at 266 nm;
The stereoisomers of rac-methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-2-(((4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate Intermediate 106 (425 mg, 0.63 mmol) were separated by chiral chromatography on a Chiralpak-IBN column (250×30 mm, 5 μm), eluted with 30% (EtOH, 20 mM DEA) in CO2, 130 bar, at a flow rate of 125 mL/min, and detected at 220 nm;
The stereoisomers of methyl 2-(((4aRS,7aSR)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 107 (495 mg, 0.75 mmol) were separated by chiral chromatography on a YMC SA (IA) column (250×30 mm, 5 μm), eluted with 15% (EtOH, 20 mM DEA) in CO2, 125 bar, at a flow rate of 140 mL/min, and detected at 220 nm;
The stereoisomers of rac-methyl 2-(((4aR,7aS)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(pyridin-3-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 108 (83 mg, 0.12 mmol) were separated by chiral chromatography on a Chiralpak IC column (250×20 mm, 5 μm), eluted with MeCN, at a flow rate of 12 mL/min, and detected at 205, 215 and 254 nm; the first eluted compound was collected and evaporated to give the title compound, Isomer 1 Intermediate 128 (20 mg, 24%); MS (ESI) m/z [M+H]+ 688.2;
The stereoisomers of methyl 4-chloro-2-(((4aRS,7aSR)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 109 (129 mg, 0.194 mmol) were separated by chiral chromatography on a YMC Chiral Art column (250×20 mm, 5 μm), eluted with hexane:IPA:MeOH (70:15:15) at a flow rate of 12 mL/min; the first eluted compound was collected and evaporated to give the title compound Isomer 1, Intermediate 131 (39 mg, 30%); MS (ESI) m/z [M+H]+ 668.2;
The stereoisomers of the second eluted compound mixture were separated by chiral chromatography on a Chiralcel OD-H column (250×20 mm, 5 μm), eluted with hexane:IPA:MeOH (70:15:15) at a flow rate of 12 mL/min;
The stereoisomers of methyl 4-chloro-2-(((4aRS,7aSR)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 110 (158 mg, 0.23 mmol) were separated by chiral chromatography on a YMC Chiral Art column (250×20 mm, 5 μm), eluted with hexane:IPA:MeOH (95:5:5), at a flow rate of 13 mL/min; the first eluted compound was collected and evaporated to give the title compound Isomer 1, Intermediate 135 (44 mg, 28%); MS (ESI) m/z [M+H]+ 685.2;
The stereoisomers of methyl 2-(((4aRS,7aSR)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 111 (114 mg, 0.17 mmol) were separated by chiral chromatography on a ChiralART YMC column (250×20 mm, 5 μm), eluted with hexane:IPA:MeOH (95:5:5), at a flow rate of 13 mL/min; the first eluted compound was collected and evaporated to give the title compound Isomer 1, Intermediate 139 (31 mg, 27%); MS (ESI) m/z [M+H]+ 667.2;
The stereoisomers of rac-methyl 2-(((4aR,7aS)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(2-(2-methyl-1H-imidazol-1-yl)ethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 112 (136 mg, 0.19 mmol) were separated by chiral chromatography on a Chiralpak IC-III column (250×20 mm, 5 μm), eluted with hexane:IPA:MeOH (75:15:15), at a flow rate of 12 mL/min;
The stereoisomers of rac-methyl 4-chloro-2-(((4aR,7aS)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(2-(2-methyl-1H-imidazol-1-yl)ethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 113 (76 mg, 0.11 mmol) were separated by chiral chromatography on a ChiralArt YMC column (250×20 mm, 5 μm), eluted with IPA:MeOH (50:50), at a flow rate of 10 mL/min; the first eluted compound was collected and evaporated to give the title compound Isomer 1, Intermediate 144 (16 mg, 21%); MS (ESI) m/z [M+H]+ 721.4.
The stereoisomers of rac-methyl 4-chloro-2-(((4aR,7aS)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(oxazol-4-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 114 (316 mg, 0.45 mmol) were separated by chiral chromatography on a Chiralpak IA III column (250×20 mm, 5 μm), eluted with hexane:IPA:MeOH (90:5:5), at a flow rate of 18 mL/min;
rel-Methyl 2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(oxazol-4-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4
The stereoisomers of rac-methyl 2-(((4aR,7aS)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(oxazol-4-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 115 (108 mg, 0.16 mmol) were separated by chiral chromatography on a Chiralcel OJ-H column (250×20 mm, 5 μm), eluted with 30% MeOH in CO2, at a flow rate of 50 mL/min;
The stereoisomers of rac-methyl 2-(((4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-(cyanomethyl)cyclopropyl)methyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate Intermediate 116 (252 mg, 0.37 mmol) were separated by chiral chromatography on a CHIRALART YMC column (250×21 mm, 5 μm) eluted with hexane:IPA:MeOH (50:25:25) at a flow rate of 14 mL/min;
The stereoisomers of rac-methyl 2-(((4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-cyanocyclopropyl)methyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate Intermediate 117 (220 mg, 0.33 mmol) were separated by chiral chromatography on a CHIRALPAK IB column (250×21 mm, 5 μm) eluted with hexane:IPA:MeOH (90:5:5) at a flow rate of 17 mL/min; the first eluted compound was collected and evaporated to give the title compound Isomer 1 Intermediate 149 (61 mg, 28%); MS (ESI) m/z [M+H]+ 659.0;
The stereoisomers of rac-methyl 4-chloro-2-(((4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-cyanocyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 118 (84 mg, 0.12 mmol) were separated on a chiral chromatography on a CHIRALPAK IB column (250×20 mm, 5 μm) eluted with hexane:IPA:MeOH (80:10:10) at a flow rate of 14 mL/min; the first eluted compound was collected and evaporated to give the title compound Isomer 1, Intermediate 152 (20 mg, 24%); MS (ESI) m/z [M+H]+ 675.2; and
The stereoisomers of rac-methyl 4-chloro-2-(((4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-(cyanomethyl)cyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 119 (76 mg, 0.11 mmol) were separated by chiral chromatography on a CHIRALPAK IC column (250×20 mm, 5 μm) eluted with hexane:IPA:MeOH (50:25:25) at a flow rate of 12 mL/min; the first eluted compound was collected and evaporated to give the title compound Isomer 1, Intermediate 154 (19 mg, 25%); MS (ESI) m/z [M+H]+ 691.0.
The stereoisomers of rac-methyl 2-(((4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-(cyanomethyl)cyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 120 (500 mg, 0.76 mmol) were separated by chiral chromatography on a Chiralcel OD-H column (250×20 mm, 5 μm) eluted with hexane:IPA:MeOH (70:15:15) at a flow rate of 12 mL/min; The first eluted compound was collected and evaporated to give the title compound Isomer 1 Intermediate 155 (137 mg, 27%); MS (ESI) m/z [M+H]+ 655.4.
The stereoisomers of rac-methyl 2-(((4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-cyanocyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 121 (400 mg, 0.62 mmol) were separated by chiral chromatography on a CHIRALPAK IC column (250×21 mm, 5 μm) eluted with hexane:IPA:MeOH (75:15:15) at a flow rate of 14 mL/min; The first eluted compound was collected and evaporated to give the title compound Isomer 1 Intermediate 156 (101 mg, 27%); MS (ESI) m/z [M+H]+ 641.2.
The stereoisomers of rac-methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-4-chloro-2-(((4aR,7aS)-4-(2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 122 (630 mg, 0.91 mmol) were separated by chiral chromatography on a Chiralpak IA column (250×50 mm, 5 μm) eluted with 35% (IPA, 20 mM DEA) in CO2, 120 bar, at a flow rate of 350 mL/min and detected at 225 nm;
2,4,5-Trimethoxybenzaldehyde (9.70 g, 49.44 mmol) was added to a solution of tert-butyl ((3R,4S)-4-aminotetrahydrofuran-3-yl)carbamate (10 g, 49.44 mmol) in MeOH (100 mL) and the reaction mixture was stirred at 80° C. for 15 min. TFA (3.81 mL, 49.44 mmol) and NaBH3CN (2.55 g, 40.54 mmol) were added and the reaction mixture was stirred at 30° C. for 5 h. The solvent was removed under reduced pressure and the residue was diluted with water (100 mL). The aqueous layer was extracted with DCM (3×100 mL), and the combined organic layer was dried over Na2SO4, filtered and evaporated. The crude product was purified by flash chromatography on silica (60-70% EtOAc in petroleum ether) to give the title compound (16.00 g, 85%) as a pale yellow gum; MS (ESI) m/z [M+H]+ 383.
4 M HCl in 1,4-dioxane (52.3 mL, 209.17 mmol) was added to a solution of tert-butyl ((3R,4S)-4-((2,4,5-trimethoxybenzyl)amino)tetrahydrofuran-3-yl)carbamate Intermediate 158 (16 g, 41.83 mmol) in DCM (150 mL) and the reaction mixture was stirred at 30° C. for 3 h. The reaction mixture was concentrated under reduced pressure to give the title compound (11.5 g, 97%) as a pale yellow solid; MS (ESI) m/z [M+H]+ 283. The product was used in the next step directly without further purification.
Dimethyloxalate (7.21 g, 61.10 mmol) was added to a solution of (3S,4R)—N3-(2,4,5-Trimethoxybenzyl)tetrahydrofuran-3,4-diamine Intermediate 159 (11.5 g, 40.73 mmol) and Et3N (17.03 mL, 122.19 mmol) in MeOH (150 mL) and the reaction mixture was stirred at 80° C. for 18 h. The solvent was removed under reduced pressure. The crude product was purified by flash chromatography on silica (70-80% MeOH in DCM) to give the title compound (12.50 g, 91%) as a pale yellow oil; MS (ESI) m/z [M+H]+ 337.
2.5 M LiAlH4 in THF (47.6 mL, 118.93 mmol) was added dropwise to a solution of (4aR,7aS)-1-(2,4,5-trimethoxybenzyl)tetrahydrofuro[3,4-b]pyrazine-2,3(1H,4H)-dione Intermediate 160 (12.5 g, 37.16 mmol) in THF (150 mL) at 0° C. The reaction mixture was stirred at 15° C. for 8 h. The reaction mixture was quenched with Na2SO4 decahydrate (40 g), and the mixture was filtered. The filtrate was collected and evaporated at reduced pressure to give the title compound (11.00 g, 96%) as a yellow oil; MS (ESI) m/z [M+H]+ 309. The product was used in the next step directly without further purification.
Di-tert-butyl dicarbonate (15.57 g, 71.34 mmol) was added to a solution of (4aR,7aS)-1-(2,4,5-trimethoxybenzyl)octahydrofuro[3,4-b]pyrazine Intermediate 161 (11 g, 35.67 mmol) in EtOH (110 mL) at 30° C., and the reaction mixture was stirred at 15° C. for 3 h. The solvent was removed under reduced pressure and the crude product was purified by flash chromatography on silica (60-70% EtOAc in petroleum ether) to give the title compound (11.00 g, 75%) as a pale yellow oil; MS (ESI) m/z [M+H]+ 409.
Pd/C (10%, 1.303 g, 1.22 mmol) was added to a solution of tert-butyl (4aS,7aR)-4-(2,4,5-trimethoxybenzyl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 162 (5 g, 12.24 mmol) in THF (50 mL) at 30° C. and the resulting mixture was hydrogenated under an atmosphere of H2 (g) (1 atm) at 15° C. for 30 h. The reaction mixture was filtered through celite and washed with MeOH (3×300 mL). The solvent was removed under reduced pressure the crude product was purified by flash chromatography on silica (70-80% EtOAc in petroleum ether) to give the title compound (2.0 g, 72%) as a pale yellow solid; MS (ESI) m/z [M+H]+ 229.
The stereoisomers of 2-(4-bromo-2-methylbenzo[d][1,3]dioxol-2-yl)-5-chloropyridine WO2020234726 (6 g, 18.37 mmol) were separated by chiral chromatography on an LUX A1 (AD) column (250×30 mm, 5 μm), eluted with 3% (IPA, 20 mM DEA) in CO2, 120 bar, at a flow rate of 150 mL/min and detected at 220 nm;
The first eluted compound was collected and evaporated to give the title compound, Intermediate 164 (2 g, 33%); [α]D20+152 (c 1.00, MeCN); 1H NMR (400 MHz, DMSO-d6) δ 2.07 (3H, d), 6.83 (1H, td), 6.97 (1H, d), 7.06 (1H, d), 7.64 (1H, dd), 8.04 (1H, dd), 8.74 (1H, d).
The absolute configuration of Intermediate 164 was determined by vibrational circular dichroism (VCD) spectroscopy. The experimental spectrum recorded in CDCl3 was compared to a simulated spectrum of the (S) enantiomer calculated using density functional theory at the B3PW91/cc-pVTZ level of theory. Based on the large number of points of agreement between the experimental and simulated spectra, the title compound was assigned as the (S) enantiomer.
TEA (26.6 g, 0.262 mol), followed by a solution of benzyl chloroformate (29.8 g, 0.175 mol) in dry DCM (70 mL), was added to a solution of rac-tert-butyl (4aS,7aR)-hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate (40 g, 0.175 mol) in dry DCM (150 mL) and the reaction mixture was stirred at rt overnight. The reaction mixture was diluted with DCM (50 mL) and washed with 10% citric acid (70 mL), sat NaHCO3 (70 mL) and brine (100 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (hexane:MTBE, 1:1) to give the title compound (63 g, 98%); 1H NMR (400 MHz, CDCl3) δ 7.41-7.17 (m, 5H), 5.16-5.01 (m, 2H), 4.54 (d, 2H), 3.98 (dd, 2H), 3.87-3.57 (m, 4H), 3.35 (ddt, 2H), 1.42 (d, 9H).
