Patent Application
20150266856
1. Field of the Invention
This invention relates to modulators of the retinoid-related orphan receptor RORγ and methods for using such modulators. The compounds described herein can be particularly useful for diagnosing, preventing, or treating a variety of diseases and disorders in humans and animals. Exemplary disorders include psoriasis, arthritis, asthma, inflammatory bowel disease and multiple sclerosis.
2. Summary of the Related Art
The retinoid-related orphan receptors RORα, RORβ, and RORγ play an important role in numerous biological processes including organ development, immunity, metabolism, and circadian rhythms. See, for example, Dussault et al. in Mech. Dev. (1998) vol. 70, 147-153; Andre et al. in EMBO J. (1998) vol. 17, 3867-3877; Sun et al. in Science (2000) vol. 288, 2369-2373; and Jetten in Nucl. Recept. Signal. (2009) vol. 7, 1-32.
RORγ is expressed in several tissues including the thymus, kidney, liver, and muscle. Two isoforms of RORγ have been identified: RORγ1 and RORγ2 (also known, respectively, as RORγ and RORγt). See, for example, Hirose et al. in Biochem. Biophys. Res. Commun. (1994) vol. 205, 1976-1983; Oritz et al. in Mol. Endocrinol. (1995) vol. 9, 1679-1691; and He et al. in Immunity (1998) vol. 9, 797-806. Expression of RORγt is restricted to lymphoid cell types including CD4+CD8+ thymocytes, IL-17 producing T helper (Th17) cells, lymphoid tissue inducer (LTi) cells, and γ7 cells. RORγt is essential for the development of lymph nodes and Peyer's patches and for the normal differentiation of Th17, γδ, and LTi cells. See, for example, Sun et al. in Science (2000) vol. 288, 2369-2373; Ivanov et al. in Cell (2006) vol. 126, 1121-1133; Eberl et al. in Nat. Immunol. (2004) vol. 5, 64-73; Ivanov et al. in Semin. Immunol. (2007) vol. 19, 409-417; and Cua and Tato in Nat. Rev. Immunol. (2010) vol. 10, 479-489.
Proinflammatory cytokines such as IL-17A (also referred to as IL-17), IL-17F, and IL-22 produced by Th17 cells and other RORγ+ lymphocytes activate and direct the immune response to extracelluar pathogens. See, for example, Ivanov et al. in Semin. Immunol. (2007) vol. 19: 409-417; and Marks and Craft in Semin. Immunol. (2009) vol. 21, 164-171. RORγ directly regulates IL-17 transcription and disruption of RORγ in mice attenuates IL-17 production. See, for example, Ivanov et al. in Cell (2006) vol. 126, 1121-1133.
Dysregulated production of IL-17 has been implicated in several human autoimmune and inflammatory diseases including multiple sclerosis, rheumatoid arthritis, psoriasis, inflammatory bowel disease (IBD), and asthma. See, for example, Lock et al. in Nat. Med. (2002) vol. 8, 500-508; Tzartos et al. in Am. J. Pathol. (2008) vol. 172, 146-155; Kotake et al. in J. Clin. Invest. (1999) vol. 103, 1345-1352; Kirkham et al. in Arthritis Rheum. (2006) vol. 54, 1122-1131; Lowes et al. in J. Invest. Dermatol. (2008) vol. 128, 1207-1211; Leonardi et al. in N. Engl. J. Med. (2012) vol. 366, 1190-1199; Fujino et al. in Gut (2003) vol. 52, 65-70; Seiderer et al. in Inflamm. Bowel Dis. (2008) vol. 14, 437-445; Wong et al. in Clin. Exp. Immunol. (2001) vol. 125, 177-183; and Agache et al. in Respir. Med. (2010) 104: 1131-1137. In murine models of these diseases, inhibition of IL-17 function by neutralizing antibodies or genetic disruption of IL-17 or IL-17 receptor ameliorates the disease course or clinical symptoms. See, for example, Hu et al. in Ann. N.Y. Acad. Sci. (2011) vol. 1217, 60-76.
