Patent Application
20230055237
Cystic fibrosis (CF) is a lethal, recessive, genetic disease affecting approximately 1 in 2500 live births among Caucasians. (Cohen-Cymberknoh, M. et al., Am. J. Respir. Crit. Care Med. 1463-1471, 2011; Boat et al., The Metabolic Basis of Inherited Disease, 6th ed., pp 2649-2680, McGraw Hill, NY (1989)). Approximately 1 in 25 persons are carriers of the disease. The major symptoms of cystic fibrosis include chronic pulmonary disease, pancreatic exocrine insufficiency, and elevated sweat electrolyte levels. The symptoms are consistent with cystic fibrosis being an exocrine disorder. (Hantash F: U.S. Patent Application No. 20060057593).
The CF gene codes for a cAMP/PKA-dependent, ATP-requiring, membrane chloride ion channel, generally found in the apical membranes of many secreting epithelia and is known as CFTR (cystic fibrosis transmembrane conductance regulator). There are currently over 1900 known mutations affecting CFTR, many of which give rise to a disease phenotype. Around 75% of CF alleles contain the ΔF508 mutation in which a triplet codon has been lost, leading to a missing phenylalanine at position 508 in the protein. This altered protein fails to be trafficked to the correct location in the cell and is generally destroyed by the proteasome. The small amount that does reach the correct location functions poorly. (Cuthbert A W, British Journal of Pharmacology, 163(1), 173-183, 2011).
Mutations in the CFTR gene result in absence or dysfunction of the protein that regulates ion transport across the apical membrane at the surface of certain epithelia. Although CFTR functions mainly as a chloride channel, it has many other roles, including inhibition of sodium transport through the epithelial sodium channel, regulation of the outwardly rectifying chloride channel, ATP channels, intracellular vesicle transport, and inhibition of endogenous calcium-activated chloride channels. CFTR is also involved in bicarbonate-chloride exchange. A deficiency in bicarbonate secretion leads to poor solubility and aggregation of luminal mucins. Obstruction of intrapancreatic ducts with thickened secretions causes autolysis of pancreatic tissue with replacement of the body of the pancreas with fat, leading to pancreatic insufficiency with subsequent malnutrition. In the lungs, CFTR dysfunction leads to airway surface liquid (ASL) depletion and thickened and viscous mucus that adheres to airway surfaces. The result is decreased mucociliary clearance (MCC) and impaired host defenses. Dehydrated, thickened secretions lead to endobronchial infection with a limited spectrum of distinctive bacteria, mainly Staphylococcus aureus and Pseudomonas aeruginosa, and an exaggerated inflammatory response leading to development of bronchiectasis and progressive obstructive airways disease. Pulmonary insufficiency is responsible for most CF-related deaths. (Cohen-Cymberknoh, M. et al., Am. J. Respir. Crit. Care Med. 1463-1471, 2011).
The prognosis for the treatment of CF has improved over the last 40 years. This was achieved by improving pancreatic enzyme supplements, drugs designed to treat pulmonary infection, reduce inflammation and enhance mucociliary clearance. Currently the therapeutic challenges are to correct the biochemical defect of CF and to identify effective treatments for chronic respiratory infection. (Frerichs C. et al., Expert Opin Pharmacother. 10(7), 1191-202, 2009).
In one embodiment, the invention relates to a compound of Formula (I)
or a pharmaceutically acceptable salt thereof, wherein:
In another embodiment, the present invention relates to a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
In another embodiment, the present invention relates to a method of treating a CFTR-mediated disease or disorder, such as cystic fibrosis, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
The present invention relates to compounds of Formula (I) and pharmaceutically salts thereof, pharmaceutical compositions comprising such compounds and methods of using such compounds for treating a CFTR-mediated disease or condition in a subject in need thereof.
In certain embodiments, the compounds of the invention have the absolute stereochemistry shown in Formula (Ia) or Formula (Ib).
In certain embodiments of the compounds of the invention, R1 is optionally substituted aryl, optionally substituted heteroaryl, optionally substituted arylalkyl, such as optionally substituted aryl-C1-C6-alkyl or optionally substituted heteroarylalkyl, such as heteroaryl-C1-C6-alkyl; preferably optionally substituted phenyl or optionally substituted 6-membered heteroaryl.
In certain embodiments of the compounds of the invention, R is hydrogen, optionally substituted C1-C6-alkyl; optionally substituted C3-C8-cycloalkyl; in certain embodiments, R is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, sec-butyl, n-pentyl, neopentyl, optionally substituted C3-C6-cycloalkyl, optionally substituted C3-C6-cycloalkylmethyl, 2-dimethylaminoethyl, or 3-hydroxycyclobutyl. In certain embodiments, R is optionally substituted C3-C12-cycloalkyl-C1-C6-alkyl, preferably optionally substituted C3-C12-cycloalkyl-methyl. In certain embodiments, R is hydrogen or C1-C6-alkyl, such as hydrogen or methyl. In certain embodiments, R is a branched C3-C10-alkyl, preferably a branched C3-C8-alkyl. In certain embodiments, R is a (β-branched C4-C10-alkyl, such as 2,2,3,3,-tetramethylbutyl or 2,2,-dimethylpropyl.
In certain embodiments of the compounds of the invention, R2 is hydrogen, optionally substituted C1-C6-alkyl, optionally substituted aryl-C1-C6-alkyl, or optionally substituted heteroaryl-C1-C6-alkyl. In certain embodiments, R2 is hydrogen, C1-C4-alkyl, halo-C1-C4-alkyl, optionally substituted arylmethyl, or optionally substituted heteroarylmethyl. In certain embodiments, R2 is hydrogen, benzyl, optionally substituted phenyl-CF2—, optionally substituted heteroaryl-CF2—, benzyl-O—CH2—, CF3, CF3CH2— or isopropyl. In certain embodiments, R2 is hydrogen, C1-C4-alkyl, halo-C1-C4-alkyl, aryl optionally substituted with 1 to 5 halogen or aryl-C1-C2-alkyl optionally substituted with 1 to 5 halogen. In certain embodiments, R2 is hydrogen, CF3, isopropyl, benzyl, benzyl-O—CH2—, 3-hydroxy-n-propyl, or α,α-difluorobenzyl.
In certain embodiments of the compounds of the invention, R3 is hydrogen, C1-C4-alkyl, halo-C1-C4-alkyl, C1-C4-alkylC(O)—, aryl-C1-C4-alkylC(O)—, aryl-C1-C4-alkyl S(O)2-, aryl-C1-C4-alkylNHC(O)—, or arylNHC(O)—. In certain embodiments, R3 is hydrogen, methyl, CF3CH2—, acetyl, propionyl, phenethylC(O)—, phenethylSO2—, benzylNHC(O)— or phenylNHC(O)—.
In certain embodiments of the compounds of the invention, at least one of R2 and R3 is hydrogen.
In certain embodiments, R2 and R3, together with the atoms to which they are attached, form an optionally substituted saturated 4 to 6-membered heterocyclyl, preferably an optionally substituted saturated 5-membered heterocyclyl, and more preferably an optionally substituted pyrollidine. In certain embodiments, R2 and R3, together with the atoms to which they are attached, form an optionally substituted saturated 6-membered heterocyclyl, such as an optionally substituted piperidinyl or optionally substituted morpholyl. In certain embodiments, the saturated 4 to 6-membered heterocyclyl is unsubstituted or substituted with one or more substituents independently selected from halogen, CN, hydroxyl, C1-C3-alkoxy, halo-C1-C3-alkoxy, C1-C3-alkyl, halo-C1-C3-alkyl, a spiro cycloalkyl, a spiro heterocyclyl or an optionally substituted C1-C3-alkylidene.
In certain embodiments of the compouns of the invention, each R4 is independently halo, such as chloro or fluoro.
In certain embodiments of the compounds of the invention, R5 is hydrogen or C1-C6-alkyl; preferably hydrogen or methyl;
In certain embodiments of the compounds of the invention, R6 is OR8, and R5 is hydrogen, optionally substituted C1-C10-alkyl or optionally substituted C2-C10-alkenyl. In certain embodiments, R8 is hydrogen or optionally substituted C1-C10-alkyl. In certain embodiments, R8 is hydrogen, C1-C4-alkyl or allyl. In certain embodiments, R8 is —CH2-O—Rc, where Rc is —C(O)—C1-C8-alkyl or
In certain embodiments of the compounds of the invention, R6 is NR9R10. In certain embodiments, R9 and R10 are both C1-C4-alkyl, preferably methyl. In certain embodiments, R9 is OH or O—C1-C2-alkyl, preferably methyl and R10 is hydrogen or C1-C3-alkyl, preferably hydrogen or methyl. In certain embodiments, R9 is SO2R8 or SO2NRaRb. In certain embodiments, R9 is —SO2-C1-C4-alkyl, —SO2-phenyl, —SO2NH2 or —SO2N(CH3)2.
In certain embodiments of the compounds of the invention, R1 is optionally substituted aryl or heteroaryl, preferably optionally substituted phenyl or optionally substituted 6-membered heteroaryl; R is hydrogen, C1-C8-alkyl or C1-C6-alkyl; preferably hydrogen,methyl or a β-branched C4-C10-alkyl; R5 is hydrogen or C1-C6-alkyl; preferably hydrogen or methyl; and R6 is OR8, and R8 is hydrogen, or optionally substituted C1-C10-alkyl; or R8 is hydrogen, optionally substituted C1-C10-alkyl; or optionally substituted C2-C6-alkenyl.
In certain embodiments, the compound of Formula (I) is represented by Formula (II),
wherein m is 0, 1, 2, 3, 4, 5 or 6; and
In certain embodiments, the compounds of Formula (II) have the absolute stereochemistry shown in Formula (IIa) or Formula (IIb).
In certain embodiments, the compound of Formula I is represented by Formula (III),
wherein X is O or C(Ra)2, and each Ra is independently hydrogen, hydroxyl, protected hydroxyl, cyano, amino, protected amino, halogen, optionally substituted alkoxy, or optionally substituted alkyl.
In certain embodiments, the compounds of Formula (III) have the absolute stereochemistry shown in Formula (IIIa) or Formula (IIIb).
In certain embodiments, the compound of Formula I is represented by Formula (IV),
or a pharmaceutically acceptable salt thereof, wherein R14 is as previously defined and p is 0, 1 or 2.
In certain embodiments, the compounds of Formula (IV) have the absolute stereochemistry shown in Formula (IVa) or Formula (IVb).
In certain embodiments, the compound of Formula I is represented by Formula (V),
or a pharmaceutically acceptable salt thereof, wherein R14 and p are as previously defined.
In certain embodiments, the compounds of Formula (V) have the absolute stereochemistry shown in Formula (Va) or Formula (Vb).
In certain embodiments of the compounds of the invention, R1 is optionally substituted aryl, or optionally substituted 5- or 6-membered heteroaryl, for example, phenyl, naphthyl, pyridyl, pyrazinyl, pyrimidyl, pyrazolyl, oxazolyl, oxadiazolyl, thiazolyl, thiadiazolyl, triazolyl or pyrrolyl. In certain embodiments, R1 is optionally substituted fused bicyclic heteroaryl, for example, quinolyl, quinazolyl, naphthyl, benzimidazolyl, isoquinolyl, pyrazopyridyl, benzothiazolyl, naphthyridyl, indolyl, or indazolyl. In certain embodiments, R1 is optionally substituted phenyl-C1-C6-alkyl, optionally substituted heteroaryl-C1-C6-alkyl, or an optionally substituted biaryl group, such as optionally substituted biphenyl, phenylheteroaryl or heteroarylphenyl, including phenylpyrazyl.
Preferably, R1 is unsubstituted or substituted with 1, 2 or 3 substituents independently selected from C1-C4-alkyl, halo-C1-C4-alkyl, halogen, C1-C4-alkoxy and halo-C1-C4-alkoxy. More preferably, the substituents are independently selected from methyl, methoxy, fluoro, chloro, methoxy, CHF2, CF3, CHF2O— and CF30—.
In certain embodiments, R1 is selected from the groups below.
In certain embodiments, R1 is represented by
where X1-X4 are each independently N or CR17, where each R17 is independently hydrogen, optionally substituted alkyl, optionally substituted alkoxy or halogen. In certain embodiments, each R17 is independently H, CF3, CH3, OCH3, OCF3 or halogen. Preferably no more than two of X1, X2, X3 and X4 are N. More preferably, no more than one of X1, X2, X3 and X4 is N.
In certain embodiments, R1 is selected from the groups shown below:
In certain embodiments of the compounds of the invention, R1 is represented by
where one of Y1, Y2, Y3 and Y4 is O, S or NR16, and the remainder are independently N or CR17, where R16 is hydrogen, optionally substituted alkyl, R7C(O)—, R7SO2— or R7NHC(O)— and R17 is hydrogen, optionally substituted alkyl, optionally substituted alkoxy, CN or halogen. Preferably R16 is hydrogen or methyl. Preferably R17 is H; CF3; CN; C1-C4-alkyl, such as CH3; OCH3; OCF3 or halogen. Preferably at least one of Y1 to Y4 is CR17. In certain embodiments, Y3 is C—CF3, one of Y1, Y2 and Y4 is O, S or NR16, and the remainder are independently N or CR17. In certain embodiments, R1 is selected from the groups shown below:
In certain embodiments of the compounds of Formula II,
is selected from the groups shown below:
In other embodiments of the compounds of Formula II,
is selected from the groups shown below:
In preferred embodiments of the compounds of Formula II,
is selected from the groups shown below:
In certain embodiments of the compounds of the invention,
is selected from the groups below:
Representative compounds of the invention include the compounds set forth in the table below and pharmaceutically acceptable salts thereof.
In compounds illustrated herein in which the stereochemistry is not indicated, the compound is preferably the stereoisomer having the absolute stereochemistry indicated in Formulas (Ia), (IIa), (IIIa), (IVa) and (Va) or Formulas (Ib), (llb), (IIIb), (IVb) and (Vb). In certain embodiments, the preferred stereoisomer has the absolute stereochemistry indicated in Formulas (Ia), (IIa), (IIIa), (IVa) and (Va).
The compounds of the invention are useful as modulators of CFTR and treating diseases or disorders mediated by CFTR. The present invention, thus, provides methods of treating a disease or disorder mediated by CFTR in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the invention. Diseases or disorders mediated by CFTR include cystic fibrosis, Asthma, Constipation, Pancreatitis, Gastrointestinal diseases or disorders, Infertility, Hereditary emphysema, Hereditary hemochromatosis, Coagulation-Fibrinolysis deficiencies, such as Protein C deficiency, Type 1 hereditary angioedema, Lipid processing deficiencies, such as Familial hypercholesterolemia, Type 1 chylomicronemia, Abetalipoproteinemia, Lysosomal storage diseases, such as I-cell disease/Pseudo-Hurler, Mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II, Polyendocrinopathy/Hyperinsulemia, Diabetes mellitus, Laron dwarfism, Myeloperoxidase deficiency, Primary hypoparathyroidism, Melanoma, Glycanosis CDG type 1, Hereditary emphysema, Congenital hyperthyroidism, Osteogenesis imperfecta, Hereditary hypofibrinogenemia, ACT deficiency, Diabetes insipidus (DI), Neurophyseal DI, Neprogenic DI, Charcot-Marie Tooth syndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Progressive supranuclear palsy, Pick's disease, several polyglutamine neurological disorders such as Huntington's disease, Spinocerebullar ataxia type I, Spinal and bulbar muscular atrophy, Dentororubal pallidoluysian, and Myotonic dystrophy, as well as spongiform encephalopathies such as Hereditary Creutzfeldt-Jakob disease, Fabry disease, and Straussler-Scheinker disease; secretory diarrhea, polycystic kidney disease, chronic obstructive pulmonary disease (COPD), dry eye disease, Sjogren's Syndrome, congenital bilateral absence of vas deferens (CBAVD), disseminated bronchiectasis, allergic pulmonary aspergillosis, chronic sinusitis, protein C deficiency, A-lipoproteinemia, mild pulmonary disease, lipid processing deficiencies, coagulation fibrinolyis, CFTR-related metabolic syndrome, chronic bronchitis, constipation, pancreatic insufficiency, melanoma, glycanosis CDG type 1, ACT deficiency, allergic pulmonary aspergillosis; celiac disease; vascular inflammation-atherosclerotic disease, increased glucagon production, cholestatic liver disease (e.g. Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC)).
In certain embodiments, the disease or disorder mediated by CFTR is selected from congenital bilateral absence of vas deferens; acute, recurrent or chronic pancreatitis; disseminated bronchiectasis; asthma; allergic pulmonary aspergillosis; smoking related lung disease (e.g., chronic obstructive pulmonary disease, COPD); dry eye disease; Sjogren's syndrome; chronic sinusitis; cholestatic liver disease, such as primary biliary cirrhosis and primary sclerosing cholangitis; and polycystic kidney disease (autosomal dominant).
In certain embodiments, the disease or disorder mediated by CFTR is selected from celiac disease; vascular inflammation-atherosclerotic disease; dry eye (keratoconjunctivitis sicca) with or without associated autoimmune disease; polycystic kidney disease; cystic fibrosis-related diabetes mellitus; increased glucagon production; non-atopic asthma; non-CF bronchiectasis; and constipation.
The compounds of the invention can be administered in combination with one or more additional therapeutic agents, such as antibiotics, anti-inflammatory medicines, bronchodilators, or mucus-thinning medicines. In particular, antibiotics for the treatment of bacteria mucoid Pseudomonas can be used in combination with compounds of the invention. Inhaled antibiotics such as tobramycin, colistin, and aztreonam can be used in combination with treatment with compounds of the invention. Anti-inflammatory medicines can also be used in combination with compounds of the invention to treat CFTR related diseases. Bronchodilators can be used in combination with compounds of the invention to treat CFTR related diseases. In certain embodiments, the compound of the invention is administered in combination with a second compound which is a CFTR modulator.
In one embodiment, the invention provides a method of treating cystic fibrosis or a symptom thereof, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of the invention. The compound of the invention is optionally administered in combination with one or more additional pharmaceutical agents useful for the treatment of cystic fibrosis, such as compounds which are CFTR modulators, for example, compounds which are modulators of CFTR expression, activity and/or function. Suitable additional pharmaceutical agents include, but are not limited to, gentamicin ataluren, ivacaftor (KALYDECO™), lumacaftor, tezacaftor, VX-445 PTI-428, PTI-801, PTI-808, GLPG1837, GLPG2222, GLPG2737, FDL169, and FDL176. In certain embodiments, the compound of the invention is administered in combination with two or more additional CFTR modulators. For example, in one embodiment, a compound of the invention is administered in combination with FDL169 and/or FDL176. In one embodiment, the compound of the invention is administered in combination with both FDL169 and FDL176. In one embodiment, the invention relates to a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient or carrier. The compositions can include one or more compounds of the invention, and a pharmaceutically acceptable carrier, adjuvant or vehicle. In certain embodiments, these compositions further comprise one or more additional therapeutic agents useful for the treatment of CFTR mediated diseases or disorders.
The pharmaceutical compositions of the present invention comprise a compound of the present invention formulated together with one or more pharmaceutically acceptable carriers or excipients.
As used herein, the term “pharmaceutically acceptable carrier or excipient” means a non-toxic, inert solid, semi-solid, gel or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose and sucrose; cyclodextrins such as alpha—(α), beta—(β) and gamma—(γ) cyclodextrins; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
The pharmaceutical compositions of this invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. In a preferred embodiment, administration is oral administration. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
The pharmaceutical compositions of this invention can contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
In another embodiment, administration is parenteral administration by injection. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable suspension or emulsion, such as INTRALIPID®, LIPOSYN® or OMEGAVEN®, or solution, in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol. INTRALIPID® is an intravenous fat emulsion containing 10-30% soybean oil, 1-10% egg yolk phospholipids, 1-10% glycerin and water. LIPOSYN® is also an intravenous fat emulsion containing 2-15% safflower oil, 2-15% soybean oil, 0.5-5% egg phosphatides 1-10% glycerin and water. OMEGAVEN® is an emulsion for infusion containing about 5-25% fish oil, 0.5-10% egg phosphatides, 1-10% glycerin and water. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, USP and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.
Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
Powders and sprays can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
For pulmonary delivery, a therapeutic composition of the invention is formulated and administered to the patient in solid or liquid particulate form by direct administration e.g., inhalation into the respiratory system. Solid or liquid particulate forms of the active compound prepared for practicing the present invention include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. Delivery of aerosolized therapeutics is known in the art (see, for example U.S. Pat. No. 5,767,068 to Van Devanter et al., U.S. Pat. No. 5,508,269 to Smith et al., and WO 98/43650 by Montgomery).
The compositions described herein can be formulated in a unit dosage form. The term “unit dosage form” refers to physically discrete units suitable as unitary dosage for subjects undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form can be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form can be the same or different for each dose. The amount of the active compound in a unit dosage form will vary depending upon, for example, the host treated, and the particular mode of administration. In one embodiment, the unit dosage form can have one of the compounds of the invention as an active ingredient in an amount of about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 750 mg, 800 mg, 900 mg, 1000 mg, or 1,250 mg.
In some embodiments, the compounds of the invention can be administered in a dose of at least about 10 mg/day to at least about 1500 mg/day. In some embodiments, the compounds of the invention are administered in a dose of at least about 300 mg (e.g., at least about 450 mg, at least about 500 mg, at least about 750 mg, at least about 1,000 mg, at least about 1250 mg, or at least about 1500 mg).
Dose adjustments can be made for patients with mild, moderate or severe hepatic impairment (Child-Pugh Class A). Furthermore, dosage adjustments can be made for patients taking one or more Cytochrome P450 inhibitors and inducers, in particular CYP3A4, CYP2D6, CYP2C9, CYP2C19 and CYP2B6 inhibitors and inducers. Dose adjustments can also be made for patients with impaired Cytochrome P450 function such as poor, intermediate, extensive and ultra-rapid metabolizers.
Listed below are definitions of various terms used to describe this invention. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group. The term “alkyl” is intended to include both branched and straight chain, substituted or unsubstituted saturated aliphatic hydrocarbon radicals/groups having the specified number of carbons. Preferred alkyl groups comprise about 1 to about 24 carbon atoms (“C1-C24”). Other preferred alkyl groups comprise at about 1 to about 8 carbon atoms (“C1-C8”) such as about 1 to about 6 carbon atoms (“C1-C6”), or such as about 1 to about 3 carbon atoms (“C1-C3”). Examples of C1-C6 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tent-butyl, n-pentyl, neopentyl and n-hexyl radicals.
The term “alkenyl” refers to linear or branched radicals having at least one carbon-carbon double bond. Such radicals preferably contain from about two to about twenty-four carbon atoms (“C2-C24”). Other preferred alkenyl radicals are “lower alkenyl” radicals having two to about ten carbon atoms (“C2-C10”) such as ethenyl, allyl, propenyl, butenyl and 4-methylbutenyl. Preferred lower alkenyl radicals include 2 to about 6 carbon atoms (“C2-C6”). The terms “alkenyl”, and “lower alkenyl”, embrace radicals having “cis” and “trans” orientations, or alternatively, “E” and “Z” orientations.
The term “alkynyl” refers to linear or branched radicals having at least one carbon-carbon triple bond. Such radicals preferably contain from about two to about twenty-four carbon atoms (“C2-C24”). Other preferred alkynyl radicals are “lower alkynyl” radicals having two to about ten carbon atoms such as propargyl, 1-propynyl, 2-propynyl, 1-butyne, 2-butynyl and 1-pentynyl. Preferred lower alkynyl radicals include 2 to about 6 carbon atoms (“C2-C6”).
The term “aryl,” as used herein, refers to a mono- or polycyclic carbocyclic ring system comprising at least one aromatic ring, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, and indenyl. A polycyclic aryl is a polycyclic ring system that comprises at least one aromatic ring. Polycyclic aryls can comprise fused rings, covalently attached rings or a combination thereof.
The term “heteroaryl,” as used herein, refers to a mono- or polycyclic aromatic radical having one or more ring atom selected from S, O and N; and the remaining ring atoms are carbon, wherein any N or S contained within the ring may be optionally oxidized. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolyl, quinoxalinyl. A polycyclic heteroaryl can comprise fused rings, covalently attached rings or a combination thereof.
As used herein, the term “arylalkyl” means a functional group wherein an alkylene chain is attached to an aryl group, e.g., —CH2CH2-phenyl. The term “substituted arylalkyl” means an arylalkyl functional group in which the aryl group is substituted. Similarly, the term “heteroarylalkyl” means a functional group wherein an alkylene chain is attached to a heteroaryl group. The term “substituted heteroarylalkyl” means a heteroarylalkyl functional group in which the heteroaryl group is substituted.
As used herein, the term “alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. Preferred alkoxy are (C1-C3) alkoxy.
The term “cycloalkyl” refers to saturated carbocyclic radicals having three to about twelve carbon atoms (“C3-C12”). The term “cycloalkyl” embraces saturated carbocyclic radicals having three to about twelve carbon atoms. Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
The term “alkoxy” is intended to refer to an alkyl-O-radical.
The term “cycloalkenyl” refers to partially unsaturated carbocyclic radicals having three to twelve carbon atoms. Cycloalkenyl radicals that are partially unsaturated carbocyclic radicals that contain two double bonds (that may or may not be conjugated) can be called “cycloalkyldienyl”. More preferred cycloalkenyl radicals are “lower cycloalkenyl” radicals having four to about eight carbon atoms. Examples of such radicals include cyclobutenyl, cyclopentenyl and cyclohexenyl.
The terms “heterocyclyl”, “heterocycle” “heterocyclic” or “heterocyclo” refer to saturated, partially unsaturated and unsaturated heteroatom-containing ring-shaped radicals, which can also be called “heterocyclyl”, “heterocycloalkenyl” and “heteroaryl” correspondingly, where the heteroatoms may be selected from nitrogen, sulfur and oxygen. Examples of saturated heterocyclyl radicals include saturated 3 to 6-membered heteromonocyclic group containing 1 to 4 nitrogen atoms (e.g. pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl, etc.); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms (e.g. morpholinyl, etc.); saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms (e.g., thiazolidinyl, etc.). Examples of partially unsaturated heterocyclyl radicals include dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole. Heterocyclyl radicals may include a pentavalent nitrogen, such as in tetrazolium and pyridinium radicals. The term “heterocycle” also embraces radicals where heterocyclyl radicals are fused with aryl or cycloalkyl radicals. Examples of such fused bicyclic radicals include benzofuran, benzothiophene, and the like.
The terms “halogen” or “halo” as used herein, refers to an atom selected from fluorine, chlorine, bromine and iodine. Preferred halogens are fluorine and chlorine.
The term “haloalkyl” refers to an alkyl group which includes one or more halogen substituents.
The term “haloalkoxy” refers to an alkoxy group which includes one or more halogen substituents.
The term “substituted” refers to substitution by independent replacement of one, two, or three or more of the hydrogen atoms with substituents including, but not limited to, —F, —Cl, —Br, —I, —OH, C1-C12-alkyl; C2-C12-alkenyl, C2-C12-alkynyl, —C3-C12-cycloalkyl, protected hydroxy, —NO2, —N3, —CN, —NH2, protected amino, oxo, thioxo, —NH—C2-C8-alkenyl, —NH—C2-C8-alkynyl, —NH—C3-C12-cycloalkyl, —NH-aryl, —NH-heteroaryl, —NH-heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino, —O-C1-C12-alkyl, —O—C2-C8-alkenyl, —O—C2-C8-alkynyl, —O—C3-C12-cycloalkyl, —O-aryl, —O-heteroaryl, —O-heterocycloalkyl, —C(O)—C1-C12-alkyl, —C(O)—C2-C8-alkenyl, —C(O)—C2-C8-alkynyl, —C(O)—C3-C12-cycloalkyl, —C(O)-aryl, —C(O)-heteroaryl, —C(O)-heterocycloalkyl, —CONH2, —CONH—C1-C12-alkyl, —CONH—C2-C8-alkenyl, —CONH—C2-C8-alkynyl, —CONH—C3-Cu-cycloalkyl, —CONH-aryl, —CONH-heteroaryl, —CONH-heterocycloalkyl, —OCO2-C1-C12-alkyl, —OCO2-C2-C8-alkenyl, —OCO2-C2-C8-alkynyl, —OCO2-C3-C12-cycloalkyl, —OCO2-aryl, —OCO2-heteroaryl, —OCO2-heterocycloalkyl, -0O2-C1-C12 alkyl, —CO2-C2-C8 alkenyl, —OCO2-C2-C8 alkynyl, CO2-C3-C12-cycloalkyl, —CO2- aryl, CO2-heteroaryl, CO2-heterocyloalkyl, —OCONH2, —OCONH—C1-C12-alkyl, —OCONH—C2-C8-alkenyl, —OCONH—C2-C8-alkynyl, —OCONH—C3-C12-cycloalkyl, —OCONH-aryl, —OCONH-heteroaryl, —OCONH-heterocyclo-alkyl, —NHC(O)H, —NHC(O)—C1-C12-alkyl, —NHC(O)—C2-C8-alkenyl, —NHC(O)—C2-C8-alkynyl, —NHC(O)—C3-C12-cycloalkyl, —NHC(O)-aryl, —NHC(O)-heteroaryl, —NHC(O)-heterocyclo-alkyl, —NHCO2-C1-C12-alkyl, —NHCO2-C2-C8-alkenyl, —NHCO2- C2-C8-alkynyl, —NHCO2-C3-C12-cycloalkyl, —NHCO2-aryl, —NHCO2-heteroaryl, —NHCO2- heterocycloalkyl, —NHC(O)NH2, —NHC(O)NH—C1-C12-alkyl, —NHC(O)NH—C2-C8-alkenyl, —NHC(O)NH—C2-C8-alkynyl, —NHC(O)NH—C3-C12-cycloalkyl, —NHC(O)NH-aryl, —NHC(O)NH-heteroaryl, —NHC(O)NH-heterocycloalkyl, NHC(S)NH2, —NHC(S)NH—C1-C12-alkyl, —NHC(S)NH—C2-C8-alkenyl, —NHC(S)NH—C2-C8-alkynyl, —NHC(S)NH—C3-C12-cycloalkyl, —NHC(S)NH-aryl, —NHC(S)NH-heteroaryl, —NHC(S)NH-heterocycloalkyl, —NHC(NH)NH2, —NHC(NH)NH—C1-C12-alkyl, —NHC(NH)NH—C2-C8-alkenyl, —NHC(NH)NH—C2-C8-alkynyl, —NHC(NH)NH—C3-C12-cycloalkyl, —NHC(NH)NH-aryl, —NHC(NH)NH-heteroaryl, —NHC(NH)NH-heterocycloalkyl, —NHC(NH)-C1-C12-alkyl, —NHC(NH)-C2-C8-alkenyl, —NHC(NH)-C2-C8-alkynyl, —NHC(NH)-C3-C12-cycloalkyl, —NHC(NH)-aryl, —NHC(NH)-heteroaryl, —NHC(NH)-heterocycloalkyl, —C(NH)NH—C1-C12-alkyl, —C(NH)NH—C2-C8-alkenyl, —C(NH)NH—C2-C8-alkynyl, —C(NH)NH—C3-C12-cycloalkyl, —C(NH)NH-aryl, —C(NH)NH-heteroaryl, —C(NH)NH-heterocycloalkyl, —S(O)—C1-C12-alkyl, —S(O)—C2-C8-alkenyl, —S(O)—C2-C8-alkynyl, —S(O)—C3-C12-cycloalkyl, —S(O)-aryl, —S(O)-heteroaryl, —S(O)-heterocycloalkyl, —SO2NH2, —SO2NH—C1-—SO2NH—C2-C8-alkenyl, —SO2NH—C2-C8-alkynyl, —SO2NH—C3-C12-cycloalkyl, —SO2NH-aryl, —SO2NH-heteroaryl, —SO2NH-heterocycloalkyl, —NHSO2-C1-C12-alkyl, —NHSO2-C2-C8-alkenyl, —NHSO2-C2-C8-alkynyl, —NHSO2-C3-C12-cycloalkyl, —NHSO2-aryl, —NHSO2-heteroaryl, —NHSO2-heterocycloalkyl, —CH2NH2, —CH2SO2CH3, -aryl, -arylalkyl, -heteroaryl, -heteroarylalkyl, -heterocycloalkyl, —C3-C12-cycloalkyl, polyalkoxyalkyl, polyalkoxy, -methoxymethoxy, -methoxyethoxy, —SH, —S—C1-C12-alkyl, —S—C2-C8-alkenyl, —S—C2-C8-alkynyl, —S—C3-C12-cycloalkyl, -S-aryl, -S-heteroaryl, -S-heterocycloalkyl, or methylthio-methyl. In certain embodiments, the substituents are independently selected from halo, preferably Cl and F; C1-C4-alkyl, preferably methyl and ethyl; halo-C1-C4-alkyl, such as fluoromethyl, difluoromethyl, and trifluoromethyl; C2-C4-alkenyl; halo-C2-C4-alkenyl; C3-C6-cycloalkyl, such as cyclopropyl; C1-C4-alkoxy, such as methoxy and ethoxy; halo-C1-C4-alkoxy, such as fluoromethoxy, difluoromethoxy, and trifluoromethoxy; —CN; —OH; NH2; C1-C4-alkylamino; di(C1-C4-alkyl)amino; and NO2. It is understood that the aryls, heteroaryls, alkyls, cycloalkyls, heterocyclyls and the like can be further substituted. In some cases, each substituent in a substituted moiety is additionally optionally substituted when possible with one or more groups, each group being independently selected from C1-C4-alkyl; —CF3, —OCH3, —OCF3, —F, —Cl, —Br, —I, —OH, —NO2, —CN, and —NH2. Preferably, a substituted alkyl group, such as a substituted methyl group, is substituted with one or more halogen atoms, more preferably one or more fluorine or chlorine atoms.
The term “optionally substituted”, as used herein, means that the referenced group may be substituted or unsubstituted. In one embodiment, the referenced group is optionally substituted with zero substituents, i.e., the referenced group is unsubstituted. In another embodiment, the referenced group is optionally substituted with one or more additional group(s) individually and independently selected from groups described herein.
The compounds of the invention can occur in various forms, including salt forms, particularly pharmaceutically acceptable salts, co-crystals, solvates, hydrates, polymorphs, enantiomers, diastereoisomers, racemates and the like of the compounds having a formula as set forth herein. In certain embodiments, the compounds of the invention occur as a racemic mixture, for example of stereoisomers having the stereochemistry of Formulas (Ia), (1Ia), (IIIa), and (IVa) and Formulas (Ib), (IIb), (IIIb), and (IVb). In other embodiments, the compounds exist as mixtures of two enantiomers, with an enantiomeric excess of one enantiomer. In still other embodiments, the compounds exists as substantially pure single enantiomers, for example with an enatiomeric excess of one enantiomer of at least 90%, 95%, 98% or 99%. As used herein, the term “pharmaceutically acceptable salt,” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Examples of pharmaceutically acceptable salts include, but are not limited to, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane-propionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include salts of an acid drug with nontoxic ammonium, quaternary ammonium, and amine cations.
The term “hydroxy protecting group,” as used herein, refers to a labile chemical moiety which is known in the art to protect a hydroxyl group against undesired reactions during synthetic procedures. After said synthetic procedure(s) the hydroxy protecting group as described herein may be selectively removed. Hydroxy protecting groups as known in the art are described generally in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, New York (1999). Examples of hydroxyl protecting groups include benzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, tert-butoxy-carbonyl, isopropoxycarbonyl, diphenylmethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, allyloxycarbonyl, acetyl, formyl, chloroacetyl, trifluoroacetyl, methoxyacetyl, phenoxyacetyl, benzoyl, methyl, t-butyl, 2,2,2-trichloroethyl, 2-trimethylsilyl ethyl, allyl, benzyl, triphenyl-methyl (trityl), methoxymethyl, methylthiomethyl, benzyloxymethyl, 2-(trimethylsilyl)-ethoxymethyl, methanesulfonyl, trimethylsilyl, triisopropylsilyl, and the like.
The term “protected hydroxy,” as used herein, refers to a hydroxy group protected with a hydroxy protecting group, as defined above, including benzoyl, acetyl, trimethylsilyl, triethylsilyl, methoxymethyl groups, for example.
The term “amino protecting group,” as used herein, refers to a labile chemical moiety which is known in the art to protect an amino group against undesired reactions during synthetic procedures. After said synthetic procedure(s) the amino protecting group as described herein may be selectively removed. Amino protecting groups as known in the art are described generally in T. H. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd pnedition, John Wiley & Sons, New York (1999). Examples of amino protecting groups include, but are not limited to, methoxycarbonyl, t-butoxycarbonyl, 9-fluorenyl-methoxycarbonyl, benzyloxycarbonyl, and the like.
The term “protected amino,” as used herein, refers to an amino group protected with an amino protecting group as defined above.
The present invention includes all pharmaceutically acceptable isotopically-labeled or enriched compounds of the invention. These compounds include at one or more positions an isotopic abundance or the indicated element which differs from the natural isotopic distribution for that element. For example, a position at which a hydrogen atom is depicted can include deuterium at a higher abundance than the natural abundance of deuterium.
Examples of isotopes suitable for inclusion in the compounds of the invention comprises isotopes of hydrogen, such as 2H and 3H, carbon, such as 11C 13C and 14C, nitrogen, such as 13N and 15N, oxygen, such as 15O, 17O and 18O, chlorine, such as 36Cl, fluorine, such as18F, iodine, 123I and 125I, phosphorus, such as 32P, and sulfur, such as 35S.
Substituents indicated as attached through variable points of attachments can be attached to any available position on the ring structure.
As used herein, the term “therapeutically effective amount of the subject compounds,” with respect to the subject method of treatment, refers to an amount of the subject compound which, when delivered as part of desired dose regimen, brings about management of the disease or disorder to clinically acceptable standards.
“Treatment” or “treating” refers to an approach for obtaining beneficial or desired clinical results in a patient. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of symptoms, diminishment of extent of a disease, stabilization (i.e., not worsening) of a state of disease, preventing spread (i.e., metastasis) of disease, preventing occurrence or recurrence of disease, delay or slowing of disease progression, amelioration of the disease state, and remission (whether partial or total).
The compounds and processes of the present invention will be better understood in connection with the following examples, which are intended as an illustration only and not limiting of the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, derivatives, formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the appended claims.
List of Abbreviations:
All temperatures are in degrees Centigrade
BF3.Et2O—boron trifluoride etherate
CDCl3—deuterated chloroform
CF—cystic fibrosis
CFTR—cystic fibrosis transmembrane conductance regulator
CH3CN—acetonitrile
CH3NO2—nitromethane
CH2Cl2—methylene chloride
DIPEA—N,N-diisopropylethylamine
DMF—dimethylformamide
DMSO-d6—deuterated dimethylsulfoxide
ENaC—epithelial sodium channel
Et2O—diethyl ether
EtOAc—ethyl acetate
H20—water
HATU—(1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate)
FIBS—Hepes-buffered saline
HCl—hydrochloric acid
HCOOH—formic acid
HPLC—high pressure liquid chromatography
hr—hours
HTS—high throughput screen
ms—milliseconds
Na2SO4—sodium sulfate
NaBH4—sodium borohydride
NaOH—sodium hydroxide
NaHCO3—sodium bicarbonate
NAUC—normalized area under the curve
NH4OAc—ammonium acetate
NMR—nuclear magnetic resonance
Pet. Ether—petroleum ether
PBS—Phosphate buffered saline
Pd(PPh3)4—palladium tetrakis
s—seconds
rt—RT
TFA—trifluoroacetic acid
THF—tetrahydrofuran
YFP—yellow fluorescent protein
Compounds of the invention were synthesized via either method A or method B below. Method A proceeds via ring opening of the corresponding succinimide compounds. These succinimide compounds were prepared as described in WO 2017/117239, which is incorporated herein by reference in its entirety.