The stereoisomers of rac-1-benzyl 4-(tert-butyl) (4aR,7aS)-hexahydrofuro[3,4-b]pyrazine-1,4-dicarboxylate Intermediate 165 were separated by chiral chromatography on a CHIRALPAK IA column (250×30 mm, 5 μm), eluted with hexane:IPA:MeOH (70:15:15), at a flow rate of 25 mL/min;
The first eluted compound was collected and evaporated to give the title compound Isomer 1, Intermediate 166 (24.95 g); MS (ESI) m/z [(M-t-Bu)+H]+ 307.2 [(M-Boc)+H]+ 263.2.
The second eluted compound was collected and evaporated to give the title compound Isomer 2, Intermediate 167; (24.68 g) MS (ESI) m/z [(M-t-Bu)+H]+ 307.2 [(M-Boc)+H]+ 263.2.
A solution of rel-1-benzyl 4-(tert-butyl) (4aR,7aS)-hexahydrofuro[3,4-b]pyrazine-1,4-dicarboxylate Isomer 2 Intermediate 167 (24.68 g 0.068 mol) and Pd/C (10%, 2.4 g) in MeOH (100 mL) was degassed under vacuum and then purged with H2(g) (×3). The reaction mixture was then stirred at rt under an atmosphere of H2(g) (2 atm) until complete reaction (monitoring by NMR). The catalyst was carefully removed by filtration and washed with MeOH (2×20 mL). The filtrate was concentrated in vacuo to give the title compound Intermediate 168 (14.5 g, 93%); [α]D20+74.12 (c 0.5, MeOH); 1H NMR (500 MHz, CDCl3) δ 4.48 (d, J=89.8 Hz, 1H), 4.00-3.64 (m, 4H), 3.26 (t, J=4.6 Hz, 1H), 2.94 (d, J=46.6 Hz, 2H), 2.72 (td, J=12.3, 3.2 Hz, 1H), 1.79 (s, 2H), 1.44 (s, 9H).
The absolute configuration of the title compound was determined by converting the title compound in two steps to the 4-chlorobenzoyl derivative Intermediate 170 as described below in the experimental descriptions for Intermediate 169 and Intermediate 170. Based on the X-ray data of Intermediate 170 the title compound Intermediate 168 was assigned as the (4aS, 7aR) enantiomer.
TEA (115 mg, 1.1 mmol) followed by a solution of 4-chlorobenzyl carbonochloridate (173 mg, 0.75 mmol) in dry DCM (5 mL) were added to a solution of tert-butyl (4aS,7aR)-hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 168 (173 mg, 0.75 mmol) in dry DCM (10 mL) and the reaction mixture was stirred at rt overnight. The reaction mixture was diluted with DCM (5 mL) and washed with 10% citric acid (10 mL), sat NaHCO3 (20 mL) and brine (10 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography (hexane:EtOAc, 1:1) to give the title compound (175 mg, 58%); MS (ESI) m/z [M-Boc+H]+ 297.0.
2 M HCl in Et2O (2 mL) was added to a solution rel-1-(tert-butyl) 4-(4-chlorobenzyl) (4aR,7aS)-hexahydrofuro[3,4-b]pyrazine-1,4-dicarboxylate Isomer 2 Intermediate 169 (99 mg, 0.249 mmol) in DCM (15 mL). The solution was stirred at ambient temperature for 8 h and then concentrated under reduced pressure to give the HCl-salt of the title compound (85 mg, quantitative yield) as a white solid; MS (ESI) m/z [M+H]+ 297.2.
Crystals for X-ray diffraction studies from the title compound was grown from a mixture of EtOAc:MeCN (2:1). The molecular structure of Intermediate 170 is shown in
A mixture of(S)-2-(4-bromo-2-methylbenzo[d][1,3]dioxol-2-yl)-5-chloropyridine Intermediate 164 (40.8 g, 124.84 mmol), tert-butyl (4aS,7aR)-hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 168 (30 g, 131.41 mmol), BINAP (4.91 g, 7.88 mmol) and Palladium(π-cinnamyl) chloride dimer (2.042 g, 3.94 mmol) in cyclopentyl methyl ether (700 mL) was evacuated and backfilled with N2(g) (×5). Sodium tert-butoxide (25.3 g, 262.82 mmol) was added and the reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at 45° C. for 24 h under an atmosphere of N2(g). The reaction mixture was washed with brine (2×100 mL) and the organic layer was dried over MgSO4, filtered and evaporated at reduced pressure. The crude product was purified by flash chromatography (5-30% EtOAc in heptane). The product containing fractions were collected and concentrated at reduced pressure and the crude compound was purified by flash chromatography (5-30% EtOAc in heptane). The product was dissolved in EtOAc (300 mL), SiliaMetS Thiol (30 g, 40-63 μm) was added and the mixture was stirred at rt for 2 h, and then filtered through celite. The filtrate was collected and concentrated at reduced pressure to give the title compound (38.0 g, 61%); [α]D20+126 (c 1.0, MeCN); MS (ESI) m/z [M+H]+ 474.1; 1H NMR (500 MHz, DMSO, 25° C.) δ 1.41 (9H, s), 2.03 (3H, s), 2.69-2.78 (1H, m), 3.14-3.25 (1H, m), 3.4-3.47 (1H, m), 3.52-3.58 (1H, m), 3.69-3.8 (3H, m), 3.82-3.86 (1H, m), 3.90 (1H, dd), 4.5-4.6 (1H, m), 6.43-6.49 (1H, m), 6.62-6.67 (1H, m), 6.78 (1H, t), 7.61-7.65 (1H, m), 8.01 (1H, dd), 8.71 (1H, d).
pTsOH hydrate (32.7 g, 171.75 mmol) was added to a solution of tert-butyl (4aS,7aR)-4-((S)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazine-1(2H)-carboxylate Intermediate 171 (37 g, 78.07 mmol) in EtOAc (80 mL) and the reaction mixture was stirred at 55° C. for 90 min. The reaction mixture was cooled to rt, diluted with EtOAc (100 mL) and sat K2CO3 (aq, 10 mL) was added dropwise. The organic layer was washed with sat K2CO3 (aq. 3×50 mL), dried over MgSO4, filtered and concentrated at reduced pressure to give the title compound (23.60 g, 81%) as an off-white solid; MS (ESI) m/z [M+H]+ 374.2.
K2CO3 (8.72 g, 63.13 mmol) and methyl (S)-2-(chloromethyl)-4-methoxy-1-(oxetan-2-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 3 (10.76 g, 33.14 mmol) were added to a solution of (4aR,7aS)-1-((S)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)octahydrofuro[3,4-b]pyrazine Intermediate 172 (11.8 g, 31.56 mmol) in MeCN (100 mL) and the reaction mixture was heated at 60° C. for 18 h. The reaction mixture was cooled to rt and EtOAc (50 mL) was added. The organic layer was washed with NaHCO3 (aq, 2×50 mL), dried over MgSO4, filtered and concentrated at reduced pressure. The crude compound was purified by preparative HPLC, PrepMethod H (gradient: 35-100%). Relevant fractions were combined and the mixture was concentrated at reduced pressure. The water layer was extracted with EtOAc (2×30 mL). The combined organic layer was dried over MgSO4, filtered, and the filtrate was stirred with SiliaMetS Thiol (13 g, 40-63 μm) at rt for 20 h. The mixture was filtered and the filtrate was concentrated at reduced pressure. The crude compound was purified by flash chromatography (30-100% EtOAc in heptane) to give the title compound (15.00 g, 71.8%) as a colourless oil; MS (ESI) m/z [M+H]+ 662.3.
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 24 (23 mg, 0.03 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (10 mg, 0.07 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated in vacuo, dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod A, (gradient: 20-90%) to give the title compound (12 mg, 52%); HRMS (ESI) m/z [M+H]+ calcd for C33H35ClN5O7: 648.2220, found: 648.2234; 1H NMR (500 MHz, CD3OD, 25° C.) δ 2.04 (3H, s), 2.44-2.52 (1H, m), 2.57-2.65 (1H, m), 2.72-2.82 (2H, m), 3.02-3.07 (1H, m), 3.15-3.21 (2H, m), 3.39 (1H, t), 3.53 (1H, d), 3.77-3.84 (1H, m), 3.96 (1H, dd), 4.03 (3H, s), 4.38-4.5 (2H, m), 4.54 (1H, d), 4.57-4.63 (1H, m), 4.65-4.72 (1H, m), 4.79-4.84 (1H, m), 5.16-5.25 (2H, m), 6.42 (1H, dd), 6.51 (1H, dd), 6.74 (1H, t), 7.44 (1H, d), 7.66 (1H, dd), 7.89 (1H, dd), 7.92 (1H, d), 8.61 (1H, dd).
A mixture or methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 25 (35 mg, 0.05 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (15 mg, 0.11 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The mixture was concentrated at reduced pressure, dissolved in DMSO, filtered and the residue was purified by preparative HPLC, PrepMethod A, (gradient: 20-90%) to give the title compound (19 mg, 57%); HRMS (ESI) m/z [M+H]+ calcd for C33H35ClN5O7: 648.2220, found: 648.2224; 1H NMR (500 MHz, CD3OD) 2.03 (3H, s), 2.36 (1H, dq), 2.48 (1H, td), 2.66-2.73 (1H, m), 2.73-2.82 (1H, m), 3.05 (1H, s), 3.11-3.2 (2H, m), 3.36 (1H, t), 3.60 (1H, d), 3.73 (1H, t), 3.86 (1H, dd), 4.05 (3H, s), 4.17 (1H, dt), 4.23 (1H, d), 4.55 (3H, td), 4.72 (1H, td), 5.04 (1H, dd), 5.23-5.32 (1H, m), 6.39 (1H, d), 6.50 (1H, d), 6.73 (1H, t), 7.44-7.47 (1H, m), 7.64 (1H, d), 7.87 (1H, dd), 7.94 (1H, d), 8.08 (1H, s), 8.59 (1H, d).
A solution of LiOH (1.356 g, 56.63 mmol) in water (20 mL) was added slowly over 5 min to a solution of methyl 2-(((4aS,7aR)-4-((S)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Intermediate 173 (15 g, 22.65 mmol) in THF (40 mL) and the resulting mixture was stirred at 35° C. overnight. The reaction mixture was concentrated at reduced pressure. Water (300 mL) and EtOAc (100 mL) was added to the crude product and the mixture was stirred. The pH of the solution was adjusted to pH 4.1 by addition of 1 M citric acid. The layers were separated and the aqueous layer was extracted with EtOAc (2×50 mL). The combined organic layer was washed with water (2×20 mL), dried over MgSO4 and filtered. The filtrate was collected and concentrated to half the volume and the mixture was stirred at rt. After 1 h, a solid was formed and the stirring was continued for 3 days. The solids were isolated by filtration and dried in vacuo for 72 h to give the title compound (11.00 g, 74.9%); [α]D20+72 (c 1.0, MeCN); HRMS (ESI) m/z [M+H]+ calcd for C33H35ClN5O7: 648.2220, found: 648.2240; 1H NMR (500 MHz, DMSO-d6, 25° C.) δ 2.02 (3H, s), 2.2-2.3 (1H, m), 2.34-2.44 (1H, m), 2.56-2.64 (1H, m), 2.69-2.75 (1H, m), 3.02-3.15 (3H, m), 3.32-3.37 (1H, m), 3.53-3.64 (2H, m), 3.72-3.78 (1H, m), 3.97 (3H, s), 4.05-4.09 (1H, m), 4.1-4.17 (1H, m), 4.36-4.48 (2H, m), 4.49-4.57 (1H, m), 4.57-4.65 (1H, m), 4.76-4.83 (1H, m), 5.1-5.18 (1H, m), 6.37-6.43 (1H, m), 6.5-6.55 (1H, m), 6.73 (1H, t), 7.25-7.29 (1H, m), 7.58-7.63 (1H, m), 7.89 (1H, d), 8.01 (1H, dd), 8.67-8.71 (1H, m), 12.82 (1H, s).
1H NMR (500 MHz, CD3OD, 25° C.) δ 2.03 (3H, s), 2.35 (1H, ddt), 2.47 (1H, ddd), 2.63-2.8 (2H, m), 3.04 (1H, ddd), 3.11-3.17 (2H, m), 3.35 (1H, t), 3.59 (1H, d), 3.68-3.76 (1H, m), 3.86 (1H, dd), 4.04 (3H, s), 4.16 (1H, dt), 4.24 (1H, dd), 4.51-4.6 (3H, m), 4.73 (1H, ddd), 5.05 (1H, dd), 5.28 (1H, tdd), 6.39 (1H, dd), 6.49 (1H, dd), 6.73 (1H, t), 7.45 (1H, d), 7.64 (1H, dd), 7.87 (1H, dd), 7.94 (1H, d), 8.59 (1H, dd).
By comparison of 1H NMR spectra and biological assay data, the title compound 2-(((4aS,7aR)-4-((S)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylic acid was concluded to be identical to 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylic acid, Isomer 3.