Disruption of RORγ in mice also attenuates disease progression or severity in animal models of autoimmunity and inflammation including experimental autoimmune encephalomyelitis (EAE), imiquimod induced psorasis, colitis, and allergic airway disease. See, for example, Ivanov et al. in Cell (2006) vol. 126, 1121-1133; Yang et al. in Immunity (2008) vol. 28, 29-39; Pantelyushin et al. in J. Clin. Invest. (2012) vol. 122, 2252-2256; Leppkes et al. in Gastroenterology (2009) vol. 136, 257-267; and Tilley et al. in J. Immunol. (2007) vol. 178, 3208-3218.
Each of the references in this Background section is hereby incorporated herein by reference in its entirety for all purposes.
Therapeutic agents exist to treat a variety of inflammatory and autoimmune diseases, but there still remains a significant unmet medical need in these therapeutic areas. Given the role of IL-17 in human disease and the validation of IL-17 and RORγ as targets in murine disease models, compounds capable of modulating RORγt activity are contemplated to provide a therapeutic benefit in the treatment of multiple immune and inflammatory disorders.
In one aspect, the invention comprises compounds of the formula (I),
or N-oxides, pharmaceutically acceptable salts, solvates and/or hydrates thereof, wherein R1, R2, R3, R4, R5, R6, Ra, x, y, G, L, Z, the bond denoted by “q”, the ring system denoted by “A” and the ring system denoted by “B” are defined herein. The invention includes stereoisomeric forms of the compounds of formula I, including stereoisomerically-pure, scalemic and racemic forms.
In another aspect, the invention comprises pharmaceutical compositions comprising a compound according to formula (I), stereoisomeric form, N-oxide, pharmaceutically acceptable salt, solvate or hydrate as described herein, and a pharmaceutically acceptable carrier, excipient, or diluent.
In another aspect, the invention comprises methods for antagonizing RORγ in a cell comprising contacting the cell with an effective amount of a compound according to formula (I), stereoisomeric form, N-oxide, pharmaceutically acceptable salt, solvate or hydrate as described herein. This aspect may be conducted in vitro or in vivo.
In another aspect, the invention comprises methods for treating a subject suffering from a disease or disorder modulated by RORγ, the method comprising administering to a subject a therapeutically effective amount of compound according to formula (I), stereoisomeric form, N-oxide, pharmaceutically acceptable salt, solvate, hydrate or pharmaceutical composition as described herein.
In another aspect, the invention comprises a method for treating a disease or disorder selected from an inflammatory disease or disorder, an autoimmune disease or disorder, an allergic disease or disorder, a metabolic disease or disorder, and/or cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of compound according to formula (I), or a stereoisomeric form, N-oxide, pharmaceutically acceptable salt, solvate, hydrate or pharmaceutical composition thereof as described herein.
In one aspect, the invention comprises compounds of formula (I),
and stereoisomeric forms thereof, and N-oxides thereof, and pharmaceutically acceptable salts thereof, and solvates and hydrates thereof, wherein
In another aspect, the invention comprises compounds of formula (I), and stereoisomeric forms thereof, and N-oxides thereof, and pharmaceutically acceptable salts thereof, and solvates and hydrates thereof, wherein
In another aspect, the invention provides compounds of formula (Ia),
and stereoisomeric forms thereof, and N-oxides thereof, and pharmaceutically acceptable salts thereof, and solvates and hydrates thereof, wherein
The “L” moieties as described herein are presented with the connection to the central pyrimidine at the left, and the connection to the ring system denoted by “A” at the right.