Method A
This method is exemplified for the preparation of 5,7-dichloro-1′-((3,5-dichloro-4-(difluoromethoxy)phenyl)carbamoyl)-6′,6′-dimethyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-2′-carboxylic acid (Compound 8) as illustrated in the scheme below.
To a stirred solution of Compound A (100 mg, 0.165 mmol) in THF (33 mL) was added 1% aqu. NaHCO3 (66 mL) at rt, and the reaction mixture was stirred at rt for 24 hr. The reaction mixture was cooled to 0° and acidified with 1N HCl (pH ˜4), then extracted with EtOAc (2×30 mL). The combined organic layers were dried over the anhydrous Na2SO4, filtered and evaporated under vacuum to afford crude product. This was purified by preparative HPLC (0.05% HCOOH/CH3CN/H2O) to give 40 mg (39% yield) of 8 as solid. LCMS: 621.9 [M+H]+; (97.2% purity). 1H NMR (500 MHz, DMSO-d6) δ=12.28 (bs, 1H), 10.89 (s, 1H), 10.51 (s, 1H), 8.16 (d, J=2.0 Hz, 1H), 7.82 (s, 2H), 7.45 (d, J=2.0 Hz, 1H), 7.11 (t, J=72.5 Hz, 1H), 4.20-4.13 (m, 2H), 3.47 (t, J=7.0 Hz, 1H), 2.49 (d, J=8.0 Hz, 1H), 1.93 (d, J=7.5 Hz, 1H), 1.61-1.58 (m, 1H), 1.29-1.23 (m, 1H), 1.00 (s, 3H), 0.91 (s, 3H); 19F NMR (470.59 MHz, DMSO-d6): −80.26, −80.10. Chiral SFC purification (97.9% ee).
Prep HPLC Conditions
Column: SYMMETRY-C8 (300*19), 7u; Mobile phase: 0.1% FORMIC ACID IN H2O: CH3CN GRADIENT: (T%B): 0/30,8/80,8.1/98,10/98,10.1/30,13/30 Flow Rate: 20 ml/min; Diluent: CH3CN +H2O+THF.
Method B
This method is exemplified for the preparation of (1′R,2′S,3R,7a′R)-1′-((3-(tert-butyl)phenethyl)carbamoyl)-5,7-dichloro-6′,6′-difluoro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-2′-carboxylic acid (Compound 44)
A solution of maleic anhydride (15.0 g, 152 mmol) in allyl alcohol (200 mL) was stirred at rt for 16 hr. The reaction mixture was concentrated in vacuo to a residue which was dissolved in Et2O (1.0 L), washed with H2O (3×1 L), brine (500 mL) then dried over anhydrous Na2SO4 and concentrated in vacuo to give 12.0 g (50% yield) of A as a colorless liquid. LCMS: m/z 157.0 [M+H]+; (99.5% purity).
To a stirred solution of B (5 g, 32.0 mmol) in THF (70 mL) was added 5,7-dichloroisatin (C) (6.9 g, 32.0 mmol) and 4,4-difluoro-L-proline.TFA salt (D) (7.9 g, 32.0 mmol) at rt. The reaction mixture was stirred at 80° for 3 hr, then cooled to rt, and evaporated in vacuo to give the crude product as a mixture of diastereomers. This was purified by column chromatography, eluting with Pet. Ether:EtOAc (80° to give crude product as a brown solid. This brown material was triturated with CH2Cl2 and filtered to give, after drying in vacuo, 1.3 g (9% yield) of E as a white solid (single isomer). LCMS: m/z 461.0 [M+H]+; (99.2% purity). 1H NMR (500 MHz, acetone-d6) δ 11.8-10.9 (br s, 1H), 9.96 (s, 1H), 7.85 (d, J=2 Hz, 1H), 7.39 (d, J=2 Hz, 1H), 5.56-5.53 (m, 1H), 5.15-5.07 (m, 2H), 4.33-4.32 (m, 2H), 4.22-4.20 (m, 1H), 4.10-4.09 (m, 1H), 3.82-3.79 (m, 1H), 3.30-3.25 (m, 1H), 2.53-2.45 (m, 1H), 2.34-2.20 (m, 1H). The desired diastereomer (i.e., having the relative stereochemistry set forth in Formulas (Ia), (IIa) and (IIIa)) was confirmed by 2D NMR.
To a solution of NH4OAc (4.2 g, 55.1 mmol) in CH3NO2 (150 mL) was added 3-(tert-butyl)benzaldehyde (F) (3 g, 18.3 mmol) at 90° and the resulting mixture was stirred at 120° for 16 hr. The reaction mixture was cooled to rt and concentrated in vacuo to give the crude product as a brown liquid. This was purified by column chromatography, eluting with Pet. Ether:EtOAc (80:20) to give 2.9 g (65% yield) of product G as a pale brown solid. 1H NMR (500 MHz, CDCl3) δ 8.03 (d, J=13.5 Hz, 1H), 7.60 (d, J=14 Hz, 1H), 7.55-7.53 (m, 2H), 7.39-7.38 (m, 2H).
To a stirred solution of NaBH4 (847 mg, 22.4 mmol) in anhydrous THF (20 mL) was added BF3.Et2O (3.3 mL, 26.8 mmol) at 0°. After stirring at 0° for 15 minutes, the reaction mixture was warmed to rt and then a solution of (H) (920 mg, 4.48 mmol) in anhydrous THF (10 mL) was added. The resulting mixture was stirred at 85° for 5 hr, then cooled to 0°. H2O (40 mL) and 1N HCl (40 mL) were added to the reaction mixture over 20 minutes, and the resulting mixture was stirred at 85° for 2 hr. The reaction mixture was cooled to 0° and basified (pH ˜12) using 5N NaOH solution. The resulting mixture was extracted with EtOAc (2×100 mL), then the combined organic layers were washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give 700 mg (88% yield) of product (I) as liquid which was used in the next step without purification. LCMS: m/z 178.1 [M+H]+; (99.5% purity).
To a stirred solution of E (200 mg, 0.43 mmol) in DMF (10 mL) was added DIPEA (0.15 mL, 0.86 mmol), HATU (196 mg, 0.51 mmol) and I (115 mg, 0.65 mmol) at rt. The resulting reaction mixture was stirred at rt for 30 min, then poured into ice cold H2O and stirred for 10 minutes. The resulting precipitate was collected, washed with cold H2O and dried in vacuo to give 250 mg (94% yield) of product J as a white solid, which was used in the next step without purification. LCMS: m/z 620.1 [M+H]+; (10.9%+83.3% purity).
To a stirred solution of J (200 mg, 0.32 mmol) in anhydrous THF (5 mL) were added aniline (30 mg, 0.32 mmol) and Pd(PPh3)4 (37 mg, 0.032 mmol) at rt. The resulting mixture was stirred at rt for 1 hr, then diluted with EtOAc (100 mL) and washed with brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give the crude product as liquid. The crude product was purified by preparative HPLC using the following conditions to give 95 mg (50% yield) of product 44: Column—INERTSIL-ODS (250 mm×20 mm×5 uM); Mobile Phase-A—0.1% Formic Acid in H2O, B—0.05% Formic Acid in CH3CN; Time (min)/%B: 0/60, 8/85, 9/85, 12/95, 12.1/60, 15/60; Flow Rate—20 mL/min.
LCMS: m/z 580.1 [M+H]+; (98.9% de). 1H NMR (500 MHz, DMSO-d6): δ 12.40 (s, 1H), 10.95 (s, 1H), 8.25 (br s, 2H), 7.43 (d, J=1.5 Hz, 1H), 7.26 (s, 1H), 7.24-7.20 (m, 2H), 7.07-7.05 (m, 1H), 3.96-3.90 (m, 2H), 3.46-3.44 (m, 2H), 3.17- 3.13 (m, 2H), 2.77-2.71 (m, 2H), 2.58-2.51 (m, 1H), 2.10-2.07 (m, 1H), 1.99-1.85 (m, 1H), 1.28 (s, 9H).
Compounds 1-65 were prepared as described herein and characterized by LCMS. For each compound, the synthesis method used is shown in the final column.
To a stirred suspension of activated Cu powder (3.8 g, 60.9 mmol), CuI (2.3 g, 12.2 mmol) in DMSO (30 mL) was added 4-iodopyridine, 66.1 (5.0 g, 24.4 mmol) and ethyl 2-bromo-2,2-difluoroacetate (12.4 g, 60.9 mmol) at RT. The resulting reaction mixture was stirred for 16 h at RT. The reaction mixture was cooled to RT, poured into ice-water (50 mL) and extracted with diethyl ether (2×50 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (80 g Silica gel cartridge, 20% EtOAc in pet ether) to afford 66.2 (4 g, 81%) as liquid. 1H NMR (500 MHz, CDCl3): 8.81 (br s, 2H), 7.53 (d, J=5.0 Hz, 2H), 4.32 (q, J=7.0 Hz, 2H), 1.32 (t, J=7.0 Hz, 3H).
To a stirred solution of 66.2 (2.2 g, 10.9 mmol) in MeOH (50 mL) was added NaBH4 (289 mg, 7.6 mmol) portion wise at −60° C. The resulting reaction mixture was stirred for 4 h at −60° C. The reaction mixture was quenched with NH4Cl solution at −60° C. and extracted with EtOAc (2×30 mL). The combined organic layer was washed with water (30 mL), brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford 66.3 (1.8 g) as a solid, which used in the next step without purification. 1H NMR (400 MHz, DMSO-d6): 8.90 (br s, 2H), 7.56 (br s, 2H), 7.33 (d, J=6.4 Hz, 1H), 4.89 (q, J=6.0 Hz, 1H), 3.29 (s, 3H).
To a stirred solution of 66.3 (1.8 g, 9.49 mmol) in toluene (30 mL) was added (S)-2-amino-2-phenylethan-1-ol (1.3 g, 9.49 mmol), PTSA (180 mg, 0.94 mmol) at RT. The resulting mixture was refluxed for 2 h using Dean-Stark apparatus. The reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (40 g Silica gel cartridge, 10% EtOAc in pet ether) to afford mixture of diastereomers 66.4 (2.1 g, 80%) as a colorless liquid. LCMS: (37.2+52.1%, m/z [M+H]+=277.0.
To a stirred solution of 66.4 (2.0 g, 7.23 mmol) in CH2Cl2 (50 mL) was added TMSCN (1.4 g, 14.5 mmol) and BF3.Et2O (2 g, 14.5 mmol) at −78° C. over a period of 10 minutes. The resulting reaction mixture was stirred at −78° C. for 8 h. The reaction mixture was poured into sat.NaHCO3 solution (80 mL) and extracted with CH2Cl2 (2×30 mL). The combined organic layer was washed with water (50 mL), brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under vacuum to afford residue. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 30% EtOAc in pet ether) to afford mixture of diastereomers 66.5 (1.8 g, 81%) as a pale brown solid. LCMS: (30.5+66.2%), m/z [M+H]+=304.3.
To a stirred solution of 66.5 (2.4 g, 7.91 mmol) in MeOH (50 mL) and CH2Cl2 (80 mL) was added Pb(OAc)4 (5.2 g, 11.9 mmol) at 0° C. The resulting reaction mixture was stirred for 2 h at 0° C. The reaction mixture was poured in to 0.2M phosphate buffer solution (50 mL) at RT and then filtered through celite bed. The aqueous layer was extracted with CH2Cl2 (2×20 mL). The combined organic layer was washed with H2O (2×50 mL), brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under vacuum to afford mixture of diastereomers 66.6 (1.8 g) as a brown sticky material, which used in the next step without purification.
To a stirred solution of 66.6 (1.7 g, 6.26 mmol) in con. HCl (60 mL) was heated at 100° C. for 6 h. The reaction mixture was cooled to RT and concentrated under vacuum at 50° C. The resulting residue was triturated with CH2Cl2 to afford a mixture of diastereomers 66.7 (550 mg) as a pale brown solid. LCMS: 87.5%, m/z [M+H]+=203.2.
To a stirred solution of 66.7 (500 mg, 2.47 mmol) in EtOH (40 mL) was added 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione (598 mg, 2.47 mmol), 5,7-dichloroindoline-2,3-dione (534 mg, 2.47 mmol) at RT. The reaction mixture was stirred at 90° C. for 2 h. The volatile components were removed under vacuum. The resulting residue was purified by flash chromatography (Silica gel 100-200 mesh, 50% EtOAc/pet ether) to afford 66.8a (120 mg, 8%) and 66.8b (240 mg, 16%) as a solid.
66.8a: 1H NMR (500 MHz, DMSO-d6): 11.18 (s, 1H), 8.76 (d, J=5.0 Hz, 2H), 7.78 (d, J=1.5 Hz, 1H), 7.58 (d, J=6.0 Hz, 2H), 7.53 (d, J=2.0 Hz, 1H), 7.33 (d, J=2.0 Hz, 1H), 7.25 (d, J=2.0 Hz, 2H), 4.60-4.49 (m, 2H), 3.93 (s, 2H); LCMS: 97.5%, m/z [M+H]+=597.1. 66.8b: 1H NMR (500 MHz, DMSO-d6): 11.07 (br s, 1H), 8.73 (d, J=5.5 Hz, 2H), 7.78 (t, J=2.0 Hz, 1H), 7.61 (d, J=6.0 Hz, 2H), 7.49 (d, J=1.5 Hz, 1H), 7.33 (d, J=1.5 Hz, 2H), 7.05 (d, J=1.5 Hz, 1H), 5.02-4.94 (m, 1H), 4.35 (d, J=5.0 Hz, 1H), 3.90 (t, J=8.5 Hz, 1H), 3.67 (d, J=8.0 Hz, 1H); LCMS: 99.4%, m/z [M+H]+=597.1.
To a stirred solution of 66.8a (80 mg, 0.13 mmol) in THF (30 mL) was added 1% NaHCO3 solution (60 mL) at RT. The resulting reaction mixture was stirred for 96 h at RT. After completion of the reaction, the pH of the solution was adjusted to ˜6-7 with 1N HCl solution and extracted with EtOAc (2×20 mL). The combined organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by prep. HPLC [X-BRIDGE C18 (150×30) mm, 5 μ; A: 0.1% HCOOH in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/40, 8/80, 9/80, 9.1/98, 14/98, 14.1/40, 17/40 at 25 mL/min] to give 66.9a (32 mg, 39%) as a solid. 1H NMR (500 MHz, DMSO-d6): 12.91 (br s, 1H), 11.21 (br s, 1H), 11.03 (br s, 1H), 8.71 (d, J=5.5 Hz, 2H), 7.54-7.52 (m, 4H), 7.44 (br s, 1H), 7.28 (s, 1H), 7.25-7.16 (m, 1H), 4.80-4.69 (m, 1H), 4.30-4.15 (m, 2H), 3.54-3.51 (m, 1H); LCMS: 97.0%, m/z [M+H]+=615.1.
To a stirred solution of 66.8b (180 mg, 0.3 mmol) in THF (60 mL) was added 1% NaHCO3 solution (120 mL) at RT. The resulting reaction mixture was stirred for 96 h at RT. After completion of the reaction, the pH of the solution was adjusted to ˜6-7 with 1N HCl solution and extracted with EtOAc (2×20 mL). The combined organic layer was washed with brine (60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The resulting residue was purified by prep. HPLC [X-BRIDGE C18 (150×30) mm, 5 μ; A: 0.1% HCOOH in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/45, 8/80, 10/80, 10.1/98, 12/98, 12.1/45, 15/45 at 25 mL/min] to afford 66.9b.1 (7 mg, 4%) as a solid, 66.9b.2 (7 mg, 4%) as a solid and 66.9b.3 (64 mg, 35%) as a solid.
66.9b.1: LCMS: 93.8%, m/z [M+H]+=615.1;
66.9b.2: LCMS: 87.0%, m/z [M+H]+=615.1;
66.9b.3: 1H NMR (500 MHz, DMSO-d6): 12.46 (br s, 1H), 10.95 (s, 1H), 10.57 (s, 1H), 8.66 (d, J=5.0 Hz, 2H), 8.19 (d, J=1.5 Hz, 1H), 7.67 (d, J=1.5 Hz, 2H), 7.51 (d, J=6.0 Hz, 2H), 7.46 (s, 1H), 7.32 (s, 1H), 4.34-4.28 (m, 1H), 4.00-3.90 (m, 1H), 3.76-3.73 (m, 1H), 3.60-3.59 (m, 1H); LCMS: 99.4%, m/z [M+H]+=615.1.
To a stirred solution of ethyl 2-(4-chlorophenyl)-2-oxoacetate, 67.1 (5.0 g, 23.6 mmol) in DCM (50 mL) was added DAST (11 mL, 82.5 mmol) at RT. The resulting reaction mixture was stirred for 16 h at RT. The reaction mixture was cooled to 0° C. and quenched with sat. NaHCO3 solution. The organic layer was separated, washed with water (2×50 mL) and brine (15 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford 67.2 (5.0 g) as a pale brown liquid, which was used in the next step without purification.
1H NMR (400 MHz, CDCl3): 7.55 (d, J=8.8 Hz, 2H), 7.44 (d, J=8.8 Hz, 2H), 4.30 (q, J=7.2 Hz, 2H), 1.31 (t, J=7.2 Hz, 3H).
To a stirred solution of 67.2 (5 g, 21.3 mmol) in MeOH (25 mL) was added NaBH4 (800 mg, 21.3 mmol) portion wise at −60° C. The resulting reaction mixture was stirred for 1 h at −60° C. The reaction mixture was quenched with 1N HCl solution (50 mL) at 0° C. and extracted with Et2O (2×30 mL). The combined organic layer was washed with water (30 mL), brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford 67.3 (5.0 g) as pale yellow liquid, which used for next step without purification and analysis.
To a stirred solution of 67.3 (5.0 g, 22.4 mmol) in toluene (50 mL) was added (S)-2-amino-2-phenylethan-1-ol (2.99 g, 22.4 mmol), PTSA (112 mg, 0.44 mniol) at RT. The reaction mixture was refluxed for 1 h using Dean-Stark apparatus. Then the reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 20% EtOAc in pet ether] to afford mixture of diastereomers 67.4 (6.5 g, 94%) as a colorless liquid. LCMS: (47.7+40.0)%, m/z [M+H]+=310.
To a stirred solution of 67.4 (6.5 g, 21.0 mmol) in CH2Cl2 (100 mL) was added TMSCN (5.2 mL, 42.0 mmol) and BF3.Et2O (5.1 mL, 42.0 mmol) at −78° C. over a period of 10 minutes. The resulting reaction mixture was stirred at RT for 16 h. The reaction mixture was cooled to 0° C., quenched with sat.NaHCO3 solution (80 mL) and extracted with CH2Cl2 (2×30 mL). The combined organic layer was washed with water (50 mL), brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under vacuum. The residue was purified by flash column chromatography (80 g Silica gel cartridge, 20% EtOAc in pet ether) to afford mixture of diastereomers 67.5 (5.8 g, 82%) as a solid. 1H NMR (400 MHz, CDCl3): 7.52-7.22 (m, 7H), 7.02 (d, J=7.6 Hz, 2H), 4.13-3.96 (m, 1H), 3.81-3.59 (m, 2H), 3.53-3.46 (m, 1H), 2.61-2.59 (m, 1H), 1.73-1.65 (m, 1H); LCMS: (31.1 +68.3)%, m/z [M+H]+=337.1.
To a stirred solution of 67.5 (5.8 g, 17.2 mmol) in MeOH (100 mL) and CH2Cl2 (200 mL) was added Pb(OAc)4 (11.5 g, 25.8 mmol) at 0° C. The resulting reaction mixture was stirred for 30 minutes at 0° C. The reaction mixture was poured into 0.2 M phosphate buffer solution (50 mL) at RT and then filtered through celite bed. The aqueous layer was extracted with CH2Cl2 (2×40 mL). The combined organic layer was washed with H2O (2×50 mL), brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under vacuum to afford 67.6 (6.0 g) as liquid, which used in the next step without purification. 1H NMR (500 MHz, CDCl3): 8.45 (s, 1H), 7.74 (d, J=1.5 Hz, 2H), 7.54-7.42 (m, 7H), 5.22-5.19 (m, 1H).
To a stirred solution of 67.6 (1.5 g, 6.36 mmol) in con. HCl (20 mL) was heated at 110° C. for 16 h. The reaction mixture was cooled to RT and and concentrated under vacuum at 50° C. The resulting residue was triturated with CH2Cl2 to afford 67.7 (550 mg) as a pale brown solid. LCMS: 89.0%, m/z [M+H]+=236.1.
To a stirred solution of 66.7 (1.0 g, 4.25 mmol) in EtOH (20 mL) was added 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione (1.09 g, 4.25 mmol), 5,7-dichloroindoline-2,3-dione (0.91 g, 4.25 mmol) at RT. The reaction mixture was heated at 80° C. for 2 h. Then the reaction mixture was concentrated under vacuum to afford residue. The residue was purified by flash chromatography (80 g Silica gel cartridge, gradient elution of 50% EtOAc/pet ether) to afford minor diastereomer 67.8a (170 mg, 6%) as a white solid and major diastereomer 678b (600 mg, 22%) as solid.
67.8a: 1H NMR (500 MHz, DMSO-d6): 11.18 (br s, 1H), 7.78 (t, J=2.0 Hz, 1H), 7.64-7.55 (m, 4H), 7.53 (d, J=2.0 Hz, 1H), 7.30 (d, J=1.5 Hz, 1H), 7.25 (d, J=1.5 Hz, 2H), 4.52-4.49 (m, 2H), 3.91 (s, 2H); LCMS: 95.7%, m/z [M−H]−=628.1.
67.8b: 1H NMR (500 MHz, DMSO-d6): 11.07 (br s, 1H), 7.79 (t, J=2.0 Hz, 1H), 7.63 (d, J=9.0 Hz, 2H), 7.56 (d, J=9.0 Hz, 2H), 7.50 (d, J=2.0 Hz, 1H), 7.30 (d, J=2.0 Hz, 2H), 7.03 (d, J=2.0 Hz, 1H), 5.00-4.92 (m, 1H), 4.32 (d, J=4.5 Hz, 1H), 3.86 (t, J=8.5 Hz, 1H), 3.66 (d, J=6.5 Hz, 1H); LCMS: 99.1%, m/z [M−H]−=628.1.
To a stirred solution of 67.8a (170 mg, 0.269 mmol) in THF (75 mL) was added 1% NaHCO3 solution (150 mL) at RT. The resulting reaction mixture was stirred for 2 days at RT. After completion of reaction, the pH of solution was adjusted to ˜6-7 with 1N HCl solution and extracted with ethyl acetate (2×40 mL). The combined organic layer was washed with brine (30 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford residue. The residue was purified by prep. HPLC [X-BRIDGE-C18 (150×30) mm, 5 μ; A: 0.1% Formic Acid in H2O; B: Acetonitrile; Gradient: (Time/%B): 0/50, 8/80, 10/80, 13/98, 14/98, 14.1/50, 17/50 at 25 mL/min] to give 67.9a (12 mg, 7%) as a white solid 1H NMR (500 MHz, DMSO-d6): 12.91 (br s, 1H), 11.23 (br s, 1H), 10.92 (br s, 1H), 7.55-7.27 (m, 9H), 4.85-4.68 (m, 1H), 4.25-4.05 (m, 1H), 3.55-3.40 (m, 1H); LCMS: 96.9%, m/z [M−H]−=646.1.
To a stirred solution of 67.8b (400 mg, 0.633 mmol) in THF (150 mL) was added 1% NaHCO3 solution (300 mL) at RT. The resulting reaction mixture was stirred for 2 days at RT. After completion of reaction, the pH of solution was adjusted to ˜6-7 with 1N HCl solution and extracted with ethyl acetate (2×60 mL). The combined organic layer was washed with brine (60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford residue. The residue was purified by prep. HPLC [X-BRIDGE-C18 (150×30) mm, 5 μ; A: 0.1% Formic Acid in H2O; B: Acetonitrile; Gradient: (Time/%B): 0/65, 8/90, 10/90, 10.1/98, 12/98, 12.1/65, 15/65 at 25 mL/min] to give 67.9b (40 mg, 10%) as a white solid. 1H NMR (500 MHz, DMSO-d6): 12.48 (br s, 1H), 10.94 (br s, 1H), 10.52 (br s, 1H), 8.18 (d, J=1.5 Hz, 1H), 7.66 (d, J=2.0 Hz, 2H), 7.54-7.45 (m, 5H), 7.30 (br s, 1H), 4.35-4.22 (m, 1H), 3.95-3.86 (m, 1H), 3.72-3.62 (m, 1H), 3.60-3.50 (m, 1H); LCMS: 96.1%, m/z [M−H]−=646.1.
Following Examples 66 and 67, the following diastereomeric examples were made. Depending on substituents on the corresponding analogous intermediates 66.8a or 66.8b, epimerized products might be found or in the case of corresponding analogous intermediates 67.8a or 67.8b, the desired products were isolated without epimerization.
A solution of 80.1 (15 g, 69.4 mmol) in NH4OH (150 mL) was stirred in a steel bomb at 180° C. for 16 h. The reaction mixture was cooled to RT, diluted with H2O and extracted with EtOAc (3×200 mL). The combined organic layer was concentrated under reduced pressure. The residue was purified by column chromatography (Silica gel 100-200 mesh, 20% EtOAc in pet ether) to afford 80.2 (7 g, 51%) as solid.
1HNMR (400 MHz, CDCl3): 6.85 (s, 1H), 6.58 (s, 1H), 4.96 (br s, 2H); LCMS: 76.1%, m/z [M+H]+=197.0.
80.2 (12 g, 61.0 mmol) was dissolved in 25% NaOH in MeOH (120 mL) solution. After stirring in a steel bomb for 16 h at 100° C., the reaction mixture was cooled to RT, diluted with H2O and extracted with EtOAc (3×200 mL). The combined organic layer was concentrated under vacuum. The residue was purified by column chromatography (Silica gel 100-200 mesh, 20% EtOAc in pet ether) to afford 80.3 (9.8 g, 83%) as solid.
1H NMR (400 MHz, CDCl3): 6.28 (s, 1H), 6.23 (s, 1H), 4.56 (br s, 2H), 3.87 (s, 3H); LCMS: 88.1%, m/z [M+H]+=193.1.
Synthesis of 80.4:
To a stirred solution of furan-2,5-dione (4.49 g, 45.8 mmol) in MTBE (100 mL) was added 80.3 (8.8 g, 45.8 mmol) at RT. After stirring for 16 h at RT, the precipitated solid was filtered and dried under vacuum. The residue was washed with MTBE to afford 80.4 (13.0 g, 66%) as a solid.
1H NMR (500 MHz, CDCl3): 8.46 (d, J=6.5 Hz, 1H), 8.03 (s, 1H), 6.87 (s, 1H), 6.54 (d, J=12.5 Hz, 1H), 6.41 (d, J=12.5 Hz, 1H), 3.93 (s, 3H); LCMS: 98.4%, m/z [M+H]+=291.0.
To a stirred solution of 80.4 (5.0 g, 19.2 mmol) in Ac2O (50 mL) was added NaOAc (1.57 g, 19.2 mmol) at RT. After stirring at 80° C. for 2 h, the reaction mixture was cooled to RT and the excess Ac2O was evaporated under reduced pressure. The residue was diluted with DCM (50 mL) and washed with water (2×25 mL). The combined organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by flash column chromatography (80 g Silica gel cartridge, 20% EtOAc in pet ether) to afford 80.5 (3.0 g, 64%) as a solid.
1H NMR (400 MHz, CDCl3): 7.14 (s, 1H), 6.99 (s, 1H), 6.90 (s, 2H), 3.97 (s, 3H); LCMS: 98.1%, m/z [M+H]+=273.0.
To a stirred solution of 80.5 (2.5 g, 9.19 mmol) in EtOH (30 mL) was added 5,7-dichloroindoline-2,3-dione (1.98 g, 9.19 mmol), (S)-4,4-difluoropyrrolidine-2-carboxylic acid (2.27 g, 9.19 mmol) at RT. After stirring at 80° C. for 2 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (120 g Silica gel cartridge, 30% EtOAc/pet ether) to afford major diastereomer 80.6 (4.4 g, 84%) as a yellow solid.
1H NMR (500 MHz, DMSO-d6): 11.40 (s, 1H), 7.59 (d, J=2.0 Hz, 1H), 7.46 (s, 1H), 7.39 (d, J=1.0 Hz, 1H), 7.14 (d, J=1.5 Hz, 1H), 4.44-4.39 (m, 1H), 4.02 (s, 3H), 3.99-3.91 (m, 1H), 3.90 (t, J=7.5 Hz, 1H), 3.09-3.05 (m, 1H), 2.69-2.63 (m, 1H), 2.57-2.51 (m, 1H), 2.36-2.30 (m, 1H); LCMS: 96.9%, m/z [M−H]−=574.8; Chiral purity: (46.6+47.9)%. Separation of 80.6a & 80.6b (absolute stereochemistry of Enantiomer 1 & 2 not determined):
80.6 (4.4 g) was purified by chiral SFC using Chiral pack IG (250×30) mm, 5 μ; 0.2% TFA in n-Hexane: Isopropanol (85:15) at RT (Isocratic 42.0 mL/min, 13 min run time with detection at 254 nm). Pure fractions were concentrated under reduced pressure to give 480 mg of 80.6a (Enantiomer-1) as a yellow solid and 470 mg of 80.6b (Enantiomer-2) as a yellow solid.
80.6a:1H NMR (500 MHz, DMSO-d6): 11.40 (s, 1H), 7.59 (d, J=2.0 Hz, 1H), 7.46 (s, 1H), 7.39 (d, J=0.5 Hz, 1H), 7.14 (d, J=2.0 Hz, 1H), 4.44-4.39 (m, 1H), 4.02 (s, 3H), 3.98 (d, J=7.5 Hz, 1H), 3.90 (t, J=7.5 Hz, 1H), 3.09-3.07 (m, 1H), 2.71-2.65 (m, 1H), 2.60-2.51 (m, 1H), 2.36-2.29 (m, 1H); LCMS: 99.5%, m/z [M+H]+=576.9; Chiral purity: 98.0%.
80.6b:'H NMR (500 MHz, DMSO-d6): 11.04 (s, 1H), 7.59 (d, J=2.0 Hz, 1H), 7.46 (s, 1H), 7.39 (s, 1H), 7.14 (d, J=1.5 Hz, 1H), 4.44-4.39 (m, 1H), 4.02 (s, 3H), 3.98 (d, J=7.5 Hz, 1H), 3.90 (t, J=7.5 Hz, 1H), 3.11-3.05 (m, 1H), 2.69-2.63 (m, 1H), 2.57-2.51 (m, 1H), 2.35-2.30 (m, 1H); LCMS: 98.6%, m/z [M+H]+=576.9; Chiral purity: 99.9%.
To a stirred solution of 80.6a (300 mg, 0.52 mmol) in THF (100 mL) was added 1% NaHCO3 solution (200 mL) at RT. The resulting reaction mixture was stirred for 16 h at RT. After completion of reaction, the pH of the solution was adjusted to −6-7 with IN HCl solution and extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine (60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford residue. The residue was purified by prep. HPLC [X-BRIDGE C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/40, 8/80, 9/80, 9.1/98, 14/98, 14.1/40, 17/40 at 25 mL/min] to afford 80.7a.1 (11 mg) as a white solid and 80.7a.2 (208 mg) as a white solid.
80.7a.1: 1HNMR (500 MHz, DMSO-d6): 12.74 (br s, 1H), 11.11 (br s, 1H), 7.83 (s, 1H), 7.49 (s, 1H), 7.05 (br s, 1H), 6.88 (s, 1H), 4.25-4.15 (m, 2H), 3.83 (s, 3H), 3.78 (d, J=7.0 Hz, 1H), 3.55-3.50 (m, 1H), 3.24-3.15 (m, 1H), 2.85-7.73 (m, 1H), 2.40-2.30 (m, 1H); LCMS: 98.6%, m/z [M+H]+=595.2.
80.7a.2: 1HNMR (500 MHz, DMSO-d6): 12.49 (br s, 1H), 11.08 (br s, 1H), 11.04 (br s, 1H), 8.05 (s, 1H), 8.02 (s, 1H), 7.48 (d, J=1.5 Hz, 1H), 6.92 (s, 1H), 4.08-4.04 (m, 2H), 3.94 (s, 3H), 3.85-3.78 (m, 1H), 3.17-3.14 (m, 1H), 2.67-2.63 (m, 1H), 2.50-2.44 (m, 1H), 2.08-1.90 (m, 1H); LCMS: 98.2%, m/z [M+H]+=595.2.
To a stirred solution of 80.6b (300 mg, 0.519 mmol) in THF (100 mL) was added 1% NaHCO3 solution (200 mL) at RT. The resulting reaction mixture was stirred for 16 h at RT. After completion of reaction, the pH of the solution was adjusted to ˜6-7 with 1N HCl solution and extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine (60 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford residue. The residue was purified by prep. HPLC [X-BRIDGE C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/50, 9/80, 11/80, 11.1/98, 12/98, 12.1/50, 15/50 at 25 mL/min] to afford 80.7b.1 (13 mg) as a white solid and 80.7b.2 (135 mg) as a white solid.
80.7b.1: 1H NMR (500 MHz, DMSO-d6): 12.76 (br s, 1H), 11.12 (br s, 1H), 7.83 (s, 1H), 7.49 (s, 1H), 7.06 (br s, 1H), 6.89 (s, 1H), 4.26-4.05 (m, 2H), 3.83 (s, 3H), 3.78 (d, J=8.0 Hz, 1H), 3.55-3.49 (m, 1H), 3.28-3.11 (m, 1H), 2.82-2.77 (m, 1H), 2.38-2.36 (m, 1H); LCMS: 98.6%, m/z [M+H]+=595.2.
80.7b.2: 1H NMR (500 MHz, DMSO-d6): 12.50 (br s, 1H), 11.08 (br s, 1H), 11.02 (br s, 1H), 8.06 (br s, 1H), 8.02 (s, 1H), 7.49 (d, J=1.5 Hz, 1H), 6.92 (s, 1H), 4.09-4.04 (m, 2H), 3.94 (s, 3H), 3.85-3.78 (m, 1H), 3.19-3.12 (m, 1H), 2.69-2.62 (m, 1H), 2.50-2.40 (m, 1H), 2.08-1.90 (m, 1H); LCMS: 98.0%, m/z [M+H]+=595.2.
To a stirred solution of 81.1 (1 g, 6.98 mmol) in EtOH (40 mL) were added 1-(3,5-dichlorophenyl)-1H-pyrrole-2,5-dione (1.69 g, 6.98 mmol) and 5,7-dichloroindoline-2,3-dione (1.50 g, 6.98 mmol). After stirring for 3 h at 80° C., the reaction mixture was cooled to RT and concentrated. The residue was purified by flash chromatography (40 g Silica gel cartridge, 10% EtOAc in pet ether) to afford minor diastereomer 81.2_1 (400 mg, 10%) as a solid and major diastereomer 81.2_2 (2.2 g, 59%) as a solid.
81.2_1: 1H NMR (400 MHz, DMSO-d6): 11.28 (br s, 1H), 7.78 (t, J=2.0 Hz, 1H), 7.54 (d, J=2.0 Hz, 1H), 7.48 (d, J=2.0 Hz, 1H), 7.26 (d, J=2.0 Hz, 2H), 4.88 (d, J=5.2 Hz, 1H), 4.50-4.46 (m, 1H), 4.12 (d, J=10.0 Hz, 1H), 3.95-3.92 (m, 1H); LCMS: 98.8%, m/z [M-H]−=535.9.
81.2_2: 1H NMR (400 MHz, DMSO-d6): 11.17 (br s, 1H), 7.79 (t, J=2.0 Hz, 1H), 7.53 (d, J=2.0 Hz, 1H), 7.38 (d, J=2.0 Hz, 2H), 7.07 (d, J=2.0 Hz, 1H), 4.88-4.83 (m, 2H), 4.06-4.01 (m, 1H), 3.71 (d, J=8.4 Hz, 1H); LCMS: 98.3%, m/z [M-H]−=535.9.
81.2_2 (2.2 g) was purified by chiral SFC using (R, R) Whelk-01 (30×250 mm), 5 μ; 80% CO2: 20% acetonitrile at RT (Isocratic 90 g/min, with detection at 214 nm) to give 81.2_2a (Enantiomer-1, 900 mg, 82%) as a solid and 81.2_2b (Enantiomer-2, 850 mg, 77%) as a solid. (absolute stereochemistry of Enantiomer 1 & 2 were not determined)
81.2_2a: 1H NMR (400 MHz, DMSO-d6): 11.17 (br s, 1H), 7.80 (t, J=2.0 Hz, 1H), 7.54 (d, J=2.0 Hz, 1H), 7.38 (d, J=2.0 Hz, 2H), 7.07 (d, J=2.0 Hz, 1H), 4.91-4.84 (m, 2H), 4.05-4.01 (m, 1H), 3.71 (d, J=8.0 Hz, 1H); LCMS: 99.4%, m/z [M−H]−=535.9. 81.2_2b: 1H NMR (400 MHz, DMSO-d6): 11.18 (br s, 1H), 7.80 (t, J=2.0 Hz, 1H), 7.53 (d, J=2.0 Hz, 1H), 7.38 (d, J=2.0 Hz, 2H), 7.07 (d, J=1.6 Hz, 1H), 4.89-4.84 (m, 2H), 4.05-4.01 (m, 1H), 3.71 (d, J=8.0 Hz, 1H); LCMS: 99.3%, m/z [M−H]−=535.9.
To a stirred solution of 81.2_2b (200 mg, 0.37 mmol) in DCM (5 mL) was added TEA (0.3 mL, 2.14 mmol) followed by Methyltriflate (0.3 mL, 2.74 mmol) at 0° C. After stirring for 16 h at RT, the reaction mixture was quenched with ice cold water and extracted with EtOAc (2×20 mL). The combined organic layer was dried over Na2SO4, filtered and concentrated under vacuum pressure. The residue was purified by prep. HPLC [Column: INERTSIL-ODS 2 (250×19mm), 5 μ; A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient:(T%B) 0/55, 8/80, 11/90, 11.1/98, 13/98, 13.1/55, 17/55 at 18 mL/min] to afford 81.3 (14 mg, 7%) as a white solid.
1H NMR (500 MHz, DMSO-d6): 11.51 (br s, 1H), 7.80 (t, J=2.0 Hz, 1H), 7.59 (d, J=1.5 Hz, 1H), 7.40 (d, J=1.5 Hz, 2H), 7.10 (d, J=1.5 Hz, 1H), 4.57-4.54 (m, 1H), 4.11 (t, J=9.0 Hz, 1H), 3.78 (d, J=8.5 Hz, 1H), 2.13 (s, 3H); LCMS: 98.7%, m/z [M−H]−=549.9.