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 27 (36 mg, 0.05 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (15 mg, 0.11 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and filled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated in vacuo, dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod A, (gradient: 20-90%) to give the title compound (24 mg, 69%); HRMS (ESI) m/z [M+H]+ calcd for C34H37ClN5O7: 662.2376, found: 662.2384; 1H NMR (500 MHz, CD3OD) 1.54-1.63 (1H, m), 1.68-1.78 (1H, m), 1.79-1.88 (1H, m), 2.02 (3H, s), 2.04-2.11 (1H, m), 2.42-2.5 (1H, m), 2.73-2.8 (1H, m), 3.04-3.2 (3H, m), 3.37 (1H, t), 3.62 (1H, d), 3.65-3.75 (2H, m), 3.76-3.83 (1H, m), 3.89 (1H, dd), 4.02 (3H, s), 4.23-4.33 (2H, m), 4.36 (1H, dd), 4.43 (1H, d), 4.65-4.72 (1H, m), 4.93 (1H, dd), 6.34-6.42 (1H, m), 6.47-6.51 (1H, m), 6.72 (1H, t), 7.4-7.45 (1H, m), 7.61-7.64 (1H, m), 7.85 (1H, dd), 7.91 (1H, d), 8.56-8.61 (1H, m).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 28 (22 mg, 0.03 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (9 mg, 0.07 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated in vacuo, dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod A, (gradient: 20-90%) to give the title compound (15 mg, 68%); HRMS (ESI) m/z [M+H]+ calcd for C34H37ClN5O7: 662.2376, found: 662.2372; 1H NMR (500 MHz, CD3OD) 1.56-1.67 (1H, m), 1.7-1.79 (1H, m), 1.81-1.91 (1H, m), 2.01 (3H, s), 2.10 (1H, d), 2.43-2.52 (1H, m), 2.74-2.81 (1H, m), 3.07-3.12 (1H, m), 3.13-3.24 (2H, m), 3.64 (1H, d), 3.68-3.77 (2H, m), 3.77-3.83 (1H, m), 3.84-3.93 (2H, m), 4.03 (3H, s), 4.25-4.35 (2H, m), 4.39 (1H, dd), 4.45 (1H, d), 4.61-4.68 (1H, m), 4.97 (1H, dd), 6.39-6.45 (1H, m), 6.48 (1H, dd), 6.73 (1H, t), 7.43 (1H, d), 7.65 (1H, dd), 7.86 (1H, dd), 7.92 (1H, d), 8.59 (1H, dd).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 30 (58 mg, 0.09 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (24 mg, 0.17 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated in vacuo, dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod A, (gradient: 20-90%) to give the title compound (44 mg, 77%); HRMS (ESI) m/z [M+H]+ calcd for C34H35ClFN4O7: 665.2172, found: 665.2212; 1H NMR (500 MHz, CD3OD): 2.05 (3H, s), 2.3-2.41 (1H, m), 2.44-2.52 (1H, m), 2.66-2.74 (1H, m), 2.74-2.81 (1H, m), 3.02-3.07 (1H, m), 3.11-3.16 (2H, m), 3.32-3.37 (1H, m), 3.60 (1H, d), 3.72 (1H, t), 3.85 (1H, dd), 4.05 (3H, s), 4.13-4.2 (1H, m), 4.23-4.29 (1H, m), 4.52-4.61 (3H, m), 4.73-4.81 (1H, m), 5.05 (1H, dd), 5.24-5.32 (1H, m), 6.37 (1H, d), 6.47-6.53 (1H, m), 6.72 (1H, t), 7.17-7.23 (1H, m), 7.28 (1H, dd), 7.46 (1H, d), 7.55 (1H, t), 7.94 (1H, d).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 2 Intermediate 31 (48 mg, 0.07 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (20 mg, 0.14 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated in vacuo, dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod A, (gradient: 20-90%) to give the title compound (35 mg, 75%); HRMS (ESI) m/z [M+H]+ calcd for C34H35ClFN4O7: 665.2172, found: 665.2174; 1H NMR (500 MHz, CD3OD) 2.02 (3H, s), 2.31-2.42 (1H, m), 2.44-2.53 (1H, m), 2.65-2.72 (1H, m), 2.72-2.79 (1H, m), 3.02-3.08 (1H, m), 3.13-3.27 (2H, m), 3.61 (1H, d), 3.75 (1H, t), 3.82-3.92 (2H, m), 4.04 (3H, s), 4.13-4.21 (1H, m), 4.24-4.3 (1H, m), 4.53-4.59 (3H, m), 4.59-4.66 (1H, m), 5.06 (1H, dd), 5.25-5.33 (1H, m), 6.37-6.43 (1H, m), 6.43-6.49 (1H, m), 6.71 (1H, t), 7.15-7.21 (1H, m), 7.22-7.28 (1H, m), 7.46 (1H, d), 7.56 (1H, t), 7.94 (1H, d).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 32 (40 mg, 0.06 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (16 mg, 0.12 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated in vacuo, dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod A, (gradient: 20-90%) to give the title compound (27 mg, 69%); HRMS (ESI) m/z [M+H]+ calcd for C34H35ClFN4O7: 665.2172, found: 665.2192; 1H NMR (500 MHz, CD3OD) 2.03 (3H, s), 2.45-2.54 (1H, m), 2.59-2.66 (1H, m), 2.7-2.76 (1H, m), 2.75-2.84 (1H, m), 3.03-3.08 (1H, m), 3.17-3.3 (2H, m), 3.55 (1H, d), 3.81 (1H, t), 3.92-4 (2H, m), 4.03 (3H, s), 4.41-4.44 (1H, m), 4.49 (1H, dd), 4.54 (1H, d), 4.57-4.64 (1H, m), 4.64-4.75 (2H, m), 5.17-5.26 (2H, m), 6.43 (1H, dd), 6.47 (1H, dd), 6.73 (1H, t), 7.16-7.22 (1H, m), 7.26 (1H, dd), 7.44 (1H, d), 7.58 (1H, t), 7.94 (1H, d).
A mixture of methyl 4-chloro-2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 34 (50 mg, 0.07 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (20 mg, 0.15 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated in vacuo, dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod A, (gradient: 20-90%) to give the title compound (52 mg, 105%); HRMS (ESI) m/z [M+H]+ calcd for C33H34Cl2N5O6: 666.1880, found: 666.1908; 1H NMR (500 MHz, CDCl3) 1.49-1.6 (1H, m), 1.71-1.82 (1H, m), 1.82-1.94 (1H, m), 2.01 (2H, s), 2.07 (5H, s), 2.57-3.46 (5H, m), 3.54-3.67 (1H, m), 3.69-3.74 (1H, m), 3.77-3.88 (2H, m), 3.91-4.04 (1H, m), 4.17-4.25 (1H, m), 4.29-4.36 (1H, m), 4.47-4.59 (1H, m), 4.65-4.76 (1H, m), 4.94-5.09 (1H, m), 6.35 (1H, d), 6.53 (1H, d), 6.74 (1H, t), 7.55 (1H, d), 7.69 (1H, dd), 7.87-8.13 (2H, m), 8.58-8.62 (1H, m).
A mixture of methyl 4-chloro-2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 35 (50 mg, 0.07 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (20 mg, 0.15 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated in vacuo, dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod A, (gradient: 20-90%) to give the title compound (32 mg, 66%); HRMS (ESI) m/z [M+H]+ calcd for C33H34Cl2N5O6: 666.1886, found: 666.1913; 1H NMR (500 MHz, CD3OD) 1.57-1.68 (1H, m), 1.74-1.94 (2H, m), 2.02 (3H, s), 2.09-2.19 (1H, m), 2.48-2.56 (1H, m), 2.80 (1H, dt), 3.11-3.16 (1H, m), 3.16-3.27 (2H, m), 3.69-3.79 (3H, m), 3.8-3.94 (3H, m), 4.27-4.38 (2H, m), 4.39-4.53 (2H, m), 4.67 (1H, td), 5.05 (1H, dd), 6.44 (1H, dd), 6.49 (1H, dd), 6.75 (1H, t), 7.64-7.7 (1H, m), 7.88 (1H, dd), 7.94 (1H, d), 8.25 (1H, d), 8.60 (1H, dd).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 37 (34 mg, 53 mmol) and LiOH·H2O (2.5 eq) in THF:H2O (v/v, 1:3, 4 mL) was stirred at 20° C. overnight. The reaction mixture was concentrated under reduced pressure and acidified with citric acid. The product was collected by filtration, washed with water (3×75 mL), and then dried in vacuo at 60° C. to give the title compound (25 mg); HRMS (ESI) m/z [M+H]+ calcd for C32H33ClN5O6: 618.2119, found: 618.2138; 1H NMR (500 MHz, DMSO-d6) δ 12.73 (s, 1H), 8.68 (s, 1H), 8.24 (s, 1H), 8.00 (d, 1H), 7.79 (d, 1H), 7.61 (t, 2H), 6.72 (t, 1H), 6.51 (d, 1H), 6.40 (d, 1H), 5.14-4.80 (m, 2H), 4.68-4.35 (m, 5H), 4.23 (d, 1H), 3.85-3.55 (m, 2H), 3.50-3.33 (m, 3H), 3.22-2.97 (m, 3H), 2.67 (d, 3H), 2.01 (s, 3H).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 2 Intermediate 38 (33 mg, 52 mmol) and LiOH·H2O (2.5 eq) in THF:H2O (v/v, 1:3, 4 mL) was stirred at 20° C. overnight. The reaction mixture was concentrated under reduced pressure and acidified with citric acid. The product was collected by filtration, washed with water (3×75 mL), and then dried in vacuo at 60° C. to give the title compound (24 mg); HRMS (ESI) m/z [M+H]+ calcd for C32H33ClN5O6: 618.2119, found: 618.2157; 1H NMR (500 MHz, DMSO-d6) δ 12.5 (brs, 1H) 8.67 (d, 1H), 8.25 (s, 1H), 7.99 (dd, 1H), 7.80 (d, 1H), 7.62 (dd, 2H), 6.72 (t, 1H), 6.51 (d, 1H), 6.39 (d, 1H), 5.13 (d, 1H), 4.81 (d, 1H), 4.66 (m, 1H), 4.51 (q, 1H), 4.47-4.33 (m, 2H), 4.22-4.11 (m, 1H), 4.05 (d, 1H), 3.73 (dd, 1H), 3.60 (dd, 2H), 3.33 (d, 2H), 3.13-3.02 (m, 3H), 2.73 (d, 1H), 2.60 (d, 1H), 2.26 (t, 1H), 2.00 (s, 3H).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 39 (32 mg, 51 mmol) and LiOH·H2O (2.5 eq) in in THF:H2O (v/v, 1:3, 4 mL) was stirred at 20° C. overnight. The reaction mixture was concentrated under reduced pressure and acidified with citric acid. The product was collected by filtration, washed with water (3×75 mL), and then dried in vacuo at 60° C. to give the title compound (27 mg); HRMS (ESI) m/z [M+H]+ calcd for C32H33ClN5O6: 618.2119, found: 618.2151; 1H NMR (500 MHz, DMSO-d6) δ 12.65 (brs, 1H), 8.70 (d, 1H), 8.25 (d, 1H), 7.99 (dd, 1H), 7.79 (dd, 1H), 7.62 (t, 2H), 6.73 (t, 1H), 6.50 (d, 1H), 6.42 (d, 1H), 5.04 (q, 1H), 4.95 (dd, 1H), 4.51 (m, 5H), 4.26 (d, 1H), 3.83 (dd, 1H), 3.77 (t, 1H), 3.70 (t, 1H), 3.45 (d, 1H), 3.25 (d, 1H), 3.09 (t, 1H), 3.00 (d, 1H), 2.68 (m, 2H), 2.42-2.30 (m, 2H), 1.99 (s, 3H).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 40 (32 mg, 51 mmol) and LiOH·H2O (2.5 eq) was stirred in THF:H2O (v/v, 1:3, 4 mL) at 20° C. overnight. The reaction mixture was concentrated under reduced pressure and acidified with citric acid. The product was collected by filtration, washed with water (3×75 mL), and then dried in vacuo at 60° C. to give the title compound (24 mg); HRMS (ESI) m/z [M+H]+ calcd for C32H33ClN5O6: 618.2119, found: 618.2153; 1H NMR (500 MHz, DMSO-d6) δ 12.64 (brs, 1H) 8.70 (d, 1H), 8.25 (d, 1H), 7.99 (dd, 1H), 7.80 (dd, 1H), 7.63 (dd, 2H), 6.72 (t, 1H), 6.45 (dd, 2H), 5.15 (d, 1H), 4.83 (dd, 1H), 4.66 (dd, 1H), 4.61-4.31 (m, 3H), 4.23-3.98 (m, 2H), 3.85-3.59 (m, 4H), 3.21 (d, 1H), 3.15-2.95 (m, 2H), 2.78-2.61 (m, 3H), 2.26 (m, 1H), 1.98 (s, 3H).
LiOH·H2O (2 eq) was added to a solution of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 42 (38 mg, 0.059 mmol) in THF-H2O (2:1, 5 mL), and the reaction mixture was stirred at rt for 16 h. The mixture was acidified by NaH2PO4 (aq) and diluted with water. The precipitate formed was collected by filtration, washed with water and dried to afford the title compound (9.6 mg); HRMS (ESI) m/z [M+H]+ calcd for C33H33ClFN4O6: 635.2068, found: 635.2084; 1H NMR (600 MHz, DMSO-d6) δ 12.76 (s, 1H), 8.25 (d, 1H), 7.80 (dd, 1H), 7.65 (d, 1H), 7.59-7.48 (m, 2H), 7.31 (dd, 1H), 6.72 (t, 1H), 6.53 (d, 1H), 6.37 (d, 1H), 5.13 (m, 1H), 4.81 (dd, 1H), 4.65 (dd, 1H), 4.56 (td, 1H), 4.49-4.34 (m, 2H), 4.16 (dt, 1H), 4.08 (dd, 1H), 3.74 (dd, 1H), 3.66-3.52 (m, 2H), 3.17-3.01 (m, 3H), 2.73 (dt, 1H), 2.68-2.55 (m, 2H), 2.44-2.32 (m, 1H), 2.26 (dq, 1H), 2.02 (s, 3H).