The invention further comprises subgenera of formula (I) in which the substituents are selected as any and all combinations of one or more of R1, R2, R3, R4, R5, R6, Ra, x, y, G, L, Z, the bond denoted by “q”, the ring system denoted by “A” and the ring system denoted by “B” as defined herein, including without limitation, the following:
In certain embodiments, the compound has one of the formulae (Ib)-(Ie):
In certain embodiments of the compounds of any of formulae (I) and (Ia)-(1e), R1 is selected from one of the following groups (1a)-(1o):
In certain embodiments of the compounds of any of formulae (I), (Ia), (Ib) and (Id) as described above, R2 is selected from one of the following groups (2a)-(2l):
In certain embodiments of the compounds of any of formulae (I), (Ia), (Ic) and (Ie) as described above, R3 is selected from one of the following groups (3a)-(3d):
In certain embodiments of the compounds of any of formulae (I) and (Ia)-(Ie) as described above, the R4 are defined as in one of the following groups (4a)-(4f):
In certain embodiments of the compounds of any of formulae (I) and (Ia)-(Ie) as described above, R5 is selected from one of the following groups (5a)-(5e):
In certain embodiments of the compounds of any of formulae (I) and (Ia)-(Ie) as described above, the bond denoted by “q” is selected from one of the following (q-1)-(q-2):
In certain embodiments of the compounds of any of formulae (I) and (Ia)-(Ie) as described above, the combination of the bond denoted by “q”, the R4 and R5 is selected from one of the following groups (q-4-5a)-(q-4-5f):
In certain embodiments of the compounds of any of formulae (I), (Ib) and (Ic) as described above, Z is selected from one of the following groups (Za)-(Zd):
In certain embodiments of the compounds of any of formulae (I) and (Ia)-(Ie) as described above, G is selected from one of the following groups (Ga)-(Gk):
In certain embodiments of the compounds of any of formula (I) and (Ia)-(Ie) as described above, the ring system denoted by “B” is selected from one of the following groups (Ba)-(Bg):
In certain embodiments of the compounds of any of formula (I) and (Ia)-(Ie) as described above, y is selected from one of the following groups (ya)-(yc):
In certain embodiments of the compounds of any of formula (I) and (Ia)-(Ie) as described above, each R6 is selected from one of the following groups (6a)-(6m):
In certain embodiments of the compounds of any of formula (I), (Ib) and (Ic) as described above, the ring system denoted by “B” and y are selected such that the
moiety has the structure (B-ya):
in which each R18 is R6 or H provided that at least one R18 is not H.
In certain embodiments of the compounds of any of formula (I) and (Ia)-(Ie) as described above, L is selected from one of the following groups (La)-(Lv):
In certain embodiments of the compounds of any of formula (I) and (Ia)-(Ie) as described above, the ring system denoted by “A” is selected from one of the following groups (Aa)-(Ai):
In certain embodiments of the compounds of any of formula (I) and (Ia)-(Ie) as described above, x is selected from one of the following groups (xa)-(xe):
In certain embodiments of the compounds of any of formula (I) and (Ia)-(Ie) as described above, the ring system denoted by “A” and x are selected to form a residue selected from one of the following (A-xa)-(A-xs):
In certain embodiments of the compounds of any of formula (I) and (Ia)-(Ie) as described above, each Ra is selected from one of the following groups (aa)-(ah):
In certain embodiments of the compounds of any of formula (I) and (Ia)-(Ie) as described above, the combination of the ring system denoted by “A”, x and all Ra groups combine to form a residue selected from one of the following (A-x-aa)-(A-x-aad):
Particular embodiments according to this aspect of the invention include compounds of Formula (I) as defined in each of the following rows, in which each entry is a group number as defined above (e.g., (1e) indicates that R1 is —CH3), and a dash “-” indicates that the variable is as defined for formula (I) or is defined according to any applicable variable definition above (e.g., when the R1 column is “-”, R1 can be defined as for Formula (I) or any one of definitions (1a)-(1o)).
Particular embodiments according to this aspect of the invention include compounds of Formula (I) as defined above, in which
The compounds according to such embodiments can be further defined as described above, as appropriate.
Other embodiments of the invention as described herein include compounds of formula (II)
in which
The compounds according to such embodiments can be further defined as described above, as appropriate.