To a stirred solution of 81.3 (30 mg, 0.054 mmol) in THF (20 mL) was added 1% NaHCO3 solution (41 mL) at RT. After stirring for 16 h at RT, the pH of the solution was adjusted to ˜6-7 with 1N HCl solution and extracted with EtOAc (2×10 mL). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: SYMMETRY-C8 (300×19 mm),7 μ; A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient:(T%B): -0/65, 8/90, 10/90, 10.1/98, 12/98, 12.1/65, 16/65 at 18 mL/min] to afford 81 (6 mg, 19%) as a solid.
1H NMR (500 MHz, DMSO-d6): 12.61 (br s, 1H), 11.02 (br s, 1H), 10.70 (s, 1H), 8.22 (s, 1H), 7.67 (d, J=1.5 Hz, 2H), 7.39 (s, 1H), 7.27 (s, 1H), 4.13-4.03 (m, 1H), 3.81-3.70 (m, 1H), 3.62-3.55 (m, 1H), 2.05 (s, 3H); LCMS: 98.7%, m/z [M−H]−=567.9.
To a solution of (S)-pyrrolidine-2-carboxylic acid, 82.1, (10 g, 66.2 mmol) in MeCN (100 mL) was added (Z)-4-(allyloxy)-4-oxobut-2-enoic acid, 82.2, (10.3 g, 66.2 mmol) and 5,7-dichloroindoline-2,3-dione, 82.3, (14.3 g, 66.2 mmol) at RT. After refluxing for 2 h, the reaction mixture was cooled to RT. The resulting precipitate was filtered and washed with MeCN (2×20 mL) and dried under high vacuum to give 82.4_1 & 82.4_2 (LCMS ratio: 26:35).
82.4_1: rac-(1′R,2′S,3R,7a′R)-2′-((allyloxy)carbonyl)-5,7-dichloro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid. 1H NMR (500 MHz, DMSO-d6): 12.56 (br s, 1H), 11.00 (s, 1H), 7.77 (d, J=2.0 Hz, 1H), 7.46 (d, J=2.0 Hz, 1H), 5.50-5.45 (m, 1H), 5.10-5.05 (m, 2H), 4.26 (d, J=5.5 Hz, 2H), 4.06 (d, J=7.5 Hz, 1H), 3.97-3.96 (m, 1H), 3.55-3.52 (m, 1H), 2.64-2.62 (m, 1H), 2.26-2.23 (m, 1H), 1.91-1.78 (m, 3H), 1.53-1.49 (m, 1H); LCMS: 94.0%, m/z [M+H]+=425.0; Chiral purity: (49.7 +50.2)%. Regiochemistry and relative stereochemistry was confirmed by 2D NMR studies. 82.4_2: 1H NMR (500 MHz, DMSO-d6): 12.40 (br s, 1H), 10.95 (s, 1H), 7.56 (d, J=2.0 Hz, 1H), 7.51 (d, J=2.0 Hz, 1H), 5.86-5.80 (m, 1H), 5.27-5.15 (m, 2H), 4.45-4.39 (m, 2H), 4.27-4.25 (m, 1H), 3.64 (d, J=8.5 Hz, 1H), 3.47-3.44 (m, 1H), 2.51-2.48 (m, 1H), 2.42-2.35 (m, 1H), 2.10-2.00 (m, 1H), 1.90-1.80 (m, 1H), 1.73-1.71 (m, 2H); LCMS: 80.7%, m/z [M+H]+=425.0. Unknown relative regiochemistry.
82.4_1 (10 g) was separated by chiral SFC using Chiral pack IG (4.6×250) mm, 5 μ; 0.5% TFA in Isopropanol at RT (Isocratic 42.0 mL/min, 16 min run time with detection at 214 nm) to give 1.8 g of 82.4_la (Peak-1) as a white solid and 3.8 g of 82.4_1b (Peak-2) as a solid. (absolute stereochemistry of Enantiomer 1 & 2 not determined).
82.4_1a: 1H NMR (500 MHz, DMSO-d6): 12.55 (br s, 1H), 11.00 (s, 1H), 7.77 (d, J=2.0 Hz, 1H), 7.46 (d, J=2.0 Hz, 1H), 5.50-5.45 (m, 1H), 5.10-5.05 (m, 2H), 4.26 (d, J=5.5 Hz, 2H), 4.06 (d, J=8.0 Hz, 1H), 3.97-3.96 (m, 1H), 3.54-3.51 (m, 1H), 2.64-2.62 (m, 1H), 2.26-2.23 (m, 1H), 1.91-1.80 (m, 3H), 1.53-1.49 (m, 1H); LCMS: 99.0%, m/z [M+H]+=425.0; Chiral purity: 99.9%.
82.4_1b: 1H NMR (400 MHz, DMSO-d6): 12.58 (br s, 1H), 11.04 (s, 1H), 7.76 (s, 1H), 7.47 (d, J=1.6 Hz, 1H), 5.53-5.43 (m, 1H), 5.11-5.05 (m, 2H), 4.27 (d, J=5.2 Hz, 2H), 4.07 (d, J=7.6 Hz, 1H), 4.04-3.97 (m, 1H), 3.58-3.54 (m, 1H), 2.70-2.65 (m, 1H), 2.30-2.27 (m, 1H), 1.93-1.75 (m, 3H), 1.59-1.52 (m, 1H); LCMS: 97.2%, m/z [M+H]+=425.0; Chiral purity: 97.7%.
Thionyl chloride (6 mL) was added to 82.4_la (300 mg, 0.70 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. To the resulting acid chloride in CH2Cl2 (3 mL) was added a solution of 3,5-dichloro-N-neopentylaniline (245 mg, 1.05 mmol) in CH2Cl2 (2 mL). After stirring for 16 h at 55° C., the reaction mixture was quenched with water (20 mL) and extracted with CH2Cl2 (2×20 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (40 g Silica gel cartridge, 20% EtOAc/pet ether) to afford 82.5a (200 mg, 50%) as a solid.
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 10.97/10.92 (s, 1H), 8.22/7.70 (d, J=2.0 Hz, 1H), 7.64 (d, J=1.2 Hz, 1H), 7.54-7.40 (m, 3H), 5.52-5.43 (m, 1H), 5.21-5.08 (m, 2H), 4.34-4.28 (m, 1H), 4.22-4.15 (m, 2H), 3.83-3.77 (m, 1H), 3.71-3.58 (m, 3H), 2.70-2.64 (m, 1H), 2.20-2.14 (m, 1H), 1.97-1.83 (m, 1H), 1.81-1.73 (m, 2H), 1.57-1.50 (m, 1H), 0.89-0.78 (m, 9H); LCMS: 98.8%, m/z [M+H]+=638.0.
Synthesis of 82.6a
To a stirred solution of 82.5a (200 mg, 0.31 mmol) in THF (4 mL) were added aniline (30 mg, 0.31 mmol) and Pd(PPh3)4 (72 mg, 0.06 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [KROMOSIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time in minutes/%B): 0/70, 8/90, 11/95, 11.1/98, 13/98, 13.1/70, 15/70 at 20 mL/min] to afford 82.6a (58 mg, 37%) as a solid. 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.44 (br s, 1H), 10.90/10.84 (s, 1H), 8.37/7.86 (d, J=2.0 Hz, 1H), 7.62-7.61 (m, 1H), 7.53-7.38 (m, 3H), 4.08 (d, J=8.0 Hz, 1H), 3.99-3.97 (m, 1H), 3.69-3.65 (m, 1H), 3.52-3.45 (m, 2H), 2.72-2.68 (m, 1H), 2.13-2.12 (m, 1H), 1.91-1.87 (m, 1H), 1.77-1.70 (m, 2H), 1.52-1.48 (m, 1H), 0.83 (s, 9H); LCMS: 98.7%, m/z [M+H]+=598.0; Chiral purity: 98.0%.
Thionyl chloride (6 mL) was added to 82.4_1b (350 mg, 0.82 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. To the resulting acid chloride in CH2Cl2 (3 mL) was added a solution of 3,5-dichloro-N-neopentylaniline (286 mg, 1.23 mmol) in CH2Cl2 (2 mL). After stirring for 16 h at 55° C., the reaction mixture was quenched with water (20 mL) and extracted with CH2Cl2 (2×20 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (40 g Silica gel cartridge, 20% EtOAc/pet ether) to afford 82.5b (155 mg, 30%) as a solid.
1H NMR (400 MHz, CDCl3): 8.33 (d, J=2.0 Hz, 1H), 7.38 (br s, 1H), 7.33 (br s, 1H), 7.26-7.18 (m, 2H), 5.49-5.44 (m, 1H), 5.14-5.06 (m, 2H), 4.36-4.31 (m, 1H), 4.24-4.19 (m, 1H), 4.12 (d, J=8.0 Hz, 1H), 3.91-3.86 (m, 1H), 3.75-3.70 (m, 2H), 3.48-3.44 (m, 1H), 2.85-2.79 (m, 1H), 2.38-2.33 (m, 1H), 2.06-2.01 (m, 1H), 1.96-1.91 (m, 1H), 1.88-1.81 (m, 1H), 1.72-1.65 (m, 1H), 0.91 (m, 9H); LCMS: 98.1%, m/z [M+H]+=638.0.
To a stirred solution of 82.5b (155 mg, 0.24 mmol) in THF (4 mL) were added aniline (22 mg, 0.24 mmol) and Pd(PPh3)4 (56 mg, 0.05 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [KROMOSIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient: (Time in minutes/%B): 0/50, 8/90, 10/90, 10.1/98, 12/98, 12.1/50, 14/50 at 22 mL/min) to afford 82.6b (88 mg, 61%) as a solid. 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.50 (br s, 1H), 11.00/10.99 (s, 1H), 8.37/7.87 (d, J=2.0 Hz, 1H), 7.63-7.62 (m, 1H), 7.53-7.40 (m, 3H), 4.13 (d, J=8.0 Hz, 1H), 4.01-3.98 (m, 1H), 3.76-3.60 (m, 2H), 3.47-3.45 (m, 1H), 2.80-2.69 (m, 1H), 2.27-2.20 (m, 1H), 1.93-1.90 (m, 1H), 1.81-1.75 (m, 2H), 1.57-1.54 (m, 1H), 0.84 (s, 9H); LCMS: 97.4%, m/z [M+H]+=598.0; Chiral purity: 96.0%.
Using the listed anilines, the following compounds were made as in Example 82. Relative stereochemistry was assigned by 2D NMR studies. Absolute stereochemistry unknown for enantiomeric pairs (a and b).
To a stirred solution of 84a (110 mg, 0.17 mmol) in CH3CN (5 mL) was added Cs2CO3 (66 mg, 0.20 mmol) followed by Mel (72 mg, 0.50 mmol) and stirred for 6 h at RT. The reaction mixture was quenched with water (10 mL) and extracted with EtOAc (2×20 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford 85.1 (110 mg) as solid which was used in the next step without further purification. LCMS: 88.2%, m/z [M+H]+=666.0.
To a stirred solution of 85.1 (110 mg, 0.16 mmol) in THF (3 mL) were added aniline (15 mg, 0.16 mmol) and Pd(PPh3)4 (38 mg, 0.03 mmol) at RT. The resulting reaction mixture was stirred for 2 h at RT. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-BRIDGE-C18 (150×30), 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/75, 8/90, 10/95, 12/98, 12.1/75, 14/75 at 25 mL/min] to afford 85 (39 mg, 38%) as a solid. Absolute stereochemistry unknown for enantiomeric pairs (a and b).
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.40 (br s, 1H), 8.48/8.03 (d, J=2.0 Hz, 1H), 7.68/7.56 (t, J=2.0 Hz, 1H), 7.45/7.41 (d, J=2.5 Hz, 1H), 7.38/7.34 (d, J=1.5 Hz, 2H), 4.08 (d, J=8.0 Hz, 1H), 3.90-3.88 (m, 1H), 3.67-3.65 (m, 1H), 3.55-3.51 (m, 1H), 3.45/3.43 (s, 3H), 3.40-3.37 (m, 1H), 2.68-2.60 (m, 1H), 2.12-2.05 (m, 1H), 1.92-1.87 (m, 2H), 1.80-1.69 (m, 2H), 1.63-1.58 (m, 5H), 1.57-1.46 (m, 2H), 1.38-1.28 (m, 1H), 1.18-1.10 (m, 1H); LCMS: 98.1%, m/z [M+H]+=624.0; Chiral purity: 97.4%.
Compound 85b was prepared from 84b following the procedure described for Example 85a.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.39 (br s, 1H), 8.48/8.03 (d, J=2.5 Hz, 1H), 7.68/7.56 (t, J=2.0 Hz, 1H), 7.45/7.41 (d, J=2.0 Hz, 1H), 7.38-7.34 (m, 2H), 4.08 (d, J=8.0 Hz, 1H), 3.92-3.87 (m, 1H), 3.67-3.65 (m, 1H), 3.55-3.51 (m, 1H), 3.45/3.42 (s, 3H), 3.40-3.37 (m, 1H), 2.68-2.60 (m, 1H), 2.10-2.04 (m, 1H), 1.92-1.87 (m, 2H), 1.80-1.69 (m, 2H), 1.63-1.57 (m, 5H), 1.55-1.42 (m, 2H), 1.38-1.27 (m, 1H), 1.17-1.10 (m, 1H); LCMS:
99.6%, m/z [M+H]+=624.0; Chiral purity: 97.6%. Absolute stereochemistry unknown for enantiomeric pairs (a and b).
To a stirred solution of CH3MgBr (3M in Et2O, 728 mL, 2.18 mol) in dry THF (6 L) was added drop wise a solution of 90.1 (200 g, 872 mmol) in dry THF (2 L) through additional funnel over a period of 2 h at −40° C. After slowly warming to RT and stirring for 16 h at RT, the reaction mixture was cooled to −5° C., quenched with 1N HCl until the reaction mixture turned into a clear solution and then extracted with EtOAc (3×3 L). The combined organic layer was washed with H2O and dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography (Silica gel 100-200 mesh, 2% MeOH in DCM) to afford 90.2 (85 g, 40%) as an off white solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.50-12.30 (br s, 1H), 4.95-4.75 (br s, 1H), 4.14-4.08 (m, 1H), 3.26-3.18 (m, 2H), 2.15-2.08 (m, 1H), 1.95-1.91 (m, 1H), 1.39/1.34 (s, 9H), 1.21 (s, 3H); LCMS: 99.3%, m/z [M−H]−=244.1.
To a stirred solution of 90.2 (15 g, 61.1 mmol) in THF (150 ml) was added NaH (14.6 g, 367 mmol) at 0° C. After stiurring at 65° C. for 30 minutes, benzyl bromide (10.9 mL, 91.7 mmol) was added at 65° C. After stirring at 65° C. for 16 h, the reaction mixture was cooled to 0° C., quenched with 10% citric acid solution and extracted with EtOAc (3×100 mL). The combined organic layer was washed with H2O and dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by column chromatography (Silica gel 100-200 mesh, 30% EtOAc in pet ether) to afford 90.3 (12 g, 58%) as a light brown solid.
1H NMR (400 MHz, DMSO-d6): 12.44 (br s, 1H), 7.33-7.22 (m, 5H), 4.42-4.34 (m, 2H), 4.24-4.17 (m, 1H), 3.47 (d, J=11.2 Hz, 1H), 3.32-3.27 (m, 1H), 2.28-2.16 (m, 2H), 1.04-1.36 (m, 12 H); LCMS: 94.8%, m/z [M−H]−=334.1.
To a stirred solution of 90.3 (12 g, 35.7 mmol) in CH2Cl2 (240 mL) was added TFA (12 mL) at RT. After stirring for 16 h at RT, the reaction mixture was concentrated under reduced pressure to afford 90.4 (12 g) as a brown solid. LCMS: 86.2%, [M-TFA+H]+=236.1.
To a stirred solution of 90.4 (3.2 g, 13.6 mmol) in MTBE (100 mL) were added Et3N (1.9 mL, 13.6 mmol), 90.5 (2.12 g, 13.6 mmol) and 90.6 (2.93 g, 13.6 mmol) and at RT. After stirring at 80° C. for 16 h, the reaction mixture was cooled to RT and concentrated under reduced pressure. The residue was purified by column chromatography (Silica gel 100-200 mesh, 30% EtOAc in pet ether) to afford 90.7 (2.1 g, 30%) as a solid. The regio and relative stereochemistry of 90.7 was confirmed by 2D NMR analysis.
1H NMR (400 MHz, DMSO-d6): 12.65 (br s, 1H), 11.00 (s, 1H), 7.68 (s, 1H), 7.46 (s, 1H), 7.27-7.22 (m, 5H), 5.55-5.42 (m, 1H), 5.10-5.04 (m, 2H), 4.38-4.26 (m, 5H), 4.00 (d, J=7.6 Hz, 1H), 3.64-3.60 (m, 1H), 2.67 (d, J=9.6 Hz, 1H), 2.54-2.48 (m, 1H), 2.16-2.09 (m, 1H), 1.61-1.53 (m, 1H), 1.26 (s, 3H); LCMS: 80.2%, m/z [M+H]+=545.1.
SOCl2 (5 mL) was added to 90.7 (1.0 g, 1.83 mmol). After stirring for 2 h at RT, SOCl2 was evaporated under reduced pressure at 40° C. to afford acid chloride intermediate. To a solution of 90.8 (638 mg, 2.75 mmol) in CH2Cl2 (10 mL) was added the acid chloride intermediate at RT. After stirring at 50° C. for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (40 g Silica gel cartridge, 10% EtOAc in pet ether) to afford 90.9 (950 mg, 70%) as a yellow solid.
1H NMR (500 MHz, DMSO-d6): 10.95/10.91 (s, 1H), 8.14/7.69 (d, J=2.0 Hz, 1H), 7.64-7.63 (m, 1H), 7.54-7.42 (m, 3H), 7.33-7.21 (m, 5H), 5.67-5.45 (m, 1H), 5.12-5.10 (m, 2H), 4.37-4.30 (m, 4H), 4.24-4.23 (m, 1H), 4.11 (d, J=8.0 Hz, 1H), 3.95-3.88 (m, 1H), 3.74-3.66 (m, 2H), 2.73 (d, J=9.5 Hz, 1H), 2.50-2.46 (m, 1H), 2.15-2.05 (m, 1H), 1.69-1.59 (m, 1H), 1.28 (s, 3H), 0.84/0.82 (s, 9H); LCMS: 96.0%, m/z [M+H]+=760.6.
To a stirred solution of 90.9 (200 mg, 0.26 mmol) in THF (5 mL) were added aniline (29 mg, 0.32 mmol) and Pd(PPh3)4 (61 mg, 0.05 mmol) at RT. After stirring at RT for 2 h, the reaction mixture was diluted with EtOAc (15 mL). The organic solution was collected, washed with brine, dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by flash chromatography (24 g Silica gel cartridge, 20% EtOAc in pet ether) followed by trituration with diethyl ether to afford 90 (45 mg, 24%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.49 (br s, 1H), 10.90/10.88 (s, 1H), 8.31/7.85 (d, J=2.0 Hz, 1H), 7.61-7.40 (m, 4H), 7.32-7.20 (m, 5H), 4.39-4.29 (m, 2H), 4.05 (d, J=8.0 Hz, 1H), 3.96-3.89 (m, 2H), 3.61-3.59 (m, 1H), 3.51 (d, J=13.5 Hz, 1H), 2.74 (d, J=8.5 Hz, 1H), 2.43 (d, J=9.0 Hz, 1H), 2.03-2.00 (m, 1H), 1.62-1.58 (m, 1H), 1.26 (s, 3H), 0.87/0.85 (s, 9H); LCMS: 99.0%, m/z [M+H]+=718.0; Chiral Purity: 98.8%.
To a stirred solution 90 (150 mg, 0.21 mmol) in CH2Cl2 (3 mL) was added TFA (0.3 mL) and CF3SO3H (0.3 mL) at RT. After stirring for 16 h, the reaction mixture was concentrated under reduced pressure. The resulting residue was diluted with EtOAc (25 mL), washed with water (15 mL), brine (15 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep HPLC [Column: X-SELECT-C18 (150×19), 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/50, 8/90, 10/90, 10.1/98, 11/98, 11.1/50, 14/50 at 20 mL/minute] to afford 91 (45 mg, 38%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.38/12.32 (br s, 1H), 10.90/10.82 (br s, 1H), 8.30/7.79 (s, 1H), 7.62-7.39 (m, 4H), 4.65/4.54 (s, 1H), 4.17-3.80 (m, 3H), 3.62-3.49 (m, 2H), 2.63 (d, J=8.0 Hz, 1H), 2.20/2.10 (d, J=8.0 Hz, 1H), 1.71-1.69 (m, 1H), 1.55-1.51 (m, 1H), 1.15 (s, 3H), 0.83 (s, 9H); LCMS: 99.1%, m/z [M+H]+=628.0; Chiral Purity: 99.6%.
1H NMR (300 MHz, DMSO-d6) (Exist in rotameric form): 12.38 (br s, 1H), 10.95/10.85 (s, 1H), 8.35/7.87 (d, J = 1.5 Hz, 1H), 7.66/7.53 (t, J = 1.8 Hz, 1H), 7.45-7.42 (m, 3H), 7.31-7.19 (m, 5H), 4.39-4.28 (m, 2H), 4.18-3.90 (m, 2H), 3.57-3.52 (m, 1H), 3.40/3.24 (s, 3H), 2.71 (d, J = 9.3 Hz, 1H), 2.45 (d, J = 9.3 Hz, 1H), 2.07-2.01 (m, 1H), 1.63-1.56 (m, 1H), 1.28/1.24 (s, 3H).
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form) 12.37 (br s, 1H), 10.90/10.85 (s, 1H), 8.34/7.93 (d, J = 1.5 Hz, 1H), 7.69/7.59 (t, J = 2.0 Hz, 1H), 7.45-7.20 (m, 8H), 4.37-4.30 (m, 2H), 4.02 (d, J = 8.0 Hz, 1H), 3.98-3.90 (m, 1H), 3.75-3.68 (m, 1H), 3.52-3.48 (m, 1H), 3.45-3.42 (m, 1H), 2.71 (d, J = 9.0 Hz, 1H), 2.44 (d, J = 9.0 Hz, 1H), 2.10-2.02 (m, 1H), 1.68-1.64 (m, 1H), 1.28 (s, 3H), 0.98-0.84 (m, 1H), 0.43-0.41 (m, 2H), 0.17-0.09 (m, 2H).
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.38 (s, 1H), 10.90/10.85 (s, 1H), 8.33/7.90 (d, J = 2.0 Hz, 1H), 7.67/7.56 (m, 1H), 7.45-7.40 (m, 3H), 7.37-7.22 (m, 5H), 4.41-4.30 (m, 2H), 4.30 (d, J = 8.0 Hz, 1H), 3.93-3.89 (m, 2H), 3.56-3.52 (m, 1H), 3.47-3.46 (m, 1H), 2.71 (d, J = 9.0 Hz, 1H), 2.44 (d, J = 9.0 Hz, 1H), 2.07-1.99 (m, 1H), 1.93-1.90 (m, 1H), 1.66-1.58 (m, 5H), 1.49-1.45 (m, 2H), 1.38-1.30 (m, 1H), 1.27 (s, 3H), 1.20-1.11 (m, 1H).
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.37 (br s, 1H), 10.90/10.85 (s, 1H), 8.27/7.78 (s, 1H), 7.61-7.40 (m, 4H), 7.33-7.20 (m, 5H), 4.36-4.29 (m, 2H), 4.10 (d, J = 14.0 Hz, 1H), 3.99 (d, J = 7.6 Hz, 1H), 3.91-3.85 (m, 1H), 3.73-3.63 (m, 2H), 2.74 (d, J = 9.2 Hz, 1H), 2.44 (d, J = 9.2 Hz, 1H), 2.04-1.99 (m, 1H), 1.67-1.61 (m, 1H), 1.27 (s, 3H), 0.89 (s, 9H), 0.85 (s, 3H), 0.67 (s, 3H).
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.35 (br s, 1H), 10.90/10.82 (s, 1H), 8.29/7.73 (d, J = 2.0 Hz, 1H), 7.62 (t, J = 1.6 Hz, 1H), 7.52-7.49 (m, 2H), 7.43/7.39 (d, J = 2.0 Hz, 1H), 4.56 (s, 1H), 4.08-4.00 (m, 2H), 3.93-3.87 (m, 1H), 3.71 (d, J = 13.6 Hz, 1H), 3.59 (t, J = 7.6 Hz, 1H), 2.61 (d, J = 8.4 Hz, 1H), 2.12 (d, J = 8.4 Hz, 1H), 1.68-1.64 (m, 1H), 1.57-1.52 (m, 1H), 1.14/1.10 (s, 3H), 0.91/0.88 (s, 9H), 0.81 (s, 3H), 0.65 (s, 3H).
Using the listed anilines, the following compounds were made as in Example 90 or 91 with intermediate 90.7 and listed aniline.
SOCl2 (10 mL) was added to 90.7 (1.2 g, 2.20 mmol). After stirring for 2 h at RT, SOCl2 was evaporated under reduced pressure to afford acid chloride intermeidate. To a solution of 97.1 (805 mg, 3.30 mmol) in CH2Cl2 (15 mL) was added to the above prepared acid chloride intermediate at RT. After stirring at RT for 16 h, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (40 g Silica gel cartridge, 15% EtOAc in pet ether) to afford 97.2 (800 mg, 50%) as a solid.
1H NMR (400 MHz, CDCl3): 8.35 (d, J=1.6 Hz, 1H), 7.42 (s, 1H), 7.36 (t, J=1.6 Hz, 1H), 7.33-7.17 (m, 7H), 5.51-5.44 (m, 1H), 5.14-5.06 (m, 2H), 4.40-4.32 (m, 3H), 4.27-4.17 (m, 2H), 4.11 (d, J=8.0 Hz, 1H), 3.89-3.84 (m, 1H), 3.67-3.61 (m, 1H), 3.36 (t, J=8.0 Hz, 1H), 2.86 (d, J=9.2 Hz, 1H), 2.64 (d, J=8.8 Hz, 1H), 2.10-2.00 (m, 2H), 1.80-1.66 (m, 5H), 1.39 (s, 3H), 1.29-1.24 (m, 3H), 0.93-0.83 (m, 1H); LCMS: 97.3%, m/z [M+H]+=772.49.
To a stirred solution of 97.2 (300 mg, 0.39 mmol) in CH3CN (5 mL) were added K2CO3 (80 mg, 0.58 mmol) and CH3I (0.05 mL, 0.78 mmol) at RT. After stirring at RT for 5 h, the reaction mixture was diluted with EtOAc (15 mL). The organic solution was washed with water (20 mL), brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (24 g Silica gel cartridge, 5% EtOAc in pet ether) to afford 97.3 (220 mg, 72%) as a solid.
1H NMR (400 MHz, CDCl3): 8.37 (d, J=2.0 Hz, 1H), 7.37-7.16 (m, 9H), 5.52-5.48 (m, 1H), 5.17-5.07 (m, 2H), 4.41-4.15 (m, 5H), 4.06 (d, J=8.0 Hz, 1H), 3.89-3.84 (m, 1H), 3.65-3.60 (m, 1H), 3.51 (s, 3H), 3.39-3.35 (m, 1H), 2.78 (d, J=8.8 Hz, 1H), 2.56 (d, J=8.8 4 Hz, 1H), 2.06-2.02 (m, 2H), 1.76-1.53 (m, 6H), 1.39 (s, 3H), 1.28-1.24 (m, 3H); LCMS: 94.4%, m/z [M+H]+=786.4.
To a stirred solution of 97.3 (220 mg, 0.28 mmol) in THF (3 mL) were added aniline (26 mg, 0.28 mmol) and Pd(PPh3)4 (64 mg, 0.06 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under pressure. The residue was purified by prep. HPLC [Column: KROMOSIL-C18 (150×25) mm, 10 μ, A: 0.1% Formic acid in H2O, B: Acetontrile; Gradient: (Time/%B): 0/80, 8/95, 11/95, 11/98, 12.1/98, 12.1/80, 15/80 at 25 mL/minute] to afford 97 (58 mg, 27%) as a solid.
1H NMR (500 MHz, DMSO-d6) (exist in rotameric form): 12.43 (s, 1H), 8.42/8.00 (d, J=2.5 Hz, 1H), 7.67/7.56 (t, J=2.0 Hz, 1H), 7.47 (d, J=2.0 Hz, 1H), 7.43-7.41 (m, 2H), 7.37-7.23 (m, 5H), 4.36-4.29 (m, 2H), 4.04 (d, J=8.0 Hz, 1H), 3.92-3.88 (m, 2H), 3.57-3.53 (m, 1H), 3.50-3.45 (m, 1H), 3.45/3.41 (s, 3H), 2.67 (d, J=9.0 Hz, 1H), 2.37 (d, J=9.0 Hz, 1H), 2.05-1.95 (m, 1H), 1.95-1.87 (m, 1H), 1.65-1.58 (m, 5H), 1.48-1.44 (m, 2H), 1.35-1.28 (m, 1H), 1.28 (s, 3H), 1.19-1.12 (m, 1H); LCMS: 99.1%, m/z [M+H]+=743.9; Chiral Purity: 99.8%.
To a solution of 100.2 (500 mg, 3.18 mmol) in EtOH (10 mL) was added 100.1 (789 mg, 3.18 mmol) and 100.3 (680 mg, 3.18 mmol) at RT. After refluxing for 2 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (80 g Silica gel cartridge, 30%-35% EtOAc/pet ether) to afford minor diastereomer 100.4a (150 mg, 10%) as a white solid and major diastereomer 100.4b (250 mg, 17%) as a white solid.
100.4a: 1H NMR (500 MHz, DMSO-d6): 12.65 (s, 1H), 11.13 (s, 1H), 7.54 (d, J=8.0 Hz, 1H), 7.28 (d, J=8.0 Hz, 1H), 5.86-5.80 (m, 1H), 5.27-5.15 (m, 2H), 4.47-4.39 (m, 3H), 3.73-3.68 (m, 2H), 3.10-2.95 (m, 1H), 2.88-2.76 (m, 1H), 2.62-2.40 (m, 2H); LCMS: 88.6%, m/z [M+H]+=461.0. Regiochemistry and relative stereochemistry was confirmed by 2D NMR studies.
100.4b: 1H NMR (400 MHz, DMSO-d6): 12.80 (s, 1H), 11.17 (s, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.25 (d, J=8.4 Hz, 1H), 5.53-5.44 (m, 1H), 5.11-5.04 (m, 2H), 4.34-4.22 (m, 2H), 4.15-4.09 (m, 1H), 3.92 (d, J=7.6 Hz, 1H), 3.76-3.72 (m, 1H), 3.32-3.24 (m, 1H), 2.75-2.67 (m, 1H), 2.51-2.40 (m, 1H), 2.35-2.15 (m, 1H); LCMS: 89.1%, m/z [M−H]−=459.0. Regiochemistry and relative stereochemistry was confirmed by 2D NMR studies.
Thionyl chloride (3 mL) was added to 100.4b (250 mg, 0.54 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure to afford residue of acid chloride. To the acid chloride in CH2Cl2 (5 mL) was added a solution of 3,5-dichloro-N-methylaniline (144 mg, 0.81 mmol) in CH2Cl2 (5 mL). After stirring for 16 h at RT, the reaction was quenched with water (10 mL). The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 (2×15 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC (X-BRIDGE-C18 (150×30) mm, 5u; A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient: (T%B):- 0/70, 8/85, 11/85, 11.1/98, 12/98, 12.1/70 ,15/70 at 20 mL/min) to afford 100.5b (80 mg, 24%) as a solid. LCMS: 96.6%, m/z [M+H]+=618.1.
To a stirred solution of 100.5b (80 mg, 0.13 mmol) in THF (2 mL) were added aniline (10 mg, 0.13 mmol) and Pd(PPh3)4 (29 mg, 0.03 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by prep. HPLC [ATLANTIS-T3 (250×20) mm, 5 μ; A: 10 mM Ammonium bicarbonate in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/55, 8/80, 11/90, 11.1/98, 13/98, 13.1/55, 16/55 at 18 mL/min] to afford 100.6b (25 mg, 34%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.44 (br s, 1H), 11.10/11.01 (s, 1H), 8.20/7.80 (d, J=8.0 Hz, 1H), 7.66-7.20 (m, 4H), 4.25-4.07 (m, 1H), 3.99 (d, J=7.5 Hz, 1H), 3.85-3.82 (m, 1H), 3.55 (t, J=7.5 Hz, 1H), 3.39/3.23 (s, 3H), 2.60-2.50 (m, 1H), 2.42-2.35 (m, 1H), 2.25-2.05 (m, 1H); LCMS: 95.2%, m/z [M+H]+=578.0; Chiral purity: (49.1+48.6)%.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.27 (br s, 1H), 10.40/10.34 (s, 1H), 8.07/7.74 (d, J = 7.5 Hz, 1H), 7.65-7.42 (m, 3H), 7.23-7.20 (m, 1H), 7.01-6.79 (m, 2H), 4.28-4.20/4.00-3.96 (m, 1H), 3.82-3.64 (m, 2H), 3.55-3.48 (m, 1H), 3.39/3.23 (s, 3H), 2.50-2.44 (m, 1H), 2.40-2.20 (m, 2H).
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.30 (br s, 1H), 10.25/10.17 (br s, 1H), 7.83-7.44 (m, 4H), 6.89-6.46 (m, 2H), 4.25-4.00 (m, 1H), 3.85-3.59 (m, 2H), 3.69/3.64 (s, 3H), 3.52-3.44 (m, 1H), 3.39/3.23 (s, 3H), 2.60-2.50 (m, 1H), 2.40-2.15 (m, 2H).
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.37 (br s, 1H), 10.86/10.78 (s, 1H), 8.15/7.77 (d, J = 7.5 Hz, 1H), 7.66 (t, J = 1.5 Hz, 1H), 7.55-7.39 (m, 2H), 7.31-7.26 (m, 1H), 7.04-6.91 (m, 1H), 4.21-4.06 (m, 1H), 3.94 (d, J = 8.0 Hz, 1H), 3.85-3.81 (m, 1H), 3.58 (t, J = 7.0 Hz, 1H), 3.45-3.37 (m, 1H), 3.39/3.23 (s, 3H), 2.40-2.33 (m, 1H), 2.24-2.17 (m, 1H).
Using various isatins in place of 6,7-dichloroindoline-2,3-dione, 100.3, as in Example 100, the following compounds were made.
To a solution of (S)-4,4-difluoropyrrolidine-2-carboxylic acid, 110.1, (10 g, 66.2 mmol) in MTBE (200 mL) were added (Z)-4-(allyloxy)-4-oxobut-2-enoic acid, 110.2, (10.3 g, 66.2 mmol) and 5,7-dichloroindoline-2,3-dione, 110.3, (14.3 g, 66.2 mmol) at RT. After stirring at reflux for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (silica-gel, 100-200 mesh, gradient 20%-25% EtOAc/pet ether) to give 110.4_1, 110.4_2, 110.4_3 & 110.4_4. (LCMS ratio: 25:15:6:5).
110.4_1: rac-(1′R,2′S,3R,7a′R)-2′-((allyloxy)carbonyl)-5,7-dichloro-6′,6′-difluoro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 1H NMR (500 MHz, DMSO-d6): 12.89 (br s, 1H), 11.15 (s, 1H), 7.69 (d, J=2.0 Hz, 1H), 7.50 (d, J=2.0 Hz, 1H), 5.47-5.44 (m, 1H), 5.11-5.06 (m, 2H), 4.28-4.27 (m, 2H), 4.07-4.02 (m, 2H), 3.64 (t, J=7.0 Hz, 1H), 3.23-3.15 (m, 1H), 2.75-2.65 (m, 1H), 2.51-2.49 (m, 1H), 2.19-2.08 (m, 1H), 19F NMR (376.49 MHz, DMSO-d6): −89.57 (d, J=226 Hz), −94.10 (d, J=226 Hz); LCMS 98.0%, m/z [M+H]+=461.0. Relative regiochemistry was confirmed by 2D NMR studies.
110.4_2: rac-(1′R,2′R,3R,7a′R)-2′-((allyloxy)carbonyl)-5,7-dichloro-6′,6′-difluoro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid 1H NMR (400 MHz, DMSO-d6): 8.96 (br s, 1H), 7.62 (d, J=2.0 Hz, 1H), 7.54 (d, J=2.0 Hz, 1H), 5.85-5.79 (m, 1H), 5.30-5.13 (m, 2H), 4.48-4.35 (m, 3H), 3.56-3.53 (m, 2H), 3.00-2.79 (m, 2H), 2.51-2.40 (m, 2H); 19F NMR (376.49 MHz, DMSO-d6): −92.55 (d, J=228 Hz), −100.21 (d, J=228 Hz); LCMS 97.8%, m/z [M+H]+=461.0. Relative regiochemistry was confirmed by 2D NMR studies.
110.4_3: 1H NMR (400 MHz, DMSO-d6): 12.75 (br s, 1H), 11.09 (s, 1H), 7.77 (d, J=2.0 Hz, 1H), 7.48 (d, J=2.0 Hz, 1H), 6.01-5.91 (m, 1H), 5.42-5.37 (m, 1H), 5.26 (dd, J=10.4 Hz, 1.6 Hz, 1H), 4.71-4.61 (m, 2H), 4.06-3.94 (m, 2H), 3.62 (t, J=6.4 Hz, 1H), 3.18-3.09 (m, 1H), 2.70-2.61 (m, 1H), 2.49-2.32 (m, 1H), 2.09-1.98 (m, 1H); 19F NMR (376.49 MHz, DMSO-d6): −89.13 (d, J=231 Hz), −92.96 (d, J=231 Hz); LCMS 99.6%, m/z [M+H]+=460.9. Unknown relative regiochemistry.
110.4_4: 1H NMR (400 MHz, DMSO-d6): 12.87 (br s, 1H), 11.42 (s, 1H), 7.54 (d, J=2.0 Hz, 1H), 6.87 (d, J=2.0 Hz, 1H), 5.76-5.66 (m, 1H), 5.23-5.16 (m, 2H), 4.51-4.35 (m, 3H), 3.87 (t, J=8.0 Hz, 1H), 3.58 (d, J=7.2 Hz, 1H), 3.08-3.01 (m, 1H), 2.89-2.67 (m, 1H), 2.58-2.32 (m, 2H); 19F NMR (376.49 MHz, DMSO-d6): −92.33 (d, J=230 Hz), −101.91 (d, J=230 Hz); LCMS 95.2%, m/z [M+H]+=461.0. Unknown relative regiochemistry.
110.4_1 (45 g) was purified by chiral SFC using Chiral pack IG (250×30) mm, 5 μ; 0.2% TFA in n-hexane: Isopropanol (85:15) at rt (isocratic 42.0 mL/min, 13 min run time with detection at 254 nm). Pure fractions were concentrated under reduced pressure to give 20 g of 110.4_1a (Peak-1) and 14.7 g of 110.4_1b (Peak-2) as white solids.