LiOH·H2O (2 eq) was added to a solution of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 2 Intermediate 43 (27 mg, 0.042 mmol) in a mixture of THF-H2O (2:1, 5 mL), and the reaction mixture was stirred at rt for 16 h. The mixture was acidified by NaH2PO4 (aq) and diluted with water. The precipitate formed was collected by filtration, washed with water and dried to afford the title compound (5 mg); HRMS (ESI) m/z [M+H]+ calcd for C33H33ClFN4O6: 635.2068, found: 635.2064; 1H NMR (600 MHz, DMSO-d6) δ 12.77 (s, 1H), 8.25 (d, 1H), 7.80 (dd, 1H), 7.66 (d, 1H), 7.62-7.47 (m, 2H), 7.32 (dd, 1H), 6.71 (t, 1H), 6.49 (d, 1H), 6.40 (d, 1H), 5.26-5.04 (m, 1H), 4.84 (dd, 1H), 4.66 (dd, 1H), 4.50 (td, 1H), 4.44 (td, 2H), 4.24-4.02 (m, 2H), 3.82-3.65 (m, 3H), 3.57 (d, 1H), 3.16-2.95 (m, 2H), 2.83-2.64 (m, 1H), 2.45-2.32 (m, 3H), 2.26 (tt, 1H), 1.98 (s, 3H).
LiOH·H2O (2 eq) was added to a solution of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 44 (29 mg, 0.044 mmol) in THF-H2O (2:1, 5 mL), and the reaction mixture was stirred at rt for 16 h. The mixture was acidified by NaH2PO4 (aq) and diluted with water. The precipitate formed was collected by filtration, washed with water and dried to afford the title compound (5 mg); HRMS (ESI) m/z [M+H]+ calcd for C33H33ClFN4O6: 635.2068, found: 635.2074; 1H NMR (600 MHz, DMSO-d6) δ 12.75 (brs, 1H), 8.24 (d, 1H), 7.79 (dd, 1H), 7.69-7.60 (m, 1H), 7.61-7.48 (m, 2H), 7.32 (dd, 1H), 6.72 (t, 1H), 6.54 (d, 1H), 6.39 (d, 1H), 5.02 (q, 1H), 4.94 (dd, 1H), 4.65 (td, 1H), 4.55-4.49 (m, 2H), 4.49-4.40 (m, 1H), 4.26 (d, 1H), 3.81 (dd, 1H), 3.78-3.55 (m, 2H), 3.46 (d, 1H), 3.21-3.11 (m, 1H), 3.12-2.96 (m, 3H), 2.76-2.63 (m, 2H), 2.41-2.31 (m, 2H), 2.02 (s, 3H).
LiOH·H2O (2 eq) was added to a solution of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 45 (25 mg, 0.038 mmol) in THF-H2O (2:1, 5 mL), and the reaction mixture was stirred at rt for 16 h. The mixture was acidified by NaH2PO4 (aq) and diluted with water. The precipitate formed was collected by filtration, washed with water and dried to afford the title compound (5 mg); HRMS (ESI) m/z [M+H]+ calcd for C33H33ClFN4O6: 635.2068, found: 635.2066; 1H NMR (600 MHz, DMSO-d6) δ 12.75 (brs, 1H), 8.25 (d, 1H), 7.79 (dd, 1H), 7.70-7.56 (m, 1H), 7.56-7.47 (m, 2H), 7.32 (dd, 1H), 6.72 (t, 1H), 6.46 (dd, 2H), 5.16-4.90 (m, 2H), 4.66-4.38 (m, 5H), 4.28 (d, 1H), 3.93-3.69 (m, 3H), 3.46 (d, 1H), 3.19-2.94 (m, 2H), 2.79-2.64 (m, 3H), 2.44-2.29 (m, 2H), 1.99 (s, 3H).
A solution of rel-methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Isomer 1 Intermediate 47 (129 mg, 0.19 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (59 mg, 0.42 mmol) in a mixture of MeCN (1 mL) and water (0.2 mL) was stirred at rt overnight. The reaction mixture was concentrated and the residue was purified by preparative HPLC, PrepMethod E (gradient: 5-95%) to give the title compound (34 mg, 27%); HRMS (ESI) m/z [M+H]+ calcd for C34H33ClFN6O5: 659.2180, found: 659.2178; 1H NMR (600 MHz, DMSO-d6) 2.02 (3H, s), 2.35-2.38 (1H, m), 2.71-2.75 (1H, m), 3.02-3.05 (1H, m), 3.09-3.15 (2H, m), 3.29-3.35 (2H, m), 3.60 (1H, t), 3.74-3.84 (2H, m), 4.16 (1H, d), 4.53-4.62 (3H, m), 4.72-4.85 (2H, m), 6.06-6.18 (1H, m), 6.38 (1H, d), 6.54 (1H, d), 6.72 (1H, t), 7.26-7.37 (2H, m), 7.44 (1H, s), 7.48-7.59 (2H, m), 7.62 (1H, d), 7.79 (1H, d), 7.90 (1H, s).v
A solution of rel-methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 2 Intermediate 48 (33 mg, 0.05 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (19 mg, 0.14 mmol) in a mixture of MeCN (1 mL) and water (0.2 mL) was stirred at rt overnight. The reaction mixture was concentrated, and the residue was dissolved in DMSO and purified by preparative HPLC, PrepMethod A, (gradient: 40-80%) to give the title compound (22 mg, 68%) as a white powder; HRMS (ESI) m/z [M+H]+ calcd for C34H33ClFN6O5: 659.2180, found: 659.2198; 1H NMR (500 MHz, DMSO) 2.00 (3H, s), 2.34-2.44 (1H, m), 2.67-2.77 (1H, m), 2.98-3.03 (1H, m), 3.12 (1H, t), 3.29-3.33 (2H, m), 3.69-3.76 (2H, m), 3.76-3.81 (1H, m), 3.84 (1H, d), 4.18 (1H, d), 4.51 (1H, td), 4.61 (2H, t), 4.73-4.92 (2H, m), 6.11 (1H, t), 6.42 (1H, d), 6.51 (1H, d), 6.73 (1H, t), 7.33 (1H, dd), 7.35 (1H, d), 7.44 (1H, d), 7.51-7.59 (2H, m), 7.63 (1H, d), 7.78 (1H, dd), 7.91 (1H, d), 12.74 (1H, s).
A solution of rel-methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 49 (16 mg, 0.02 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (10 mg, 0.07 mmol) in a mixture of MeCN (1 mL) and water (0.2 mL) was stirred at rt overnight. The reaction mixture was concentrated and the residue was dissolved in DMSO and purified by preparative HPLC, PrepMethod A, (gradient: 40-80%) to give the title compound (0.011 g, 70%) as a white powder; HRMS (ESI) m/z [M+H]+ calcd for C34H33ClFN6O5: 659.2180, found: 659.2188; 1H NMR (500 MHz, DMSO) 2.00 (3H, s), 2.33-2.44 (1H, m), 2.69-2.79 (1H, m), 3.01 (1H, t), 3.05-3.17 (1H, m), 3.3-3.32 (2H, m), 3.69-3.76 (2H, m), 3.79 (1H, dd), 3.84 (1H, d), 4.18 (1H, d), 4.51 (1H, td), 4.61 (2H, t), 4.7-4.88 (2H, m), 6.11 (1H, t), 6.42 (1H, d), 6.51 (1H, d), 6.73 (1H, t), 7.33 (1H, dd), 7.35 (1H, d), 7.44 (1H, d), 7.49-7.59 (2H, m), 7.62 (1H, d), 7.78 (1H, dd), 7.91 (1H, d), 12.74 (1H, s).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 51 (94 mg, 0.15 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (40 mg, 0.29 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated in vacuo, and the residue was dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod F, (gradient: 20-70%), to give the title compound (54 mg, 58%); HRMS (ESI) m/z [M+H]+ calcd for C33H35ClN5O6: 632.2270, found: 632.2284; 1H NMR (600 MHz, DMSO-d6) 1.48-1.58 (1H, m), 1.7-1.81 (2H, m), 1.96-2.06 (4H, m), 2.5-2.51 (1H, m), 2.69-2.77 (1H, m), 3.10 (3H, q), 3.37 (1H, t), 3.55-3.66 (3H, m), 3.69-3.81 (2H, m), 4.11 (1H, dd), 4.19-4.27 (1H, m), 4.27-4.42 (2H, m), 4.48-4.56 (1H, m), 4.76 (1H, dd), 6.40 (1H, dd), 6.53 (1H, dd), 6.7-6.76 (1H, m), 7.60 (1H, dd), 7.62-7.66 (1H, m), 7.80 (1H, dd), 7.99 (1H, dd), 8.17-8.22 (1H, m), 8.68 (1H, dd).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 52 (60 mg, 0.09 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (26 mg, 0.19 mmol) in a sealed flask was evacuated and backfilled with N2(g) (×3). A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2(g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated in vacuo, and the residue was dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod F, (gradient: 20-70%), to give the title compound (39 mg, 67%); HRMS (ESI) m/z [M+H]+ calcd for C33H35ClN5O6: 632.2270, found: 632.2292; 1H NMR (600 MHz, DMSO-d6) 1.54 (1H, dq), 1.68-1.81 (2H, m), 1.97-2.06 (4H, m), 2.39-2.47 (2H, m), 2.7-2.78 (1H, m), 3.04-3.13 (2H, m), 3.21 (1H, dt), 3.58-3.65 (2H, m), 3.68 (1H, t), 3.71-3.77 (2H, m), 3.80 (1H, dd), 4.1-4.16 (1H, m), 4.21-4.29 (1H, m), 4.35-4.44 (2H, m), 4.5-4.56 (1H, m), 4.79 (1H, dd), 6.42 (1H, dd), 6.52 (1H, dd), 6.74 (1H, t), 7.58-7.67 (2H, m), 7.80 (1H, dd), 7.99 (1H, dd), 8.21-8.25 (1H, m), 8.7-8.73 (1H, m).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 54 and LiOH·H2O (3 eq) in THF:H2O (1:3, 4 mL) was stirred at 20° C. overnight. The reaction mixture was concentrated under reduced pressure and acidified with citric acid. The product was collected by filtration, washed with water (3×75 mL), and then dried in vacuo at 60° C. to give the title compound (28 mg); HRMS (ESI) m/z [M+H]+ calcd for C32H32ClFN5O6: 636.2025, found: 636.2036; 1H NMR (600 MHz, DMSO-d6) δ 13.05 (s, 1H), 8.67 (d, 1H), 8.14 (d, 1H), 7.99 (dd, 1H), 7.59 (d, 1H), 7.51 (dd, 1H), 6.72 (t, 1H), 6.51 (d, 1H), 6.39 (d, 1H), 5.18-5.07 (m, 1H), 4.84 (dd, 1H), 4.69 (dd, 1H), 4.52 (m, 1H), 4.48-4.35 (m, 2H), 4.17 (dt, 1H), 4.05 (dd, 1H), 3.73 (dd, 1H), 3.66-3.55 (m, 2H), 3.35 (t, 1H), 3.14-3.03 (m, 3H), 2.72 (dt, 1H), 2.66-2.56 (m, 1H), 2.45-2.33 (m, 1H), 2.29-2.18 (m, 1H), 2.00 (s, 3H).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 2 Intermediate 55 and LiOH·H2O (3 eq) in THF:H2O (1:3, 4 mL) was stirred at 20° C. overnight. The reaction mixture was concentrated under reduced pressure and acidified with citric acid. The product was collected by filtration, washed with water (3×75 mL), and then dried in vacuo at 60° C. to give the title compound (21 mg); HRMS (ESI) m/z [M+H]+ calcd for C32H32ClFN5O6: 636.2025, found: 636.2036; 1H NMR (600 MHz, DMSO-d6) δ 13.07 (s, 1H), 8.70 (d, 1H), 8.14 (d, 1H), 7.99 (dd, 1H), 7.61 (d, 1H), 7.51 (dd, 1H), 6.73 (t, 1H), 6.50 (d, 1H), 6.41 (d, 1H), 5.20-5.10 (m, 1H), 4.87 (dd, 1H), 4.70 (dd, 1H), 4.52 (td, 1H), 4.44 (dt, 2H), 4.16 (dt, 1H), 4.07 (d, 1H), 3.76 (dd, 1H), 3.72 (t, 1H), 3.66 (t, 1H), 3.59 (d, 1H), 3.22 (dt, 1H), 3.12-3.01 (m, 1H), 2.75-2.68 (m, 2H), 2.66-2.57 (m, 1H), 2.45-2.34 (m, 1H), 2.25 (m, 1H), 1.98 (s, 3H).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 56 and LiOH·H2O (3 eq) in THF:H2O (1:3, 4 mL) was stirred at 20° C. overnight. The reaction mixture was concentrated under reduced pressure and acidified with citric acid. The product was collected by filtration, washed with water (3×75 mL), and then dried in vacuo at 60° C. to give the title compound (35 mg); HRMS (ESI) m/z [M+H]+ calcd for C32H32ClFN5O6: 636.2025, found: 636.2064; 1H NMR (600 MHz, DMSO-d6) δ 13.05 (s, 1H), 8.68 (d, 1H), 8.14 (s, 1H), 8.01 (dd, 1H), 7.60 (d, 1H), 7.49 (d, 1H), 6.73 (t, 1H), 6.51 (d, 1H), 6.41 (d, 1H), 5.07-5.00 (m, 1H), 4.97 (dd, 1H), 4.61 (td, 1H), 4.58-4.49 (m, 2H), 4.49-4.39 (m, 2H), 4.23 (d, 1H), 3.80 (dd, 1H), 3.65 (t, 1H), 3.49 (d, 1H), 3.37 (t, 1H), 3.19-3.12 (m, 1H), 3.13-3.05 (m, 1H), 3.03 (d, 1H), 2.73-2.63 (m, 2H), 2.44-2.37 (m, 2H), 2.01 (s, 3H).