As noted above, each alkyl moiety (i.e., defined as “alkyl”) is optionally substituted with one or more substituents (e.g., in various embodiments, from 1-5, from 1-3, from 1-2 or one) selected from the group consisting of -D, -halogen, —CN, —NO2, —O—R20, —N(R21)2 and —S(R20), in which each R20 is independently selected from the group consisting of —H, —CH3, —CH2CH3 and —CF3, and each R21 is independently selected from the group consisting of —H, —CH3 and —CH2CH3. In certain embodiments of the invention as described herein, each alkyl is optionally substituted with one or more substituents selected from the group consisting of -D, -halogen, —O—R20 and —N(R21)2 and —S(R20), in which each R20 is independently selected from the group consisting of —H, and —CH3, and each R21 is independently selected from the group consisting of —H and —CH3. In other embodiments, each alkyl is optionally substituted with one or more substituents selected from the group consisting of -D, -halogen, —OH, —NH2 and —SH. In other embodiments, each alkyl is unsubstituted.
As used herein, the “alkyl” groups are defined as having a given number of carbons. Accordingly, “(C1-C4)alkyl” is an alkyl group having from one to four carbons. An alkyl group can be branched or unbranched. Thus, “(C1-C4)alkyl” encompasses methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl and t-butyl, in unsubstituted and substituted forms as described above. In certain embodiments, each —(C1-C6)alkyl is a (C1-C4)alkyl. In certain embodiments, each —(C1-C6)alkyl and —(C1-C4)alkyl is a —(C1-C2)alkyl.
The term “fluoroalkyl” as used herein, means an alkyl group substituted with one or more fluorines and no other substituents. In certain embodiments, one or more carbons of the fluoroalkyl group is persubstituted with fluorine. Examples of fluoroalkyl moieties include, without limitation, fluoromethyl, difluromethyl, trifluoromethyl, pentafluoroethyl, 2,2,2-trifluoroethyl, and 1,1,1,3,3,3-hexafluoroisopropyl. “Fluoroalkyl” is encompassed within optionally-substituted alkyl as described above. In certain embodiments, each —(C1-C6)fluoroalkyl is a (C1-C4)fluoroalkyl. In certain embodiments, each —(C1-C6)fluoroalkyl and —(C1-C4)fluoroalkyl is a —(C1-C2)fluoroalkyl.
The term “heterocycloalkyl” as used herein means a cyclic unsaturated or partially unsaturated group that includes one or more (e.g., 1, 2 or 3, for example, 1 or 2) heteroatoms selected from N, O and S in the ring. The heterocycloalkyl can be, for example, a (C3-C6)heterocycloalkyl (i.e., having 3-6 members in the ring, inclusive of heteroatoms). In certain embodiments, each heterocycloalkyl is selected from the group consisting of piperidine, piperazine and morpholine. In other embodiments, each heterocycloalkyl is selected from the group consisting of piperidine, piperazine, morpholine, 1H-tetrahydropyran, pyrrolidine and tetrahydrofuran. In other embodiments, each heterocycloalkyl is selected from the group consisting of piperidine, piperazine, morpholine, 1H-tetrahydropyran, pyrrolidine, tetrahydrofuran, azetidine, aziridine and oxetane.
In general, any hydrogen atom of the compounds described herein (whether described explicitly as “—H” or as part of another moiety such as an alkyl or a phenyl) can be provided as a protium, or a deuterium. Thus, while deuterium is often described herein as a “substituent,” the person of skill in the art will understand that deuterium can be used as the hydrogen atom species at any position in the compound. However, in certain embodiments of the compounds described herein, every hydrogen atom, unless otherwise explicitly specified, is a protium.
The term “deuteroalkyl” as used herein means an alkyl group substituted with one or more deuteria and no other substituents. Examples of “deuteroalkyl” include deuteromethyl and dideuteromethyl.
Individual compounds of certain embodiments of the present invention are provided in Tables 1-15 below.
The compounds disclosed herein can be made using procedures familiar to the person of ordinary skill in the art and as described herein. For example, compounds of structural formula (I) can be prepared according to Schemes 1-50, below, or analogous synthetic schemes:
Additional schemes are provided in the Examples, below.