110.4_1a: (1′R,2′S,3R,7a′R)-2′-((allyloxy)carbonyl)-5,7-dichloro-6′,6′-difluoro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid. [α]25D+78.8 (c 1.0, MeOH); 1H NMR (500 MHz, DMSO-d6): 12.86 (s, 1H), 11.16 (s, 1H), 7.68 (d, J=2.0 Hz, 1H), 7.51 (d, J=2.0 Hz, 1H), 5.49-5.43 (m, 1H), 5.11-5.06 (m, 2H), 4.28-4.27 (m, 2H), 4.07-4.02 (m, 2H), 3.64 (t, J=6.5 Hz, 1H), 3.21-3.18 (m, 1H), 2.69-2.52 (m, 1H), 2.51-2.50 (m, 1H), 2.15-2.05 (m, 1H); 19F NMR (376.49 MHz, DMSO-d6): −89.56 (d, J=226 Hz), −94.08 (d, J=226 Hz); LCMS 98.9%, m/z [M+H]+=461.2; Chiral purity: 99.8%. Absolute stereochemistry was determined by single crystal x-ray diffraction analysis.
110.4_1b: (1′S,2′R,3S,7a′S)-2′-((allyloxy)carbonyl)-5,7-dichloro-6′,6′-difluoro-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro[indoline-3,3′-pyrrolizine]-1′-carboxylic acid [α]25D−73.2 (c 1.0, MeOH); 1H NMR (400 MHz, DMSO-d6): 12.88 (br s, 1H), 11.16 (s, 1H), 7.69 (d, J=2.0 Hz, 1H), 7.50 (d, J=2.0 Hz, 1H), 5.50-5.42 (m, 1H), 5.11-5.06 (m, 2H), 4.28-4.26 (m, 2H), 4.09-4.01 (m, 2H), 3.64 (t, J=7.2 Hz, 1H), 3.23-3.14 (m, 1H), 2.74-2.65 (m, 1H), 2.53-2.44 (m, 1H), 2.16-2.08 (m, 1H); 19F NMR (376.49 MHz, DMSO-d6): −89.56 (d, J=226 Hz), −94.08 (d, J=226 Hz); LCMS 98.7%, m/z [M+H]+=461.0; Chiral purity: 99.9%.
Thionyl chloride (8 mL) was added to 110.4_1a (500 mg, 1.08 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. To the resulting residue dissolved in CH2Cl2(10 mL) was a solution of 3,5-dichloro-N-neopentylaniline (500 mg, 2.16 mmol) in CH2Cl2 (5 mL). After stirring for 16 h at 55° C., the reaction mixture was quenched with water (20 mL) and extracted with CH2Cl2 (2×20 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (40 g silica gel cartridge, gradient 10% EtOAc/pet ether) to afford 110.5a (410 mg, 53%) as a solid.
1H NMR (400 MHz, CDCl3): 8.28 (d, J=2.0 Hz, 1H), 7.43 (s, 1H), 7.34 (t, J=1.6 Hz, 1H), 7.27-7.26 (m, 2H), 5.48-5.42 (m, 1H), 5.14-5.06 (m, 2H), 4.36-4.31 (m, 1H), 4.24-4.19 (m, 1H), 4.04-3.97 (m, 2H), 3.81-3.68 (m, 2H), 3.51 (t, J=7.6 Hz, 1H), 3.33-3.26 (m, 1H), 2.77-2.69 (m, 1H), 2.31-2.17 (m, 2H), 0.91 (s, 9H); LCMS 99.3%, m/z [M+H]+=674.0.
To a stirred solution of 110.5a (400 mg, 0.59 mmol) in THF (10 mL) were added aniline (55 mg, 0.59 mmol) and Pd(PPh3)4 (136 mg, 0.11 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (KROMOSIL-C18 (150×30) mm, 10 μ; A: 0.1% Formic acid in H2O; B: acetonitrile; gradient: (Time in minutes/%B): 0/70, 8/90, 10/95 at 24 mL/min) to afford 110 (161 mg, 42%) as a solid.
[α]25D −6.41 (c 0.5, MeOH); 1H NMR (500 MHz, DMSO-d6): 12.65 (br s, 1H), 10.98 (s, 1H), 8.26 (d, J=2.0 Hz, 1H), 7.81-7.43 (m, 4H), 4.05 (d, J=7.5 Hz, 1H), 4.01 (d, J=14 Hz, 1H), 3.84-3.69 (m, 1H), 3.60 (dd, J=7.5 Hz, J=6.5 Hz, 1H), 3.44 (d, J=14.0 Hz, 1H), 3.32-3.24 (m, 1H), 2.63-2.52 (m, 1H), 2.40-2.32 (m, 1H), 2.11-2.04 (m, 1H), 0.84/0.82 (s, 9H); 19F NMR (470.59 MHz, DMSO-d6): −89.21 (d, J=226 Hz), −97.17 (d, J=226 Hz); LCMS 99.3%, m/z [M+H]+=633.9; Chiral purity: 98.9%.
Thionyl chloride (5 mL) was added to 110.4_1b (300 mg, 0.65 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure. To the resulting residue dissolved in CH2Cl2(3 mL) was a solution of 3,5-dichloro-N-neopentylaniline (217 mg, 0.93 mmol) in CH2Cl2 (2 mL). After stirring for 16 h at 55° C., the reaction mixture was quenched with water (10 mL) and extracted with CH2Cl2 (2×20 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (40 g silica gel cartridge, gradient 15% EtOAc/pet ether) to afford 111.1b (125 mg, 30%) as a solid.
1H NMR (400 MHz, DMSO-d6): 11.10/11.04 (s, 1H), 8.13 (d, J=2.0 Hz, 1H), 7.71-7.44 (m, 4H), 5.46-5.36 (m, 1H), 5.18-5.05 (m, 2H), 4.28-4.19 (m, 2H), 4.14 (d, J=7.6 Hz, 1H), 3.87-3.78 (m, 2H), 3.70 (t, J=7.2 Hz, 1H), 3.61 (d, J=14.0 Hz, 1H), 3.28-3.26 (m, 1H), 2.67-2.57 (m, 1H), 2.44-2.38 (m, 1H), 2.16-2.07 (m, 1H), 0.84 (s, 9H); LCMS 94.5%, m/z [M+H]+=674.0.
To a stirred solution of 111.1b (125 mg, 0.18 mmol) in THF (3 mL) were added aniline (17 mg, 0.18 mmol) and by Pd(PPh3)4 (42 mg, 0.03 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep-HPLC (XSELECT-C18 (150×30), 5 μ; A: 0.1% Formic Acid in H2O; B: acetonitrile; Gradient: (Time in minutes/%B): 0/70, 8/95, 12/98 at 24 mL/min) to afford 111 (40 mg, 34%) as a solid.
[α]25D+9.70 (c 0.5, MeOH); 1H NMR (500 MHz, DMSO-d6): 12.69 (br s, 1H), 11.05/10.99 (s, 1H), 8.27 (d, J=2.0 Hz, 1H), 7.82-7.43 (m, 4H), 4.05 (d, J=7.5 Hz, 1H), 4.01 (d, J=14 Hz, 1H), 3.84-3.80 (m, 1H), 3.60 (dd, J=7.5 Hz, J=6.5 Hz, 1H), 3.45 (d, J=14.0 Hz, 1H), 3.29-3.27 (m, 1H), 2.60-2.54 (m, 1H), 2.38-2.34 (m, 1H), 2.10-2.05 (m, 1H), 0.85/0.82 (s, 9H); 19F NMR (376.49 MHz, DMSO-d6): −89.21 (d, J=226 Hz), −97.18 (d, J=226 Hz); LCMS 95.9%, m/z [M+H]+=634.0; Chiral purity: 97.6%.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.54/12.50 (br s, 1H), 11.05/10.99 (s, 1H), 8.30/7.92 (d, J = 2.0 Hz, 1H), 7.68-7.40 (m, 4H), 4.00-3.95 (m, 2H), 3.81-3.79 (m, 1H), 3.48-3.42 (m, 2H), 3.36-3.25 (m, 1H), 2.64-2.55 (m, 1H), 2.50-2.34 (m, 1H), 2.20-2.05 (m, 1H), 1.49-1.47 (m, 1H), 1.36-1.32 (m, 1H), 0.88/0.87 (s, 9H).
1H NMR(500 MHz, DMSO-d6) (Exist in rotameric form): 12.55 (br s, 1H), 11.01/10.99 (s, 1H), 8.30/7.95 (d, J = 2.0 Hz, 1H), 7.79-7.73 (m, 2H), 7.70-7.61 (m, 2H), 7.49/7.43 (d, J = 2.0 Hz, 1H), 3.91 (d, J = 7.5 Hz, 1H), 3.79-3.76 (m, 2H), 3.53-3.45 (m, 1H), 3.32-3.24 (m, 2H), 2.61-2.55 (m, 1H), 2.45-2.30 (m, 1H), 2.29-2.15 (m, 1H), 0.90-0.87 (m, 1H), 0.41-0.38 (m, 2H), 0.15-0.12 (m, 1H), 0.03-0.01 (m, 1H).
1H NMR(500 MHz, DMSO-d6) (Exist in rotameric form): 12.52 (br s, 1H), 10.99 (br s, 1H), 8.33 (d, J = 2.0 Hz, 1H), 7.59-7.48 (m, 2H), 7.33/6.65 (d, J = 7.5 Hz, 1H), 7.12/6.58 (m, 1H), 5.85/5.15 (m, 1H), 3.91-3.86 (m, 2H), 3.75-3.42 (m, 3H), 3.32-3.25 (m, 1H), 2.58-2.56 (m, 1H), 2.44-2.32 (m, 1H), 2.31-2.18 (m, 1H), 1.58 (s, 3H), 1.56 (s, 3H), 0.93-0.91 (m, 1H), 0.43-0.39 (m, 2H), 0.20-0.15 (m, 1H), 0.07-0.02 (m, 1H).
1H NMR(500 MHz, DMSO-d6) (Exist in rotameric form): 12.58 (br s, 1H), 11.05/10.99 (s, 1H), 8.28 (d, J = 2.0 Hz, 1H), 7.68-7.44 (m, 4H), 4.25-4.17 (m, 1H), 4.03 (d, J = 7.5 Hz, 1H), 3.81-3.77 (m, 1H), 3.66-3.62 (m, 2H), 3.29-3.25 (m, 1H), 2.60-2.55 (m, 1H), 2.36-2.31 (m, 1H), 2.12-2.07 (m, 1H), 0.89 (s, 9H), 0.87 (s, 3H), 0.64 (s, 3H).
1H NMR(500 MHz, DMSO-d6) (Exist in rotameric form): 12.55 (br s, 1H), 11.05/10.99 (s, 1H), 8.30/7.91 (d, J = 1.5 Hz, 1H), 7.71 (t, J = 2.0 Hz, 1H), 7.49 (d, J = 2.0 Hz, 1H), 7.44-7.33 (m, 2H), 4.13-4.09 (m, 1H), 4.01 (d, J = 7.5 Hz, 1H), 3.84-3.80 (m, 1H), 3.48-3.42 (m, 2H), 3.30-3.26 (m, 1871871H), 2.62-2.57 (m, 1H), 2.49-2.36 (m, 1H), 2.20-2.05 (m, 1H), 0.98 (s, 3H), 0.92 (s, 3H), 0.77 (s, 3H), 0.54 (s, 3H), 0.32-0.30 (m, 1H).
1H NMR(500 MHz, DMSO-d6) (Exist in rotameric form): 12.60 (br s, 1H), 11.00/10.99 (s, 1H), 8.30 (dd, J = 8.5 Hz, J = 2.0 Hz, 1H), 7.71-7.69 (m, 1H), 7.49 (s, 1H), 7.44-7.34 (m, 2H), 4.36-4.12 (m, 1H), 4.02-4.00 (m, 1H), 3.83-3.74 (m, 2H), 3.49-3.40 (m, 1H), 3.30-3.15 (m, 1H), 2.66-2.50 (m, 1H), 2.48-2.35 (m, 1H), 2.20-2.06 (m, 1H), 1.32-1.15 (m, 1H), 0.93-0.89 (m, 1H), 0.69-0.49 (m, 5H).
1H NMR(500 MHz, DMSO-d6) (Exist in rotameric form): 12.67/12.55 (br s, 1H), 11.06/11.00 (br s, 1H), 8.28/7.85 (d, J = 2.0 Hz, 1H), 7.66/7.58 (t, J = 1.5 Hz, 1H), 7.53-7.45 (m, 3H), 4.42 (d, J = 14.5 Hz, 1H), 4.07 (d, J = 7.5 Hz, 1H), 3.85-3.83 (m, 1H), 3.72 (d, J = 14.5 Hz, 1H), 3.50-3.47 (m, 1H), 3.37-3.32 (m, 1H), 2.60-2.51 (m, 1H), 2.40-2.36 (m, 1H), 2.18-2.03 (m, 1H), 2.02-1.90 (m, 6H).
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.50 (br s, 1H), 11.00/10.99 (s, 1H), 8.31 (d, J = 2.0 Hz, 1H), 7.54-7.32 (m, 4H), 3.91-3.88 (m, 1H), 3.76-3.65 (m, 2H), 3.58-3.45 (m, 1H), 3.40-3.22 (m, 2H), 2.61-2.50 (m, 1H), 2.42-2.17 (m, 2H), 1.39-1.33 (m, 2H), 1.22-1.12 (m, 2H), 0.94-0.88 (m, 1H), 0.43-0.38 (m, 2H), 0.17-0.07 (m, 2H).
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.22 (br s, 1H), 10.99 (br s, 1H), 8.21/8.09 (d, J = 2.0 Hz, 1H), 7.44-7.43 (m, 1H), 4.09-3.96 (m, 3H), 3.23-3.00 (m, 3H), 3.06/2.92 (s, 3H), 2.62-2.54 (m, 1H), 2.50-2.43 (m, 1H), 2.11-2.00 (m, 1H), 1.96-1.90 (m, 3H), 1.75-1.50 (m, 12H).
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.55 (br s, 1H), 11.05/10.98 (s, 1H), 8.29/7.90 (d, J = 2.0 Hz, 1H), 7.68/7.59 (t, J = 2.0 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.44-7.38 (m, 2H), 4.14/4.01 (d, J = 7.6 Hz, 1H), 3.95-3.88 (m, 1H), 3.84-3.79 (m, 1H), 3.54-3.44 (m, 2H), 3.32-3.28 (m, 1H), 2.61-2.50 (m, 1H), 2.44-2.34 (m, 1H), 2.15-2.07 (m, 1H), 1.47-1.36 (m, 2H), 1.28-1.22 (m, 6H), 0.87-0.82 (m, 3H).
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.53 (br s, 1H), 11.05/10.98 (s, 1H), 8.29/7.90 (d, J = 2.0 Hz, 1H), 7.68/7.59 (t, J = 2.0 Hz, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.44-7.38 (m, 2H), 4.14/4.00 (d, J = 7.2 Hz, 1H), 3.92-3.79 (m, 2H), 3.56-3.44 (m, 2H), 3.32-3.23 (m, 1H), 2.68-2.50 (m, 1H), 2.43-2.32 (m, 1H), 2.15-2.07 (m, 1H), 1.46-1.18 (m, 12H), 0.86-0.83 (m, 3H).
1H NMR(500 MHz, DMSO-d6) (Exist in rotameric form): 12.55 (br s, 1H), 11.02/10.99 (s, 1H), 8.29/7.90 (d, J = 2.0 Hz, 1H), 7.68/7.59 (br s, 1H), 7.49-7.38 (m, 3H), 4.12/4.01 (d, J = 7.5 Hz, 1H), 3.89-3.79 (m, 2H), 3.55-3.37 (m, 3H), 2.64-2.50 (m, 1H), 2.42-2.35 (m, 1H), 2.14-2.08 (m, 1H), 1.50-1.23 (m, 20H), 0.88-0.83 (m, 3H).
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.63 (br s, 1H), 11.02/10.99 (s, 1H), 8.25/7.81 (d, J = 2.0 Hz, 1H), 7.59-7.43 (m, 4H), 4.05 (d, J = 7.6 Hz, 1H), 3.88 (d, J = 14.4 Hz, 1H), 3.84-3.78 (m, 1H), 3.60 (t, J = 6.8 Hz, 1H), 3.37-3.27 (m, 2H), 2.60-2.50 (m, 1H), 2.38-2.30 (m, 1H), 2.13-2.04 (m, 1H), 1.90-1.82 (m, 3H), 1.63-1.47 (m, 6H), 1.50-1.40 (m, 6H).
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.60 (s, 1H), 11.05/10.98 (s, 1H), 8.30/7.91 (d, J = 1.6 Hz, 1H), 7.66/7.56 (br s, 1H), 7.48-7.42 (m, 3H), 4.42-4.37 (m, 1H), 4.02 (d, J = 7.6 Hz, 1H), 3.86-3.80 (m, 1H), 3.53-3.48 (m, 1H), 3.43-3.45 (m, 2H), 2.60-2.50 (m, 1H), 2.36-2.33 (m, 1H), 2.17-2.14 (m, 1H), 1.99-1.93 (m, 1H), 1.88-1.51 (m, 12H), 1.50-1.45 (m, 2H).
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.51 (br s, 1H), 10.97 (br s, 1H), 8.30/7.82 (d, J = 1.6 Hz, 1H), 7.51-7.35 (m, 5H), 4.20-4.00 (m, 1H), 4.04-3.95 (m, 1H), 3.78-3.73 (m, 2H), 3.63-3.53 (m, 1H), 3.26-3.22 (m, 1H), 2.60-2.50 (m, 1H), 2.45-2.28 (m, 1H), 2.21-2.07 (m, 1H), 0.89 (s, 9H), 0.84 (s, 3H), 0.66 (s, 3H).
1H NMR (400 MHz, DMSO-d6): 12.41 (br s, 1H), 10.96 (br s, 1H), 8.34 (br s, 1H), 7.46-7.33 (m, 6H), 4.09-4.01 (m, 1H), 3.87-3.69 (m, 3H), 3.61-3.54 (m, 1H), 3.27-3.17 (m, 1H), 2.61-2.50 (m, 1H), 2.42-2.31 (m, 1H), 2.24-2.07 (m, 1H), 0.88 (s, 9H), 0.82 (s, 3H), 0.68 (s, 3H).
1H NMR (400 MHz, DMSO-d6): 12.40 (br s, 1H), 10.96 (s, 1H), 8.33 (d, J = 2.0 Hz, 1H), 7.47 (d, J = 2.0 Hz, 1H), 7.07-6.96 (m, 3H), 4.03 (d, J = 14.0 Hz, 1H), 3.87 (d, J = 7.6 Hz, 1H), 3.74-3.62 (m, 3H), 3.28-3.19 (m, 1H), 2.67-2.50 (m, 1H), 2.41-2.33 (m, 1H), 2.30 (br s, 6H), 2.24-2.07 (m, 1H), 0.88 (s, 9H), 0.82 (s, 3H), 0.70 (s, 3H).
1H NMR (400 MHz, DMSO-d6): 12.60 (br s, 1H), 11.10 (br s, 1H), 7.77 (br s, 1H), 7.53 (s, 1H), 7.28 (s, 1H), 4.18 (br s, 2H), 3.92-3.86 (m, 1H), 3.87 (s, 3H), 2.89-2.73 (m, 3H), 2.68-2.55 (m, 1H), 1.34 (s, 9H).
Using either 110.4_1a or 110.4_1b and following the amide coupling procedures (as used to make 110.5a or 111.1b) and deprotection (110.6a or 111.2b), the following examples were made.
Thionyl chloride (6 mL) was added to 110.4_1a (300 mg, 0.65 mmol) at RT and stirred for 2 h. The excess thionyl chloride was concentrated under reduced pressure to afford the acid chloride derivative. To this acid chloride in CH2Cl2 (5 mL) was added a solution of 3,5-dichloro-N-methylaniline (228 mg, 1.3 mmol) in CH2Cl2 (5 mL). After stirring for 16 h at RT, the reaction mixture was quenched with water (10 mL) and the organic layer was separated. The aqueous layer was extracted with CH2Cl2 (2×10 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (40 g Silica gel cartridge, 20% EtOAc in pet ether) to afford 200.1 (280 mg, 69%) as a solid. LCMS: 62.1%, m/z [M+H]+=620.0.
To a stirred solution of 200.1 (280 mg, 0.45 mmol) in MeCN (10 mL) was added K2CO3 (62 mg, 0.45 mmol) followed by Mel (69 mg, 0.48 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was quenched with water (10 mL) and extracted with EtOAc (15 mL). The organic layer was washed with water (15 mL), brine (15 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford 200.2 (270 mg) as a pale brown solid. The residue was used in the next step without purification. LCMS: 58.1%, m/z [M+H]+=634.2.
To a stirred solution of 200.2 (260 mg, 0.41 mmol) in THF (10 mL) were added aniline (38 mg, 0.41 mmol) and Pd(PPh3)4 (95 mg, 0.08 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was triturated with diethyl ether: n-pentane. The resulting residue was purified by prep. HPLC [Column: X-BRIDGE C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: acetonitrile; Gradient: (Time/%B): 0/60, 8/85, 10/90, 10.1/98, 13/98, 13.1/60, 16/60 at 18 mL/min] to afford 200 (45 mg, 17%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.53 (br s, 1H), 8.40/7.98 (d, J=2.0 Hz, 1H), 7.67-7.42 (m, 4H), 4.11-4.02 (m, 1H), 3.82-3.78 (m, 1H), 3.58-3.55 (m, 1H), 3.43 (s, 3H), 3.23 (s, 3H), 3.22-3.16 (m, 1H), 2.57-2.50 (m, 1H), 2.38-2.30 (m, 1H), 2.11-2.04 (m, 1H); LCMS: 95.0%, [M+H]+=592.0.
Using 110.4_1a and the listed anilines, the following compounds were made as in Example 200.
208.1 was synthesized from 110.4_1a following procedure described for the synthesis of 200.1.
To a stirred solution of 208.1 (300 mg, 0.45 mmol) in DCM (10 mL) was added cyclopropylboronic acid (78 mg, 0.91 mmol) and TEA (0.12 mL, 0.91 mmol). After purging with oxygen for 10 minutes, Cu(OAc)2 (82 mg, 0.45 mmol) was added and purged again with oxygen for 5 minutes. After stirring at RT for 16 h, the reaction mixture was diluted with DCM (20 mL), filtered through a small pad of Celite and the pad was washed with DCM (50 mL). The filtrate was washed with water (20 mL) and brine (20 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography using (40 g Silica gel cartridge, 20% EtOAc in pet ether) to afford 208.2 (120 mg, 37%) as solid.
1H NMR (400 MHz, CDCl3): 8.32 (d, J=2.4 Hz, 1H), 7.41-7.40 (m, 1H), 7.30-7.26 (m, 1H), 7.24-7.17 (br s, 2H), 5.48-5.40 (m, 1H), 5.16-5.07 (m, 2H), 4.28-4.23 (m, 1H), 3.97-3.93 (m, 2H), 3.72-3.67 (m, 1H), 3.60-3.55 (m, 1H), 3.51-3.45 (m, 1H), 3.35-3.32 (m, 1H), 3.22-3.13 (m, 1H), 2.94-2.92 (m, 1H), 2.65-2.57 (m, 1H), 2.29-2.15 (m, 2H), 1.12-1.10 (m, 2H), 0.98-0.86 (m, 3H), 0.52-0.50 (m, 2H), 0.21-0.19 (m, 2H); LCMS: 98.6%, m/z [M+H]+=700.0.
To a stirred solution of 208.2 (100 mg, 0.14 mmol) in THF (10 mL) were added aniline (13 mg, 0.14 mmol) and Pd(PPh3)4 (33 mg, 0.02 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: KROMOSIL-C18 (150×25 mm), 10 u; A: 0.1% Formic in H2O, Acetonitrile; Gradient:(Time/%B): −0/60, 8/85, 12/95, 12.1/98, 14/98, 14.1/60, 16/60 at 22 mL/min] to afford 208 (80 mg, 85%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.53 (br s, 1H), 8.36/7.98 (d, J=2.5 Hz, 1H), 7.68/7.60 (t, J=2.0 Hz, 1H), 7.51-7.38 (m, 3H), 4.12-3.98 (m, 1H), 3.79-3.75 (m, 2H), 3.44-3.40 (m, 2H), 3.18 (m, 1H), 2.96-2.94 (m, 1H), 2.51-2.50 (m, 1H), 2.41-2.32 (m, 1H), 2.15-1.93 (m, 1H), 1.13-0.96 (m, 2H), 0.88-0.85 (m, 2H), 0.80-0.65 (m, 1H), 0.42-0.40 (m, 2H), 0.17- 0.16 (m, 1H), 0.07-0.02 (m, 1H); LCMS: 98.2% , m/z [M+H]+=657.9.
To a stirred solution of 216.1 (3.0 g, 18.2 mmol) in conc. H2SO4 (15 mL) and CH3SO3H (15 mL) was added trichloroisocyanuric acid (2.1 g, 9.08 mmol) at 0° C. After stirring for 4 h at RT, the reaction was cooled to 0° C. and quenched with ice cold H2O (100 mL). The resulting precipitate was filtered, washed with H2O (200 mL), collected and dried under vacuum to afford 216.2 (3.5 g, 97%) as an orange solid.
1H NMR (400 MHz, DMSO-d6): 11.66 (s, 1H), 7.77 (dd, J=2.0 Hz, 10.0 Hz, 1H), 7.47 (d, J=2.0 Hz, 1H); GCMS: 97.7%, m/z [M+H]+=200.8.
To a stirred solution of 216.3 (3 g, 12.1 mmol) in MTBE (100 mL) were added 216.2 (2.4 g, 12.1 mmol) and 216.4 (1.88 g, 12.1 mmol) at RT. After stirring at 80° C. for 16 h, the reaction mixture was cooled to RT and then concentrated under reduced pressure. The diastereomeric mixture (dr 7%:26%:27%:5% by LCMS) was purified by column chromatography (Silica gel 100-200 mesh, 20-30% EtOAc/pet ether) to afford required diastereomer 216.5 (1.7 g, 32%) as an off white solid. Regio and relative stereochemistry were confirmed by 2D NMR studies.
1H NMR (500 MHz, DMSO-d6): 12.86 (br s, 1H), 11.23 (s, 1H), 7.58 (d, J=2.0 Hz, 1H), 7.42 (dd, J=1.5 Hz, 9.5 Hz, 1H), 5.50-5.45 (m, 1H), 5.11-5.06 (m, 2H), 4.28-4.27 (m, 2H), 4.09-4.05 (m, 1H), 4.01 (d, J=7.5 Hz, 1H), 3.65 (t, J=7.0 Hz, 1H), 3.21-3.18 (m, 1H), 2.69-2.68 (m, 1H), 2.50-2.46 (m, 1H), 2.19-2.09 (m, 1H); LCMS: 97.9%, m/z [M+H]+=445.0; Chiral purity: (50.5+49.4)%.
216.5 (1.7 g) was separated by chiral SFC using Chiralcel OX-H (30×250) mm, 5 μ; A: 75% CO2%, B: 25% (0.5% DEA in Methanol at RT (Isocratic 90 g/min, with detection at 214 nm). Pure fractions were concentrated under reduced pressure to afford 216.5a (Enantiomer-1, 760 mg, 89%) as an off-white solid and 216.5b (Enantiomer-2, 670 mg, 79%) as an off-white solid.
216.5a: 1H NMR (500 MHz, DMSO-d6): 12.88 (br s, 1H), 11.23 (s, 1H), 7.58 (d, J=1.5 Hz, 1H), 7.42 (dd, J=2.0 Hz, 10.0 Hz, 1H), 5.50-5.45 (m, 1H), 5.12-5.06 (m, 2H), 4.28-4.27 (m, 2H), 4.10-4.05 (m, 1H), 4.01 (d, J=7.5 Hz, 1H), 3.65 (t, J=7.0 Hz, 1H), 3.21-3.18 (m, 1H), 2.74-2.65 (m, 1H), 2.50-2.45 (m, 1H), 2.20-2.09 (m, 1H); LCMS: 97.5%, m/z [M+H]+=445.0; Chiral purity: 99.7%.
216.5b: 1H NMR (500 MHz, DMSO-d6): 12.86 (br s, 1H), 11.23 (s, 1H), 7.58 (d, J=2.0 Hz, 1H), 7.42 (dd, J=2.0 Hz, 10.0 Hz, 1H), 5.50-5.45 (m, 1H), 5.12-5.06 (m, 2H), 4.29-4.27 (m, 2H), 4.08-4.06 (m, 1H), 4.01 (d, J=7.5 Hz, 1H), 3.65 (t, J=7.0 Hz, 1H), 3.21-3.18 (m, 1H), 2.74-2.65 (m, 1H), 2.50-2.44 (m, 1H), 2.20-2.09 (m, 1H); LCMS: 98.7%, m/z [M+H]+=445.0; Chiral purity: 99.8%.
To 216.5a (200 mg, 0.44 mmol) was added SOCl2 (3 mL). After stirring for 2 h at RT, SOCl2 was evaporated under reduced pressure to afford intermediate acid chloride. To above prepared intermediate acid chloride at RT was added solution of 216.6 (150 mg, 0.64 mmol) in CH2Cl2. After stirring at 50° C. for 16 h, the reaction mixture was evaporated and resulting residue was purified by flash column chromatography (24 g Silica gel cartridge, 15% EtOAc in pet ether) to afford 216.7a (150 mg, 53%) as an off-white solid.
1H NMR (400 MHz, CDCl3): 8.18 (d, J=2.0 Hz, 1H), 7.60 (br s, 1H), 7.34 (t, J=1.6 Hz, 1H), 7.26 (s, 1H), 7.08 (dd, J=2.0 Hz, 9.6 Hz, 1H), 5.50-5.42 (m, 1H), 5.15-5.06 (m, 2H), 4.37-4.32 (m, 1H), 4.25-4.20 (m, 1H), 4.05-3.98 (m, 2H), 3.81-3.68 (m, 2H), 3.54-3.49 (m, 1H), 3.34-3.29 (m, 1H), 2.75-2.70 (m, 1H), 2.31-2.10 (m, 2H), 0.92 (s, 9H); LCMS: 96.1%, m/z [M+H]+=660.0.
To a stirred solution of compound 216.7a (130 mg, 0.19 mmol) in THF (3 mL) were added aniline (18 mg, 0.19 mmol) and Pd(PPh3)4 (46 mg, 0.04 mmol) at RT. After stirring for 1 h, the reaction mixture was diluted with EtOAc (15 mL). The organic solution was collected, washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-BRIDGE-C18 (150×30) mm, 5 μ, A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/70, 8/90, 9/90, 9.1/98, 11/98, 11.1/70, 14/70 at 20 mL/min] to afford 216.8a (47 mg, 38%) as solid.
1H NMR (500 MHz, DMSO-d6) (exist in rotameric form): 12.59/12.45 (s, 1H), 11.10/11.06 (s, 1H), 8.16/7.71 (d, J=1.5 Hz, 1H), 7.61-7.34 (m, 4H), 4.06-4.00 (m, 2H), 3.84-3.80 (m, 1H), 3.61-3.58 (m, 1H), 3.44 (d, J=14 Hz, 1H), 3.36-3.29 (m, 1H), 2.59-2.50 (m, 1H), 2.38-2.34 (m, 1H), 2.13-2.06 (m, 1H), 0.85/0.82 (s, 9H); LCMS: 98.1%, m/z [M+H]+=618.0; Chiral Purity: 99.9%.
To 216.5b (200 mg, 0.44 mmol) was added SOCl2 (3 mL). After stirring for 2 h at RT, SOCl2 was evaporated under reduced pressure to afford intermediate acid chloride. To above prepared intermediate acid chloride at RT was added a solution of compound 216.6 (150 mg, 0.64 mmol) in CH2Cl2. After stirring at 50° C. for 16 h, the reaction mixture was evaporated and the residue was purified by flash column chromatography (24 g Silica gel cartridge, 15% EtOAc in pet ether) to afford 216.7b (180 mg, 63%) as an off white solid.
1H NMR (400 MHz, CDCl3): 8.18 (d, J=2.0 Hz, 1H), 7.65 (br s, 1H), 7.34 (t, J=1.6 Hz, 1H), 7.26 (s, 1H), 7.08 (dd, J=1.6 Hz, 9.2 Hz, 1H), 5.50-5.42 (m, 1H), 5.14-5.06 (m, 2H), 4.37-4.32 (m, 1H), 4.25-4.20 (m, 1H), 4.05-3.98 (m, 2H), 3.81-3.68 (m, 2H), 3.54-3.50 (m, 1H), 3.36-3.27 (m, 1H), 2.76-2.68 (m, 1H), 2.31-2.05 (m, 2H), 0.92 (s, 9H); LCMS: 90.2%, m/z [M+H]+=660.0.
To a stirred solution of compound 216.7b (160 mg, 0.24 mmol) in THF (3 mL) were added aniline (23 mg, 0.24 mmol) and Pd(PPh3)4 (56 mg, 0.05 mmol) at RT. After stirring for 1 h, the reaction mixture was diluted with EtOAc (15 mL). The organic solution was collected, washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-BRIDGE-C18 (150×30) mm, 5 μ, A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/60, 8/90, 10/90, 10.1/98, 11/98, 11.1/60, 14/60 at 20 mL/min] to afford 216.8b (93 mg, 62%) as solid.
1H NMR (500 MHz, DMSO-d6) (exist in rotameric form): 12.59/12.45 (s, 1H), 11.10/11.06 (s, 1H), 8.16/7.71 (d, J=1.5 Hz, 1H), 7.61-7.34 (m, 4H), 4.06-4.00 (m, 2H), 3.85-3.80 (m, 1H), 3.60 (t, J=7.0 Hz, 1H), 3.44 (d, J=14 Hz, 1H), 3.36-3.28 (m, 1H), 2.59-2.50 (m, 1H), 2.38-2.34 (m, 1H), 2.11-2.06 (m, 1H), 0.85/0.82 (s, 9H); LCMS: 97.4%, m/z [M+H]+=618.0; Chiral Purity: 99.6%.
1H NMR (500 MHz, DMSO-d6) (Exist in rotamcric form): 12.56/12.50 (br s, 1H), 10.90/10.86 (s, 1H), 8.10 (d, J = 7.0 Hz, 1H), 7.66-7.45 (m, 3H), 7.33/7.28 (d, J = 8.0 Hz, 1H), 4.05 (d, J = 7.5 Hz, 1H), 3.97 (d, J = 14.0 Hz, 1H), 3.84-3.80 (m, 1H), 3.62-3.61 (m, 1H), 3.49 (d, J = 14.0 Hz, 1H), 3.32-3.21 (m, 1H), 2.60-2.50 (m, 1H), 2.41-2.33 (m, 1H), 2.15-2.03 (m, 1H), 0.84/0.82 (s, 9H).
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.56/12.50 (br s, 1H), 10.90/10.86 (s, 1H), 8.10 (dd, J = 2.0 Hz, 9.0 Hz, 1H), 7.66-7.44 (m, 3H), 7.33/7.28 (d, J = 8.0 Hz, 1H), 4.05 (d, J = 7.5 Hz, 1H), 3.97 (d, J = 14.0 Hz, 1H), 3.84-3.80 (m, 1H), 3.63-3.60 (m, 1H), 3.49 (d, J = 14.0 Hz, 1H), 3.32-3.21 (m, 1H), 2.60-2.50 (m, 1H), 2.41-2.33 (m, 1H), 2.15-2.03 (m, 1H), 0.84/0.82 (s, 9H).
1H NMR (400 MHz. DMSO-d6) (Exist in rotameric form): 12.55/12.45 (br s, 1H), 10.98/10.93 (s, 1H), 7.99 (dd, J = 2.0 Hz, 8.8 Hz, 1H), 7.61-7.44 (m, 3H), 7.26- 7.21 (m, 1H), 4.04 (d, J = 7.6 Hz, 1H), 3.97 (d, J = 14.0 Hz, 1H), 3.85-3.80 (m, 1H), 3.63-3.60 (m, 1H), 3.49 (d, J = 13.6 Hz, 1H), 3.32-3.26 (m, 1H), 2.60-2.50 (m, 1H), 2.42-2.32 (m, 1H), 2.14-2.05 (m, 1H), 0.84/0.83 (s, 9H).
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.54 (br s, 1H), 10.98/10.93 (s, 1H), 7.99 (dd, J = 2.0 Hz, 8.8 Hz, 1H), 7.61-7.44 (m, 3H), 7.26- 7.21 (m, 1H), 4.04 (d, J = 7.6 Hz, 1H), 3.97 (d, J = 14.0 Hz, 1H), 3.85-3.80 (m, 1H), 3.61-3.60 (m, 1H), 3.49 (d, J = 13.6 Hz, 1H), 3.32-3.26 (m, 1H), 2.60-2.50 (m, 1H), 2.42-2.32 (m, 1H), 2.15-2.04 (m, 1H), 0.84/0.83 (s, 9H).
The following examples was made as in Example 216 with the listed isatins in place of 5-chloro-7-fluoroindoline-2,3-dione, 216.2. Regiochemistry and relative stereochemistry was assigned by 2D NMR studies. Absolute stereochemistry unknown for enantiomeric pairs (a and b).
To a solution of 219.1 (3.0 g, 23.2 mmol) in acetontrile (50 mL) were added 219.2 (5.0 g, 23.2 mmol) and 219.3 (3.62 g, 23.2 mmol) at RT. After stirring at 90° C. for 16 h, the reaction mixture was concentrated. The residue was purified by column chromatography (Silica gel 100-200 mesh, 30% EtOAc/pet ether) to afford a mixture of diastereomers (dr=7:10:10:2). The mixture of diasteromers was purified by prep. HPLC [Column: X-SELECT-C18 (150×30) mm, 5 μ, A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/30, 8/70, 11/70, 12/98, 12.1/98, 15/9 at 22 mL/min] to afford 219.4 (200 mg, 2%) as an off-white solid. The regio chemistry and relative stereochemistry was confirmed by 2D NMR analysis.
1H NMR (500 MHz, DMSO-d6, at 100° C.): 11.93 (br s, 1H), 10.62 (br s, 1H), 7.85 (d, J=2.0 Hz, 1H), 7.28 (d, J=2.0 Hz, 1H), 5.51-5.45 (m, 1H), 5.08-5.03 (m, 2H), 4.23-4.21 (m, 2H), 3.72 (d, J=7.5 Hz, 1H), 3.36-3.33 (m, 1H), 3.27-3.24 (m, 1H), 2.29-2.20 (m, 2H), 1.83-1.72 (m, 2H), 1.48-1.46 (m, 1H), 1.22-1.17 (m, 3H); LCMS: 95.5%, m/z [M+H]+=439.0; Chiral purity: (50.8+49.2)%.
Thionyl chloride (2 mL) was added to 219.4 (110 mg, 0.25 mmol) at RT. After stirring for 2 h, the thionyl chloride was evaporated under reduced pressure to afford acid chloride. To the above intermediate acid chloride at RT was added a solution of 219.5 (66 mg, 0.38 mmol) in CH2Cl2 (3 mL). After stirring at 50° C. for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (Silica gel 100-200 mesh, 20% EtOAc/pet ether) to afford 219.6 (30 mg, 20%) as a light brown solid. LCMS: 69.9%, m/z [M+H]+=598.0.