A mixture of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 57 and LiOH·H2O (3 eq) in THF:H2O (1:3, 4 mL) was stirred at 20° C. overnight. The reaction mixture was concentrated under reduced pressure and acidified with citric acid. The product was collected by filtration, washed with water (3×75 mL), and then dried in vacuo at 60° C. to give the title compound (23 mg); HRMS (ESI) m/z [M+H]+ calcd for C32H32ClFN5O6: 636.2025, found: 636.2051; 1H NMR (600 MHz, DMSO-d6) δ 13.06 (s, 1H), 8.70 (d, 1H), 8.14 (d, 1H), 7.99 (dd, 1H), 7.61 (d, 1H), 7.50 (dd, 1H), 6.73 (t, 1H), 6.51 (d, 1H), 6.43 (d, 1H), 5.10-4.90 (m, 2H), 4.63-4.41 (m, 5H), 4.26 (d, 1H), 3.82 (dd, 1H), 3.77 (t, 1H), 3.71 (t, 1H), 3.48 (d, 1H), 3.28-3.23 (m, 2H), 3.09 (t, 1H), 3.00 (t, 1H), 2.68 (m, 2H), 2.44-2.37 (m, 1H), 1.99 (s, 3H).
A solution of rel-methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 124 (131 mg, 0.20 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (60 mg, 0.43 mmol) in a mixture of MeCN (1 mL) and water (0.2 mL) was stirred at rt overnight. The reaction mixture was concentrated and the crude compound was purified by preparative HPLC, PrepMethod A, (gradient: 35-75%) to give the title compound Isomer 1 (97 mg, 76%) as a white powder; HRMS (ESI) m/z [M+H]+ calcd for C33H33ClN7O5: 642.2226, found: 642.2230; 1H NMR (500 MHz, CD3OD) 2.03 (s, 3H), 2.44-2.52 (m, 1H), 2.79-2.87 (m, 1H), 3.03-3.11 (m, 1H), 3.13-3.20 (m, 1H), 3.20-3.28 (m, 1H), 3.37-3.46 (m, 2H), 3.73 (t, 1H), 3.84-3.96 (m, 2H), 4.21 (dd, 1H), 4.63-4.75 (m, 3H), 4.78 (dt, 1H), 4.97 (ddd, 1H), 6.11 (s, 1H), 6.42 (d, 1H), 6.51 (d, 1H), 6.74 (t, 1H), 7.15 (d, 1H), 7.50 (d, 1H), 7.64 (dd, 2H), 7.84-7.90 (m, 2H), 7.93 (dd, 1H), 8.60 (d, 1H).
A solution of rel-methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 125 (92 mg, 0.14 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (42 mg, 0.30 mmol) in a mixture of MeCN (1 mL) and water (0.2 mL) was stirred at rt overnight. The reaction mixture was concentrated under reduced pressure and the residue was partitioned between DCM (2 mL) and 1 M HCl (aq, 2 mL). The aqueous phase was extracted with another portion of DCM (2 mL) and the combined organic layer was concentrated and dried under reduced pressure to give the title compound Isomer 1 (87 mg, 97%) as a white solid; HRMS (ESI) m/z [M+H]+ calcd for C33H32ClFN7O5: 660.2132, found: 660.2160; 1H NMR (500 MHz, CD3OD) 2.02 (s, 3H), 2.75-2.88 (m, 1H), 3.07-3.17 (m, 1H), 3.24-3.29 (m, 1H), 3.32-3.39 (m, 1H), 3.39-3.46 (m, 1H), 3.49 (t, 1H), 3.7-3.79 (m, 2H), 3.95 (dd, 1H), 4.06 (d, 1H), 4.23 (dd, 1H), 4.65-4.78 (m, 3H), 4.79-4.85 (m, 1H), 4.92-5.00 (m, 1H), 6.15 (s, 1H), 6.44 (d, 1H), 6.54 (d, 1H), 6.75 (t, 1H), 7.28 (d, 1H), 7.48 (d, 1H), 7.58-7.68 (m, 2H), 7.76 (d, 1H), 7.86 (dd, 1H), 8.58 (d, 1H).
A sealed flask with methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 126 (81 mg, 0.12 mmol) was evacuated and backfilled with N2(g) (×3) and then 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (34 mg, 0.24 mmol) was added. A mixture of MeCN (3 mL) and water (0.6 mL) was degassed by N2 (g) bubbling for 15 min, and then added. The reaction mixture was evacuated and backfilled with N2(g) (×3) and then stirred at rt overnight. The reaction mixture was concentrated at reduced pressure, the residue was dissolved in DMSO, filtered and purified by preparative HPLC, PrepMethod A (gradient: 20-90%) to give the title compound Isomer 3 (64 mg, 80%); MS (ESI) m/z [M+H]+ 650.53; 1H NMR (500 MHz, CD3OD) 1.51 (dq, 1H), 1.65-1.78 (m, 1H), 1.78-1.9 (m, 1H), 1.96-2.09 (m, 4H), 2.54 (dt, 1H), 2.79 (d, 1H), 3.09-3.2 (m, 3H), 3.50 (t, 1H), 3.62-3.79 (m, 4H), 3.90 (dd, 1H), 4.20-4.34 (m, 3H), 4.44 (d, 1H), 4.64 (td, 1H), 4.97 (dd, 1H), 6.30 (d, 1H), 6.47 (d, 1H), 6.70 (t, 1H), 7.54 (d, 1H), 7.63 (d, 1H), 7.70 (dd, 1H), 7.97 (d, 1H), 8.53 (d, 1H).
The title compound was prepared as described for Example 12a starting from methyl 2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 127 (83 mg, 0.12 mmol) to give the title compound Isomer 4 (63 mg, 78%); MS (ESI) m/z [M+H]+ 648.5; 1H NMR (500 MHz, CD3OD) 1.59 (dq, 1H), 1.77 (td, 1H), 1.82-1.91 (m, 1H), 2.05-2.14 (m, 1H), 2.15 (s, 3H), 2.52 (dt, 1H), 2.76-2.83 (m, 1H), 3.12 (s, 1H), 3.18-3.24 (m, 2H), 3.63-3.84 (m, 4H), 3.84-3.94 (m, 2H), 4.26-4.34 (m, 2H), 4.39 (dd, 1H), 4.46 (dd, 1H), 4.64 (td, 1H), 5.01 (dd, 1H), 6.36 (dd, 1H), 6.44-6.5 (m, 1H), 6.69-6.76 (m, 1H), 7.58-7.66 (m, 2H), 7.79 (dt, 1H), 8.05-8.1 (m, 1H), 8.55 (d, 1H).
LiOH monohydrate (3.2 mg, 72 μmol) was added to a solution of rel-methyl 2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(pyridin-3-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 128 (20 mg, 29 μmol) in THF:H2O (2:1, 1 mL) and the reaction mixture was stirred at rt for 16 h. The reaction mixture was purified by preparative HPLC, PrepMethod D (gradient: 0-40%) to give the lithium salt of the title compound Isomer 1 (13.7 mg, 73%); HRMS (ESI) m/z [M+H]+ calcd for C35H31ClF2N5O5: 674.1976, found: 674.1972; 1H NMR (600 MHz, DMSO-d6) δ 8.46-8.28 (m, 2H), 7.64 (s, 1H), 7.56-7.44 (m, 3H), 7.38 (d, 1H), 7.35-7.23 (m, 2H), 6.70 (t, 1H), 6.52 (d, 1H), 6.25 (d, 1H), 5.64 (d, 2H), 4.45-4.40 (m, 1H), 4.33 (d, 1H), 4.12 (d, 1H), 3.71 (dd, 1H), 3.52 (d, 1H), 3.15-3.10 (m, 2H), 3.02-2.89 (m, 2H), 2.74-2.62 (m, 2H), 2.37-2.30 (m, 1H), 2.00 (s, 3H).
The title compound was prepared as described for Example 13a from rel-methyl 2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(pyridin-3-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 2 Intermediate 129 (5.3 mg, 7.7 μmol) to give the lithium salt of the title compound Isomer 2 (4.6 mg, 82%); HRMS (ESI) m/z [M+H]+ calcd for C35H31ClF2N5O5: 674.1976, found: 674.1988; 1H NMR (600 MHz, DMSO-d6) δ 8.41 (dd, 1H), 8.35 (d, 1H), 7.64 (s, 1H), 7.56-7.44 (m, 3H), 7.38 (d, 1H), 7.34-7.23 (m, 2H), 6.70 (t, 1H), 6.52 (d, 1H), 6.25 (d, 1H), 5.64 (d, 2H), 4.42 (td, 1H), 4.32 (d, 1H), 4.12 (d, 1H), 3.71 (dd, 1H), 3.52 (d, 1H), 3.15-3.10 (m, 2H), 3.02-2.90 (m, 2H), 2.76-2.62 (m, 2H), 2.33 (t, 1H), 2.01 (s, 3H).
The title compound was prepared as described for Example 13a from rel-methyl 2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(pyridin-3-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 130 (7.5 mg, 11 μmol) to give the lithium salt of the title compound Isomer 4 (3.9 mg, 56%); MS (ESI) m/z [M+H]+ 674.0; 1H NMR (600 MHz, DMSO-d6) δ 8.41 (dd, 1H), 8.35 (d, 1H), 7.64 (s, 1H), 7.56-7.42 (m, 3H), 7.43-7.34 (m, 1H), 7.35-7.24 (m, 2H), 6.70 (t, 1H), 6.51 (d, 1H), 6.26 (d, 1H), 5.64 (s, 2H), 4.41 (q, 1H), 4.32 (d, 1H), 4.15-4.05 (m, 1H), 3.71 (dd, 1H), 3.52 (d, 1H), 3.18-3.08 (m, 2H), 3.03-2.91 (m, 2H), 2.68 (q, 2H), 2.35-2.30 (m, 1H), 2.00 (s, 3H).
A mixture of methyl 4-chloro-2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 131 (39 mg, 58 μmol) and LiOH monohydrate (7.6 mg, 0.17 mmol) in THF:H2O (1:3, 4 mL) was stirred at 20° C. overnight. The reaction mixture was concentrated under reduced pressure and acidified with 1 M citric acid (aq). The product was collected by filtration, washed with water (3×75 mL), and then dried in vacuo at 60° C. to give the title compound Isomer 1 (19 mg, 51%); HRMS (ESI) m/z [M+H]+ calcd for C32H32Cl2N5O6: 652.1724, found: 652.1796; 1H NMR (600 MHz, DMSO-d6) δ 13.08 (s, 1H), 8.67 (d, 1H), 8.25 (s, 1H), 7.99 (dd, 1H), 7.79 (s, 1H), 7.59 (d, 1H), 6.72 (t, 1H), 6.51 (d, 1H), 6.39 (d, 1H), 5.13 (q, 1H), 4.85 (dd, 1H), 4.70 (dd, 1H), 4.53 (q, 1H), 4.47-4.39 (m, 2H), 4.17 (dd, 1H), 4.06 (d, 1H), 3.73 (dd, 1H), 3.67-3.56 (m, 2H), 3.35 (t, 1H), 3.14-3.04 (m, 3H), 2.70 (d, 1H), 2.64-2.61 (m, 1H), 2.45-2.38 (m, 1H), 2.24 (p, 1H), 2.00 (s, 3H).
The title compound was prepared as described in Example 14a from methyl 4-chloro-2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 2 Intermediate 132 (35 mg, 53 μmol) to give the title compound Isomer 2 (14 mg, 42%); HRMS (ESI) m/z [M+H]+ calcd for C32H32Cl2N5O6: 652.1724, found: 652.1742; 1H NMR (600 MHz, DMSO-d6) δ 13.13 (s, 1H), 8.68 (d, 1H), 8.24 (s, 1H), 8.01 (dd, 1H), 7.78 (s, 1H), 7.61 (d, 1H), 6.73 (t, 1H), 6.51 (d, 1H), 6.40 (d, 1H), 5.05-4.95 (m, 2H), 4.66-4.40 (m, 5H), 4.24 (d, 1H), 3.80 (dd, 1H), 3.65 (t, 1H), 3.50 (d, 1H), 3.37 (t, 1H), 3.21-2.97 (m, 3H), 2.70-2.62 (m, 2H), 2.38 (d, partly overlapping with solvent), 2.01 (s, 3H).
The title compound was prepared as described in Example 14a from methyl 4-chloro-2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 133 (29 mg, 44 μmol) to give the title compound Isomer 3 (24 mg, 85%); HRMS (ESI) m/z [M+H]+ calcd for C32H32Cl2N5O6: 652.1724, found: 652.1722; 1H NMR (600 MHz, DMSO-d6) δ 13.13 (s, 1H), 8.70 (d, 1H), 8.25 (s, 1H), 7.99 (dd, 1H), 7.79 (s, 1H), 7.61 (d, 1H), 6.72 (t, 1H), 6.50 (d, 1H), 6.41 (d, 1H), 5.15 (q, 1H), 4.96-4.81 (m, 1H), 4.70 (dd, 1H), 4.59-4.36 (m, 3H), 4.21-4.02 (m, 2H), 3.85-3.50 (m, 4H), 3.22 (d, 1H), 3.10-2.97 (m, 2H), 2.70 (d, 1H), 2.63 (s, 1H), 2.41 (d, 1H), 2.24 (p, 1H), 1.98 (s, 3H).