One of skill in the art can adapt the reaction sequences of Schemes 1-50 and of the Examples to fit the desired target molecule. For example, use of a 2-ethylpyrimidine will result in compounds in which R1 is ethyl, instead of methyl as in many of the example compounds. Similarly, while the schemes generally depict the -(“B” ring system)-(Rb)y moiety as ortho-disubstituted phenyl, the person of skill will appreciate that use of different starting materials will provide different rings and/or different patterns of substitution. Of course, in certain situations one of skill in the art will use different reagents to affect one or more of the individual steps or to use protected versions of certain of the substituents. Additionally, one skilled in the art would recognize that compounds of structural formula (I) can be synthesized using different routes altogether.
The compounds of the present invention can be provided in a number of stereoisomeric forms. Accordingly, another aspect of the invention is a stereoisomeric form of a compound as described herein. For example, a compound of the present invention can be provided in racemic form. In other embodiments, a compound of the present invention is provided in scalemic form, or in a stereoisomerically pure form (e.g., substantially as a single enantiomer).
Another aspect of the invention is an N-oxide of a compound or stereoisomeric form as described herein.
Another aspect of the invention is a pharmaceutically acceptable salt of a compound, stereoisomeric form, or N-oxide as described herein. As used herein, the phrase “pharmaceutically acceptable salt” refers to both pharmaceutically acceptable acid and base addition salts. Such pharmaceutically acceptable salts include salts of acids such as hydrochloric, phosphoric, hydrobromic, sulfuric, sulfinic, formic, trifluoroacetic, toluenesulfonic, methanesulfonic, nitric, benzoic, citric, tartaric, maleic, hydroiodic, alkanoic such as acetic, HOOC—(CH2)n—COOH where n is 0-4, and the like. Non-toxic pharmaceutical base addition salts include salts of bases such as sodium, potassium, calcium, ammonium, and the like. Those skilled in the art will recognize a wide variety of non-toxic pharmaceutically acceptable addition salts.
Another aspect of the invention is a solvate or hydrate of a compound, stereoisomeric form, N-oxide or pharmaceutically acceptable salt as described herein. The person of skill in the art can determine whether a particular compound will form a solvate or a hydrate.
Another aspect of the invention is a pharmaceutical composition including a compound, stereoisomeric form, N-oxide, pharmaceutical salt, solvate or hydrate as described herein The pharmaceutical compositions described herein generally comprise a combination of a compound described herein and a pharmaceutically acceptable carrier, diluent, or excipient. Such compositions are substantially free of non-pharmaceutically acceptable components, i.e., contain amounts of non-pharmaceutically acceptable components lower than permitted by U.S. regulatory requirements at the time of filing this application. In some embodiments of this aspect, if the compound is dissolved or suspended in water, the composition further optionally comprises an additional pharmaceutically acceptable carrier, diluent, or excipient. In other embodiments, the pharmaceutical compositions described herein are solid pharmaceutical compositions (e.g., tablet, capsules, etc.).
These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
Also, pharmaceutical compositions can contain, as the active ingredient, one or more of the compounds described herein above in combination with one or more pharmaceutically acceptable carriers. In making the compositions described herein, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.
Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions described herein can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the subject by employing procedures known in the art.
The active compound can be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the like.
For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound described herein. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above containing from, for example, 0.1 to about 500 mg of the active ingredient of a compound described herein.
The tablets or pills can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
The liquid forms in which the compounds and compositions can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face masks tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner.
The amount of compound or composition administered to a subject will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the subject, the manner of administration, and the like. In therapeutic applications, compositions can be administered to a subject already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Effective doses will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as the severity of the disease, the age, weight and general condition of the subject, and the like.
The compositions administered to a subject can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations typically will be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.
The therapeutic dosage of the compounds can vary according to, for example, the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the subject, and the judgment of the prescribing physician. The proportion or concentration of a compound described herein in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds described herein can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dose ranges are from about 1 g/kg to about 1 g/kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular subject, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.