To a stirred solution of 219.6 (30 mg, 0.05 mmol) in THF (2 mL) were added aniline (5 mg, 0.05 mmol) and Pd(PPh3)4 (11 mg, 0.01 mmol) at RT. After stirring at RT for 2 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: KROMOSIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/60, 8/80, 9/80, 9.1/98, 12/98, 12.1/60, 14/60 at 22 mL/min] to afford 219 (3 mg, 10%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) (exist in rotameric form): 12.40/12.30 (s, 1H), 10.94/10.90 (s, 1H), 8.32/8.01 (d, J=2.0 Hz, 1H), 7.63/7.53 (t, J=1.6 Hz, 1H), 7.46-7.35 (m, 3H), 3.64 (d, J=7.6 Hz, 1H), 3.53 (t, J=6.0 Hz, 1H), 3.43/3.22 (s, 3H), 3.01-2.99 (m, 1H), 2.20-2.13 (m, 2H), 1.68-1.66 (m, 1H), 1.54-1.51 (m, 1H), 1.43-1.40 (m, 1H), 1.19-1.05 (m, 3H); LCMS: 90.1%, m/z [M+H]+=556.1; Chiral purity: (46.7+45.2)%.
To a stirred solution of 220.1 (3.0 g, 22.9 mmol) in THF (100 mL) were added 220.2 (4.94 g, 22.9 mmol), 220.3 (3.57 g, 22.9 mmol) and DIPEA (2.95 g, 22.9 mmol) at RT. After stirring at 80° C. for 16 h, the reaction mixture was diluted with water (30 mL) and extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine (25 mL), dried over anhydrous Na2SO4, filtered and concentrate under reduced pressure to afford crude mixture of diastreomers (dr=5+3+6%). The crude mixture of diastereomers was purified by column chromatography (Silica gel 100-200 mesh, 30% EtOAc in pet ether) to afford 220.4a (250 mg), 220.4b (60 mg), 220.4c (450 mg) as solids.
220.4a: 1H NMR (500 MHz, Acetone-d6): 11.11 (s, 1H), 9.90 (s, 1H), 7.98 (d, J=2.0 Hz, 1H), 7.33 (d, J=2.0 Hz, 1H), 5.61-5.52 (m, 1H), 5.14-5.05 (m, 2H), 4.31-4.28 (m, 2H), 4.09-4.06 (m, 1H), 3.85 (d, J=7.0 Hz, 1H), 3.71-3.68 (m, 1H), 3.62-3.55 (m, 2H), 3.29-3.24 (m, 2H), 2.60-2.59 (m, 1H), 2.28-2.26 (m, 1H); LCMS: 93.0%, m/z [M+H]+441.0.
220.4b: LCMS: 80%, m/z [M+H]+=441.1
220.4c: LCMS: 68%, m/z [M+H]+=441.1
SOCl2(5 mL) was added to 220.4a (250 mg, 0.59 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure to afford intermediate acid chloride. To above acid chloride was added a solution of 220.5 (158 mg, 0.68 mmol) in DCM (10 mL). After stirring at 55° C. for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (Silica gel 100-200 mesh, 30% EtOAc in pet ether) to afford 220.6_1 (40 mg, 11%) as solid.
1H NMR (500 MHz, Acetone-d6): 9.82 (s, 1H), 8.43 (d, J=1.5 Hz, 1H), 7.58-7.54 (m, 2H), 7.48 (d, J=2 Hz, 1H), 7.33 (d, J=2.0 Hz, 1H), 5.63-5.50 (m, 1H), 5.15-5.11 (m, 2H), 4.42-4.40 (m, 1H) 4.29-4.21 (m, 1H), 3.89-3.83 (m, 4H), 3.62-3.60 (m, 2H), 3.31-3.24 (m, 3H), 2.52-2.49 (m, 1H), 2.25-2.23 (m, 1H), 0.91 (s, 9H); LCMS: 93.5%, m/z [M+H]+=656.
To a stirred solution of 220.6a (40 mg, 0.06 mmol) in THF (2 mL) were added aniline (6 mg, 0.06 mmol) and Pd(PPh3)4 (14 mg, 0.01 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The resulting residue was purified by prep. HPLC [Column: KROMOSIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/60, 8/70, 8.1/98, 9/98, 9.1/60, 12/60 at 25 mL/min] to afford 220.7_1 (3.0 mg, 8%) as solid.
LCMS: 95.1%, m/z [M+H]+=614.1
SOCl2 (10 mL) was added to 220.4c (450 mg, 1.02 mmol) at RT. After stirring under nitrogen atmosphere for 2 h, the reaction mixture was concentrated under reduced pressure to afford intermediate acid chloride. To the above prepared acid chloride was added 220.5 (273 mg, 1.18 mmol) in DCM (15 mL). After stirring at 55° C. for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (100-200 Silica gel, 30% EtOAc/pet ether) to afford 220.6c (250 mg, 37%) as an orange solid.
1H NMR (400 MHz, DMSO-d6): 11.24 (s, 1H), 7.81-7.68 (m, 3H), 7.49 (d, J=2.0 Hz, 1H), 6.65 (s, 1H), 5.34-5.26 (m, 1H), 5.06-5.00 (m, 2H), 4.26-4.21 (m, 1H), 4.15-3.97 (m, 3H), 3.87 (d, J=10.0 Hz, 1H), 3.57 (d, J=10.0 Hz, 1H), 3.27-3.03 (m, 4H), 2.50-2.45 (m, 2H), 2.15-2.11 (m, 1H), 0.80 (s, 9H). LCMS: 85.3%, m/z [M+H]+=656.
To a stirred solution of 220.6a (250 mg, 0.38 mmol) in THF (10 mL) were added aniline (35 mg, 0.38 mmol) and Pd(PPh3)4 (88 mg, 0.08 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-SELECT-C18 (150×19) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/10, 8/80, 11/89, 11.1/98, 13.98, 15/98 at 18 mL/minute] to afford 220.7c (35 mg, 15%) as solid.
1H NMR (500 MHz, DMSO-d6): 12.80 (s, 1H), 11.18 (s, 1H), 7.79 (s, 2H), 7.74 (s, 1H), 7.48 (s, 1H), 6.68 (s, 1H), 4.14 (d, J=14.0 Hz, 1H), 3.94 (m, J=10.0 Hz, 1H), 3.79 (d, J=10.5 Hz, 1H), 3.56 (d, J=9.5 Hz, 1H), 3.30-3.20 (m, 2H), 3.09-3.01 (m, 2H), 2.50-2.46 (m, 1H), 2.42-2.36 (m, 1H), 2.10-2.07 (m, 1H), 0.77 (s, 9H). LCMS: 97.7%, m/z [M+H]+=614.0; Chiral purity: (51.5+48.4)%.
To a stirred solution of 225.1 (350 mg, 0.64 mmol) in DMF (15 mL) were added TEA (1.33 mL, 9.58 mmol) and N-methylhydroxylamine hydrochloride (534 mg, 6.39 mmol) at 0° C. in sealed tube. After warming slowly allowed to RT and stirring for 16 h, the reaction mixture was diluted with EtOAc (20 mL) and washed with H2O (2×20 mL). The organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography followed by SFC purification [Column: Chiralpak IC (4.6×250) mm, 5 μ; 75% CO2: 25% Methanol at RT (Isocratic 20 mL/min, with detection at 214 nm)] to afford 225a (20 mg, 5%) as an off-white solid and 225b (45 mg, 12%) as an off-white solid. Absolute stereochemistry was not established for 225a and 225b.
225a: 1H NMR (500 MHz, DMSO-d6): 11.20 (br s, 1H), 10.06 (br s, 1H), 10.03 (br s, 1H), 7.55 (d, J=2.0 Hz, 1H), 7.41 (d, J=1.5 Hz, 2H), 7.29 (t, J=2.0 Hz, 1H), 7.10 (br s, 1H), 4.58-4.52 (m, 1H), 4.47-4.42 (m, 1H), 3.83-3.74 (m, 1H), 3.34-3.32 (m, 1H), 3.08 (s, 3H), 3.01-2.90 (m, 1H), 2.83-2.78 (m, 1H), 2.37-2.31 (m, 1H); LCMS: 94.8%, m/z [M+H]+=593.1.
225b: 1H NMR (500 MHz, DMSO-d6): 10.85 (br s, 1H), 10.32 (s, 1H), 9.86 (br s, 1H), 7.68 (d, J=2.0 Hz, 3H), 7.49 (d, J=2.0 Hz, 1H), 7.30 (t, J=2.0 Hz, 1H), 4.25 (d, J=7.5 Hz, 1H), 4.13-4.09 (m, 1H), 3.49-3.38 (m, 2H), 2.69 (s, 3H), 2.60-2.50 (m, 1H), 2.47-2.39 (m, 1H), 2.26-2.13 (m, 1H); LCMS: 95.8%, m/z [M+H]+=593.1.
To a stirred solution of 225.1 (200 mg, 0.37 mmol) in DMF (3 mL) were added NH2OH.HCl (127 mg, 1.83 mmol) and TEA (0.25 mL, 1.83 mmol) at RT. After stirring for 4 h at RT, the reaction mixture was poured into ice cold water (15 mL) and stirred well for 10 minutes. The resulting precipitate was filtered, washed with cold water (10 mL) and dried under high vacuum. The residue was purified by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/45, 8/70, 9.4/70, 9.5/98, 11/98, 11.1/45, 13/45 at 25 mL/min] to afford 226a (30 mg, 14%) as a white solid and 226b (17 mg, 8%) as a white solid. Absolute stereochemistry was not established for 226a and 226b.
226a: 1H NMR (500 MHz, DMSO-d6): 11.14 (br s, 1H), 10.53 (br s, 1H), 10.06 (s, 1H), 8.88 (s, 1H), 7.53 (d, J=2.0 Hz, 1H), 7.44-7.35 (m, 2H), 7.30-7.29 (m, 2H), 4.29-4.24 (m, 1H), 4.00-3.97 (m, 1H), 3.65-3.60 (m, 1H), 3.50 (d, J=7.5 Hz, 1H), 3.00-2.81 (m, 1H), 2.79-2.76 (m, 1H), 2.34-2.27 (m, 1H); LCMS: 97.5%, m/z [M−H]−=576.9.
226b: 1H NMR (500 MHz, DMSO-d6): 11.08 (br s, 1H), 10.40 (br s, 1H), 10.17 (br s, 1H), 8.92 (s, 1H), 7.70-7.65 (m, 2H), 7.57 (d, J=2.0 Hz, 1H), 7.42 (d, J=1.5 Hz, 1H), 7.29 (t, J=2.0 Hz, 1H), 4.40-4.35 (m, 1H), 4.24-4.21 (m, 1H), 3.79-3.64 (m, 1H), 3.26 (d, J=8.0 Hz, 1H), 3.10-2.95 (m, 1H), 2.75-2.70 (m, 1H), 2.37-2.28 (m, 1H); LCMS: 95.6%, m/z [M−H]−=576.9.
To a stirred solution of 227.1 (300 mg, 0.53 mmol) in THF (10 mL) were added N-methylmorpholine (87 μL, 0.79 mmol) and isobutyl chloroformate (62 μL, 0.64 mmol) at 0° C. After stirring for 5 minutes, N,O-dimethylhydroxylamine hydrochloride (103 mg, 1.06 mmol) was added at 0° C. After stirring for 16 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: SYMMETRY-C8 (300×19) mm, 7 u; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/50, 8/80, 8.1/98, 10/98, 10.1/50, 13/50 at 20 mL/min] followed by normal phase prep. HPLC [Column: Chiracel OX—H (250×30) mm, 5 u, Mobile Phase: Acetonitrile at RT (Isocratic 42.0 mL /min, with detection at 215 nm)] to afford 227 (59 mg, 18%) as a white solid.
1H NMR (400 MHz, DMSO-d6): 10.89 (s, 1H), 10.38 (s, 1H), 7.70-7.68 (m, 3H), 7.51 (d, J=2.0 Hz, 1H), 7.31 (t, J=2.0 Hz, 1H), 4.26 (d, J=7.2 Hz, 1H), 4.13 (dd, J=7.2 Hz, J=6.8 Hz, 1H), 3.54-3.40 (m, 2H), 3.44 (s, 3H), 2.67 (s, 3H), 2.61-2.54 (m, 1H), 2.49-2.46 (m, 1H), 2.25-2.16 (m, 1H); LCMS: 98.3%, m/z [M+H]+=606.9.
To a stirred solution of 227.1 (200 mg, 0.35 mmol) in THF (20 mL) were added N-methylmorpholine (58 μL, 0.53 mmol) and isobutyl chloroformate (41 μL, 0.42 mmol) at 0° C. After stirring for 5 minutes, NH3 gas was purged into the reaction mixture for 10 minutes at 0° C. After stirring for 16 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-BRIDGE C18 (250×19) mm, 5; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/50, 8/80, 8.1/98, 10/98, 10.1/50, 13/50 at 20 mL/min] to afford 228 (96 mg, 47%) as a white solid.
1H NMR (500 MHz, DMSO-d6): 10.89 (s, 1H), 10.74 (s, 1H), 7.71-7.70 (m, 3H), 7.53 (d, J=2.0 Hz, 1H), 7.33 (t, J=2.0 Hz, 1H), 7.00 (br s, 1H), 6.66 (br s, 1H), 4.55-4.50 (m, 1H), 4.25 (t, J=10.5 Hz, 1H), 3.99 (d, J=11.0 Hz, 1H), 3.49-3.41 (m, 1H), 2.84 (t, J=11.5 Hz, 1H), 2.41-2.35 (m, 1H), 2.16-2.06 (m, 1H); LCMS: 96.9%, m/z [M+H]+=562.9.
229a and 229b were synthesized from 250.2 following the procedure described for the synthesis of 260a and 260b. Absolute stereochemistry was not established for 229a and 229b.
229a: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 10.88/10.79 (s, 1H), 10.07/9.95 (s, 1H), 7.72/7.54 (s, 1H), 7.51-7.30 (m, 4H), 4.55/4.42 (d, J=8.0 Hz, 1H), 4.17-4.05 (m, 1H), 3.51-3.36 (m, 2H), 3.36/3.22 (s, 3H), 2.99/2.97 (s, 3H), 2.74-2.50 (m, 2H), 2.23-2.10 (m, 1H); LCMS: 96.0%, m/z [M+H]+=607.0.
229b: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 10.75/10.69 (s, 1H), 9.90/9.82 (s, 1H), 7.95/7.82 (s, 1H), 7.77/7.63 (s, 1H), 7.60-7.45 (m, 3H), 4.22/4.07 (d, J=7.5 Hz, 1H), 3.85-3.77 (m, 1H), 3.56-3.35 (m, 2H), 3.24 (s, 3H), 2.72 (s, 3H), 2.50-2.32 (m, 2H), 2.22-2.10 (m, 1H); LCMS: 95.1%, m/z [M+H]+=607.0.
To a stirred solution of 250.2 (200 mg, 0.34 mmol) in DMF (5 mL) were added DIPEA (0.12 mL, 0.69 mmol) and HATU (196 mg, 0.51 mmol) at RT. After stirring 15 minutes, benzenesulfonamide (81 mg, 0.51 mmol) was added. After stirring for 12 h at RT, the reaction mixture was quenched with ice cold water (5 mL) and extracted with EtOAc (2×10 mL). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated. The resulted residue was purified by prep. HPLC [Column: X-BRIDGE-C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/40, 8/80, 11/90, 11.1/98, 12/98, 12.1/40, 15/40 at 23 mL/min] to afford 230 (24 mg, 10%) as an off-white solid.
1H NMR (500 MHz, DMSO-d6) (exist in rotameric form): 12.00 (br s, 1H), 10.86/10.66 (s, 1H), 7.74-7.39 (m, 10H), 4.60/4.21 (m, 1H), 4.00-3.92 (m, 1H), 3.67-3.63 (m, 1H), 3.27-3.23 (m, 1H), 3.20 (s, 3H), 2.96-2.87 (m, 1H), 2.64-2.50 (m, 1H), 2.14-2.02 (m, 1H); LCMS: 93.0%, m/z [M-H]−=715.0.
To a stirred solution of 252.2 (1.0 g, 1.72 mmol) in DMF (15 mL) were added DIPEA (0.47 mL, 2.59 mmol) and HATU (0.98 g, 2.59 mmol) at RT. After stirring for 30 minutes, methansulphonamide (0.27 g, 2.59 mmol) was added. After stirring for 16 h at RT, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (2×20 mL). The combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 30% EtOAc in pet ether) followed by prep. HPLC [Column: X-BRIDGE-C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/50, 8/80, 9/80, 9.1/98, 10/98, 10.1/50, 12/50 at 23 mL/min] to afford 231 (71 mg, 6%) as an off-white solid.
1H NMR (500 MHz, DMSO-d6) (exist in rotameric form): 11.32 (br s, 1H), 11.10/11.93 (s, 1H), 7.75-7.48 (m, 5H), 4.58/4.25 (m, 1H), 4.00 (d, J=10.5 Hz, 1H), 3.75-3.71 (m, 1H), 3.39-3.33 (m, 1H), 3.22 (s, 3H), 3.01/2.98 (s, 3H), 2.92-2.86 (m, 1H), 2.50-2.43 (m, 1H), 2.15-2.07 (m, 1H); LCMS: 99.0, m/z [M+H]+=655.0; Chiral purity: 96.4%.
To a stirred solution of 250.2 (200 mg, 0.34 mmol) in DMF (5 mL) were added HATU (196 mg, 0.52 mmol) and Et3N (0.14 mL, 1.04 mmol) at RT. After stirred for 15 minutes, 2,2,2-trifluoroethanamine hydrochloride (92 mg, 0.69 mmol) was added. After stirring for 3 h at RT, the reaction mixture was diluted with cold water (50 mL) and extracted with EtOAc (2×60 mL). The combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [KROMOSIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient:(T%B): −0/30, 8/80, 11/85, 11.1/98, 13/98, 13.1/30, 15/30 at 22 mL/min] to afford 232 (110 mg, 50%) as an off-white solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 10.95/10.74 (s, 1H), 7.82-7.45 (m, 6H), 4.26 (t, J=10.5 Hz, 1H), 4.00-3.97 (m, 1H), 3.88-3.84 (m, 1H), 3.77-3.72 (m, 1H), 3.65-3.60 (m, 1H), 3.46-3.39 (m, 1H), 3.39/3.21 (s, 3H), 2.89-2.85 (m, 1H), 2.60-2.50 (m, 1H), 2.25-2.10 (m, 1H); LCMS: 96.4%, m/z [M−H]−=657.0; Chiral purity: (49.9%+50.0%).
232 (100 mg) was separated by chiral SFC [Column: (R,R) Whelk-01 (30×250 mm), 5 μ; 90% CO2: 10% Acetonitrile at RT (Isocratic 70 g/min, with detection at 214 nm)] to afford 232a (Enantiomer-1, 17 mg, 34%) as an off-white solid and 232b (Enantiomer-2, 20 mg, 40%) as an off white solid. Absolute stereochemistry was not determined.
232a: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 10.95/10.74 (s, 1H), 7.82-7.44 (m, 6H), 4.26 (t, J=10 Hz, 1H), 3.99-3.97 (m, 1H), 3.90-3.84 (m, 1H), 3.77-3.72 (m, 1H), 3.67-3.62 (m, 1H), 3.46-3.39 (m, 1H), 3.39/3.21 (s, 3H), 2.89-2.85 (m, 1H), 2.60-2.50 (m, 1H), 2.25-2.10 (m, 1H); LCMS: 99.1%, m/z [M−H]−=657.0; Chiral purity: 98.65%.
232b: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 10.95/10.74 (s, 1H), 7.76-7.44 (m, 6H), 4.26 (t, J=10 Hz, 1H), 3.99-3.97 (m, 1H), 3.90-3.84 (m, 1H), 3.77-3.72 (m, 1H), 3.65-3.62 (m, 1H), 3.48-3.37 (m, 1H), 3.41/3.21 (s, 3H), 2.89-2.85 (m, 1H), 2.60-2.50 (m, 1H), 2.25-2.10 (m, 1H); LCMS: 99.1%, m/z [M−H]−=657.0; Chiral purity: 99.7%.
To a stirred solution of 233.1 (WO2017117239) (200 mg, 0.39 mmol) in DMF (15 mL) were added methoxyamine hydrochloride (326 mg, 3.9 mmol) and TEA (1.6 mL, 11.7 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was quenched with ice cold water and extracted with EtOAc (2×30 mL). The combined organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-BRIDGE-C8 (150×19) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/40, 8/80, 9/80, 9.1/98, 11/98, 11.1/40, 14/40 at 25 mL/min] to obtain 233 (7 mg, 3%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6): 10.96 (br s, 1H), 10.79 (br s, 1H), 10.12 (br s, 1H), 7.67 (s, 2H), 7.57-7.49 (m, 2H), 7.26 (t, J=2.0 Hz, 1H), 4.22-4.10 (m, 1H), 4.10-4.01 (m, 1H) 3.40 (s, 3H), 3.25-3.12 (m, 2H), 2.07-1.98 (m, 1H), 1.97-1.65 (m, 4H); LCMS: 94.6%, m/z [M+H]+=557.0. Regiochemistry is unknown.
To a stirred solution of 233.1 (200 mg, 0.39 mmol) in DMF (5 mL) were added NH2OH (137 mg, 1.96 mmol) and NEt3 (0.27 mL, 1.96 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was poured into ice cold water (15 mL) and stirred well. The resulting precipitate was filtered, washed with water (10 mL) and dried under high vacuum. The residue was purified by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/10, 9/80, 9.1/98, 11/98, 11.1/10, 13/10 at 25 mL/min] to obtain 234a (40 mg, 19%) as an off-white solid and 234b (150 mg, 71%) as an off white solid.
234a: 1H NMR (500 MHz, DMSO-d6): 10.99 (br s, 1H), 10.44 (br s, 1H), 9.88 (s, 1H), 8.81 (s, 1H), 7.47 (d, J=2.0 Hz, 2H), 7.38 (d, J=2.0 Hz, 2H), 7.25 (t, J=2.0 Hz, 1H), 4.07-4.02 (m, 1H), 3.91-3.88 (m, 1H), 3.55-3.51 (m, 1H), 3.09-3.05 (m, 1H), 2.37-2.30 (m, 1H), 2.09-2.05 (m, 1H), 1.92-1.85 (m, 1H), 1.84-1.77 (m, 1H), 1.70-1.62 (m, 1H); LCMS: 84.6%, m/z [M+H]+=542.9. Regiochemistry is unknown.
234b: 1H NMR (500 MHz, DMSO-d6): 10.93 (br s, 1H), 10.22 (br s, 1H), 10.13 (s, 1H), 8.71 (s, 1H), 7.68 (d, J=1.5 Hz, 2H), 7.60 (s, 1H), 7.49 (d, J=1.5 Hz, 1H), 7.27 (t, J=2.0 Hz, 1H), 4.16-4.12 (m, 1H), 4.02-3.99 (m, 1H), 3.16-3.11 (m, 1H), 2.27-2.23 (m, 1H), 2.02-1.99 (m, 1H), 1.92-1.85 (m, 1H), 1.81-1.65 (m, 3H); LCMS: 85.1%, m/z [M+H]+=542.9. Regiochemistry is unknown.
To a stirred solution of 233.1 (200 mg, 0.39 mmol) in DMF (10 mL) were added NEt3 (0.27 mL, 1.96 mmol) and N-methylhydroxylamine hydrochloride (164 mg, 1.96 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was poured into ice cold water (15 mL) and stirred for 15 minutes. The resulting precipitate was filtered, washed with water (10 mL) and dried under high vacuum. The residue was purified by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/30, 8/80, 10/90, 10.1/98, 12/98, 12.1/30, 14/30 at 25 mL/min] to obtain 235a (15 mg, 7%) as a white solid and 235b (30 mg, 14%) as a white solid.
235a: 1H NMR (400 MHz, DMSO-d6): 11.01 (br s, 1H), 9.85 (br s, 2H), 7.48 (d, J=2.0 Hz, 1H), 7.42 (d, J=2.0 Hz, 2H), 7.25-7.24 (m, 2H), 4.54-4.50 (m, 1H), 4.27-4.18 (m, 1H), 3.35-3.25 (m, 2H), 3.08 (s, 3H), 2.37-2.33 (m, 1H), 2.12-2.05 (m, 1H), 1.92-1.83 (m, 2H), 1.69-1.60 (m, 1H); LCMS: 86.30%, m/z [M+H]+=557.0. Regiochemistry is unknown.
235b:1HNMR (400 MHz, DMSO-d6): 10.67 (s, 1H), 10.20 (s, 1H), 9.72 (s, 1H), 7.78 (d, J=1.6 Hz, 1H), 7.69 (d, J=2.0 Hz, 2H), 7.41 (d, J=2.0 Hz, 1H), 7.26 (t, J=2.0 Hz, 1H), 4.26 (d, J=7.6 Hz, 1H), 3.96-3.94 (m, 1H), 3.35-3.30 (m, 1H), 2.82-2.80 (m, 1H), 2.69 (s, 3H), 2.17-2.09 (m, 1H), 1.88-1.77 (m, 3H), 1.58-1.48 (m, 1H); LCMS: 99.7%, m/z [M+H]+=557.0. Regiochemistry is unknown.
To a stirred solution of 233.1 (500 mg, 0.97 mmol) in THF (5 mL) was added hydrazine mono hydrate (98 mg, 1.85 mmol) at RT. After stirring at RT for 4 h, the reaction mixture was quenched with water and extracted with EtOAc (2×10 mL). The combined organic layer was washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with CH2Cl2 (5 mL) to get 236 (320 mg, 60%) as a white solid.
1H NMR (400 MHz, DMSO-d6): 10.92 (s, 1H), 10.13 (s, 1H), 8.81 (s, 1H), 7.68 (d, J=1.6 Hz, 2H), 7.52 (s, 1H), 7.48 (s, 1H), 7.26 (s, 1H), 4.19-4.13 (m, 1H), 4.08-4.06 (m, 3H), 3.40 (d, J=7.6 Hz, 1H), 3.16-3.10 (m, 1H), 2.30-2.26 (m, 1H), 1.99-1.88 (m, 2H), 1.78-1.68 (m, 2H); LCMS: 92.3%, m/z [M+H]+=542.3; Chiral purity: 98.7%. Regiochemistry is unknown.
To a stirred solution of 237.2 (1 g, 8.84 mmol) in DMF (20 mL) was added NaH (0.7 g, 17.7 mmol) portion wise at 0° C. over a period of 10 minutes. After stirring at 0° C. for 1 h, MeI (1.5 g, 10.6 mmol) was added. After stirring at rt for 16 h, the reaction mixture was quenched with ice cold water and extracted with EtOAc (3×200 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford 237.3 (300 mg, 27%) as a gum, which was carried to next step without purification. LCMS: 95.7%, m/z [M+H]+=128.1.
To a stirred solution of 237.3 (1 g, 7.86 mmol) in EtOH (20 mL) was added 10% Pd/C (0.2 g) at RT. After hydrogenating at rt for 16 h using balloon pressure, the reaction mixture was filtered through Celite pad. The filtrate was concentrated under reduced pressure to afford 237.4 (800 mg) as an off-white solid. LCMS: 40.9%, m/z [M+H]+=98.1.
To a stirred solution of 237.1 (0.3 g, 0.57 mmol) in THF (10 mL) were added N-methyl morpholine (86 mg, 0.85 mmol) and isobutyl chloroformate (93 mg, 0.68 mmol) at 0° C. After stirring at 0° C. for 30 minutes, 237.4 (69 mg, 0.68 mmol) was added. After stirring for 16 h at 60° C., the reaction mixture was diluted with water and extracted with EtOAc (3×60 mL). The combined organic layer was washed with brine and dried over anhydrous Na2SO4 and concentrated. The residue was purified by column chromatography (Silica gel 100-200 mesh, 3% MeOH in DCM) to obtain 237 (18 mg, 5%) as a brown solid.
1H NMR (400 MHz, DMSO-d6): 10.94 (s, 1H), 10.07 (s, 1H), 9.94 (s, 1H), 7.64 (br s, 2H), 7.45 (s, 1H), 7.30-7.20 (m, 4H), 4.34-4.22 (m, 2H), 3.62-3.57 (m, 1H), 3.61 (s, 3H), 3.26-3.20 (m, 1H), 2.35-2.33 (m, 1H), 2.18-2.14 (m, 1H), 1.90-1.80 (m, 2H), 1.72-1.62 (m, 1H); LCMS: 95.2%, m/z [M+H]+=607.0. Absolute stereochemistry is unknown.
To a stirred solution of 238.1 (0.2 g, 1.76 mmol) in DMF (10 mL) was added 60% NaH (0.14 g, 3.54 mmol) portion wise at 0° C. After stirring for 1 h, MeI (0.25 g, 1.77 mmol) was added at 0° C. After stirring at RT for 16 h, the reaction mixture was quenched with ice cold water and extracted with EtOAc (3×50mL). The combined organic layer was washed with brine and dried over anhydrous Na2SO4 and concentrated to obtain 238.2 (0.14 g) as a gummy material.
1H NMR (500 MHz, CDCl3): 7.14 (d, J=1.0 Hz, 1H), 7.06 (s, 1H), 4.08 (s, 3H); LCMS: 57.04%, m/z [M+H]+=127.9.
To a stirred solution of 238.2 (0.1 g, 0.79 mmol) in 1,4-dioxane (5 mL) was added 10% Pd/C (50% wet, 20 mg). After hydrogenating for16 hat RT using balloon pressure, the reaction mixture was filtered through Celite pad. The filtrate was concentrated under reduced pressure to obtain 238.3 (70 mg) as an off-white solid. LCMS: 98.2%, m/z [M+H]+=98.2.
To a stirred solution of 237.1 (0.25 g, 0.47 mmol) in THF (10 mL) were added N-methyl morpholine (72 mg, 0.71 mmol) and isobutyl chloroformate (77 mg, 0.57 mmol) at 0° C. After stirring at 0° C. for 30 minutes, 238.3 (69 mg, 0.71 mmol) was added. After stirring for 24 h at 60° C., the reaction mixture was diluted with water and extracted with EtOAc (3×50 mL). The combined organic layer was washed with brine and dried over anhydrous Na2SO4 and concentrated. The residue was purified by column chromatography (Silica gel 100-200 mesh, 3% MeOH in DCM) followed by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/30, 7/70, 7.1/98, 9/98, 9.1/30, 11/30 at 25 mL/min] to obtain 238 (11 mg, 4%) as a pale pink solid.
1H NMR (400 MHz, DMSO-d6): 11.82 (br s, 1H), 10.61 (s, 1H), 10.54 (s, 1H), 7.76 (s, 2H), 7.45 (s, 2H), 7.25 (s, 1H), 6.76 (s, 1H), 6.60 (s, 1H), 4.21-4.11 (m, 1H), 4.06 (d, J=3.2 Hz, 2H), 2.96 (s, 3H), 2.91-2.83 (m, 1H), 2.50-2.40 (m, 1H), 1.81-1.72 (m, 2H), 1.61-1.48 (m, 2H); LCMS: 96.2%, m/z [M−H]−=605.2. Absolute stereochemistry is unknown.
N-Methyl morpholine (151 mg, 1.50 mmol) was added to 237.1 (400 mg, 0.75 mmol) in THF (40 mL) at −10° C. followed by isobutyl chloroformate (204 mg, 1.50 mmol). After stirring for 20 minutes at −10° C., 1-methyl-1H-pyrazol-5-amine (220 mg, 2.26 mmol) was added and stirred for 1 h at the same temperature. The reaction mixture was concentrated under reduced pressure to obtain residue which was purified by reverse phase chromatography [Column: Buchi Reveleris C18 (40 g); B: 0.05% Formic acid in H2O, B: Acetonitrile]. Pure fractions were lyophilized to get 239 (40 mg, 8%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6): 11.07 (s, 1H), 10.16 (s, 1H), 9.63 (s, 1H), 7.66 (d, J=1.6 Hz, 2H), 7.56 (d, J=2.0 Hz, 1H), 7.35 (d, J=2.0 Hz, 1H), 7.31 (d, J=1.6 Hz, 1H), 7.27 (t, J=2.0 Hz, 1H), 5.90 (d, J=1.6 Hz, 1H), 4.31-4.26 (m, 2H), 3.63 (d, J=7.2 Hz, 1H), 3.51 (s, 3H), 3.20-3.18 (m, 1H), 2.36-2.33 (m, 1H), 2.12-2.02 (m, 1H), 1.92-1.79 (m, 2H), 1.71-1.63 (m, 1H); LCMS: 96.9%, m/z [M+H]+=607.0. Absolute stereochemistry is unknown.
To a stirred solution of 237.1 (100 mg, 0.18 mmol) in THF (10 mL) were added at 0° C. N-methyl morpholine (38 mg, 0.37 mmol) and isobutyl chloroformate (51 mg, 0.37 mmol). After stirring for 15 minutes, 1-methyl-1H-pyrazol-3-amine (36 mg, 0.37 mmol) was added. After stirring for 1 h at 0° C., the reaction mixture was concentrated under reduced pressure. The residue was purified by reverse phase chromatography [Column: Buchi Reveleris C18 (40 g); B: 0.05% Formic acid in H2O, B: Acetonitrile] to afford 240 (20 mg, 17%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6): 10.97 (s, 1H), 10.10 (s, 1H), 10.05 (s, 1H), 7.65 (d, J=2.0 Hz, 2H), 7.50 (d, J=2.0 Hz, 1H), 7.47 (d, J=2.0 Hz, 1H), 7.33 (d, J=2.0 Hz, 1H), 7.25 (t, J=2.0 Hz, 1H), 6.38 (d, J=2.0 Hz, 1H), 4.35-4.25 (m, 2H), 3.70 (s, 3H), 3.57 (d, J=8.0 Hz, 1H), 3.26-3.20 (m, 1H), 2.36-2.32 (m, 1H), 2.15-2.09 (m, 1H), 1.92-1.88 (m, 1H), 1.83-1.80 (m, 1H), 1.68-1.66 (m, 1H); LCMS: 93.1%, m/z [M+H]+=607.0. Absolute stereochemistry is unknown.
To a stirred solution of 237.1 (300 mg, 0.56 mmol) in DMF (10 mL) were added N-methylmorpholine (0.09 mL, 0.85 mmol) and isobutyl chloroformate (0.1 mL, 0.73 mmol) at 0° C. After 15 minutes, N,O-dimethylhydroxylamine hydrochloride (276 mg, 2.83 mmol) was added at 0° C. After stirring for 16 h at RT, the reaction mixture was poured into ice cold water (20 mL) and stirred for 20 minutes. The resulting precipitate was filtered. The solid was collected, washed with water (20 mL) and dried under high vacuum. The residue was purified by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/50, 8/80, 10/80, 10.1/98, 11/98, 11.1/50, 13/50 at 25 mL/min] to obtain 241 (17 mg, 5% yield) as a white solid.
1H NMR (400 MHz, DMSO-d6): 10.72 (br s, 1H), 10.24 (br s, 1H), 7.81 (d, J=2.0 Hz, 1H), 7.70 (d, J=1.6 Hz, 2H), 7.44 (d, J=2.0 Hz, 1H), 7.27 (t, J=2.0 Hz, 1H), 4.25 (d, J=7.2 Hz, 1H), 4.01-3.95 (m, 1H), 3.43 (s, 3H), 3.37-3.35 (m, 1H), 2.90-2.82 (m, 1H), 2.66 (s, 3H), 2.17-2.10 (m, 1H), 1.90-1.72 (m, 3H), 1.61-1.53 (m, 1H); LCMS: 95.2%, m/z [M+H]+=571.0. Absolute stereochemistry is unknown.
To a stirred solution of 237.1 (100 mg, 0.18 mmol) in THF (3 mL) were added triethyl amine (0.1 mL, 0.75 mmol) and acetichydrazide (28 mg, 0.37 mmol) at RT. After 10 minutes, propylphosphonic anhydride solution (50% wt in EtOAc, 0.24 mL, 0.75 mmol) was added at RT. After stirring for 16 h at RT, the reaction mixture was concentrated under reduced. The residue was purified by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/40, 8/90, 8.1/98, 10/98, 10.1/40, 12/40 at 25 mL/min] to afford 242 (27 mg, 24%) as a white solid.
1H NMR (400 MHz, DMSO-d6): 10.89 (s, 1H), 10.22 (s, 1H), 9.83 (s, 1H), 9.71 (s, 1H), 7.71-7.68 (m, 2H), 7.59 (d, J=2.0 Hz, 1H), 7.42 (d, J=2.0 Hz, 1H), 7.27 (t, J=2.0 Hz, 1H), 4.18-4.12 (m, 1H), 4.08-4.04 (m, 1H), 3.68 (d, J=8.0 Hz, 1H), 2.96-2.90 (m, 1H), 2.28-2.24 (m, 1H), 1.88-1.68 (m, 4H), 1.77 (s, 3H); LCMS: 95.6%, m/z [M+H]+=584.0. Absolute stereochemistry is unknown.
To a stirred solution of 237.1 (200 mg, 0.37 mmol) in DMF (10 mL) were added at 0° C. N-methyl morpholine (0.05 mL, 0.49 mmol) and isobutyl chloroformate (0.07 mL, 0.56 mmol). After 10 mintues, morpholin-4-amine hydrochloride (0.07 mL, 0.56 mmol) was added at 0° C. After stirring for 2 h at RT, the reaction mixture was poured into ice cold water (15 mL) and stirred for 15 minutes. The resulting precipitate was filtered, washed with water (10 mL) and dried under high vacuum. The compound material was purified by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 10 mM NH4OAc in H2O, B: Acetonitrile; Gradient: (T%B): −0/50, 8/80, 9/80, 9.1/50, 11/50 at 25 mL/min] to obtain 243 (35 mg, 15%) as a white solid.
1H NMR (400 MHz, DMSO-d6): 10.81 (br s, 1H), 10.15 (br s, 1H), 8.18 (s, 1H), 7.90 (d, J=1.6 Hz, 1H), 7.62 (d, J=1.2 Hz, 2H), 7.37 (d, J=1.6 Hz, 1H), 7.18 (s, 1H), 4.12 (d, J=7.2 Hz, 1H), 3.79-3.74 (m, 1H), 3.57-3.47 (m, 2H), 3.39-3.25 (m, 3H), 2.71-2.60 (m, 2H), 2.26-2.10 (m, 2H), 2.02-2.00 (m, 1H), 1.75-1.69 (m, 3H), 1.41-1.37 (m, 1H), 1.02-1.00 (m, 1H); LCMS: 99.6%, m/z [M+H]+=612.0. Absolute stereochemistry is unknown.
Methanolic ammonia (10 mL) was added to 244.1 (WO2017117239) (250 mg, 0.46 mmol) at RT. After stirring for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: YMC TRIART-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/20, 8/60, 10/60, 10.1/20, 12/20 at 25 mL/min] to obtain 244 (32 mg, 12%) as a white solid.
1H NMR (500 MHz, DMSO-d6): 10.74 (s, 1H), 10.61 (s, 1H), 7.71 (d, J=1.5 Hz, 2H), 7.65 (d, J=1.5 Hz, 1H), 7.48 (d, J=2.0 Hz, 1H), 7.30 (t, J=2.0 Hz, 1H), 6.91 (s, 1H), 6.57 (s, 1H), 4.50-4.47 (m, 1H), 4.43 (s, 1H), 4.32-4.28 (m, 1H), 3.81 (d, J=11.5 Hz, 1H), 2.92 (d, J=8.5 Hz, 1H), 2.30 (d, J=8.0 Hz, 1H), 1.69-1.65 (m, 1H), 1.50-1.45 (m, 1H), 1.21 (s, 3H); LCMS: 99.5%, m/z [M+H]+=557.0; Chiral Purity: 99.9%. Regiochemistry is unknown.