The title compound Isomer 4 was prepared as described in Example 14a from methyl 4-chloro-2-(((4aR*,7aS*)-4-((R*)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 134 (23 mg, 35 μmol) to give the title compound Isomer 4 (21 mg, 94%); HRMS (ESI) m/z [M+H]+ calcd for C32H32Cl2N5O6: 652.1724, found: 652.1770; 1H NMR (600 MHz, DMSO-d6) δ 13.12 (s, 1H), 8.70 (d, 1H), 8.26 (d, 1H), 7.99 (dd, 1H), 7.78 (d, 1H), 7.62 (t, 1H), 6.73 (t, 1H), 6.51 (t, 1H), 6.42 (d, 1H), 5.14-4.91 (m, 2H), 4.68-4.41 (m, 5H), 4.26 (d, 1H), 3.93-3.66 (m, 3H), 3.50 (d, 1H), 3.15-2.94 (m, 2H), 2.77-2.57 (m, 4H), 2.42-2.36 (m, 1H), 1.99 (s, 3H).
LiOH hydrate (4.2 mg, 96 μmol, 1.5 eq) was added to a solution of methyl 4-chloro-2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 135 (44 mg, 64 μmol) in THF:H2O (2:1, 5 mL) and the reaction mixture was stirred at rt for 16 h. The reaction mixture was concentrated under reduced pressure and acidified with 1 M citric acid (aq). The product was collected by filtration, washed with water (3×75 mL), and then dried in vacuo at 60° C. to give the title compound Isomer 1 (32 mg, 74%); HRMS (ESI) m/z [M+H]+ calcd for C33H32Cl2FN4O6: 669.1678, found: 669.1708; 1H NMR (600 MHz, DMSO-d6) δ 13.09 (s, 1H), 8.25 (d, 1H), 7.79 (d, 1H), 7.57-7.48 (m, 2H), 7.31 (dd, 1H), 6.72 (t, 1H), 6.53 (d, 1H), 6.37 (d, 1H), 5.17-5.08 (m, 1H), 4.86 (dd, 1H), 4.70 (dd, 1H), 4.57 (td, 1H), 4.49-4.36 (m, 2H), 4.16 (dt, 1H), 4.11-4.03 (m, 1H), 3.74 (dd, 1H), 3.66-3.54 (m, 2H), 3.17-3.00 (m, 3H), 2.71 (dt, 1H), 2.64-2.58 (m, 1H), 2.43 (td, 1H), 2.27-2.20 (m, 1H), 2.02 (s, 3H).
The title compound was prepared as described in Example 15a from methyl 4-chloro-2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 2 Intermediate 136 (14 mg, 20 μmol) to give the title compound Isomer 2 (12 mg, 90%) HRMS (ESI) m/z [M+H]+ calcd for C33H32Cl2FN4O6: 669.1678, found: 669.1712; 1H NMR (600 MHz, DMSO-d6) δ 13.11 (s, 1H), 8.26 (s, 1H), 7.80 (s, 1H), 7.60-7.47 (m, 2H), 7.32 (dd, 1H), 6.71 (t, 1H), 6.50 (d, 1H), 6.40 (d, 1H), 5.16 (q, 1H), 4.89 (d, 1H), 4.71 (dd, 1H), 4.57-4.38 (m, 3H), 4.15 (dt, 1H), 4.10 (d, 1H), 3.80-3.66 (m, 3H), 3.61 (d, 1H), 3.11-2.99 (m, 2H), 2.70 (d, 1H), 2.63 (s, 1H), 2.44 (t, 1H), 2.30-2.18 (m, 1H), 1.99 (s, 3H).
The title compound was prepared as described in Example 15a from methyl 4-chloro-2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 137 (46 mg, 67 μmol) to give the title compound Isomer 3 (28 mg, 62%); HRMS (ESI) m/z [M+H]+ calcd for C33H32Cl2FN4O6: 669.1678, found: 669.1712; 1H NMR (600 MHz, DMSO-d6) δ 13.08 (s, 1H), 8.25 (d, 1H), 7.78 (d, 1H), 7.58-7.51 (m, 2H), 7.32 (dd, 1H), 6.73 (t, 1H), 6.54 (d, 1H), 6.39 (d, 1H), 5.00 (ddd, 2H), 4.67 (td, 1H), 4.59-4.41 (m, 4H), 4.27 (d, 1H), 3.81 (dd, 1H), 3.64 (t, 1H), 3.51 (d, 1H), 3.22-3.10 (m, 1H), 3.11-2.99 (m, 2H), 2.74-2.62 (m, 2H), 2.46-2.34 (m, 2H), 2.02 (s, 3H).
The title compound Isomer 4 was prepared as described in Example 15a from methyl 4-chloro-2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 138 (13 mg, 19 μmol) to give the title compound Isomer 4 (10 mg, 86%); HRMS (ESI) m/z [M+H]+ calcd for C33H32Cl2FN4O6: 669.1678, found: 669.1698; 1H NMR (600 MHz, DMSO-d6) δ 13.08 (s, 1H), 8.23 (s, 1H), 7.78 (s, 1H), 7.62-7.46 (m, 2H), 7.32 (dd, 1H), 6.72 (t, 1H), 6.50 (d, 1H), 6.42 (d, 1H), 5.09-4.93 (m, 2H), 4.66-4.42 (m, 5H), 4.28 (d, 1H), 3.89-3.67 (m, 3H), 3.50 (d, 1H), 3.34 (d, 2H), 3.08 (t, 1H), 3.00 (t, 1H), 2.67 (dd, 2H), 2.45-2.38 (m, 1H), 1.99 (s, 3H).
LiOH hydrate (5.1 mg, 0.12 mmol) was added in one portion to a solution of methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 139 (31 mg, 46 μmol) in a mixture of THF:H2O (1:1, 5 mL) and the reaction mixture was stirred at rt for 24 h. The reaction mixture was concentrated, and the residue were acidified using 1 M citric acid (aq, 4 mL). The reaction mixture was stirred at ambient temperature overnight. The obtained solid was filtered off and dried in vacuo for 2 h to give the title compound Isomer 1 (29 mg, 96%); HRMS (ESI) m/z [M+H]+ calcd for C33H32ClF2N4O6: 653.1972, found: 653.1988; 1H NMR (600 MHz, DMSO-d6) δ 13.05 (s, 1H), 8.14 (s, 1H), 7.58-7.45 (m, 3H), 7.31 (dd, 1H), 6.72 (t, 1H), 6.53 (d, 1H), 6.37 (d, 1H), 5.13 (dt, 1H), 4.84 (d, 1H), 4.69 (dd, 1H), 4.57 (t, 1H), 4.48-4.37 (m, 2H), 4.19-4.12 (m, 1H), 4.08 (d, 1H), 3.74 (dd, 1H), 3.65-3.53 (m, 2H), 3.15-3.01 (m, 3H), 2.77-2.68 (m, 1H), 2.61 (d, 1H), 2.44-2.39 (m, 2H), 2.25 (p, 1H), 2.02 (s, 3H).
The title compound was prepared as described for Example 16a from methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 2 Intermediate 140 (12 mg, 18 μmol) to give the title compound Isomer 2 (11 mg, 97%); HRMS (ESI) m/z [M+H]+ calcd for C33H32ClF2N4O6: 653.1972, found: 653.1954; 1H NMR (600 MHz, DMSO-d6) δ 13.05 (s, 1H) 8.15 (s, 1H), 7.61-7.46 (m, 3H), 7.36-7.27 (m, 1H), 6.71 (t, 1H), 6.50 (d, 1H), 6.40 (d, 1H), 5.16 (q, 1H), 4.93-4.83 (m, 1H), 4.70 (dd, 1H), 4.57-4.40 (m, 3H), 4.16 (q, 1H), 4.09 (d, 1H), 3.81-3.66 (m, 3H), 3.59 (d, 1H), 3.12-3.00 (m, 2H), 2.71 (d, 1H), 2.61 (d, 2H), 2.43 (d, 1H), 2.25 (p, 1H), 1.99 (s, 3H).
The title compound was prepared as described for Example 16a from methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 141 (28 mg, 42 μmol) to give the title compound Isomer 3 (24 mg, 88%); HRMS (ESI) m/z [M+H]+ calcd for C33H32ClF2N4O6: 653.1972, found: 653.1996; 1H NMR (600 MHz, DMSO-d6) δ 13.04 (s, 1H), 8.13 (s, 1H), 7.60-7.46 (m, 3H), 7.32 (dd, 1H), 6.73 (t, 1H), 6.54 (d, 1H), 6.39 (d, 1H), 5.08-4.91 (m, 2H), 4.66 (td, 1H), 4.60-4.41 (m, 4H), 4.26 (d, 1H), 3.81 (dd, 1H), 3.64 (t, 1H), 3.48 (d, 1H), 3.16 (d, 1H), 3.12-2.99 (m, 2H), 2.70-2.65 (m, 2H), 2.45-2.35 (m, 3H), 2.02 (s, 3H).
The title compound was prepared as described for Example 16a from methyl 2-(((4aR*,7aS*)-4-((R*)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(((S)-oxetan-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 142 (11 mg, 16 μmol) to give the title compound Isomer 4 (10 mg, 86%); HRMS (ESI) m/z [M+H]+ calcd for C33H32ClF2N4O6: 653.1972, found: 653.1996; 1H NMR (600 MHz, DMSO-d6) δ 13.04 (s, 1H), 8.14 (s, 1H), 7.64-7.45 (m, 3H), 7.32 (dd, 1H), 6.72 (t, 1H), 6.50 (d, 1H), 6.42 (d, 1H), 5.11-4.90 (m, 2H), 4.63-4.41 (m, 6H), 4.28 (d, 1H), 3.87-3.69 (m, 3H), 3.48 (d, 1H), 3.14-2.95 (m, 2H), 2.74-2.62 (m, 2H), 2.45-2.38 (m, 2H), 1.99 (s, 3H).
LiOH monohydrate (3.2 mg, 74 μmol) was added to a solution of rel-methyl 2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(2-(2-methyl-1H-imidazol-1-yl)ethyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 143 (26 mg, 37 μmol) in THF:H2O (2:1, 5 mL) and the reaction mixture was stirred at rt for 16 h. The reaction mixture was acidified to pH 3 with 1 M NaH2PO4 (aq) and diluted with water. The precipitate was collected by filtration, washed with water and dried in vacuo to give the title compound Isomer 1 (10 mg, 47%); HRMS (ESI) m/z [M+H]+ calcd for C35H34ClF2N6O5: 691.2242, found: 691.2302; 1H NMR (500 MHz, DMSO-d6) δ 13.01 (s, 1H), 7.63 (s, 1H), 7.58-7.43 (m, 3H), 7.31 (dd, 1H), 6.96 (s, 1H), 6.72 (dd, 2H), 6.54 (d, 1H), 6.38 (d, 1H), 4.74 (d, 2H), 4.57 (q, 1H), 4.38 (t, 2H), 4.13 (d, 1H), 3.99 (d, 1H), 3.76 (dd, 1H), 3.59 (t, 1H), 3.41 (dd, 2H), 3.17-3.02 (m, 2H), 2.78-2.68 (m, 1H), 2.66-2.56 (m, 1H), 2.40 (d, 1H), 2.02 (s, 3H), 1.80 (s, 3H).
LiOH hydrate (2.4 mg, 54 μmol) was added in one portion to a solution of rel-methyl 4-chloro-2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(2-(2-methyl-1H-imidazol-1-yl)ethyl)-1H-benzo[d]imidazole-6-carboxylate Isomer 1 Intermediate 144 (16 mg, 22 μmol) in THF:H2O (1:1, 5 mL) and the reaction mixture was stirred at rt for 24 h. The reaction mixture was concentrated in vacuo and the residue was dissolved in DMSO (5 mL) and purified by preparative HPLC, PrepMethod D (gradient: 60-85%) to give the lithium salt of the title compound Isomer 1 (8 mg, 50%); HRMS (ESI) m/z [M+H]+ calcd for C35H34Cl2FN6O5: 707.1946, found: 707.1946 1H NMR (600 MHz, DMSO-d6) δ 7.88 (s, 1H), 7.79 (d, 1H), 7.58-7.50 (m, 2H), 7.31 (dd, 1H), 6.84 (d, 1H), 6.74-6.66 (m, 2H), 6.53 (d, 1H), 6.37 (d, 1H), 4.67 (t, 2H), 4.52 (td, 1H), 4.38-4.25 (m, 2H), 4.02 (d, 1H), 3.72 (dd, 1H), 3.66 (d, 1H), 3.57 (t, 1H), 3.09 (td, 2H), 3.01 (d, 2H), 2.64 (d, 1H), 2.33 (t, 2H), 2.01 (s, 3H), 1.85 (s, 3H).