The compounds of the present invention are useful to prevent, diagnose, and treat various medical disorders in humans or animals. The compounds are used to inhibit or reduce one or more activities associated with RORγ receptors, relative to RORγ receptors in the absence of the same compounds. Thus, in one aspect of the invention, a method for treating a disease or disorder selected from an autoimmune disease or disorder, asthma, an allergic disease or disorder, a metabolic disease or disorder, and cancer in a subject comprises administering to the subject a therapeutically effective amount of compound according to formula (I), stereoisomeric form, N-oxide, pharmaceutically acceptable salt, solvate, hydrate or pharmaceutical composition as described herein. See, e.g., L. A. Solt et al., “Action of RORs and their ligands in (patho)physiology,” Trends Endocrinol Metab., preprint available online Jul. 11, 2012 at http://www.sciencedirect.com/science/article/pii/S1043276012000926; M. S. Maddur et al., “Th17 cells: biology, pathogenesis of autoimmune and inflammatory diseases, and therapeutic strategies,” Am. J. Pathol. 2012 July; 181(1):8-18; and A. M. Jetten, “Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism,” Nucl. Recept. Signal. 2009; 7:e003, each of which is hereby incorporated herein by reference in its entirety, as well as the references discussed in the Background section. In certain embodiments, the autoimmune disease or disorder is selected from rheumatoid arthritis, ankylosing spondylitis, psoriasis and psoriatic arthritis, multiple sclerosis, inflammatory bowel diseases and lupus. In certain embodiments, the allergic disease or disorder is selected from allergic rhinitis and dermatitis. In certain embodiments, the metabolic disease or disorder is selected from obesity, obesity-induced insulin resistance and type II diabetes.
In certain embodiments, the disease or disorder is rheumatoid arthritis. See, e.g., L. A. Solt et al., referenced above, as well as the references discussed in the Background section.
In other embodiments, the disease or disorder is multiple sclerosis. See, e.g., L. Codarri et al., “RORγt drives production of the cytokine GM-CSF in helper T cells, which is essential for the effector phase of autoimmune neuroinflammation,” Nat. Immunol., 2011 June; 12(6):560-7, which is hereby incorporated herein by reference in its entirety, as well as the references discussed in the Background section.
In other embodiments, the disease or disorder is ankylosing spondylitis. See, e.g., E. Toussirot, “The IL23/Th17 pathway as a therapeutic target in chronic inflammatory diseases,” Inflamm. Allergy Drug Targets, 2012 April; 11(2):159-68, which is hereby incorporated herein by reference in its entirety, as well as the references discussed in the Background section.
In other embodiments, the disease or disorder is inflammatory bowel disease. See, e.g., M. Leppkes et al., “RORgamma-expressing Th17 cells induce murine chronic intestinal inflammation via redundant effects of IL-17A and IL-17F,” Gastroenterology, 2009 January; 136(1):257-67, which is hereby incorporated herein by reference in its entirety, as well as the references discussed in the Background section.
In other embodiments, the disease or disorder is lupus. See, e.g., K. Yoh et al., “Overexpression of RORγt under control of the CD2 promoter induces polyclonal plasmacytosis and autoantibody production in transgenic mice,” Eur. J. Immunol., 2012 August; 42(8):1999-2009, which is hereby incorporated herein by reference in its entirety, as well as the references discussed in the Background section.
In other embodiments, the disease or disorder is psoriasis. See, e.g., S. Pantelyushin et al., “RORγt+ innate lymphocytes and γδ T cells initiate psoriasiform plaque formation in mice,” J. Clin. Invest., 2012 Jun. 1; 122(6):2252-6; and S. P. Raychaudhuri, “Role of IL-17 in Psoriasis and Psoriatic Arthritis,” Clin. Rev. Allergy Immunol., preprint available online Feb. 24, 2012 at http://rd.springer.com/article/10.1007/s12016-012-8307-1 (PubMed PMID: 22362575), each of which is hereby incorporated herein by reference in its entirety, as well as the references discussed in the Background section.
In other embodiments, the disease or disorder is psoriatic arthritis. See, e.g., S. P. Raychaudhuri, referenced above, as well as the references discussed in the Background section.