To a stirred solution of 244.1 (500 mg, 0.92 mmol) in DMF (15 mL) was added NH2OH.HCl (320 mg, 4.61 mmol)) and Et3N (0.64 mL, 4.61 mmol) at RT and the resulting reaction mixture was stirred for 16 h. The reaction mixture was diluted with ice cold water (20 mL) and the resulting precipitate was filtered to obtain the residue which was purified by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/20, 8/60, 8/98, 10/98, 10.1/20, 13/20 at 25 mL/min] to obtain 245 (30 mg, 5%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6): 10.91 (br s, 1H), 10.21 (br s, 1H), 10.08 (br s, 1H), 8.75 (br s, 1H), 7.67 (d, J=2.0 Hz, 2H), 7.50 (s, 1H), 7.47 (s, 1H), 7.25 (t, J=1.6 Hz, 1H), 4.51-4.42 (m, 1H), 4.00 (s, 1H), 4.22-4.18 (m, 1H), 3.23 (d, J=9.2 Hz, 1H), 3.17 (d, J=7.6 Hz, 1H), 2.20-2.14 (m, 2H), 1.69-1.65 (m, 1H), 1.20 (s, 3H); LCMS: 90.5%, m/z [M+H]+=573.0. Regiochemistry is unknown.
To a stirred solution of 244.1 (300 mg, 0.55 mmol) in DMF (15 mL) were added Et3N (1.1 mL, 8.31 mmol) and CH3ONH2.HCl (462 mg, 5.54 mmol) at RT. After stirring at RT for 48 h, the reaction mixture was diluted with water (50 mL) and extracted with EtOAc (2×30 mL). The combined organic layer was dried over Na2SO4, filtered and evaporated by nitrogen bubbling. The resulting residue was purified by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/40, 7/65, 7.1/98, 9/98, 9.1/40, 11/40 at 25 mL/min] to obtain 246 (25 mg, 12%) as an off-white solid.
1H NMR (500 MHz, DMSO-d6): 11.00 (s, 1H), 10.80 (s, 1H), 10.13 (s, 1H), 7.66 (d, J=1.5 Hz, 2H), 7.56 (s, 1H), 7.42 (s, 1H), 7.26 (d, J=2.0 Hz, 1H), 4.57-4.48 (m, 1H), 4.43 (s, 1H), 4.27-4.23 (m, 1H), 3.45 (s, 3H), 3.32-3.24 (m, 1H), 3.01 (d, J=8.0 Hz, 1H), 2.24-2.14 (m, 2H), 1.68-1.61 (m, 1H); LCMS: 96.4%, m/z [M+H]+=587.0. Regiochemistry is unknown.
To a stirred solution of 247.1 (1 g, 4.13 mmol) in DMF (5 mL) were added methanesulfonamide (393 mg, 4.13 mmol) and TEA (1.72 mL, 12.4 mmol) at RT. After stirring at RT for 2 h, the reaction mixture was concentrated under reduced pressure to obtain 247.2 (1.39 g) as a thick brown liquid, which used in the next step without any purification. LCMS: 57.2%, m/z [M−H]−=334.7.
To a stirred solution of 247.2 (1.2 g, 3.55 mmol) in THF (20 mL) were added TFA salt of (2S,4S)-4-hydroxy-4-methyl-1-(2,2,2-trifluoroacetyl)-114-pyrrolidine-2-carboxylic acid (860 mg, 3.55 mmol) and 5,7-dichloroindoline-2,3-dione (767 mg, 3.55 mmol) at RT. After stirring for 2 h at 80° C., the reaction mixture was cooled to RT and diluted with EtOAc. The organic solution was collected, washed with ice cold water, dried over Na2SO4, filtered and concentrated using a stream of nitrogen gas. The residue was purified by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/45, 7/55, 7.1/98, 10/98, 10.1/45, 12/45 at 25 mL/min] to obtain 247 (26 mg) as an off-white solid. Regiochemistry was not confirmed.
1H NMR (400 MHz, DMSO-d6): 11.75 (br s, 1H), 11.02 (br s, 1H), 9.98 (s, 1H), 7.49 (s, 1H), 7.41 (d, J=2 Hz, 2H), 7.26 (s, 1H), 7.12 (br s, 1H), 4.56-4.42 (m, 2H), 4.28-4.19 (m, 1H), 3.39 (d, J=7.6 Hz, 1H), 3.22-3.10 (m, 4H), 2.25-2.10 (m, 2H), 1.80-1.75 (m, 1H), 1.24 (s, 3H); LCMS: 90.9%, m/z [M+H]+=634.9.
To a stirred solution of 247.1 (500 mg, 2.06 mmol) and 248.1 (256 mg, 2.06 mmol) in DMF (3 mL) was added Et3N (0.86 mL, 6.19 mmol) at RT. After stirring at RT for 2 h, the reaction mixture was evaporated under reduced pressure to afford 248.2, which used as such in the next step. LCMS: (13+39)%, m/z [M−H]−=364.0.
To a stirred solution of 248.2 (300 mg, 0.81 mmol) in THF (10 mL) were added TFA salt of (2S,4S)-4-hydroxy-4-methyl-1-(2,2,2-trifluoroacetyl)-114-pyrrolidine-2-carboxylic acid (197 mg, 0.81 mmol) and 5,7-dichloroindoline-2,3-dione (176 mg, 0.81 mmol) at RT. After stirring at 80° C. for 2 h, the reaction mixture was cooled to RT and diluted with EtOAc (20 mL). The organic solution was collected, washed with ice cold water, dried over Na2SO4 filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: KROMASIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/40, 8/70, 8.1/40, 10/40 at 25 mL/min] to afford 248 (16 mg, 3%) as an off-white solid. Regiochemistry was not confirmed. LCMS: 92.6%, m/z [M+H]+=664.0.
249a and 249b were synthesized from 110.4_1 following the procedure described for the synthesis of 265a and 265b.
249a: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.55 (br s, 1H), 10.97 (br s, 1H), 8.34 (s, 1H), 7.47-7.44 (m, 1H), 7.05-6.86 (m, 3H), 4.15-3.95 (m, 1H), 3.83-3.79 (m, 1H), 3.79 (s, 3H), 3.60-3.50 (m, 1H), 3.32-3.25 (m, 1H), 3.23 (s, 3H), 2.60-2.49 (m, 1H), 2.45-2.30 (m, 1H), 2.14-2.07 (m, 1H); LCMS: 93.1%, m/z [M+H]+=574.1.
249b:1HNMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.51 (br s, 1H), 11.04/10.97 (br s, 1H), 8.34 (s, 1H), 7.47-7.44 (m, 1H), 7.05-6.85 (m, 3H), 4.15-3.95 (m, 1H), 3.83-3.79 (m, 1H), 3.79/3.68 (s, 3H), 3.60-3.50 (m, 1H), 3.37-3.24 (m, 1H), 3.23 (s, 3H), 2.60-2.50 (m, 1H), 2.45-2.30 (m, 1H), 2.14-2.07 (m, 1H); LCMS: 92.2%, m/z [M+H]+=574.1.
Thionyl chloride (20 mL) was added to 110.4_1 (1.5 g, 3.25 mmol) at RT. After stirring for 2 h, the excess thionyl chloride was removed under reduced pressure to give an acid chloride. To this acid chloride in CH2Cl2 (25 mL) was added a solution of 3,5-dichloro-N-methylaniline (1.24 g, 7.08 mmol) in CH2Cl2 (5 mL) at RT. After stirring for 16 h at RT, the reaction mixture was quenched with water (10 mL). The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 (2×20 mL). The combined organic layer was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 30% EtOAc in pet ether) to afford 250.1 (1.4 g, 73%) as a solid.
1H NMR (400 MHz, DMSO-d6) (exist in rotameric form): 11.10/11.05 (br s, 1H), 8.20/7.76 (d, J=2.0 Hz, 1H), 7.68/7.57 (t, J=2.0 Hz, 1H), 7.50-7.42 (m, 3H), 5.45-5.36 (m, 1H), 5.10-5.06 (m, 2H), 4.29-4.18 (m, 2H), 4.13 (d, J=7.6 Hz, 1H), 3.84-3.80 (m, 1H), 3.64-3.60 (m, 1H), 3.41/3.24 (s, 3H), 3.35-3.24 (m, 1H), 2.67-2.55 (m, 1H), 2.43-2.33 (m, 1H), 2.17-2.09 (m, 1H); LCMS: 95.1%, m/z [M+H]+=620.2.
To a stirred solution of 250.1 (1.4 g, 2.26 mmol) in THF (20 mL) were added aniline (210 mg, 2.26 mmol) and Pd(PPh3)4 (522 mg, 0.45 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-SELECT-C18 (150×30) mm, 5 μ; A: 0.1% HCOOH in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/45, 8/80, 10/80, 10.1/98, 13/98, 13.1/45, 15/45 at 18 ml/min] to afford 250.2 (430 mg, 33%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form):12.54 (br s, 1H), 11.07/10.99 (br s, 1H), 8.30/7.88 (d, J=2.0 Hz, 1H), 7.66-7.37 (m, 4H), 4.01 (d, J=7.5 Hz, 1H), 3.84-3.80 (m, 1H), 3.56-3.53 (m, 1H), 3.40/3.24 (s, 3H), 3.30-3.24 (m, 1H), 2.64-2.55 (m, 1H), 2.42-2.32 (m, 1H), 2.19-2.03 (m, 1H); LCMS: 91.4%, m/z [M−H]−=576.2.
To a stirred solution of 250.2 (300 mg, 0.51 mmol) and 4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (100 mg, 0.77 mmol) in THF (10 mL) were added triphenyl phosphine (160 mg, 0.62 mmol) and DIAD (130 mg, 0.62 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was concentrated under reduced pressure to afford residue. The residue was purified by prep. HPLC [X-SELECT-C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/45, 8/80, 10/80, 10.1/98, 13/98, 13.1/45, 15/45 at 18 mL/min] to afford 250.3a (81 mg, 23%) as a solid and 250.3b (21 mg, 5%) as a solid. 250.3a: 1H NMR (500 MHz, DMSO-d6) (Exist as a rotamer): 11.14/11.06 (s, 1H), 8.18/7.76 (d, J=2.0 Hz, 1H), 7.69/7.57 (t, J=2.0 Hz, 1H), 7.45-7.37 (m, 3H), 4.91-4.87 (m, 1H), 4.66/4.59 (d, J=14.5 Hz, 1H), 4.15 (d, J=7.5 Hz, 1H), 3.82-3.79 (m, 1H), 3.66-3.63 (m, 1H), 3.32-3.19 (m, 1H), 3.21 (s, 3H), 2.61-2.56 (m, 1H), 2.38-2.36 (m, 1H), 2.13-2.03 (m, 1H), 2.03/2.02 (s, 3H); LCMS: 98.7%, m/z [M+H]+=689.9. 250.3b: 1H NMR (500 MHz, DMSO-d6) (Exist as a Rotamer): 8.33/7.91 (d, J=2.0 Hz, 1H), 7.69/7.57 (t, J=2.0 Hz, 1H), 7.53-7.42 (m, 3H), 5.12-5.03 (m, 2H), 4.94-4.91 (m, 1H), 4.54/4.42 (d, J=14.5 Hz, 1H), 4.31/4.23 (d, J=7.5 Hz, 1H), 3.82-3.80 (m, 1H), 3.67-3.65 (m, 1H), 3.41/3.24 (s, 3H), 3.19-3.12 (m, 1H), 2.58-2.50 (m, 1H), 2.41-2.30 (m, 1H), 2.17/2.13 (s, 3H), 2.07-2.04 (m, 1H), 2.02/2.01 (s, 3H); LCMS: 95.5%, m/z [M+H]+=801.9.
To a stirred solution of 250.2 (200 mg, 0.35 mmol) and chloromethyl pivalate (80 mg, 0.51 mmol) in CH3CN (10 mL) was added K2CO3 (100 mg, 0.69 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was quenched with water and extracted with EtOAc (10 mL). The combined organic layer was washed with water (5 mL), brine solution (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [X- BRIDGE-C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/70, 8/90, 10/95, 14/98, 14.1/70, 17/70 at 22 mL/min] to afford 251a (19 mg, 8%) as a solid, 251b (20 mg, 8%) as a solid and 251c (49 mg, 17%) as a solid.
251a: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.01/10.91 (br s, 1H), 7.84-7.68 (m, 2H), 7.57-7.30 (m, 3H), 5.54 (s, 2H), 4.34 (d, J=10.5 Hz, 1H), 4.03-3.99 (m, 1H), 3.75-3.73 (m, 1H), 3.48-3.41 (m, 1H), 3.23 (s, 3H), 2.75-2.70 (m, 1H), 2.64-2.50 (m, 1H), 2.37-2.20 (m, 1H), 1.07/1.05 (s, 9H); LCMS: 96.9%, m/z [M+H]+=692.0;
251b: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.10 (br s, 1H), 8.20 (br s, 1H), 7.71 (t, J=2.0 Hz, 1H), 7.56-7.44 (m, 3H), 5.54-5.47 (m, 2H), 4.14 (d, J=7.5 Hz, 1H), 3.79-3.76 (m, 1H), 3.68-3.66 (m, 1H), 3.40/3.21 (s, 3H), 3.18-3.05 (m, 1H), 2.64-2.51 (m, 1H), 2.42-2.32 (m, 1H), 2.13-2.00 (m, 1H), 1.15-1.03 (m, 9H); LCMS: 95.5%, m/z [M+H]+=692.0; (epimerized material-absolute stereochemistry not determined).
251c: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 8.01 (d, J=2.0 Hz, 1H), 7.77 (br s, 1H), 7.68-7.65 (m, 1H), 7.55-7.45 (m, 2H), 5.78-5.71 (m, 2H), 5.56-5.43 (m, 2H), 4.45 (d, J=10.0 Hz, 1H), 3.96-3.92 (m, 1H), 3.76-3.71 (m, 1H), 3.48-3.37 (m, 1H), 3.24 (s, 3H), 2.72-2.67 (m, 1H), 2.55-2.50 (m, 1H), 2.38-2.23 (m, 1H), 1.10-1.06 (m, 18H); LCMS: 93.7%, m/z [M+H]+=806.0.
Thionyl chloride (10 mL) was added to 110.4_1a (500 mg, 1.08 mmol) at RT. After stirring for 2 h, the excess thionyl chloride was removed under reduced pressure to afford intermediate acid chloride. To the intermediate acid chloride in CH2Cl2 (5 mL) was added a solution of 3,5-dichloro-N-methylaniline (380 mg, 2.16 mmol) in CH2Cl2 (5 mL) at RT. After stirring for 16 h at RT, the reaction mixture was quenched with water (10 mL). The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 (2×10 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 30% EtOAc in pet ether) to afford 252.1 (510 mg, 74%) as a solid.
1H NMR (500 MHz, DMSO-d6) (exist in rotameric form): 11.10/11.05 (br s, 1H), 8.20/7.76 (d, J=1.5 Hz, 1H), 7.68/7.57 (t, J=2.0 Hz, 1H), 7.50-7.42 (m, 3H), 5.44-5.38 (m, 1H), 5.10-5.06 (m, 2H), 4.26-4.19 (m, 2H), 4.13 (d, J=7.5 Hz, 1H), 3.84-3.81 (m, 1H), 3.63-3.61 (m, 1H), 3.41/3.24 (s, 3H), 3.32-3.24 (m, 1H), 2.64-2.58 (m, 1H), 2.40-2.36 (m, 1H), 2.19-2.09 (m, 1H); LCMS: 88.4%, m/z [M+H]+=620.2.
To a stirred solution of 252.1 (500 mg, 0.80 mmol) in THF (10 mL) were added aniline (46 mg, 0.50 mmol) and Pd(PPh3)4 (144 mg, 0.12 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-SELECT-C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/45, 8/80, 10/80, 10.1/98, 13/98, 13.1/45, 15/45 at 18 mL/min] to afford 252.2 (165 mg, 35%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form):12.54 (br s, 1H), 11.07/10.99 (br s, 1H), 8.30/7.88 (d, J=1.5 Hz, 1H), 7.66-7.43 (m, 4H), 4.01 (d, J=7.5 Hz, 1H), 3.84-3.80 (m, 1H), 3.55-3.53 (m, 1H), 3.40/3.24 (s, 3H), 3.32-3.24 (m, 1H), 2.64-2.50 (m, 1H), 2.42-2.32 (m, 1H), 2.14-2.06 (m, 1H); LCMS: 94.7%, m/z [M+H]+=578.0; Chiral purity: 99.8%.
To a stirred solution of 252.2 (300 mg, 0.51 mmol) and 4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (101 mg, 0.77 mmol) in THF (5 mL) were added triphenylphosphine (163 mg, 0.62 mmol) and DIAD (125 mg, 0.62 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (40 g Silica gel cartridge, 30% EtOAc in pet ether) followed by prep. HPLC [X-SELECT-C18 (150×25) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/65, 8/80, 10/90, 10.1/65, 13/65 at 22 mL/min] to afford 252 (120 mg, 33%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.14/11.06 (s, 1H), 8.18 (d, J=2.0 Hz, 1H), 7.76-7.69 (m, 1H), 7.57-7.37 (m, 3H), 4.91-4.87 (m, 1H), 4.68-4.65 (m, 1H), 4.23-4.14 (m, 1H), 3.82-3.81 (m, 1H), 3.66-3.63 (m, 1H), 3.32-3.19 (m, 1H), 3.24 (s, 3H), 2.61-2.54 (m, 1H), 2.41-2.36 (m, 1H), 2.09-2.03 (m, 1H), 2.03 (s, 3H); LCMS: 98.1%, m/z [M+H]+=689.9; Chiral purity: 97.2%.
Thionyl chloride (8 mL) was added to 110.4_1b (400 mg, 0.86 mmol) at RT. After stirring for 2 h, the excess thionyl chloride was removed under reduced pressure to afford intermediate acid chloride. To this acid chloride in CH2Cl2 (5 mL) was added a solution of 3,5-dichloro-N-methylaniline (308 mg, 1.75 mmol) in CH2Cl2 (5 mL) at RT. After stirring for 16 h at RT, the reaction was quenched with water (10 mL). The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 (10 mL×2). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 30% ethyl acetate in pet ether) to afford 253.1 (410 mg, 74%) as a solid.
1H NMR (500 MHz, DMSO-d6) (exist in rotameric form): 11.13/11.05 (br s, 1H), 8.20/7.76 (d, J=2.0 Hz, 1H), 7.68/7.57 (t, J=2.0 Hz, 1H), 7.50-7.42 (m, 3H), 5.42-5.38 (m, 1H), 5.10-5.06 (m, 2H), 4.26-4.19 (m, 2H), 4.13 (d, J=7.5 Hz, 1H), 3.84-3.81 (m, 1H), 3.63-3.61 (m, 1H), 3.41/3.24 (s, 3H), 3.32-3.24 (m, 1H), 2.64-2.58 (m, 1H), 2.42-2.34 (m, 1H), 2.20-2.09 (m, 1H); LCMS: 84.1%, m/z [M+H]+=620.2.
To a stirred solution of 253.1 (400 mg, 0.64 mmol) in THF (10 mL) were added aniline (60 mg, 0.64 mmol) and Pd(PPh3)4 (138 mg, 0.12 mmol) at RT. After stirring for 2 h at RT. After completion of the reaction, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-SELECT-C18 (150×30) mm, 5 μ; A: 0.1% HCOOH in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/45, 8/80, 10/80, 10.1/98, 13/98, 13.1/45, 15/45 at 18 mL/min] to afford 253 (140 mg, 37%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.55 (br s, 1H), 11.07/11.00 (s, 1H), 8.30/7.88 (d, J=2.0 Hz, 1H), 7.66/7.56 (t, J=2.0 Hz, 1H), 7.49-7.43 (m, 2H), 4.02 (d, J=8.0 Hz, 1H), 3.84-3.80 (m, 1H), 3.56-3.53 (m, 1H), 3.40/3.24 (s, 3H), 3.32-3.24 (m, 1H), 2.64-2.54 (m, 1H), 2.41-2.31 (m, 1H), 2.17-2.02 (m, 1H); LCMS: 90.8%, [M+H]+=578.0; Chiral purity: 99.8%.
To a stirred solution of 253.2 (300 mg, 0.51 mmol) and 4-(hydroxymethyl)-5-methyl-1,3-dioxol-2-one (101 mg, 0.77 mmol) in THF (5 mL) was added triphenyl phosphine (163 mg, 0.62 mmol) and DIAD (125 mg, 0.62 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (40 g Silica gel cartridge, 30% EtOAc in pet ether) followed by prep. HPLC [KROMOSIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/50, 8/70, 12/98, 14/98, 14.1/50, 16/50 at 22 mL/min] to afford 253 (134 mg, 37%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.10/11.06 (s, 1H), 8.18 (d, J=2.0 Hz, 1H), 7.76-7.69 (m, 1H), 7.57-7.37 (m, 3H), 4.91-4.87 (m, 1H), 4.68-4.65 (m, 1H), 4.23-4.14 (m, 1H), 3.82-3.81 (m, 1H), 3.66-3.63 (m, 1H), 3.31-3.21 (m, 1H), 3.24 (s, 3H), 2.60-2.54 (m, 1H), 2.40-2.36 (m, 1H), 2.11-2.02 (m, 1H), 2.02 (s, 3H); LCMS: 99.0%, m/z [M+H]+=689.9; Chiral purity: 99.5%.
To a stirred solution of 252.2 (350 mg, 0.60 mmol) and chloromethyl pivalate (273 mg, 1.81 mmol) in CH2Cl2:CH3CN (4:1, 5 mL) was added DBU (275 mg, 1.81 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was washed with water (5 mL), brine (5 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by prep. HPLC [X BRIDGE-C18 (150×25) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/65, 8/85, 10/90, 14/98, 17/98, 17.1/65, 20/65 at 24 mL/min] to afford 254a (95 mg, 22%) as a solid and 254b (58 mg, 14%) (epimerized material) as a solid.
254a: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.15/11.06 (br s, 1H), 8.20 (d, J=1.5 Hz, 1H), 7.84-7.70 (m, 1H), 7.55-7.44 (m, 3H), 5.54-5.47 (m, 2H), 4.15-4.13 (m, 1H), 3.80-3.76 (m, 1H), 3.68-3.65 (m, 1H), 3.21 (s, 3H), 3.20-3.10 (m, 1H), 2.60-2.54 (m, 1H), 2.38-2.33 (m, 1H), 2.07-2.04 (m, 1H), 1.07/1.03 (m, 9H); LCMS: 99.4%, m/z [M+H]+=691.9; Chiral purity: 94.6%. Stereochemistry was confirmed by 2D NMR studies.
254b: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.08/10.92 (br s, 1H), 7.84 (d, J=1.5 Hz, 1H), 7.77 (s, 1H), 7.54-7.44 (m, 3H), 5.60-5.50 (m, 2H), 4.34 (d, J=10.5 Hz, 1H), 4.11-3.97 (m, 1H), 3.77-3.71 (m, 1H), 3.50-3.41 (m, 1H), 3.23 (s, 3H), 2.75-2.70 (m, 1H), 2.55-2.45 (m, 1H), 2.31-2.20 (m, 1H), 1.07 (s, 9H); LCMS: 97.8%, m/z [M+H]+=691.9; Chiral purity: 95.7%. Stereochemistry was confirmed by 2D NMR studies.
To a stirred solution of 253.2 (350 mg, 0.60 mmol) and chloromethyl pivalate (273 mg, 1.81 mmol) in CH2Cl2:CH3CN (4:1, 5 mL) was added DBU (275 mg, 1.81 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was washed with water (5 mL), brine solution (5 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [KROMOSIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/70, 8/90, 10/95, 12/98, 14/98, 14.1/70, 16/70 at 18 mL/min] to afford 255a (65 mg, 15%) as a solid and 255b (108 mg, 25%) as a solid. 255a: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.07/10.91 (s, 1H), 7.84 (s, 1H), 7.77 (s, 1H), 7.54-7.44 (m, 3H), 5.56-5.54 (m, 2H), 4.43-4.33 (m, 1H), 4.03-3.99 (m, 1H), 3.77-3.71 (m, 1H), 3.50-3.40 (m, 1H), 3.23 (s, 3H), 2.75-2.64 (m, 1H), 2.54-2.50 (m, 1H), 2.47-2.25 (m, 1H), 1.07 (s, 9H); LCMS: 99.2%, m/z [M+H]+=692.0; Chiral purity: 99.6%.
255b: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.06/10.90 (br s, 1H), 8.20 (d, J=1.5 Hz, 1H), 7.84-7.70 (m, 1H), 7.55-7.45 (m, 3H), 5.54-5.47 (m, 2H), 4.18-4.13 (m, 1H), 3.80-3.76 (m, 1H), 3.68-3.65 (m, 1H), 3.21 (s, 3H), 3.21-3.10 (m, 1H), 2.65-2.50 (m, 1H), 2.40-2.30 (m, 1H), 2.11-1.98 (m, 1H), 1.15-1.03 (m, 9H); LCMS: 99.6%, m/z [M+H]+=691.9; Chiral purity: 97.1%.
To a stirred solution of 252.2 (300 mg, 0.52 mmol) in DCM (6 mL) and CH3CN (1.5 mL) were added DBU (87 mg, 0.52 mmol) and bromomethyl acetate (87 mg, 0.52 mmol) at 0° C. After stirring at RT for 12 h, the reaction mixture was diluted with water (30 mL) and extracted with DCM (2×50 mL). The combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC (Column: X-BRIDGE-C18 (150×30) mm, 5 μ; A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient: 0/60,8/80,10/85,13/98,15/98,15.1/60,18/60 at 23 mL/min) to afford 256a (Peak-1, 30 mg, 9%) as an off white solid and 256b (Peak-2, 60 mg, 18%) as a solid.
256a: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.10/10.93 (s, 1 H), 7.81 (d, J=1.5 Hz, 1H), 7.77 (s, 1H), 7.56-7.44 (m, 3H), 5.54 (d, J=6.0 Hz, 1H), 5.45 (d, J=6.0 Hz, 1H), 4.33 (d, J=10.5 Hz, 1H), 4.01-3.97 (m, 1H), 3.78-3.73 (m, 1H), 3.51-3.44 (m, 1H), 3.23 (s, 3H), 2.77-2.72 (m, 1H), 2.51-2.49 (m, 1H), 2.37-2.36 (m, 1H), 2.01 (s, 3H); LCMS: 96.7%, m/z [M+H]+=650.0; Chiral purity: 99.9%.
256b: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.10/11.05 (s, 1H), 8.18/7.75 (d, J=2.0 Hz, 1H), 7.70 (t, J=2.0 Hz, 1H), 7.58-7.45 (m, 3H), 5.52/5.48 (d, J=6.0 Hz, 1H), 5.40-5.38 (m, 1H), 4.14 (d, J=7.5 Hz, 1H), 3.80-3.77 (m, 1H), 3.68-3.65 (m, 1H), 3.40/3.23 (s, 3H), 3.21-3.17 (m, 1H), 2.64-2.63 (m, 1H), 2.37-2.35 (m, 1H), 2.11-2.08 (m, 1H), 1.97/1.89 (s, 3H); LCMS: 98.0%, m/z [M+H]+=650.1; Chiral purity: 99.5%.
257 was synthesized from 250.2 following the procedure described for the synthesis of 256a and 256b.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.15/11.05 (s, 1H), 8.18/7.75 (d, J=2.0 Hz, 1H), 7.69/7.57 (t, J=2.0 Hz, 1H), 7.51-7.45 (m, 3H), 5.52/5.48 (d, J=6.0 Hz, 1H), 5.40-5.38 (m, 1H), 4.14 (d, J=7.5 Hz, 1H), 3.82-3.77 (m, 1H), 3.66 (t, J=7.0 Hz, 1H), 3.40/3.23 (s, 3H), 3.21-3.17 (m, 1H), 2.61-2.51 (m, 1H), 2.42-2.30 (m, 1H), 2.15-2.00 (m, 1H), 1.97/1.89 (s, 3H); LCMS: 97.5%, m/z [M+H]+=650.
To a stirred solution of 252.2 (400 mg, 0.69 mmol) and 2-bromo-N,N-dimethylacetamide (230 mg, 1.38 mmol) in CH2Cl2:CH3CN (4:1, 8 mL) was added DBU (210 mg, 1.38 mmol) at RT. After stirring for 16 h at RT, the reaction mixture was diluted with CH2Cl2 (10 mL). The organic layer was washed with water (10 mL), brine (10 mL), dried over Na2SO4 and filtered. The organic layer was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-BRIDGE-C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/60, 8/80, 11/90, 11.1/98, 13/98, 13.1/60, 16/60 at 18 mL/min] to afford 258 (198 mg, 43%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.13/11.05 (s, 1H), 8.17/7.75 (d, J=2.0 Hz, 1H), 7.68-7.67 (m, 1H), 7.56-7.45 (m, 3H), 4.69-4.44 (m, 2H), 4.25-4.07 (m, 1H), 3.84-3.81 (m, 1H), 3.74-3.71 (m, 1H), 3.22 (s, 3H), 3.19-3.00 (m, 1H), 2.83/2.82 (s, 3H), 2.77 (s, 3H), 2.63-2.57 (m, 1H), 2.35-2.34 (m, 1H), 2.13-2.10 (m, 1H); LCMS: 99.2%, m/z [M+H]+=663.2; Chiral purity: 97.3%.
To a stirred solution of 252.2 (400 mg, 0.69 mmol) in THF (8 mL) was added N-methylmorpholine (140 mg, 1.38 mmol) and isobutyl chloroformate (141 mg, 1.38 mmol) at 0° C. After 30 minutes, N-(2-mercaptoethyl)acetamide (246 mg, 2.07 mmol) was added at 0° C. After stirring for 16 h at RT, the reaction mixture was quenched with water and extracted with EtOAc (2×10 mL). The combined organic layer was washed with water (20 mL), brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: KROMASIL C18 (150×25) mm, 10 μ; A: 10 mM Ammonium bicarbonate in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/50, 8/80, 10/80, 10.1/98, 11/98, 11.1/50, 14/50 at 20 mL/min] to afford 259 (38 mg, 8%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.00 (br s, 1H), 7.93-7.76 (m, 3H), 7.57-7.46 (m, 3H), 4.68-4.35 (m, 1H), 4.07-4.03 (m, 1H), 3.80-3.79 (m, 1H), 3.54-3.49 (m, 1H), 3.27 (s, 3H), 3.00-2.98 (m, 2H), 2.78-2.73 (m, 3H), 2.57-2.50 (m, 1H), 2.36-2.23 (m, 1H). 1.75 (s, 3H); LCMS: 98.6%, m/z [M+H]+=679.2.
To a stirred solution of 252.2 (900 mg, 1.55 mmol) in DMF (20 mL) were added HATU (709 mg, 1.86 mmol) and DIPEA (0.5 mL, 3.10 mmol,) at RT. After 15 minutes, N-methylhydroxylamine (193 mg, 2.33 mmol) was added. After stirring for 16 h at RT, the reaction mixture was diluted with ice-water (20 mL) and extracted with EtOAc (2×20 mL). The combined organic layer was washed with cold water (20 mL), brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: KROMASIL C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/50, 8/65, 11/65, 11.1/98, 12/98, 12.1/50, 14/50 at 23 mL/min] to afford 260a (83 mg) as off-white solid and 260b (55 mg, 6%) as off-white solid.
260a: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 10.88/10.78 (br s, 1 H), 10.07/9.95 (br s, 1H), 7.72 (s, 1H), 7.54-7.30 (m, 4H), 4.63-4.41 (m, 1H), 4.12 (m, 1H), 3.51-3.39 (m, 2H), 3.37/3.22 (s, 3H), 2.99/2.97 (s, 3H), 2.74-2.69 (m, 1H), 2.60-2.50 (m, 1H), 2.22-2.10 (m, 1H); LCMS: 98.3%, m/z [M+H]+=607.2; Chiral Purity: 98.9%.
260b: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 10.69 (s, 1H), 9.81 (s, 1H), 7.93 (s, 1H), 7.63 (s, 1H), 7.51 (s, 2H), 7.45 (d, J=2.0 Hz, 1H), 4.07 (d, J=7.5 Hz, 1H), 3.83-3.79 (m, 1H), 3.50-3.44 (m, 1H), 3.36 (t, J=6.5 Hz, 1H), 3.24 (s, 3H), 2.72 (s, 3H), 2.50-2.33 (m, 2H), 2.21-2.12 (m, 1H); LCMS: 95.4%, m/z [M+H]+=607.1; Chiral Purity: 87.9%.
To a stirred solution of 252.2 (400 mg, 0.69 mmol) in DMF (6 mL) were added HATU (395 mg, 1.04 mmol) and DIPEA (0.23 mL, 1.39 mmol) at RT. After 15 minutes, benzenesulfonamide (163 mg, 1.04 mmol) was added. After stirring at RT for 48 h, the reaction mixture was diluted with ice-cold water (80 mL) and extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine (50 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X BRIDGE C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: 0/60, 8/75, 10/75, 10.1/98, 12/98, 12.1/60, 15/60 at 23 mL/min] to afford 261 (102 mg, 21%) as a solid.
1H NMR (300 MHz, DMSO-d6) (Exist in rotameric form): 12.00 (br s, 1H), 10.85/10.65 (br s, 1H), 7.73-7.40 (m, 10H), 4.65-4.52 (m, 1H), 4.28-4.10 (m, 1H), 4.01-3.92 (m, 1H), 3.70-3.60 (m, 1H), 3.20 (s, 3H), 3.00-2.80 (m, 1H), 2.51-2.40 (m, 1H), 2.15-2.00 (m, 1H); LCMS: 94.2%, m/z [M+H]+=716.9; Chiral purity: 94.5%.
To a stirred solution of 265.1 (500 mg, 2.77 mmol) in MeOH (10 mL) were added paraformaldehyde (0.083 g, 2.77 mmol) and AcOH (0.1 mL, cat.) at RT. After stirring for 3 h at RT, NaCNBH3 (523 mg, 8.33 mmol) was added. After stirring for 16 h at 60° C., the reaction mixture was evaporated under reduced pressure, diluted with H2O (25 mL) and extracted with EtOAc (2×15 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by (Silica gel 100-200 mesh, 2% EtOAc/hexane) to afford 265.2 (200 mg, 37%) as a yellow liquid.
1H NMR (400 MHz, CDCl3): 6.69-6.63 (m, 1H), 6.55-6.49 (m, 1H), 4.12 (br s, 1H), 2.87 (d, J=5.2 Hz, 3H); LCMS: 94.7%, m/z [M+H]+=194.0.
Thionyl chloride (5 mL) was added to compound 110.4_1 (350 mg, 0.76 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure. To the intermediate acid chloride in CH2Cl2 (3 mL) was added a solution of 265.2 (221 mg, 1.13 mmol) in CH2Cl2 (2 mL). After stirring for 16 h at RT, the reaction mixture was quenched with water (25 mL) and extracted with CH2Cl2 (2×15 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by (Silica gel 100-200 mesh, 10% EtOAc/hexane) to afford 265.3 (200 mg, 41%) as a solid. LCMS: 57.1%, m/z [M−H]−=636.2.
To a stirred solution of 265.3 (200 mg, 0.31 mmol) in THF (5 mL) were added aniline (29.0 mg, 0.31 mmol) and Pd(PPh3)4 (72.0 mg, 0.062 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by chiral SFC [Chiralpak IE (30×250) mm, 5 μ; 80% CO2: 20% of 0.5% isopropylamine in isopropanol at RT (Isocratic 70 g/min, with detection at 214 nm) to afford 265a (7 mg) and 265b (7 mg) as a solid.
265a: 1H NMR (300 MHz, DMSO-d6) (Exist in rotameric form): 12.83-12.14 (br s, 1H), 10.99 (br s, 1H), 8.26/8.22 (s, 1H), 7.88-7.82 (m, 2H), 7.70-7.43 (m, 1H), 4.15-3.88 (m, 3H), 3.31-3.26 (m, 1H), 3.20 (s, 3H), 2.73-2.43 (m, 2H), 2.10-1.95 (m, 1H); LCMS: 98.1%, m/z [M+H]+=595.9, Chiral purity: 96.9%. Absolute stereochemistry was not determined.
265b: 1H NMR (300 MHz, DMSO-d6) (Exist in rotameric form): 11.19-10.24 (br s, 1H), 8.28/8.22 (br s, 1H), 7.83-7.38 (m, 3H), 4.13-3.83 (m, 4H), 3.32-3.25 (m, 1H), 3.18 (s, 3H), 2.60-2.44 (m, 1H), 2.30-2.25 (m, 1H); LCMS: 95.7%, m/z [M+H]+=595.9; Chiral purity: 90.9%. Absolute stereochemistry was not determined.
Thionyl chloride (10 mL) was added to 82.4_1 (500 mg, 1.17 mmol). After stirring at RT for 2 h, the reaction mixture was concentrated under reduced pressure to afford acid chloride intermediate. To the acid chloride intermediate was added a solution of 3,5-dichloro-N-methylaniline (395 mg, 2.26 mmol) in CH2Cl2 (10 mL). After stirring at RT for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash chromatography (Silica gel, 15% EtOAc/pet ether) to afford 266.1 (200 mg, 29%) as a pale brown solid. LCMS: 96.1%, m/z [M−H]−=581.8.
To a stirred solution of 266.1 (100 mg, 0.17 mmol) in THF (5 mL) were added aniline (16.1 mg, 0.17 mmol) and Pd(PPh3)4 (39.6 mg, 0.03 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Kromasil C8 (150×25), 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient:(T%B): −0/60, 8/90, 9/80, 9.1/80, 10/98, 10.1/60, 14/60 at 25 mL/min] to afford 266 (60 mg, 9% yield) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.47 (brs, 1H), 10.98 (br s, 1H), 8.41/7.91 (d, J=1.5 Hz, 1H), 7.68-7.54 (m, 1H), 7.47-7.40 (m, 3H), 4.09 (d, J=8.0 Hz, 1H), 3.80-3.77 (m, 1H), 3.59-3.50 (m, 1H), 3.41/3.23(s, 3H), 2.88-2.79 (m, 1H), 2.30-2.19 (m, 1H), 1.98-1.88 (m, 1H), 1.75-1.70 (m, 2H), 1.60-1.50 (m, 1H); LCMS: 91.4%, m/z [M+H]+=542.1.
To a stirred solution of 267.1 (5.0 g, 19.4 mmol) in DCM (50 mL) was added DAST (7.8 mL, 58.3 mmol) dropwise at −78° C. After warming to RT and stirred for 16 h at RT, the reaction mixture was quenched with sat. NaHCO3 solution at 0° C. and extracted with DCM (2×20 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (80 g Silica gel cartridge, 20%-30% EtOAc/pet ether) to afford 267.2 (5.0 g, 92%) as liquid. 1H NMR (500 MHz, CDCl3) (Exist in rotameric form): 4.44-4.40 (m, 1H), 4.30-4.23 (m, 2H), 3.78-3.75 (m, 1H), 3.57-3.55 (m, 1H), 2.60-2.30 (m, 2H), 1.48-1.42 (m, 9H), 1.33-1.29 (m, 3H).
To 267.2 (8 g, 28.6 mmol) was added 6N HCl (80 mL) at RT. After stirring at 65° C. for 6 h, the reaction mixture was concentrated under reduced pressure. The crude residue was washed 3 times with EtOAc:DCM (1:3; 40 mL) and dried under high vacuum to afford 267.3 (5.6 g, 73%) as a brown solid.