LiOH hydrate (7.9 mg, 0.18 mmol) was added in one portion to a solution of rel-methyl 4-chloro-2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-(oxazol-4-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 145 (50 mg, 72 μmol) in THF:H2O (1:1, 5 mL) and the reaction mixture was stirred at rt for 24 h. The reaction mixture was concentrated, and the residue was acidified by addition of 1 M citric acid (aq, 4 mL) and stirred at ambient temperature for 8 h. The obtained solid was filtered off and dried in vacuo for 2 h to give the title compound Isomer 4 (44 mg, 91%); MS (ESI) m/z [M+H]+ 682.0; 1H NMR (500 MHz, DMSO-d6) δ 13.14 (s, 1H), 8.32 (s, 1H), 8.22 (s, 1H), 8.14 (s, 1H), 7.78 (s, 1H), 7.53 (dd, 2H), 7.30 (d, 1H), 6.72 (t, 1H), 6.53 (d, 1H), 6.35 (d, 1H), 5.79-5.57 (m, 2H), 4.56 (d, 2H), 4.22 (d, 1H), 3.78 (dd, 1H), 3.67 (d, 1H), 3.53 (t, 1H), 3.42 (dd, 2H), 3.10 (d, 2H), 2.99 (t, 1H), 2.71 (d, 1H), 2.02 (s, 3H).
LiOH hydrate (3.7 mg, 0.084 mmol) was added in one portion to a solution of rel-methyl 2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(oxazol-4-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 146 (23 mg, 34 μmol) in THF:H2O (1:1, 5 mL) and the reaction mixture was stirred at rt for 24 h. The reaction mixture was concentrated in vacuo and the crude product was dissolved in DMSO (5 mL) and purified by preparative HPLC, PrepMethod D (gradient: 30-50%) to give the lithium salt of the title compound (7.3 mg, 36%); HRMS (ESI) m/z [M+H]+ calcd for C33H29ClF2N5O6: 664.1768, found: 664.1780; 1H NMR (600 MHz, DMSO-d6) δ 8.32 (d, 1H), 8.10 (s, 1H), 7.85 (d, 1H), 7.59-7.50 (m, 2H), 7.44 (d, 1H), 7.32 (dd, 1H), 6.71 (t, 1H), 6.53-6.45 (m, 1H), 6.39 (d, 1H), 5.65 (d, 1H), 5.51 (d, 1H), 4.59-4.44 (m, 2H), 4.24 (d, 1H), 3.79 (dd, 1H), 3.66 (d, 2H), 3.56 (d, 1H), 3.29-3.22 (m, 1H), 3.07-2.95 (m, 2H), 2.74 (d, 1H), 2.41 (t, 1H), 1.98 (s, 3H).
The title compound was prepared as described for Example 20a from rel-methyl 2-(((4aR,7aS)-4-((R)-2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-fluoro-1-(oxazol-4-ylmethyl)-1H-benzo[d]imidazole-6-carboxylate, Isomer 4 Intermediate 147 (10 mg, 15 μmol) to give the title compound Isomer 4 (4.9 mg, 47%); HRMS (ESI) m/z [M+H]+ calcd for C33H29ClF2N5O6: 664.1768, found: 664.1766; 1H NMR (600 MHz, DMSO-d6) δ 8.31 (s, 1H), 8.07 (s, 1H), 7.84 (s, 1H), 7.58-7.48 (m, 2H), 7.43 (d, 1H), 7.30 (dd, 1H), 6.72 (t, 1H), 6.53 (d, 1H), 6.35 (d, 1H), 5.62 (d, 1H), 5.49 (d, 1H), 4.59-4.43 (m, 3H), 4.20 (d, 1H), 3.78 (dd, 1H), 3.63-3.47 (m, 2H), 3.28-3.22 (m, 1H), 3.15-2.95 (m, 2H), 2.74 (d, 1H), 2.40 (d, 1H), 2.02 (s, 3H).
LiOH hydrate (11.4 mg, 260 μmol) was added to a solution of rel-methyl 2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-(cyanomethyl)cyclopropyl)methyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate Isomer 1 Intermediate 148 (79 mg, 104 μmol) in THF:H2O (2:1, 7 mL) and the reaction mixture was stirred at rt for 16 h. The reaction mixture was acidified to pH 3 using 10% NaH2PO4 (aq). The formed precipitate was filtered off and dried in vacuo to give the title compound Isomer 1 (70 mg, 92%) as a white solid; HRMS (ESI) m/z [M+H]+ calcd for C34H33ClFN6O5: 659.2180, found: 659.2134; 1H NMR (600 MHz, DMSO-d6) δ 8.67 (d, 1H), 8.05 (s, 1H), 7.99 (dd, 1H), 7.59 (d, 1H), 7.49 (d, 1H), 6.72 (t, 1H), 6.52 (d, 1H), 6.40 (d, 1H), 4.59 (d, 1H), 4.52 (d, 1H), 4.46 (q, 1H), 4.25 (d, 1H), 4.12 (dd, 1H), 3.79 (dd, 1H), 3.67 (d, 1H), 3.57 (t, 1H), 3.40 (t, 2H), 3.15-3.06 (m, 2H), 2.84 (dd, 1H), 2.76 (d, 1H), 2.60 (d, 2H), 2.00 (s, 3H), 0.74-0.63 (m, 4H).
LiOH hydrate (10.2 mg, 0.23 mmol) was added to a solution of rel-methyl 2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-cyanocyclopropyl)methyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate, Isomer 1 Intermediate 149 (61 mg, 93 μmol) in THF:H2O (2:1, 5 mL) and the reaction mixture was stirred at rt for 16 h. The reaction mixture was acidified to pH 3 with 10% NaH2PO4 (aq). The formed precipitate was filtered off and dried in vacuo to give the title compound Isomer 1 (40 mg, 67%) as a white solid; HRMS (ESI) m/z [M+H]+ calcd for C33H31ClFN6O5: 645.2022, found: 645.2012; 1H NMR (600 MHz, DMSO-d6) δ 13.00 (s, 1H), 8.68 (d, 1H), 8.09 (s, 1H), 8.00 (dd, 1H), 7.60 (d, 1H), 7.52 (d, 1H), 6.72 (t, 1H), 6.52 (d, 1H), 6.39 (d, 1H), 4.84 (d, 1H), 4.58 (d, 1H), 4.52 (q, 1H), 4.36 (d, 1H), 4.14 (dd, 1H), 3.80 (dd, 1H), 3.68 (d, 1H), 3.62 (t, 1H), 3.48-3.36 (m, 2H), 3.19-3.04 (m, 2H), 2.73 (d, 1H), 2.42 (d, 1H), 2.00 (s, 3H), 1.46-1.27 (m, 4H).
The title compound was prepared as described for Example 22a from rel-methyl 2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-cyanocyclopropyl)methyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate, Isomer 2 Intermediate 150 (43 mg, 0.65 mmol) to give the title compound Isomer 2 (27 mg, 64%) as a white solid; HRMS (ESI) m/z [M+H]+ calcd for C33H31ClFN6O5: 645.2022, found: 645.2040; 1H NMR (600 MHz, DMSO-d6) δ 13.01 (s, 1H), 8.70 (d, 1H), 8.14 (s, 1H), 7.99 (dd, 1H), 7.61 (d, 1H), 7.53 (d, 1H), 6.73 (t, 1H), 6.51 (d, 1H), 6.41 (d, 1H), 4.87 (d, 1H), 4.61 (d, 1H), 4.52 (q, 1H), 4.40 (d, 1H), 4.16 (d, 1H), 3.82 (dd, 1H), 3.69 (d, 1H), 3.63 (t, 1H), 3.43-3.38 (m, partly overlapping with solvent), 3.19-3.07 (m, partly overlapping with solvent), 2.73 (d, 1H), 2.43 (d, 1H), 1.99 (s, 3H), 1.48-1.27 (m, 4H).
The title compound was prepared as described for Example 22a from rel-methyl 2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-cyanocyclopropyl)methyl)-4-fluoro-1H-benzo[d]imidazole-6-carboxylate, Isomer 3 Intermediate 151 (67 mg, 101 μmol) to give the title compound Isomer 3 (41 mg, 67%) as a white solid; HRMS (ESI) m/z [M+H]+ calcd for C33H31ClFN6O5: 645.2022, found: 645.2014; 1H NMR (600 MHz, DMSO-d6) δ 13.00 (s, 1H), 8.68 (d, 1H), 8.08 (s, 1H), 8.00 (dd, 1H), 7.60 (d, 1H), 7.51 (d, 1H), 6.72 (t, 1H), 6.51 (d, 1H), 6.39 (d, 1H), 4.84 (d, 1H), 4.61 (d, 1H), 4.52 (q, 1H), 4.36 (d, 1H), 4.17 (d, 1H), 3.80 (dd, 1H), 3.71-3.60 (m, 2H), 3.19-3.08 (m, 3H), 2.73 (d, 1H), 2.46-2.40 (m, 1H), 2.00 (s, 3H), 1.42-1.28 (m, 4H).
LiOH hydrate (3.3 mg, 74 μmol) was added to a solution rel-methyl 4-chloro-2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-cyanocyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Isomer 1 Intermediate 152 (20 mg, 30 μmol) in THF:H2O (1:1, 4 mL) and the reaction mixture was stirred at rt for 24 h. The reaction mixture was acidified to pH 3 with 10% NaH2PO4 (aq). The formed precipitate was filtered off and dried in vacuo to give the title compound Isomer 1 (20 mg, 98%) as a white powder; HRMS (ESI) m/z [M+H]+ calcd for C33H31Cl2N6O5: 661.1728, found: 661.1746; 1H NMR (600 MHz, DMSO-d6) δ 13.12 (s, 1H), 8.68 (d, 1H), 8.34 (d, 1H), 8.00 (dd, 1H), 7.84 (d, 1H), 7.60 (d, 1H), 6.72 (t, 1H), 6.52 (d, 1H), 6.39 (d, 1H), 4.88 (d, 1H), 4.66 (d, 1H), 4.53 (q, 1H), 4.39 (d, 1H), 4.14 (d, 1H), 3.80 (dd, 1H), 3.74 (d, 1H), 3.62 (t, 1H), 3.40 (t, 1H), 3.19-3.06 (m, 4H), 2.72 (d, 1H), 2.00 (s, 3H), 1.43-1.28 (m, 4H).
The title compound was prepared as described for Example 23a from rel-methyl 4-chloro-2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-cyanocyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Isomer 2 Intermediate 153 (14 mg, 21 μmol) to give the title compound Isomer 2 (13 mg, 95%); HRMS (ESI) m/z [M+H]+ calcd for C33H31Cl2N6O5: 661.1728, found: 661.1752; 1H NMR (600 MHz, DMSO-d6) δ 13.12 (s, 1H), 8.70 (d, 1H), 8.35 (s, 1H), 7.99 (dd, 1H), 7.84 (s, 1H), 7.61 (d, 1H), 6.73 (t, 1H), 6.51 (d, 1H), 6.41 (d, 1H), 4.90 (d, 1H), 4.67 (d, 1H), 4.53 (q, 1H), 4.42 (d, 1H), 4.16 (d, 1H), 3.82 (dd, 1H), 3.79-3.63 (m, 3H), 3.26-3.03 (m, 4H), 2.70 (t, 1H), 1.99 (s, 3H), 1.47-1.28 (m, 4H).
LiOH hydrate (3.0 mg, 67 μmol) was added to a solution rel-methyl 4-chloro-2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-(cyanomethyl)cyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Isomer 1 Intermediate 154 (19 mg, 27 μmol) in THF:H2O (2:1, 4 mL) and the reaction mixture was stirred at rt 16 h. The reaction mixture was acidified to pH 3 with 10% NaH2PO4 (aq). The formed precipitate was filtered off and dried in vacuo to give the title compound Isomer 1 (16 mg, 92%) as a white solid; HRMS (ESI) m/z [M+H]+ calcd for C34H33Cl2N6O5: 675.1884, found: 675.1918; 1H NMR (600 MHz, DMSO-d6) δ 13.14 (s, 1H), 8.67 (d, 1H), 8.23 (d, 1H), 7.99 (dd, 1H), 7.80 (d, 1H), 7.60 (d, 1H), 6.72 (t, 1H), 6.52 (d, 1H), 6.40 (d, 1H), 4.64-4.53 (m, 2H), 4.48 (q, 1H), 4.29 (d, 1H), 4.13 (dd, 1H), 3.79 (dd, 1H), 3.71 (d, 1H), 3.57 (t, 1H), 3.40 (t, 1H), 3.19-3.07 (m, 3H), 2.82 (d, 1H), 2.76 (d, 1H), 2.62-2.58 (m, 2H), 2.01 (s, 3H), 0.76-0.63 (m, 4H).
LiOH hydrate (27.7 mg, 0.63 mmol) was added in one portion to a solution of rel-methyl 2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-(cyanomethyl)cyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Isomer 1 Intermediate 155 (137 mg, 0.21 mmol) in THF:H2O (1:1, 2 mL) and the reaction mixture was stirred at rt for 24 h. The reaction mixture was concentrated in vacuo, diluted with water (3 mL) and acidified to pH 3 with 10% NaH2PO4 (aq). The formed precipitate was filtered off and dried in vacuo to give the title compound Isomer 1 (101 mg, 87%); HRMS (ESI) m/z [M+H]+ calcd for C34H34ClN6O5: 641.2274, found: 641.2308; 1H NMR (500 MHz, DMSO-d6) δ 12.80 (s, 1H), 8.67 (dd, 1H), 8.23 (d, 1H), 8.10-7.94 (m, 1H), 7.81 (dd, 1H), 7.66 (d, 1H), 7.59 (dd, 1H), 6.72 (t, 1H), 6.52 (dd, 1H), 6.39 (d, 1H), 4.64-4.50 (m, 2H), 4.46 (q, 1H), 4.25 (d, 1H), 4.15-4.06 (m, 1H), 3.79 (dd, 1H), 3.67 (d, 1H), 3.57 (t, 1H), 3.39 (t, 1H), 3.19-3.06 (m, 3H), 2.84 (d, 1H), 2.73 (d, 1H), 2.65-2.56 (m, 1H), 2.44 (s, 1H), 2.00 (s, 3H), 0.73-0.62 (m, 4H).