1H NMR (400 MHz, D2O): 4.64-4.58 (m, 1H), 3.66 (t, J=7.6 Hz, 2H), 2.74-2.62 (m, 2H).
To a solution of (Z)-4-(allyloxy)-4-oxobut-2-enoic acid (4.5 g, 28.8 mmol) in EtOH (50 mL) were added 267.3 (5.39 g, 28.8 mmol) and 5,7-dichloroindoline-2,3-dione (6.23 g, 28.8 mmol) at RT. After stirring at reflux for 2 h, the reaction mixture was concentrated. The residue was purified by flash chromatography (80 g Silica gel cartridge, 100% EtOAc) to afford 267.4 (4.2 g, 32%) as a brown solid.
LCMS: (28+32+10+6) %, m/z [M+H]+=461.0).
To a stirred solution of 267.4 (1.5 g, 3.25 mmol) in DMF (10 mL) were added DIPEA (1.7 mL, 9.75 mmol) and HATU (1.48 g, 3.90 mmol) at RT. After stirring for 10 minutes, 2-(3-(tert-butyl)phenyl)ethan-1-amine (0.86 g, 4.87 mmol) was added. After stirring for 3 h at RT, the reaction mixture was poured into ice cold water and stirred for 10 minutes. The resulting precipitate was filtered, washed with cold water (2×20 mL) and dried under vacuum (1.3 g). The residue was purified by prep. HPLC [Column: X-SELECT-C18 (150×19) mm, 5 μ; A: 10 mM Ammonium bicarbonate in H2O, B:Acetonitrile; Gradient: (T%B): −0/50, 8/80, 10/80, 14/98, 16/98, 16.1/50, 18/50 at 18 mL/min] to afford major diastereomer (600 mg) which was further purified by flash chromatography (80 g Silica gel cartridge, 20%-30% EtOAc/pet ether) to afford 267.5 (200 mg, 10%) as a solid.
1HNMR (400 MHz, DMSO-d6): 11.20 (s, 1H), 8.57-8.54 (m, 1H), 7.58 (d, J=1.6 Hz, 1H), 7.23-7.14 (m, 3H), 7.07-7.01 (m, 2H), 5.33-5.24 (m, 1H), 5.06-5.02 (m, 2H), 4.21-4.16 (m, 2H), 4.01-3.96 (m, 3H), 3.67-3.61 (m, 2H), 2.72-2.50 (m, 4H), 2.50-2.33 (m, 2H), 1.27 (s, 9H); LCMS: 95.5%, m/z [M+H]+=620.0).
To a stirred solution of 267.5 (200 mg, 0.33 mmol) in THF (2 mL) were added aniline (30 mg, 0.32 mmol) and Pd(PPh3)4 (74 mg, 0.064 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [X BRIDGE-C18 (150×25), 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/60, 8/90, 8.1/98, 10/98, 10.1/60, 13/60 at 24 mL/min] to afford 267 (160 mg, 85%) as a solid.
1H NMR (500 MHz, DMSO-d6): 12.65 (br s, 1H), 11.13 (br s, 1H), 8.49 (br s, 1H), 7.57 (s, 1H), 7.23-7.18 (m, 3H), 7.11 (s, 1H), 7.07-7.01 (m, 1H), 4.02-3.90 (m, 2H), 3.60-3.56 (m, 1H), 3.32-3.22 (m, 2H), 2.71-2.67 (m, 2H), 2.50-2.22 (m, 4H), 1.27 (s, 9H); LCMS: 97.8%; m/z [M+H]+=580.1; absolute stereochemistry not known. Tested as racemate.
To a stirred solution of 267.4 (500 mg, 1.08 mmol) in DCM (8 mL) were added 3,5-dichloro-N-methylaniline (230 mg, 1.30 mmol), TEA (0.36 mL, 2.61 mmol) and DMAP (39 mg, 0.32 mmol) at RT. After stirring for 30 min, POCl3 (0.10 mL, 1.084 mmol) dissolved in DCM (1 mL) was added. After stirring for 16 h at RT, the reaction mixture was quenched with water (5 mL) and extracted with DCM (2×10 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced. The residue was purified by prep. HPLC [X-BRIDGE C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/70, 8/90, 10/90, 10.1/98, 12/98, 12.1/70, 15/70 at 20 mL/min] to afford 268.1a (Peak-1, 390 mg, 58%) as a white solid and 268.1b (Peak-2, 140 mg, 21%) as a white solid.
268.1a: 1H NMR (400 MHz, DMSO-d6): 11.05 (br s, 1H), 7.90-7.44 (m, 5H), 5.80-5.64 (m, 1H), 5.25-4.90 (m, 2H), 4.90-4.75 (m, 1H), 4.39 (br s, 2H), 3.90-3.50 (m, 2H), 3.40-3.10 (m, 4H), 2.57-2.50 (m, 1H), 2.32-2.25 (m, 2H); LCMS: 95.8%, m/z [M+H]+=620.0.
268.1b: 1H NMR (400 MHz, DMSO-d6): 11.24 (s, 1H), 7.78 (s, 1H), 7.58-7.56 (m, 3H), 6.42 (s, 1H), 5.35-5.27 (m, 1H), 5.07-5.03 (m, 2H), 4.31-4.19 (m, 3H), 3.93-3.88 (m, 1H), 3.78-3.73 (m, 1H), 3.40-3.20 (m, 4H), 2.63-2.50 (m, 1H), 2.30-2.21 (m, 2H); LCMS: 86.9%, m/z [M+H]+=620.2.
To a stirred solution of 268.1a (400 mg, 0.65 mmol) in THF (8 mL) were added aniline (60 μL, 0.65 mmol) and Pd(PPh3)4 (149 mg, 0.13 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [X-SELECT-C18 (150×19), 5 μ; A: 10 mM NH4HCO3 in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/20, 8/70, 8.1/98, 11/98, 11.1/20, 13/20 at 18 mL/min] to afford 268a (86 mg, 23%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.90-12.70 (br s, 1H), 11.00-10.80 (br s, 1H), 7.61-6.50 (m, 5H), 4.84-4.79 (m, 1H), 4.60-4.30 (m, 1H), 3.85-3.68 (m, 1H), 3.32 (s, 3H), 3.30-3.10 (m, 1H), 2.60-2.50 (m, 1H), 2.40-2.15 (m, 2H); LCMS: 86.1%; m/z [M+H]+=578.0; absolute stereochemistry not known. Tested as racemate.
To a stirred solution of 268.1b (150 mg, 0.24 mmol) in THF (3 mL) were added aniline (20 μL, 0.24 mmol) and Pd(PPh3)4 (55 mg, 0.05 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [KROMOSIL-C18 (150×25 mm), 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/50, 8/75, 9/75, 9.1/98, 11/98, 11.1/50, 13/50 at 23 mL/min] to afford 268b (50 mg, 35%) as a solid.
1H NMR (500 MHz, DMSO-d6): 13.01 (br s, 1H), 11.17 (s, 1H), 7.76-7.55 (m, 4H), 6.43 (s, 1H), 4.10 (d, J=11.0 Hz, 1H), 3.88-3.84 (m, 1H), 3.73-3.68 (m, 1H), 3.27 (s, 3H), 2.64-2.59 (m, 1H), 2.50-2.36 (m, 1H), 2.25-2.14 (m, 2H); LCMS: 99.3%, m/z [M+H]+=578.1; absolute stereochemistry not known. Tested as racemate.
To a stirred solution of 269.1 (2 g, 13.9 mmol) in EtOH (70 mL) were added (Z)-4-(allyloxy)-4-oxobut-2-enoic acid (2.18 g, 13.9 mmol) and 5,7-dichloroindoline-2,3-dione (3 g, 13.9 mmol) at RT. After stirring for 3 h at 80° C., the reaction mixture was cooled to RT and concentrated under vacuum. The residue was purified by flash chromatography (80 g Silica gel cartridge, 40% EtOAc in pet ether) to afford major diastereomer 269.2_1 (750 mg, 12%) as a solid and minor diastereomer 269.2_2 (36 mg) as a solid.
269.2_1: 1H NMR (500 MHz, DMSO-d6): 12.78 (br s, 1H), 11.04 (s, 1H), 7.89 (d, J=2.0 Hz, 1H), 7.42 (d, J=2.5 Hz, 1H), 5.47-5.39 (m, 1H), 5.08-5.04 (m, 2H), 4.40-4.35 (m, 2H), 4.25 (d, J=5.5 Hz, 2H), 3.94 (d, J=7.5 Hz, 1H), 3.65 (t, J=7.0 Hz, 1H). Regio chemistry and relative stereochemistry was confirmed by 2D NMR studies.
269.2_2: 1H NMR (500 MHz, DMSO-d6): 12.65 (br s, 1H), 10.98 (br s, 1H), 7.98 (d, J=2.0 Hz, 1H), 7.42 (d, J=2.0 Hz, 1H), 5.96-5.89 (m, 1H), 5.42-5.37 (m, 2H), 4.65-4.57 (m, 2H), 4.42-4.38 (m, 2H), 3.88-3.71 (m, 1H), 3.69-3.65 (m, 1H); LCMS: 90.5%, m/z [M+H]+=453.2.
To a stirred solution of 269.2 (700 mg, 1.54 mmol) in pyridine (10 mL) was added EDC-HCl (592 mg, 3.09 mmol) at RT. After stirring for 10 minutes, 3,5-dichloro-N-methylaniline (408 mg, 2.31 mmol) was added. After stirring for 2 h at RT, the reaction mixture was evaporated under reduced pressure. The residue was purified by column chromatography (Silica gel 100-200 mesh, 20% EtOAc in pet ether) to afford 269.3 (180 mg, 19%) as a solid.
269.3 (280 mg) was purified by chiral SFC (Chiralcel OX-H (30 ×250) mm, 5 μ; 50% CO2: 50% Acetonitrile at RT (Isocratic 90 g/min, with detection at 214 nm). Pure fractions were concentrated under reduced pressure to give of 269.3a (Enantiomer-1, 90 mg, 64%) as a solid and of 269.3b (Enantiomer-2, 90 mg, 64%) as a solid.
269.3a: 1H NMR (500 MHz, DMSO-d6): 11.24 (s, 1H), 7.78 (br s, 1H), 7.61-7.55 (m, 2H), 7.49 (br s, 1H), 6.51 (br s, 1H), 5.33-5.28 (m, 1H), 5.06-5.03 (m, 2H), 4.57 (d, J=6.0 Hz, 1H), 4.24-4.18 (m, 3H), 3.85 (d, J=11.0 Hz, 1H), 3.76-3.72 (m, 1H), 3.25 (s, 3H); LCMS: 90.5%, m/z [M+H]+=611.9; Chiral purity: 99.8%.
269.3b: 1H NMR (500 MHz, DMSO-d6): 11.24 (s, 1H), 7.78 (br s, 1H), 7.66-7.55 (m, 2H), 7.49 (br s, 1H), 6.51 (br s, 1H), 5.33-5.28 (m, 1H), 5.06-5.03 (m, 2H), 4.57 (d, J=5.5 Hz, 1H), 4.24-4.18 (m, 3H), 3.85 (d, J=11.0 Hz, 1H), 3.77-3.72 (m, 1H), 3.26 (s, 3H); LCMS: 90.2%, m/z [M+H]+=611.9.
To a stirred solution of 269.3a (80 mg, 0.13 mmol) in THF (3 mL) were added aniline (18 mg, 0.19 mmol) and Pd(PPh3)4 (30 mg, 0.026 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was diluted with water (10 mL) and acidified with 1N HCl. The reaction mixture was extracted with EtOAc (20 mL). The organic layer was washed with brine (10 mL), dried over Na2SO4, filtered and evaporated. The residue was purified by prep. HPLC [Column: X BRIDGE-C18 (150×25), 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient (T%B): −0/50, 8/80, 10/80, 10.1/98, 12/98, 12.1/50, 15/50 at 22 mL/min] to afford 269a (40 mg, 38%) as a solid.
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 13.08 (br s, 1H), 11.20 (br s, 1H), 7.76-7.32 (m, 4H), 6.53 (br s, 1H), 4.49-4.46 (m, 1H), 4.14-4.11 (m, 1H), 3.81-3.58 (m, 2H), 3.27 (s, 3H); LCMS: 99.4%, [M−H]−=567.9. (absolute stereochemistry was not determined).
To a stirred solution of 269.3b (90 mg, 0.15 mmol) in THF (3 mL) were added aniline (21 mg, 0.22 mmol) and Pd(PPh3)4 (17 mg, 0.015 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was diluted with water (10 mL) and acidified with 1N HCl. The reaction mixture was extracted with EtOAc (20 mL) and the orgaic layer was washed with brine (10 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X BRIDGE-C18 (150×25), 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient (T%B): —0/50, 8/70, 10/70, 11.4/80, 11.15/98, 13/98, 13.1/50, 16/50 at 22 mL/min] to afford 269b (40 mg, 38%) as a sold.
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 13.04 (br s, 1H), 11.20 (br s, 1H), 7.76-7.33 (m, 4H), 6.52 (br s, 1H), 4.55-4.42 (m, 1H), 4.20-4.10 (m, 1H), 3.82-3.68 (m, 2H), 3.27 (s, 3H); LCMS: 99.0% [M−H]−=568.0. (absolute stereochemistry was not determined)
270a and 270b were synthesized from 269.2_1 following procedure described for the synthesis of 269a and 269b.
270a: 1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.69 (br s, 1H), 11.04 (br s, 1H), 8.23/7.93 (d, J=1.5 Hz, 1H), 7.69-7.26 (m, 4H), 4.06-3.98 (m, 2H), 3.87 (d, J=7.0 Hz, 1H), 3.80-3.76 (m, 1H), 3.66 (t, J=6.5 Hz, 1H), 3.44-3.40 (m, 1H), 0.89-0.85 (m, 1H), 0.42-0.36 (m, 2H), 0.19-0.12 (m, 1H), 0.02-0.00 (m, 1H); LCMS: 98.1% [M+H]+=609.9; Chiral purity: 99.0%. Absolute stereo chemistry is unknown.
270b:
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.69 (br s, 1H), 11.04 (s, 1H), 8.23/7.93 (d, J=1.5 Hz, 1H), 7.69-7.26 (m, 4H), 4.06-3.98 (m, 2H), 3.87 (d, J=7.5 Hz, 1H), 3.80-3.76 (m, 1H), 3.66 (t, J=6.5 Hz, 1H), 3.44-3.40 (m, 1H), 0.89-0.85 (m, 1H), 0.42-0.37 (m, 2H), 0.19-0.16 (m, 1H), 0.03-0.00 (m, 1H); LCMS: 96.2% [M+H]+=609.9; Chiral purity: 97.6%. Absolute stereo chemistry is unknown.
To a stirred solution of 269.3 (250 mg, 0.41 mmol) in acetonitrile (5 mL) was added K2CO3 (169 mg, 1.22 mmol) at RT. After stirring for 30 minutes at RT, MeI (0.05 mL, 0.81 mmol) was added. After stirring for 16 h at RT, the reaction mixture was quenched with water (25 mL) and extracted with EtOAc (50 mL). The organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (Silica gel 100-200 mesh, 10% EtOAc in pet ether) to afford 271.1 (130 mg, 51%) as a brown solid.
1H NMR (400 MHz, DMSO-d6): 7.78 (br s, 1H), 7.66-7.58 (m, 2H), 7.52 (d, J=1.6 Hz, 1H), 7.36 (br s, 1H), 6.54 (br s, 1H), 5.39-5.32 (m, 1H), 5.13-5.05 (m, 2H), 4.53 (d, J=6.4 Hz, 1H), 4.27-4.15 (m, 3H), 3.90-3.80 (m, 1H), 3.78-3.69 (m, 1H), 3.44 (s, 3H), 3.26 (s, 3H); LCMS: 93.9%, m/z [M+H]+=626.00.
To a stirred solution of 271.1 (120 mg, 0.19 mmol) in THF (3 mL) were added aniline (21 mg, 0.23 mmol) and Pd(PPh3)4 (22 mg, 0.019 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was diluted with water (10 mL) and acidified with 1N HCl. The reaction mixture was extracted with EtOAc (20 mL). The organic layer was washed with brine (10 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-BRIDGE-C18 (150×30), 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/70, 8/90, 10/90, 10.1/98, 12/98, 12.1/70, 15/70 at 20 mL/min) to afford 271 (40 mg, 38%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 13.11 (br s, 1H), 7.82-7.33 (m, 4H), 6.57 (s, 1H), 4.55-4.45 (m, 1H), 4.26-4.16 (m, 1H), 3.80 (d, J=11.5 Hz, 1H), 3.72-3.68 (m, 1H), 3.45 (s, 3H), 3.26 (s, 3H); LCMS: 96.6%, m/z [M+H]+=584.0;
To a stirred solution of 272.1 (3.0 g, 18.5 mmol) in CH3NO2 (150 mL) was added NH4OAc at RT. After stirring for 16 h at 120° C., the reaction mixture was cooled to RT and concentrated under reduced pressure. The residue was purified by column chromatography (Silica gel 100-200 mesh, 2% EtOAc in pet ether) to afford 272.2 (3.4 g, 89%) as liquid.
1H NMR (500 MHz, CDCl3): 8.03 (d, J=13.5 Hz, 1H), 7.60 (d, J=13.5 Hz, 1H), 7.55-7.54 (m, 2H), 7.41-7.37 (m, 2H), 1.35 (s, 9H); LCMS: 95.2%, m/z [M+H]+=206.2.
To a stirred solution of t-BuOK (4.09 g, 36.5 mmol) in DMSO (50 mL) was added TMSOI (8.03 g, 36.5 mmol) portion wise at RT. After stirring for 10 minutes, 272.2 (3.0 g, 14.6 mmol) was added dropwise at RT. After stirring for 30 minutes, the reaction mixture was poured into ice cold water and extracted with EtOAc (2×50 mL). The combined organic layer was washed with brine (25 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified column chromatography (Silica gel 100-200 mesh, 20% EtOAc in pet ether) to afford 272.3 (500 mg, 15%). LCMS: 82.8%, m/z [M+H]+=220.2.
To a stirred solution of 272.3 (140 mg, 0.63 mmol) in isopropanol (15 mL) were added 1N HCl (4.2 mL) and Zn (823 mg, 12.6 mmol) at RT. After stirring for 2 h, the reaction mixture was filtered through a small pad of Celite and the filtrate was concentrated under reduced pressure to afford 272.4 (110 mg) as a pale brown liquid which was used for the next step without purification. LCMS: 70.1%, m/z [M+H]+=190.3.
To a stirred solution of 110.4_1 (364 mg, 0.79 mmol) in DMF (5 mL) were added DIPEA (0.16 mL, 0.94 mmol), HATU (300 mg, 0.79 mmol) and 272.4 (150 mg, 0.79 mmol) at RT. After stirring at RT for 30 minutes. The reaction mixture was diluted with ice cold water. After stirring for 10 minutes, the resulting precipitate was filtered, washed with cold water and dried under vacuum to afford 272.5 (450 mg) which was used in the next step without purification. LCMS: 38.4%, m/z [M+H]+=632.1.
To a stirred solution of 272.5 (450 mg, 0.71 mmol) in THF (10 mL) were added aniline (79 mg, 0.85 mmol) and Pd(PPh3)4 (82 mg, 0.038 mmol) at RT. After stirring at RT for 2 h, the reaction mixture was diluted with water (10 mL) and pH adjusted to pH-6 with 1N HCl. The reaction mixture was extracted with EtOAc (2×20 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-BRIDGE-C18 (150×30), 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (T%B): −0/50, 8/80, 10/80, 12/98, 14/98, 14.1/50, 17/50 at 20 mL/min] to afford mixture of diastereomers 272 (90 mg, 21%) as an off white solid.
1H NMR (500 MHz, DMSO-d6): 12.35 (br s, 1H), 10.98 (s, 1H), 8.51 (br s, 1H), 8.26 (d, J=6.5 Hz, 1H), 7.44-6.88 (m, 5H), 4.03-3.90 (m, 2H), 3.32-3.22 (m, 2H), 2.87-2.85 (m, 1H), 2.61-2.56 (m, 1H), 2.45-2.34 (m, 1H), 2.20-1.90 (m, 2H), 1.28 (s, 9H), 1.28-1.09 (m, 2H); LCMS: 95.48%, m/z [M+H]+=592.2.
To a stirred solution of 273.1 (12.0 g, 118 mmol) in sulfolane (75 mL) was added 273.2 (24.4 g, 58.4 mmol) at RT. After stirring at 120° C. for 16 h, the reaction mixture was cooled to RT, poured into ice cold water and stirred well. The resulting precipitate was filtered, washed with cold water (2×20 mL) and dried under vacuum to afford 273.3 (18 g, 56%) as a white solid.
1H NMR (500 MHz, DMSO-d6): 13.03 (s, 1H), 8.00 (s, 1H), 7.83 (s, 1H), 7.62 (s, 1H); LCMS: 98.2%, m/z [M+H]+=275.0.
To 273.3 (36 g, 131 mmol) was added concentrated H2SO4 (80 mL) and H2O (54 mL) at RT. After stirring at 170° C. for 12 h, the reaction mixture was cooled to RT, poured into ice cold water and stirred well. The resulting precipitate was filtered, washed with cold water (2×50 mL) and dried under vacuum to afford 273.4 (20 g, 66%) as a white solid. LCMS: 99.6%, m/z [M+H]+=232.
To a stirred solution of 273.4 (20 g, 86.6 mmol) in DCM (200 mL) was added DIPEA (16 mL, 95.2 mmol) at 0° C. After stirring for 20 minutes, triflic anhydride (14.5 mL, 86.6 mmol) was dropwise added at 0° C. After stirring for 10 minutes at RT, the reaction mixture was quenched with water (50 mL) and the organic layer was separated. The aq. layer was extracted with DCM (50 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (Silica gel 100-200 mesh; 2%-4% EtOAc/pet ether) to afford 273.5 (20 g, 64%) as solid. 1H NMR (500 MHz, CDCl3): 7.98 (s, 1H), 7.64 (s, 1H); 19F NMR (470 MHz, CDCl3): −64.56, −68.26, −72.05.
To a stirred solution of 273.5 (10 g, 27.5 mmol) in THF (60 mL) was added DIPEA (9.6 mL, 55.1 mmol) at 0° C. After stirring for 30 minutes, 273.6 (4.15 g, 30.3 mmol) in THF (40 mL) was added at RT. After stirring for 16 h at RT, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (2×60 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (80 g Silica gel cartridge; 10% EtOAc/pet ether) to afford 273.7 (8.5 g, 88%) as solid.
1H NMR (500 MHz, CDCl3): 7.29-7.26 (m, 2H), 7.09 (s, 1H), 6.90-6.87 (m, 2H), 6.71 (s, 1H), 4.50 (d, J=6.0 Hz, 2H), 3.81 (s, 3H); 19F NMR (470 MHz, CDCl3): −65.35.−69.07; LCMS: 87.6%, m/z [M+H]+=351.0.
To a stirred solution of 273.7 (1.0 g, 2.85 mmol) in DMF (15 mL) were added dropwise NaHMDS (1M, 4.2 mL, 4.28 mmol) at 0° C. and MeI (0.18 mL, 2.85 mmol). After stirring for 2 h at RT, the reaction mixture was quenched with water (5 mL) and extracted with EtOAc (2×15 mL). The combined organic layer was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (24 g Silica gel cartridge; 20% EtOAc/pet ether) to afford 273.8 (0.85 g, 85%) as a solid.
1H NMR (400 MHz, CDCl3): 7.21 (d, J=8.4 Hz, 2H), 7.05 (s, 1H), 6.87-6.85 (m, 2H), 6.80 (s, 1H), 4.79 (s, 2H), 3.79 (s, 3H), 3.11 (s, 3H); LCMS: 94.3%, m/z [M+H]+=365.1.
To a stirred solution of 273.8 (0.700 g, 1.92 mmol) in DCM (5 mL) was added TFA (3.5 mL) at RT. After stirring for 1 h at RT, the reaction mixture was diluted with DCM (20 mL). The organic layer was collected, washed with saturated NaHCO3 (2×10 mL) and water (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford 273.9 (0.35 g, 74%) as a solid.
1H NMR (500 MHz, CDCl3): 7.08 (s, 1H), 6.71 (s, 1H), 5.07 (br s, 1H), 3.01 (d, J=5.5 Hz, 3H).
Thionyl chloride (5 mL) was added to 110.4_1a (0.500 g, 1.08 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure to afford intermediate acid chloride. To this acid chloride in CH2Cl2 (5 mL) was added 273.9 (0.45 g, 1.87 mmol) in CH2Cl2 (5 mL). After stirring for 16 h at RT, the reaction mixture was quenched with water (10 mL). The organic layer was separated, and the aqueous layer was extracted with CH2Cl2 (2×15 mL). The combined organic layer was washed with brine (10 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-BRIDGE C18 (150×30), 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/70, 8/80, 10/98, 14/98, 16/98, 16.1/70, 20/70 at 23 mL/min] to afford 273.10a (Peak-1, 35 mg, 5%) as a white solid and 273.10b (Peak-2, 25 mg, 3%) as a white solid.
273.10a: 1H NMR (400 MHz, DMSO-d6): 11.22 (s, 1H), 8.38 (s, 1H), 8.27 (s, 1H), 7.55 (s, 1H), 7.33 (d, J=2.0 Hz, 1H), 5.30-5.21 (m, 1H), 5.02-4.98 (m, 2H), 4.24-4.11 (m, 3H), 4.09-3.99 (m, 3H), 3.60 (s, 3H), 2.91-2.72 (m, 2H), 2.54-2.50 (m, 1H); LCMS: 98.9%, m/z [M+H]+=687.2. Absolute stereochemistry was not determined.
273.10b: 1H NMR (400 MHz, DMSO-d6): 11.20-11.10 (br s, 1H), 8.52 (s, 1H), 8.26 (s, 2H), 7.50 (s, 1H), 5.45-5.39 (m, 1H), 5.06-5.02 (m, 2H), 4.22-3.90 (m, 5H), 3.50 (s, 3H), 3.22-3.12 (m, 1H), 2.69-2.67 (m, 1H), 2.15-2.09 (m, 2H); LCMS: 97.1%, m/z [M+H]+=687.2. Absolute stereochemistry was not determined.
To a stirred solution of 273.10a (30 mg, 0.043 mmol) in THF (2 mL) were added aniline (4 μL, 0.043 mmol) and Pd(PPh3)4 (9 mg, 0.008 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: KROMOSIL-C18 (150×25 mm), 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/20, 8/80, 10/80, 10.1/98, 12/98, 12.1/20, 14/20 at 20 mL/min] to afford 273a (14 mg, 50%) as solid.
1H NMR (500 MHz, DMSO-d6): 12.82 (br s, 1H), 11.15 (br s, 1H), 8.39 (br s, 1H), 8.28 (s, 1H), 7.55 (s, 1H), 7.27 (br s, 1H), 4.13-4.10 (m, 1H), 4.01-3.93 (m, 2H), 3.57 (s, 3H), 2.86-2.83 (m, 1H), 2.78-2.72 (m, 1H), 2.60-2.50 (m, 2H). LCMS: 99.3%, m/z [M+H]+=647.0; Chiral purity: 97.1%.
To a stirred solution of 273.10b (20 mg, 0.029 mmol) in THF (2 mL) were added aniline (2 μL, 0.029 mmol) and Pd(PPh3)4 (6 mg, 0.005 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: KROMOSIL-C18 (150×25 mm), 10 μ; A: 0.1% Formic acid in H2O; B: Acetonitrile; Gradient: (Time/%B): 0/20, 8/80, 10/80, 10.1/98, 11/98, 11.1/40, 13/40 at 24 mL/min] to afford 273b (5 mg, 27%) as a solid. LCMS: 94.1%, m/z [M+H]+=647.0.
To a solution of 274.1 (5 g, 20.8 mmol) in MTBE (50 mL) were added 274.2 (3.2 g, 20.8 mmol) and 274.3 (4.4 g, 20.8 mmol) at RT. After stirring at 80° C. for 2 h, the reaction mixture was cooled to RT. The precipitate was filtered and washed with EtOAc. The filtrate was concentrated under reduced pressure. The residue was isolated by column chromatography (Silica-gel 100-200 mesh, 50% EtOAc/pet ether) to afford major diastereomer 274.4_1 (racemate, 1.65 g, 17%) as a white solid.
274.4_1: 1H NMR (500 MHz, DMSO-d6): 12.53 (s, 1H), 10.96 (s, 1H), 7.78 (d, J=2.0 Hz, 1H), 7.46 (d, J=2.0 Hz, 1H), 5.51-5.45 (m, 1H), 5.10-5.05 (m, 2H), 4.27-4.26 (m, 2H), 4.17-4.10 (m, 1H), 4.01 (d, J=7.5 Hz, 1H), 3.55 (t, J=7.5 Hz, 1H), 2.57 (d, J=7.5 Hz, 1H), 1.98 (d, J=7.0 Hz, 1H), 1.63-1.61 (m, 1H), 1.47-1.45 (m, 1H), 1.01 (s, 3H), 0.97 (s, 3H); LCMS: 99.1%, m/z [M+H]+=453.0; HPLC Purity: 98.9%; Chiral Purity: (49.8 +50.1)%. Regiochemistry and relative stereochemistry was confirmed by 2D NMR studies.
274.4_2: 1H NMR (400 MHz, DMSO-d6): 12.35 (br s, 1H), 10.90 (s, 1H), 7.55 (d, J=1.6 Hz, 1H), 7.50 (d, J=2.0 Hz, 1H), 5.87-5.80 (m, 1H), 5.27-5.14 (m, 2H), 4.45-4.37 (m, 3H), 3.74 (d, J=8.8 Hz, 1H), 3.46-3.42 (m, 1H), 2.51-2.48 (m, 1H), 2.08 (d, J=8.0 Hz, 1H), 1.92-1.87 (m, 1H), 1.60-1.55 (m, 1H), 1.02 (s, 6H); LCMS: 92.6%, m/z [M+H]+=453.1. Regiochemistry and relative stereochemistry was confirmed by 2D NMR studies.
274.4_1 (1.6 g) was purified by chiral SFC using Lux Cellulose-4 (30×250) mm, 5 μ; 70% CO2: 30% Isopropanol at RT (Isocratic 100 g/min, with detection at 214 nm). Pure fractions were concentrated under reduced pressure to give 274.4_1a (Peak-1, 625 mg, 78%) as a white solid and 274.4_1b (Peak-2, 650 mg, 81%) as a solid. (absolute stereochemistry of Enantiomer 1 & 2 not determined).
274.4_1a: 1H NMR (500 MHz, DMSO-d6): 12.50 (s, 1H), 10.96 (s, 1H), 7.78 (d, J=2.0 Hz, 1H), 7.46 (d, J=2.0 Hz, 1H), 5.51-5.45 (m, 1H), 5.10-5.05 (m, 2H), 4.28-4.26 (m, 2H), 4.17-4.10 (m, 1H), 4.01 (d, J=8.0 Hz, 1H), 3.55 (t, J=7.5 Hz, 1H), 2.57 (d, J=7.5 Hz, 1H), 1.98 (d, J=7.5 Hz, 1H), 1.63-1.61 (m, 1H), 1.48-1.45 (m, 1H), 1.01 (s, 3H), 0.97 (s, 3H); LCMS: 99.2%, m/z [M+H]+=453.1; Chiral Purity: 99.7%.
274.4_1b: 1H NMR (500 MHz, DMSO-d6): 12.53 (s, 1H), 10.96 (s, 1H), 7.79 (d, J=1.5 Hz, 1H), 7.46 (d, J=2.0 Hz, 1H), 5.49-5.46 (m, 1H), 5.11-5.05 (m, 2H), 4.28-4.27 (m, 2H), 4.14-4.13 (m, 1H), 4.00 (d, J=6.5 Hz, 1H), 3.55 (t, J=7.5 Hz, 1H), 2.57 (d, J=7.5 Hz, 1H), 1.97 (d, J=7.5 Hz, 1H), 1.64-1.61 (m, 1H), 1.48-1.44 (m, 1H), 1.01 (s, 3H), 0.97 (s, 3H); LCMS: 98.9%, m/z [M+H]+=453.1; Chiral Purity: 99.7%.
Thionyl chloride (6 mL) was added to 274.4_1a (300 mg, 0.66 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure to afford intermediate acid chloride. To this acid chloride in CH2Cl2 (5 mL) was added a solution of 3,5-dichloro-N-neopentylaniline (296 mg, 1.27 mmol) in CH2Cl2 (5 mL). After stirring at 55° C. for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 20% to 30% EtOAc/pet ether) to afford 274.5a (230 mg, 53%) as solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 10.90/10.84 (s, 1H), 8.26 (s, 1H), 7.63 (s, 1H), 7.44-7.39 (m, 3H), 5.48-5.39 (m, 1H), 5.09-5.05 (m, 2H), 4.27-4.13 (m, 3H), 3.84-3.68 (m, 3H), 3.54 (t, J=7.5 Hz, 1H), 2.68 (d, J=7.0 Hz, 1H), 1.86 (d, J=7.0 Hz, 1H), 1.54-1.49 (m, 2H), 0.97 (s, 6H), 0.84/0.82 (s, 9H); LCMS: 98.8%, m/z [M+H]+=668.0.
To a stirred solution of 274.5a (230 mg, 0.34 mmol) in THF (10 mL) were added aniline (48 mg, 0.68 mmol) and Pd(PPh3)4 (80 mg, 0.06 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: X-BRIDGE-C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/70, 8/90, 10/70, 10.1/98, 12/98, 12.1/70, 15/70 at 20 mL/min) to afford 274.6a (118 mg, 55%) as a solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.40 (br s, 1H), 10.85/10.78 (s, 1H), 8.41/7.91 (d, J=2.0 Hz, 1H), 7.61/7.52 (t, J=2.0 Hz, 1H), 7.47-7.39 (m, 3H), 4.07 (d, J=8.0 Hz, 1H), 3.94 (d, J=14.0 Hz, 1H), 3.84-3.78 (m, 1H), 3.51 (d, J=14.0 Hz, 1H), 3.45 (t, J=7.0 Hz, 1H), 2.68 (d, J=7.0 Hz, 1H), 1.82 (d, J=7.0 Hz, 1H), 1.50-1.41 (m, 2H), 0.96 (s, 6H), 0.84/0.82 (s, 9H); LCMS: 99.3%, m/z [M+H]+=626.0; Chiral purity: 99.8%.
Thionyl chloride (5 mL) was added to 274.4_1b (100 mg, 0.22 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure to afford acid chloride intermediate. To this acid chloride in CH2Cl2 (5 mL) was added a solution of 3,5-dichloro-N-neopentylaniline (98 mg, 0.42 mmol) in CH2Cl2 (5 mL). After stirring at 55° C. for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 20% to 30% EtOAc/pet ether) to afford 274.5b (100 mg, 68%) as a white solid.
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 10.90/10.84 (s, 1H), 8.26/7.78 (d, J=2.0 Hz, 1H), 7.63/7.54 (t, J=2.0 Hz, 1H), 7.44-7.39 (m, 3H), 5.48-5.39 (m, 1H), 5.09-5.04 (m, 2H), 4.30-4.12 (m, 3H), 3.85-3.67 (m, 3H), 3.54 (t, J=7.6 Hz, 1H), 2.68-2.66 (m, 1H), 1.86 (d, J=6.8 Hz, 1H), 1.54-1.49 (m, 2H), 0.97 (s, 6H), 0.84/0.81 (s, 9H); LCMS: 96.0%, m/z [M+H]+=667.9.
To a stirred solution of 274.5b (100 mg, 0.15 mmol) in THF (5 mL) were added aniline (17 mg, 0.18 mmol) and Pd(PPh3)4 (34.6 mg, 0.03 mmol) at RT. After stirring for 3 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: KROMOSIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/70, 8/95, 10/98, 12/98, 12.1/70, 14/70 at 25 mL/min] to afford 274.6b (65 mg, 69%) as a solid.
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.38 (br s, 1H), 10.80/10.77 (s, 1H), 8.41/7.92 (d, J=2.0 Hz, 1H), 7.60/7.52 (t, J=1.6 Hz, 1H), 7.47-7.38 (m, 3H), 4.06 (d, J=8.0 Hz, 1H), 3.93 (d, J=14.0 Hz, 1H), 3.84-3.77 (m, 1H), 3.52 (d, J=14.0 Hz, 1H), 3.45 (t, J=7.2 Hz, 1H), 2.68 (d, J=6.8 Hz, 1H), 1.82 (d, J=6.8 Hz, 1H), 1.48-1.43 (m, 2H), 0.96 (s, 6H), 0.84/0.82 (s, 9H); LCMS: 99.0%, m/z [M+H]+=626.0; Chiral purity: 99.4%.
To a stirred solution of 275.1 (10 g, 41.4 mmol) in DCM (100 mL) was added TFA (16 mL, 207 mmol) at 0° C. After stirring at RT for 16 h, the reaction mixture was concentrated under reduced pressure. The resulting residue was triturated with diethyl ether (200 mL) to afford 275.2 (7.4 g, 75%) as a solid.
1H NMR (400 MHz, DMSO-d6): 9.47 (br s, 1H), 4.43 (dd, J=8.4 Hz, J=6.8 Hz, 1H), 3.16 (d, J=11.2 Hz, 2H), 3.12 (d, J=11.2 Hz, 2H), 2.24-2.18 (m, 1H), 2.03-1.98 (m, 1H), 0.68-0.61 (m, 4H); LCMS: 87.6%, m/z [M+H]+=142.2.
To a solution of 275.2 (4.2 g, 17.6 mmol) in MTBE (60 mL) were added 275.3 (2.75 g, 17.6 mmol) and 275.4 (3.81 g, 17.6 mmol) at RT. After stirring at 80° C. for 2 h, the reaction mixture was cooled to RT. The resulting precipitate was filtered and washed with EtOAc. The filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (Silica gel 100-200 mesh, 10%-30% EtOAc/pet ether). The major diastereomer was triturated with DCM (50 mL) to afford 275.5_1 (racemate, 3.0 g, 38%) as a white solid.
1H NMR (500 MHz, DMSO-d6): 12.60 (br s, 1H), 11.03 (s, 1H), 7.74 (d, J=2.0 Hz, 1H), 7.46 (d, J=2.0 Hz, 1H), 5.51-5.48 (m, 1H), 5.11-5.06 (m, 2H), 4.28 (d, J=5.5 Hz, 2H), 4.22-4.16 (m, 1H), 4.04 (d, J=7.5 Hz, 1H), 3.63-3.58 (m, 1H), 2.75 (d, J=7.5 Hz, 1H), 2.04 (d, J=8.0 Hz, 1H), 1.93-1.85 (m, 1H), 1.52-1.49 (m, 1H), 0.50-0.36 (m, 4H); LCMS: 96.8%, m/z [M+H]+=451.1; Chiral Purity: (49.9%+50.0%). The regio and relative stereochemistry was confirmed by 2D NMR analysis.
275.5_2: 1H NMR (400 MHz, DMSO-d6): 12.45 (br s, 1H), 10.98 (s, 1H), 7.58 (d, J=1.6 Hz, 1H), 7.51 (d, J=2.0 Hz, 1H), 5.89-5.79 (m, 1H), 5.28-5.15 (m, 2H), 4.51-4.37 (m, 3H), 3.65-3.57 (m, 2H), 2.72 (d, J=8.8 Hz, 1H), 2.04-1.99 (m, 2H), 1.77-1.72 (m, 1H), 0.65-0.35 (m, 4H); LCMS: 91.9%, m/z [M+H]+=451.0. The regiochemistry and relative stereochemistry was confirmed by 2D NMR analysis.