LiOH hydrate (17.3 mg, 394 μmol) was added in one portion to a solution of rel-methyl 2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1-((1-cyanocyclopropyl)methyl)-1H-benzo[d]imidazole-6-carboxylate Isomer 1 Intermediate 156 (101 mg, 158 μmol) in THF:H2O (1:1, 2 mL) and the reaction mixture was stirred at rt for 24 h. The reaction mixture was acidified to pH 3 with 10%. NaH2PO4 (aq). The formed precipitate was filtered off and dried in vacuo to give the title compound Isomer 1 (82 mg, 87%) as a beige solid; HRMS (ESI) m/z [M+H]+ calcd for C33H32ClN6O5: 627.2118, found: 627.2176; 1H NMR (400 MHz, DMSO-d6) δ 12.85 (s, 1H), 8.69 (d, 1H), 8.35 (s, 1H), 8.01 (dd, 1H), 7.85 (dd, 1H), 7.70 (d, 1H), 7.60 (d, 1H), 6.73 (t, 1H), 6.53 (d, 1H), 6.40 (d, 1H), 4.86 (d, 1H), 4.67-4.48 (m, 2H), 4.38 (d, 1H), 4.14 (d, 1H), 3.89-3.75 (m, 1H), 3.75-3.58 (m, 2H), 3.40 (t, 1H), 3.20-3.09 (m, 3H), 2.75 (d, 1H), 2.44 (s, 1H), 2.01 (s, 3H), 1.44-1.29 (m, 4H).
A solution of rel-methyl 1-(2-(1H-pyrazol-1-yl)ethyl)-4-chloro-2-(((4aR,7aS)-4-((R)-2-(5-chloropyridin-2-yl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-1H-benzo[d]imidazole-6-carboxylate Isomer 1 Intermediate 157 (130 mg, 0.19 mmol) and 1,3,4,6,7,8-hexahydro-2H-pyrimido[1,2-a]pyrimidine (53 mg, 0.38 mmol) in a mixture of MeCN (1 mL) and H2O (0.2 mL) was stirred at rt overnight The reaction mixture was concentrated under reduced pressure and the residue was partitioned between DCM (2 mL) and 1 M HCl (aq, 2 mL). The aqueous phase was extracted with another portion of DCM (2 mL) and the combined organic layer was concentrated under reduced pressure to give the title compound (122 mg, 96%) as a white solid; HRMS (ESI) m/z [M+H]+ calcd for C33H32Cl2N7O5: 676.1836, found: 676.1866; 1H NMR (500 MHz, CD3OD) δ 2.04 (s, 3H), 2.74-2.87 (m, 1H), 3.05-3.20 (m, 1H), 3.26-3.29 (m, 1H), 3.33-3.45 (m, 2H), 3.49 (t, 1H), 3.73-3.81 (m, 2H), 3.95 (dd, 1H), 4.09 (d, 1H), 4.24 (dd, 1H), 4.62-4.74 (m, 2H), 4.74-4.84 (m, 2H), 4.94-5.04 (m, 1H), 6.16 (t, 1H), 6.46 (dd, 1H), 6.56 (dd, 1H), 6.77 (t, 1H), 7.28 (d, 1H), 7.48 (d, 1H), 7.66 (dd, 1H), 7.79-7.87 (m, 1H), 7.89 (dd, 1H), 7.96 (d, 1H), 8.61 (dd, 1H).
The stereoisomers of 2-(((4aS,7aR)-4-(2-(4-chloro-2-fluorophenyl)-2-methylbenzo[d][1,3]dioxol-4-yl)hexahydrofuro[3,4-b]pyrazin-1(2H)-yl)methyl)-4-methoxy-1-(((S)-tetrahydrofuran-2-yl)methyl)-1H-benzo[d]imidazole-6-carboxylic acid Isomer 1 Intermediate 123 (700 mg, 1.03 mmol) were separated by chiral chromatography on a CHIRAL ART Cellulose-SA column (250×20 mm ID, 5 μm) eluted with 5% IPA in hexane:DCM (3:1, 0.1% FA) at a flow rate of 20 mL/min and detected at 220 and 254 nm; the first eluted compound was collected and evaporated to give the title compound Isomer 1 (148 mg, 21%) as a white solid; HRMS (ESI) m/z [M+H]+ calcd for C35H37ClFN4O7: 679.2330, found: 679.2372; 1H NMR (400 MHz, DMSO-d6) δ 7.81 (s, 1H), 7.60-7.49 (m, 2H), 7.38-7.24 (m, 2H), 6.73 (t, 1H), 6.55 (d, 1H), 6.39 (d, 1H), 4.71 (d, 1H), 4.56 (q, 1H), 4.34 (dd, 2H), 4.23 (q, 1H), 4.14 (d, 1H), 3.94 (s, 3H), 3.86-3.65 (m, 2H), 3.64-3.53 (m, 3H), 3.32 (t, 2H), 3.08 (d, 3H), 2.78-2.63 (m, 1H), 2.40 (t, 1H), 2.03 (s, 3H), 1.98 (dd, 1H), 1.73 (q, 1H), 1.52 (dq, 1H).
CHOK1 GLP-1R cAMP Assay
A cell line stably expressing the human GLP-1R receptor (NM_002062.5, including the naturally-occurring variant Leu260Phe) in a CHO-K1 (ATCC® CCL-61™) was used for assay.
GLP-1 Receptor mediated agonist activity was determined in a cell based assay measuring cAMP levels in cells using Homogeneous Time-Resolved Fluorescence (HTRF) cAMP detection kit (CisBio catalog #62AM4PEC, cAMP Gs Dynamic range kit). The cAMP detection method is based on a competitive immunoassay, in which cAMP produced by the cells and cAMP labeled with the dye d2 compete for binding to a Europium-Cryptate-labeled anti-cAMP antibody. The specific HTRF signal is inversely proportional to the concentration of cAMP.
Compounds were added to individual well in 384 well-assay plates (Greiner #784076) using an Echo (LabCyte) dispenser from 10 mM stocks. Varying concentration of compounds were added to wells, and dimethyl sulfoxide was used to normalize each well to a volume of 100 nanoliter. A dose response curve of GLP1(7-36)NH2 (Bachem H-6795) was included in each run. 5 μL of cAMP concentration response standards are applied in specified wells in the assay plates.
Cryo-preserved cells are thawed and resuspended in assay buffer pre-heated to 37° C. (20 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) pH 7.4, lx Hank's Balanced Salt Solution (HBSS, Life Technologies #14065) supplemented with 0.1% (w/v) bovine serum albumin (Sigma, A-7030)). Cells were centrifuged at 250*g for 5 min at room temperature, and resuspend in room tempered assay buffer to a final density of 0.16*106 cell/mL, to deliver 800 cells/well. 5 μL of assay buffer with 1 mM 3-isobutyl-1-methylxanthin (IBMX; Sigma cat I-7018) was dispensed per well in assay plates using a multidrop combi (Thermo Scientific) subsequently 5 μL of cell suspension was distributed to relevant wells in the assay using a multidrop dispenser. Assay plates were incubated 20 min at room temperature.
Detection reagents, Europium-Cryptate-labeled anti-cAMP antibody and cAMP labeled with the dye d2, are diluted in lysis buffer, provided by the manufacturer. 5 μL of each detection reagent is supplemented to each assay well using a multidrop dispenser. Assay plates are incubated in the dark for at least one hour. The HTRF signal is measured using the HTRF module (excitation: 337 nm, emission A: 665 nm and emission B: 620 nm) in Pherastar FSX (BMG Labtech).
Raw data were converted to pM cAMP using the cAMP standard curve included in each run. Converted data were further analyzed in Genedata Screener (Genedata) and EC50 determinations were made from agonist dose-response curves analyzed with a curve fitting program using a 4-parameter logistic dose response equation (Equation y=A+((B−A)/1+((C/x){circumflex over ( )}D))) where A is no stimulation, B is full stimulation, C is the EC50 and D is the Hill slope). The percent effect was determined relative to a saturating concentration of a full GLP-1R agonist (GLP1(7-36)NH2 has 100% effect in this assay setup).
The GLP-1R EC50 values for the Example compounds are set forth in Table 1 herein below.
EndoC cAMP Accumulation Assay
A HTRF (Homogeneous Time-Resolved Fluorescence) cAMP assay (cAMP Gs dynamic kit; CisoBio Cat #62AM4PEJ) was used to identify agonists of the endogenous human glucagon-like peptide 1 receptor (GLP-1R) in a pancreatic insulinoma cell line (EndoC-βH1). The EndoC-βH1 cell line was sourced from Univercell Biosolutions and is a genetically engineered human pancreatic β cell line which exhibits glucose-inducible insulin secretion. EndoC-βH1 cells have detectable GLP-1R mRNA as detected by qPCR1. The functionality of GLP-1R signalling in EndoC-βH1 has been demonstrated by Exendin-4 treatment leading to augmented insulin secretion; an effect which is blunted with shRNA-mediated knockdown of GLP-1R. The EndoC-βH1 cell line is a valid model of human beta cells and applicable for screenings to identify novel drug target candidates (Mol. Metab., 2018, 8, 144-157). CisBio HTRF cAMP kits are based on a competitive immunoassay using cryptate-labelled anti-cAMP antibody and d2-labeled cAMP. The detection kit is intended for the direct quantitative determination of cAMP. The specific signal (i.e. energy transfer) is inversely proportional to the concentration of cAMP in the standard or sample. Test compounds (10 mM in DMSO) were diluted into assay buffer (HBSS (Sigma #H8264) supplemented with 25 mM HEPES (Gibco #15630, pH 7.4), 0.1% BSA (Sigma #A3059) and 0.5 mM IBMX (Sigma #I7018) included fresh on the day of the assay) into 96 well U-bottom plates (Greiner #650201). Diluted compounds were transferred to ECHO source PP plates (Labcyte #P-05525) and dose response curves were dispensed acoustically using ECHO 550 into black shallow-well u-bottom 384-well HTRF Assay Plates (Corning 4514).
Cryovials of EndoC-H1 (supplied at 1×10e7 cells/vial) were used directly for screening. The cryovials and were removed from LN2 and thawed rapidly in a 37° C. water bath. The cells were resuspended in assay buffer and centrifuged at 300 g for 5 min. Cells were resuspended in assay buffer at the appropriate concentration, typically at 12e5 cells per mL (3000 cells per well, dependent on cell batch) and 2.5 μL diluted cells were added to all wells of destination plate by Multidrop combi reagent dispenser (Thermofisher). The plates were incubated at room temperature for 30 min. The assay was stopped by adding 2.5 μL anti-cAMP cryptate solution to all wells and 2.5 μL cAMP-d2 solution (both diluted 1:20 in lysis buffer) to columns 1-22 by Combi drop. A volume of 2.5 μL cAMP-d2 solution was added to wells E23 to P24 and 2.5 μL lysis buffer added to wells A23 to D24 by multichannel pipette. The plates were incubated at room temperature for 1 hr and read on an Envision plate reader using excitation wavelength of 320 nm and emission of 590 nm and 660 nm.
Raw data from Envision is converted to % DeltaF according to the manufacturer's instructions. Dose response curves are analysed via 4-Parameter Logistical Analysis and assay plate Z′ values obtained. Samples are graphed as percentage (%) activation plots compared to GIP (1-42, Bachem H-5645) with assay window defined by negative control as basal cell cAMP levels and positive control are defined by maximum GIP (82.5 nM) signal. GLP1 (7-36 amide, Bachem H-6795) dose response curve was included on all plates.
The EndoC EC50 values for the Example compounds are set forth in Table 2 herein below.
Inhibition of PDE3 has been shown to result in an increase in cardiovascular mortality in clinical trials (Movsesian M. A., Kukreja R. C. (2011) Phosphodiesterase Inhibition in Heart Failure. In: Francis S., Conti M., Houslay M. (eds) Phosphodiesterases as Drug Targets. Handbook of Experimental Pharmacology, vol 204. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-17969-3_10). Chronic treatment with PDE3 inhibitors has been shown to result in increased mortality, primarily as a result of arrhythmias and sudden death (Expert Opinion on Investigational Drugs, 2002, 11, 1529-1536; J. of Cardiovasc. Trans. Res., 2010, 3, 507-515) and it may therefore be an advantage to as far as possible avoid PDE3 inhibitory activity.
Evaluation of the effects of compounds on the activity of the human phosphodiesterase-3A is quantified by measuring the formation of 5′AMP from cAMP using a human recombinant enzyme expressed in Sf9 cells.
The test compound, reference compound or water (control) are added to a buffer containing 40 mM Tris/HCl (pH 7.4) and 8 mM MgCl2, 450 nM cAMP and 0.25 μCi [3H]cAMP.
Thereafter, the reaction is initiated by addition of the enzyme (about 1 U) and the mixture is incubated for 20 min at 22° C.
For basal control measurements, the enzyme is omitted from the reaction mixture.
Following incubation SPA beads are added. After 30 min at 22° C. under shaking, the amount of [3H]5′AMP is quantified with a scintillation counter (Topcount, Packard).
The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is milrinone (CAS number 78415-72-2), which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC50 value is calculated.
The PDE3 IC50 values for Example compounds and reference compounds are set forth in Table 3 herein below.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/077508 | 10/4/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63262109 | Oct 2021 | US |