275.5_1 (3 g) was purified by chiral SFC using Chiralpak AD-H (30×250) mm, 5 μ; A: 80% CO2%, B: 20% of 0.5% TFA in Isopropanol at RT (Isocratic 100 g/min, with detection at 214 nm). Pure fractions were concentrated under reduced pressure to give 275.5_1a (Peak-1, 1.2 g, 80%) as a solid and 275.5_1b (Peak-2, 1.4 g, 93%) as a solid. Absolute stereochemistry of Enantiomer 1 & 2 not determined.
275.5_1a: 1H NMR (500 MHz, DMSO-d6): 12.60 (br s, 1H), 11.10 (s, 1H), 7.71 (d, J=1.5 Hz, 1H), 7.49 (d, J=2.0 Hz, 1H), 5.53-5.49 (m, 1H), 5.13-5.07 (m, 2H), 4.31 (d, J=5.5 Hz, 2H), 4.26-4.24 (m, 1H), 4.05 (d, J=7.5 Hz, 1H), 3.69-3.63 (m, 1H), 2.88-2.82 (m, 1H), 2.09 (d, J=8.0 Hz, 1H), 1.97-1.90 (m, 1H), 1.55-1.52 (m, 1H), 0.52-0.36 (m, 4H); LCMS: 98.3%, m/z [M+H]+=451.0; Chiral Purity: 99.1%.
275.5_1b: 1H NMR (500 MHz, DMSO-d6): 12.65 (br s, 1H), 11.10 (s, 1H), 7.71 (s, 1H), 7.49 (s, 1H), 5.55-5.48 (m, 1H), 5.12-5.07 (m, 2H), 4.31 (d, J=5.5 Hz, 2H), 4.27-4.23 (m, 1H), 4.05 (d, J=8.0 Hz, 1H), 3.69-3.66 (m, 1H), 2.85 (d, J=7.0 Hz, 1H), 2.09 (d, J=8.0 Hz, 1H), 1.97-1.93 (m, 1H), 1.55-1.52 (m, 1H), 0.51-0.37 (m, 4H); LCMS: 98.0%, m/z [M+H]+=451.0; Chiral Purity: 95.4%.
Thionyl chloride (5 mL) was added to 275.5_1a (300 mg, 0.66 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure to afford acid chloride intermediate. To this acid chloride in CH2Cl2 (5 mL) was added a solution of 3,5-dichloro-N-neopentylaniline (296 mg, 1.27 mmol) in CH2Cl2 (5 mL). After stirring at 55° C. for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 20% to 30% EtOAc/pet ether) to afford 275.6a (300 mg, 71%) as solid.
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 10.91/10.98 (s, 1H), 8.21/7.71 (d, J=1.6 Hz, 1H), 7.63/7.54 (t, J=1.6 Hz, 1H), 7.43-7.39 (m, 3H), 5.67-5.46 (m, 1H), 5.23-5.08 (m, 2H), 4.34-4.19 (m, 3H), 4.14 (d, J=8.0 Hz, 1H), 3.89-3.80 (m, 2H), 3.68-3.60 (m, 2H), 2.80 (d, J=7.6 Hz, 1H), 1.93-1.88 (m, 1H), 1.43-1.39 (m, 1H), 0.83/0.81 (s, 9H), 0.47-0.33 (m, 4H); LCMS: 98.2%, m/z [M+H]+=665.9.
To a stirred solution of 275.6a (300 mg, 0.44 mmol) in THF (10 mL) wereadded aniline (50 mg, 0.53 mmol) and Pd(PPh3)4 (104 mg, 0.08 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: KROMOSIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/70, 8/95, 10/98, 12/98, 12.1/70, 14/70 at 25 mL/min] to afford 275.7a (113 mg, 40%) as a solid.
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.43/12.30 (br s, 1H), 10.90/10.85 (s, 1H), 8.37/7.87 (d, J=2.0 Hz, 1H), 7.61/7.53 (t, J=2.0 Hz, 1H), 7.48 (d, J=1.6 Hz, 2H), 7.42/7.39 (d, J=2.0 Hz, 1H), 4.07 (d, J=8.0 Hz, 1H), 4.00 (d, J=14.0 Hz, 1H), 3.88-3.86 (m, 1H), 3.55 (t, J=7.2 Hz, 1H), 3.44 (d, J=14.0 Hz, 1H), 2.87 (d, J=7.2 Hz, 1H), 1.87-1.82 (m, 2H), 1.36-1.33 (m, 1H), 0.83/0.82 (s, 9H), 0.45-0.32 (m, 4H); LCMS: 99.0%, m/z [M+H]+=624.0; Chiral purity: 98.3%.
Thionyl chloride (5 mL) was added to 275.5_1b (300 mg, 0.66 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure to afford acid chloride intermediate. To this acid chloride in CH2Cl2 (5 mL) was added a solution of 3,5-dichloro-N-neopentylaniline (296 mg, 1.27 mmol) in CH2Cl2 (5 mL). After stirring at 55° C. for 16 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 20% to 30% EtOAc/pet ether) to afford 275.6b (200 mg, 45%) as solid.
LCMS: 92.9%, m/z [M+H]+=666.1.
To a stirred solution of 275.6b (200 mg, 0.30 mmol) in THF (10 mL) were added aniline (56 mg, 0.60 mmol) and Pd(PPh3)4 (104 mg, 0.08 mmol) at RT. After stirring for 3 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [Column: KROMOSIL-C18 (150×25) mm, 10 μ, A: 0.1% Formic Acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/70, 8/95, 10/98, 12/98, 12.1/70, 14/70 at 25 mL/min] to afford 275.7b (30 mg, 16%) as a solid.
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 12.42/12.30 (br s, 1H), 10.90/10.85 (s, 1H), 8.37/7.86 (d, J=2.0 Hz, 1H), 7.61/7.53 (t, J=2.0 Hz, 1H), 7.48 (d, J=1.6 Hz, 2H), 7.42/7.39 (d, J=2.0 Hz, 1H), 4.07 (d, J=7.6 Hz, 1H), 4.00 (d, J=14.0 Hz, 1H), 3.88-3.86 (m, 1H), 3.55 (t, J=7.2 Hz, 1H), 3.44 (d, J=14.0 Hz, 1H), 2.87 (d, J=7.6 Hz, 1H), 1.89-1.82 (m, 2H), 1.36-1.32 (m, 1H), 0.83/0.82 (s, 9H), 0.45-0.32 (m, 4H); LCMS: 95.8%, m/z [M+H]+=624.0; Chiral purity: 95.8%.
280 was synthesized from 280.1b following the procedure described for the synthesis of 110. Absolute stereochemistry is unknown.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.66 (br s, 1H), 10.91 (br s, 1H), 7.92-7.38 (m, 5H), 4.62-4.55 (m, 1H), 3.67-3.59 (m, 1H), 3.48-3.38 (m, 2H), 3.20-3.07 (m, 2H), 2.74-2.64 (m, 1H), 2.50-2.41 (m, 1H), 2.30-2.15 (m, 1H), 0.90-0.80 (m, 1H), 0.38-0.36 (m, 2H), 0.06-0.04 (m, 2H); LCMS: 92.6%, m/z [M+H]+=618.0; Chiral Purity: 95.1%.
281 was synthesized from 110.4_2 following the procedure described for the synthesis of 111. Absolute stereochemistry is unknown.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.66 (br s, 1H), 10.91 (br s, 1H), 7.92-7.38 (m, 5H), 4.61-4.53 (m, 1H), 3.68-3.58 (m, 1H), 3.49-3.39 (m, 2H), 3.22-3.07 (m, 2H), 2.72-2.65 (m, 1H), 2.50-2.40 (m, 1H), 2.30-2.15 (m, 1H), 0.90-0.81 (m, 1H), 0.38-0.36 (m, 2H), 0.06-0.04 (m, 2H); LCMS: 90.3%, m/z [M+H]+=618.0.
To a stirred solution of 282.1 (2.5 g, 22.3 mmol) in THF (25 mL) were added 282.2 (5.5 g, 22.3 mmol) and 282.3 (4.8 g, 22.3 mmol) at RT. After stirring at RT for 8 h, the reaction mixture was evaporated under reduced pressure to afford 282.4 (8.5 g) as a brown solid, which was used as such in the next step without purification. LCMS: 16%, m/z [M+H]+=416.9.
A solution of 282.4 (8.5 g, 20.4 mmol) in allyl alcohol (50 mL) was heated at 90° C. for 16 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by flash column chromatography (120 g Silica gel cartridge, 30% EtOAc/pet ether) to afford mixture of regio-isomers. The regio isomers were purified by prep. HPLC to obtain 282.5a (160 mg, 2%) as an off-white solid and 282.5b (1.1 g, 11%) as an off-white solid. The regio chemistry and relative stereochemistry was confirmed by 2D NMR analysis.
282.5a data: 1H NMR (400 MHz, MeOH-d4): 7.89 (d, J=1.6 Hz, 1H), 7.29 (d, J=1.6 Hz, 1H), 5.56-5.47 (m, 1H), 5.11-5.05 (m, 2H), 4.35-4.24 (m, 2H), 3.82-3.78 (m, 1H), 3.74 (s, 1H), 3.07-2.98 (m, 1H), 2.73-2.64 (m, 1H), 2.38-2.32 (m, 1H), 2.19-2.10 (m, 1H), 1.59 (s, 3H); LCMS: 95.5%, m/z [M+H]+=475.0.
282.5b: 1H NMR (500 MHz, DMSO-d6): 13.40 (br s, 1H), 11.10 (br s, 1H), 7.61 (s, 1H), 7.22 (s, 1H), 5.95-5.87 (m, 1H), 5.34 (dd, J=17.5 Hz, J=1.5 Hz, 1H), 5.23 (dd, J=10.5 Hz, J=1.5 Hz, 1H), 4.62-4.54 (m, 2H), 4.45-4.42 (m, 1H), 4.02 (d, J=10.5 Hz, 1H), 3.88-3.72 (m, 1H), 3.20-2.98 (m, 1H), 2.80-2.75 (m, 1H), 2.31-2.26 (m, 1H), 1.30 (s, 3H); LCMS: 91.5%, m/z [M+H]+=475.0.
To a stirred solution of 282.5b (400 mg, 0.84 mmol) in methanol (8 mL) was added trimethyl silyldiazomethane (2M in hexane, 2.1 mL, 4.2 mmol) at 0° C. After stirring at RT for 16 h, the reaction mixture was cooled to 0° C. and quenched with acetic acid (0.5 mL). After stirring at RT for 1 h, the reaction mixture was concentrated under reduced pressure at low temperature. The residue was purified by flash column chromatography (24 g Silica gel cartridge, 10% to 25% EtOAc/pet ether) to obtain 282.6b (260 mg, 63%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6): 11.31 (s, 1H), 7.67 (d, J=2.0 Hz, 1H), 6.91 (d, J=2.0 Hz, 1H), 5.94-5.86 (m, 1H), 5.37-5.25 (m, 2H), 4.59 (d, J=4.8 Hz, 2H), 4.54-4.47 (m, 1H), 4.11 (d, J=10.4 Hz, 1H), 3.77-3.69 (m, 1H), 3.64 (s, 3H), 2.91-2.81 (m, 2H), 2.40-2.33 (m, 1H), 1.24 (s, 3H); LCMS: 91.0%, m/z [M−H]−=486.9.
To a stirred solution of 282.6b (240 mg, 0.49 mmol) in THF (5 mL) were added aniline (46 mg, 0.49 mmol) and Pd(PPh3)4 (114 mg, 0.09 mmol) at RT. After stirring for 4 h at RT, the reaction mixture was concentrated under reduced pressure. The residue was purified by column chromatography (Silica gel 100-200 mseh, 30% EtOAc/pet ether) to afford 282.7b (120 mg, 55%) as a yellow solid.
1H NMR (400 MHz, DMSO-d6): 12.90 (br s, 1H), 11.28 (s, 1H), 7.66 (d, J=1.6 Hz, 1H), 6.90 (d, J=2.0 Hz, 1H), 4.49-4.42 (m, 1H), 4.06-3.97 (m, 2H), 3.82-3.71 (m, 1H), 3.64 (s, 3H), 2.97-2.84 (m, 1H), 2.39-2.30 (m, 1H), 1.19 (s, 3H); LCMS: 95.1%, m/z [M+H]+=450.4.
Thionyl chloride (1 mL) was added to 282.7b (70 mg, 0.15 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure to afford intermediate acid chloride. To this acid chloride in CH2Cl2 (2 mL) were added a solution of 3,5-dichloro-N-methylaniline (38 mg, 0.21 mmol) in CH2Cl2 (3 mL) and catalytic amount of DMAP (2 mg) at RT. After stirring at 55° C. for 16 h, the reaction mixture was concentrated under reduced pressure. The reside was purified by flash column chromatography (12 g Silica gel cartridge, 15% to 20% EtOAc/pet ether) to afford 282 (50 mg, 57%) as an off-white solid.
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 11.31/11.13 (s, 1H), 7.77-7.44 (m, 4H), 6.99-6.91 (m, 1H), 4.40/4.33 (d, J=10.0 Hz, 1H), 3.96-3.72 (m, 1H), 3.65/3.58 (s, 3H), 3.46-3.39 (m, 1H), 3.34/3.17 (s, 3H), 2.88-2.77 (m, 2H), 2.17-2.07 (m, 1H), 1.10/1.04 (s, 3H); LCMS: 94.5%, m/z [M+H]+=605.9.
To a stirred solution of 110 (300 mg, 0.47 mmol) in DMF (6 mL) were added HATU (360 mg, 0.94 mmol) and DIPEA (0.51 mL, 2.83 mmol) at room temperature. After stirring for 10 minutes, NH4Cl (128 mg, 2.36 mmol) was added. After stirring for 16 h at RT, the reaction mixture was diluted with ice-cold water (50 mL) and stirred for 10 minutes. The resulting precipitate was filtered, washed with water and dried under vacuum. The crude compound was purified by prep. HPLC [KROMOSIL-C18 (150×25) mm, 10 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/55, 8/80, 8.1/98, 10/98, 10.1/55, 13/55 at 20 mL/min] to afford 284 (70 mg, 23%) as an off-white solid.
1H NMR (400 MHz, DMSO-d6) (Exist in rotameric form): 10.80/10.70 (s, 1H), 7.70 (t, J=1.6 Hz, 1H), 7.67 (d, J=1.6 Hz, 1H), 7.61-7.54 (m, 2H), 7.49 (d, J=1.6 Hz, 1H), 6.95 (br s, 1H), 6.42 (br s, 1H), 4.33-4.27 (m, 1H), 3.92 (d, J=11.6 Hz, 1H), 3.78 (d, J=14.0 Hz, 1H), 3.68-3.58 (m, 2H), 3.45-3.35 (m, 1H), 2.81-2.76 (m, 1H), 2.50-2.44 (m, 1H), 2.28-2.13 (m, 1H), 0.81 (s, 9H); LCMS: 99.1%, m/z [M+H]+=633.0; Chiral purity: 94.7%.
To a stirred solution of 110 (400 mg, 0.62 mmol) in DMF (5 mL) were added EDC.HCl (240 mg, 1.25 mmol), HOBt (255 mg, 1.88 mmol) and Et3N (0.88 mL, 6.29 mmol) at room temperature. After stirring for 5 minutes, (Me)NHOH.HCl (420 mg, 5.03 mmol) was added. After stirring for 16 h at RT, the reaction mixture was diluted with ice-cold water (50 mL) and stirred for 10 minutes. The resulting precipitate was filtered, washed with water and dried under vacuum. The crude compound was purified by prep. HPLC [X-SELECT-C18 (250×19) mm, 5 μ; A: 10 mM Ammonium bicarbonate in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/65, 8/80, 10/80, 10.1/98, 12/98, 12.1/65, 15/65 at 20 mL/min] to afford 285 (53 mg, 13%) as an off-white solid.
1H NMR (500 MHz, DMSO-d6): 10.77 (s, 1H), 10.10 (s, 1H), 7.67 (s, 1H), 7.59-7.44 (m, 3H), 7.28 (s, 1H), 4.40 (d, J=8.0 Hz, 1H), 4.15-4.08 (m, 1H), 3.78 (d, J=14.0 Hz, 1H), 3.63 (d, J=14.0 Hz, 1H), 3.52 (t, J=9.0 Hz, 1H), 3.44-3.40 (m, 1H), 2.98 (s, 3H), 2.71-2.66 (m, 1H), 2.64-2.55 (m, 1H), 2.21-2.10 (m, 1H), 0.82 (s, 9H); LCMS: 97.2%, m/z [M+H]+=662.9; Chiral purity: 93.2%.
To a stirred solution of 110 (400 mg, 0.62 mmol) in DMF (5 mL) were added HATU (360 mg, 0.94 mmol) and DIPEA (1.2 mL, 6.29 mmol) at RT. After stirring for 10 minutes, NH2OH.HCl (352 mg, 5.03 mmol) was added. After stirring for 16 h at RT, the reaction mixture was diluted with ice cold water (50 mL) and stirred for 15 minutes. The resulting precipitate was filtered, washed with water (10 mL) and dried under vacuum. The residue was purified by prep. HPLC [X-SELECT-C18 (150×30) mm, 5 μ; A: 10 mM Ammonium bicarbonate in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/60, 8/90, 11/90, 11.1/98, 15/98, 15.1/60, 19/60 at 22 mL/min] to obtain 286a (Peak-1, 37 mg, 9%) as an off-white solid and 286b (Peak-2, 38 mg, 9%) as an off-white solid.
286a: 1H NMR (500 MHz, DMSO-d6): 10.74 (br s, 1H), 9.96 (br s, 1H), 8.83 (s, 1H), 7.71-7.52 (m, 5H), 4.32 (t, J=10.5 Hz, 1H), 3.80-3.75 (m, 1H), 3.71-3.63 (m, 3H), 3.38-3.32 (m, 1H), 2.87-2.83 (m, 1H), 2.64-2.50 (m, 1H), 2.23-2.16 (m, 1H), 0.80 (s, 9H); LCMS: 97.0%, m/z [M+H]+=648.9; chiral purity: 99.8%.
286b: LCMS: 95.9%, m/z [M+H]+=649.0.
Thionyl chloride (3 mL) was added to 110.4_1a (300 mg, 0.65 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure to afford intermediate acid chloride. To the above prepared acid chloride in CH2Cl2 (5 mL) was added solution of (3s,5s,7s)-N-methyladamantan-1-amine (161 mg, 0.97 mmol) in CH2Cl2 (5 mL). After stirring for 16 h at 50° C., the reaction was quenched with water (10 mL) and extracted with CH2Cl2 (2×15 mL). The combined organic layer was washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 20%-25% EtOAc/pet ether) to obtain 287.1_1 (Peak-1, 105 mg, 26%) as a pale brown solid and 287.1_2 (Peak-2, 84 mg, 21%) as a pale brown solid.
287.1_1: LCMS: 24.6%, m/z [M+H]+=608.1.
287.1_2: LCMS: 81.9%, m/z [M+H]+=608.1.
To a stirred solution of 287.1_1 (105 mg, 0.17 mmol) in THF (5 mL) were added aniline (16 mg, 0.17 mmol) and Pd(PPh3)4 (39 mg, 0.03 mmol) at RT. After stirring for 2 h, the reaction mixture was concentrated under reduced pressure. The residue was purified by prep. HPLC [X-BRIDGE-C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/65, 8/80, 8.1/98, 10/98, 10.1/98, 13/65 at 22 mL/min] to obtain 287 (16 mg, 15%) as solid.
1H NMR (500 MHz, DMSO-d6): 12.40 (br s, 1H), 11.06 (br s, 1H), 7.68 (s, 1H), 7.52 (s, 1H), 4.13 (d, J=11.0 Hz, 1H), 3.89-3.85 (m, 1H), 3.72-3.67 (m, 1H), 3.06 (s, 3H), 2.86-2.64 (m, 3H), 2.50-2.47 (m, 1H), 2.13-2.04 (m, 9H), 1.65-1.60 (m, 6H); LCMS: 94.1%, m/z [M+H]+=568.1; Chiral purity: 90.5%.
To a stirred solution of 288.1 (5 g, 33.2 mmol) in CH2Cl2 (60 mL) were added pyridine (2.9 mL, 36.5 mmol) and trifluoromethanesulfonic anhydride (6.1 mL, 36.5 mmol) at 0° C. After stirring for 16 h at RT, the reaction mixture was quenched with ice water. The organic layer was separated and washed with water (60 mL), brine (60 mL), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash column chromatography (40 g Silica gel cartridge, 10% EtOAc in pet ether) to afford 288.2 (3.9 g, 42%) as a colorless oil.
1H NMR (500 MHz, CDCl3): 7.42-7.35 (m, 2H), 7.26-7.25 (m, 1H), 7.10-7.07 (m, 1H), 1.33 (s, 9H).
To a stirred solution of 288.3 (3 g, 21.2 mmol) in EtOH (40 mL) and water (401 mg, 22.2 mmol) at RT was added a solution of potassium tert-butoxide (2.4 g, 21.2 mmol) in EtOH (20 mL) at 60° C. over a period of 30 minutes. After stirring for 2 h at 60° C., the reaction mixture was concentrated under reduced pressure. The residue was triturated with a mixture of diethyl ether and EtOH to afford 288.4 (1.8 g, 56%) as a solid.
1H NMR (500 MHz, DMSO-d6): 1.29 (s, 6H).
A stirred suspension of 288.2 (2 g, 7.08 mmol), 288.4 (1.28 g, 8.5 mmol) and Xantphos (205 mg, 0.35 mmol) in mesitylene (20 mL) was purged with argon for 15 minutes followed by addition of bis(allyl)dichlorodipalladium (78 mg, 0.21 mmol) and purging with argon for an additional 5 minutes. After stirring at 130° C. for 6 h, the reaction mixture was cooled to RT and poured into ice water (30 mL). The organic layer was separated, washed with water (20 mL), brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by flash column chromatography (80 g Silica gel cartridge, 10% EtOAc in pet ether to afford 288.5 (1.2 g, 72%) as a colorless oil.
1H NMR (500 MHz, CDCl3): 7.51 (d, J=1.5 Hz, 1H), 7.36-7.25 (m, 3H), 1.73 (s, 6H), 1.34 (s, 9H); GCMS: 99.4%, m/z [M+H]+=202.2.
To a stirred solution of 288.5 (1 g, 4.96 mmol) in THF (15 mL) was added LiAlH4 solution (1M in THF, 7.5 mL, 7.45 mmol) at 0° C. After stirring for 4 h at 0° C., the reaction mixture was quenched with 10% NaOH solution at 0° C. and extracted with EtOAc (20 mL). The organic layer was separated, washed with water (20 mL), brine (20 mL), dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was triturated with n-pentane (10 mL) to afford 288.6 (850 mg, 84%) as a solid.
1H NMR (500 MHz, DMSO-d6): 7.34 (d, J=2.0 Hz, 1H), 7.34-7.13 (m, 3H), 3.62-3.59 (m, 1H), 2.67 (s, 2H), 1.78-1.76 (m, 1H), 1.28 (s, 9H), 1.23 (s, 6H); LCMS: 92.9%, m/z [M+H]+=206.2.
To a stirred solution of 110.4_1 (300 mg, 0.65 mmol) in DMF (3 mL) were added DIPEA (252 mg, 1.95 mmol) and HATU (297 mg, 0.77 mmol) at RT. After stirring for 15 minutes at RT, 288.6 (200 mg, 0.97 mmol) was added. After stirring for 6 h at the RT, the reaction mixture was quenched with water (10 mL) and extracted with EtOAc (10 mL). The combined organic layer was washed with water (20 mL), brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure to afford mixture of diastereomers of 288.7 (500 mg) as a brown gummy material. The residue was used in the next step without purification.
To a stirred solution of 288.7 (500 mg, 0.77 mmol) in THF (20 mL) were added aniline (70 mg, 0.77 mmol) and Pd(PPh3)4 (180 mg, 0.2 mmol) at RT. After stirring for 2 h at RT, the reaction mixture was concentrated under reduced pressure and triturated with diethyl ether and pentane. The residue was purified by prep. HPLC [X-BRIDGE C18 (150×30) mm, 5 μ; A: 0.1% Formic acid in H2O, B: Acetonitrile; Gradient: (Time/%B): 0/65, 9/80, 11/80, 11.1/65, 14/65 at 25 mL/min] to afford minor diastereomer 288a (20 mg, 5%) as a solid and major diastereomer 288b (57 mg, 14%) as a solid.
288a: 1H NMR (500 MHz, DMSO-d6): 12.50 (br s, 1H), 11.07 (br s, 1H), 7.54-7.39 (m, 2H), 7.31 (s, 1H), 7.24-7.17 (m, 3H), 7.10-7.07 (m, 1H), 4.15-4.05 (m, 2H), 3.57-3.47 (m, 2H), 3.32-3.20 (m, 2H), 2.76-2.64 (m, 2H), 2.31-2.24 (m, 1H), 1.39-1.30 (m, 9H), 1.28-1.26 (m, 6H); LCMS: 98.5%, m/z [M+H]+=608.3. (absolute stereochemistry not determined)
288b: 1H NMR (500 MHz, DMSO-d6): 12.35 (br s, 1H), 10.93 (br s, 1H), 8.19 (br s, 1H), 7.88 (br s, 1H), 7.42 (d, J=1.5 Hz, 2H), 7.23-7.19 (m, 3H), 3.92-3.86 (m, 2H), 3.63-3.59 (m, 1H), 3.47-3.40 (m, 1H), 3.16-3.01 (m, 2H), 2.56-2.50 (m, 1H), 1.94-1.87 (m, 1H), 1.71-1.58 (m, 1H), 1.33-1.26 (m, 15H); LCMS: 98.5%, m/z [M+H]+=608.3.
1H NMR (500 MHz, DMSO-d6) (Exist in rolameric form): 12.55 (br s, 1H), 11.05/10.99 (br s, 1H), 8.30/7.93 (s, 1H), 7.69-7.37 (m, 4H), 4.01-4.00 (m, 1H), 3.92- 3.89 (m, 1H), 3.84-3.79 (m, 1H), 3.57-3.53 (m, 1H), 3.45-3.44 (m, 1H), 3.32-3.26 (m, 1H), 2.60- 2.50 (m, 1H), 2.50-2.36 (m, 1H), 2.19-2.03 (m, 1H), 1.10-1.04 (m, 3H).
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.65 (br s, 1H), 11.05/10.99 (s, 1H), 8.67-7.01 (m, 4H), 4.17-4.12 (m, 1H), 4.01- 3.99 (m, 1H), 3.85-3.81 (m, 1H), 3.55 (t, J = 6.5 Hz, 1H), 3.40/3.24 (s, 3H), 3.29-3.10 (m, 1H), 2.67-2.54 (m, 1H), 2.39- 2.35 (m, 1H), 2.15-2.02 (m, 1H).
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.60 (br s, 1H), 11.05/10.97 (s, 1H), 8.31/7.89 (s, 1H), 7.64-7.45 (m, 3H), 4.15- 3.97 (m, 1H), 3.86 (s, 3H), 3.86-3.80 (m, 1H), 3.58-3.50 (m, 1H), 3.37/3.17 (s, 3H), 3.33- 3.20 (m, 1H), 2.60-2.50 (m, 1H), 2.42-2.30 (m, 1H), 2.18-2.05 (m, 1H).
1H NMR (500 MHz, DMSO-d6) (Exist in rotameric form): 12.38 (br s, 1H), 11.03 (br s, 1H), 8.21 (br s, 1H), 7.82-7.47 (m, 4H), 4.07- 3.92 (m, 1H), 3.82-3.72 (m, 1H), 3.42/3.32 (br s, 3H), 3.32-3.25 (m, 1H), 3.16-3.08 (m, 1H), 2.70- 2.60 (m, 1H), 2.50-2.40 (m, 1H), 2.17-2.00 (m, 1H).
1H NMR (500 MHz, DMSO-d6): 12.60 (br s, 1H), 11.10 (br s, 1H), 8.05 (br s, 1H), 7.60-7.50 (m, 1H), 7.22-7.17 (m, 4H), 7.03-7.02 (m, 1H), 4.70-4.60 (m, 1H), 3.90- 3.70 (m, 1H), 3.45-3.34 (m, 3H), 3.32-3.28 (m, 2H), 2.92-2.80 (m, 1H), 2.76-2.63 (m, 3H), 2.36- 2.23 (m, 2H), 1.20 (s, 6H).
1H NMR (500 MHz, DMSO-d6): 12.27 (br s, 1H), 10.96 (br s, 1H), 8.22 (br s, 1H), 8.18-8.09 (m, 1H), 7.46-7.43 (m, 1H), 7.34 (s, 1H), 7.22- 7.14 (m, 3H), 3.92-3.86 (m, 2H), 3.83-3.78 (m, 1H), 3.42-3.39 (m, 1H), 3.09-3.04 (m, 2H), 2.54- 2.50 (m, 1H), 1.97-1.86 (m, 1H), 1.75-1.60 (m, 1H), 1.27 (s, 9H), 0.95- 0.84 (m, 3H), 0.71-0.68 (m, 1H).
Using 110.4_1 and the listed aniline, the following compounds were made as in Example 288.
Assays for Detecting and Measuring the Effect of Compounds on dF508-CFTR Channels CFRT-YFP High Throughput Assay:
The following protocol is designed to selectively screen small molecule compounds for F508del CFTR corrector activities in the HTS YFP flux assay. In this protocol, the cells are incubated with test compounds for 24 hours, washed with PBS, stimulated with forskolin and a standard potentiator, and read on a 384-well HTS plate reader, such as the Hamamatsu FDDD-6000.
YFP fluorescence intensity is acquired at high speed before and after iodide buffer is injected to the assay cells. Iodide enters the cells via active CFTR channels in the plasma membrane, and quenches the YFP fluorescence. The rate of fluorescence quenching is proportionally related to the total CFTR activities in the cell membrane. dF508-CFTR corrector accelerates YFP quenching by increasing the number of CFTR molecules in the testing cell plasma membrane.
This method was initially developed for bench top plate readers (Galietta et al., 2001), and was adapted to the HTS format (Sui et al. Assay Drug Dev. Technol. 2010).
Fisher Rat Thyroid (FRT) cells stably expressing both human ΔF508-CFTR and a halide-sensitive yellow fluorescent protein (YFP-H148Q/I152L 25, 22) (Galietta et al., Am.J.Physiol Cell Physiol 281(5), C1734, 2001) were cultured on plastic surface in Coon's modified Ham's F12 medium supplemented with FBS 10%, L-glutamine 2 mM, penicillin 100 U/mL, and streptomycin 100 μg/mL. G418 (0.75-1.0 mg/mL) and zeocin (3.2 ug/mL) were used for selection of FRT cells expressing ΔF508-CFTR and YFP. For primary screening, FRT cells were plated into 384-well black wall, transparent bottom microtiter plates (Costar; Corning Inc.) at a cell density of 20,000-40,000 per well. Test compounds were applied to the cells at varying concentrations. Cells were incubated in a cell culture incubator at 37° C. with 5% CO2 for 24-26 hr. Assay plates were washed with DPBS media (Thermo, cat# SH30028.02) to remove unbound cells and compound. Stimulation media (25 μL) containing 20 μM Forskolin & 30 μM P3 [6-(Ethyl-phenyl-sulfonyl)-4-oxo-1, 4-dihydro-quinoline-3-carboxylic acid 2-methoxy-benzylamide] in Hams F-12 Coon's modified media was added to the plate wells and incubated at RT for 60-120 min. 25 μL of HEPES-PBS-I buffer (10 mM HEPES, 1 mM MgCl2, 3 mM KCl, 1 mM CaCl2, 150 mM NaI) was then added and fluorescence quench curves (Excitation 500 nm/Emission 540 nm; exposure 136 ms) were immediately recorded on an FDSS-6000 plate reader (Hamamatsu). Quench rates were derived from least squares fitting of the data as described by Sui et al., (2010).
Galietta, L. J., Jayaraman, S., and Verkman, A. S. Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists. Am.J.Physiol Cell Physiol 281(5), C1734, 2001.
Sui J, Cotard S, Andersen J, Zhu P, Staunton J, Lee M, Lin S. (2010) Optimization of a Yellow fluorescent protein-based iodide influx high-throughput screening assay for cystic fibrosis transmembrane conductance regulator (CFTR) modulators. Assay Drug Dev Technol. 2010 Dec; 8(6):656-68.
Alejandro A. Pezzulo, Xiao Xiao Tang, Mark J. Hoegger, Mahmoud H. Abou Alaiwa, Shyam Ramachandran, Thomas O. Moninger, Phillip H. Karp, Christine L. Wohlford-Lenane, Henk P. Haagsman, Martin van Eijk, Botond Ba'nfi, Alexander R. Horswill, David A. Stoltz, Paul B. McCray Jr, Michael J. Welsh, Joseph Zabner. Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature 487, 109-115 (2012).
Primary CF airway epithelial cells were obtained from the UNC Cystic Fibrosis Tissue Procurement and Cell Culture Core. The cells are grown at 37° C. in a Heracell 150i incubator using growth media (BEGM, Fischer). Cells were then transferred to differentiation media (airway liquid interface media (ALI) media; Lechner JF and LaVeck MA, J. Tissue Culture Methods 1985, 9: 43-48) for a minimum of 4 weeks on coated Costar snapwells. Two days before the Ussing assay the mucus on the apical surface of the cells was aspirated after incubating with 200 μL of differentiation media for at least thirty (30) minutes. One day before the Ussing assay test compounds were added to the basolateral surface of the cells at various test concentrations dissolved in DMSO. Duplicate wells were prepared giving a n=3 or n=4 protocol.
Cells were treated for 24 hours with various combinations and concentrations of the test articles, reference standard (3 μM VX809 or 3 μM FDL169, positive control). Compounds stock solutions were prepared in DMSO and diluted 1/1000 into ALI media to their final assay concentration. Cells were treated with combination solutions (2 mL of each dilution) and incubated at 37° C. for 24 h.
For an Ussing assay, cells on four Snapwell (6-well) plates were treated 24 hours prior to experimentation. The next day filters from individual Snapwells were removed from the plates and mounted vertically in Ussing chambers pre-equilibrated at 37° C. in 5 ml of HBS (pH 7.4) both apical and basolateral sides and bubbled with room air to facilitate mixing upon addition of compounds. The resting current was recorded for 10 min to ensure a stable baseline. Resting current was blocked by the apical addition of 3 μM benzamil, an ENaC inhibitor. After 10 min, 10 μM forskolin was added to both the apical and basolateral side to stimulate CFTR. The increase in chloride current was detected as an upward deflection of the trace. After an additional 10 min, the potentiator VX770 (1 μM) was added, further increasing the chloride current. Finally CFTR-172 (a CFTR inhibitor, 20 μM) and/or bumetanide (20 μM) was added to block CFTR mediated chloride current, resulting in a decrease in the observed current.
For the equivalent current assay, cells on four Transwell (24-well) plates were treated. Each Transwell plate was filled with 200 μl of HBS on the apical surface and 2 ml on the basolateral surface. Plates were placed horizontally in a heated mount at 37° C., and equilibrated for several minutes. Resting current was measured for 15 min and then blocked by the apical addition of 5 μM benzamil. After 20 min, 10 μM forskolin and 1 μM VX770 were added to both the apical and basolateral side to stimulate CFTR. An increase in chloride current is seen as an upward deflection of the trace. After another 30 min, CFTR-172 (a CFTR inhibitor, 20 μM) and/or bumetanide (20 uM) was added to block CFTR mediated chloride current.
The raw data, current vs. time for the Ussing chamber (sampling interval: 10 s) and voltage vs. time and resistance vs. time for the equivalent current assay (sampling interval: 5 minutes) were transferred to Excel (Microsoft Office Professional, version 14.0.7106.5003) for analysis. CFTR specific current was measured as the average amplitude of the increase in current elicited upon addition forskolin and ending upon addition of the CFTR channel specific blocker CFTR-172. This average is equivalent to the sum of the average forskolin activated and the average VX770-potentiated currents. The average current measured in vehicle (0.1% DMSO) treated cells, Iv, was subtracted from the current for the test article, ITA, or from the corrector reference standard VX809 (3 uM ISTD). For replicate measurements, the average vehicle subtracted response for the test article, was normalized to the average vehicle subtracted inhibitor response of the reference corrector VX809 (3 μM).
I
NSC=(ITA−IV)(ave)/(ISTD−IV)(ave) (Equation 1)
A second endpoint, for the equivalent current assay, evaluated was NAUC, the normalized area under the curve (AUC) measuring the response after addition of forskolin and VX770 to the time point right before the addition of the CFTR inhibitor. The AUC is effectively the average response multiplied by the duration of the response. The AUC of the test article, AUCTA was then corrected by subtracting the average vehicle response, AUCV,ave over the same time range, and normalized as for the inhibitor-sensitive current to the difference of the corrector reference standard VX809 (3 μM VX809r,ave) or FDL176 (3 μM FDL176r,ave) and the vehicle response:
NAUCTA=[AUCTA−AUCV,ave]/[AUCVx809r,ave−AUCV,ave] (Equation 2).
The normalized value for DMSO is 0.0 and for VX-809 alone is 1.0. Combinations of compounds with VX-809 that give normalized values greater than 1.0 show activity in the combination assay. A value of 2 means the test compound doubles the effect to VX-809 alone.
Experiments were run with a minimum of n=4 replicates per concentration. Since the distribution for the ratio of two normal distributions is a Cauchy distribution, the median value must be used for the average and the average deviation must be used for the error of all normalized data. Potency (EC50) and efficacy (maximum response) were determined by fitting dose response data to a sigmoid dose response model (GraphPad Prism 5.04, Manufacturer) using Equation 3:
E=E
min+(Emax−Emin)/(1+10{circumflex over ( )}((LogEC5OS)*nH)) (Equation 3)
where E is the recorded response, and S is the concentration of test compound in combination with VX-809. Since there were at most 8 points in the dose response curve only EC50 and maximum (Emax) were allowed to vary, while the minimum (Emin) was fixed equal to the VX-809 response of 1.0, and the Hill slope, nH, was fixed equal to 1.
Statistical comparisons (t-test and Mann Whitney) and calculation of averages and errors were performed in Excel.
The table below provides the results of the equivalent current assays in Primary CF airway epithelial for cells treated with combination solutions (2 mL of each dilution: either FDL169 (3 μM) or VX-809 (3 μM) as noted in column 5-1st site corrector) and incubated at 37° C. for 24 h.
NAUC “+++” refers to an observed NAUC>170% of positive control; NAUC “++” refers to an observed NAUC 170-140% of positive control; NAUC “+” refers to an observed NAUC <140% of positive control.
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
This application is a continuation of International Application No. PCT/US2020/015441, which designated the United States and was filed on Jan. 28, 2020, published in English, which claims the benefit of U.S. Provisional Application No. 62/797,743, filed on Jan. 28, 2019 and U.S. Provisional Application No. 62/931,502, filed on Nov. 6, 2019. The entire teachings of the above applications are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 62797743 | Jan 2019 | US | |
| 62931502 | Nov 2019 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2020/015441 | Jan 2020 | US |
| Child | 17383797 | US |