In other embodiments, the disease or disorder is graft-vs.-host disease (GVHD). Y. Yu et al., “Prevention of GVHD while sparing GVL effect by targeting Th1 and Th17 transcription factorT-bet and RORγt in mice,” Blood, 2011 Nov. 3; 118(18):5011-20, which is hereby incorporated herein by reference in its entirety, as well as the references discussed in the Background section.
In other embodiments, the disease or disorder is autoimmune uveitis. See, e.g., R. Horai et al., “Cytokines in autoimmune uveitis,” J. Interferon Cytokine Res., 2011 October; 31(10):733-44, which is hereby incorporated herein by reference in its entirety, as well as the references discussed in the Background section.
In other embodiments, the disease or disorder is obesity and/or insulin resistance. See, e.g., B. Meissburger et al., “Adipogenesis and insulin sensitivity in obesity are regulated by retinoid-related orphan receptor gamma,” EMBO Mol. Med., 2011 November; 3(11):637-51, which is hereby incorporated herein by reference in its entirety, as well as the references discussed in the Background section.
In other embodiments, the disease or disorder is melanoma. See, e.g., Purwar R, et al. Robust tumor immunity to melanoma mediated by interleukin-9-producing T cells. Nat. Med., 2012 July: 18:1248-53, which is hereby incorporated herein by reference in its entirety, as well as the references discussed in the Background section.
In certain aspects, the medical disorder being diagnosed, treated, or prevented by use of the presently disclosed compounds can be, for example, an autoimmune disorder. In other embodiments, the disorder being diagnosed, treated or prevented by use of the presently disclosed compounds can be an inflammatory disorder. For example, in certain embodiments, the disorder is selected from arthritis, diabetes, multiple sclerosis, uveitis, rheumatoid arthritis, psoriasis, asthma, bronchitis, allergic rhinitis, chronic obstructive pulmonary disease, atherosclerosis, H. pylori infection and inflammatory bowel disease. In other embodiments, the disorder is selected from Crohn's disease, ulcerative colitis, sprue and food allergies. In other embodiments, the disorder is experimental autoimmune encephalomyelitis, imiquimod-induced psoriasis, colitis or allergic airway disease.
As used herein, the phrase “therapeutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician.
In certain embodiments, a therapeutically effective amount can be an amount suitable for (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder; or (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease.
As used here, the terms “treatment” and “treating” means (i) ameliorating the referenced disease state, for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing or improving the pathology and/or symptomatology) such as decreasing the severity of disease; (ii) eliciting the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician; or (iii) inhibiting the referenced disease state; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder.
tert-Butyl 4-(3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate (40 g, 133 mmol) and dimethylformamide dimethylacetal (DMFDMA) (19 g, 160 mmol) were dissolved in toluene (40 mL). The solution was stirred under reflux for 5 h and cooled to room temperature. The solution was concentrated and used as is in the next reaction.
To a suspension of acetamidine hydrochloride (15.1 g, 160 mmol) in EtOH (100 mL) was added EtONa (11 g, 160 mmol). The resulting mixture was stirred for 15 min at room temperature. Crude enamine A1.1 was dissolved in EtOH (100 mL) and added to the acetamidine hydrochloride mixture. The mixture was heated to 70° C. with stirring for 2 h. The solvent was removed under reduced pressure. The residue was stirred in EtOAc (500 mL) and filtered. Concentration of the filtrate gave the crude product, which was further purified by flash column chromatography.
To a solution of A1.2 (5 g, 13.7 mmol) in EtOH (20 mL) was added 4N HCl in dioxane (12 mL). The resulting solution was stirred for 12 h at room temperature. Ether (100 mL) was added with stirring. The solid was filtered and dried under vacuum. The crude product was used in the next step without further purification.
A1.3 was mixed with 2,6-difluorophenylacetic acid (3.13 g, 18 mmol), hydroxybenzotriazole (HOBt) (2.43 g, 18 mmol) and N,N-diisopropylethylamine (DIEA) (9 g, 70 mmol) in tetrahydrofuran (THF) (50 mL). To this solution was added ethyl(dimethylaminopropyl) carbodiimide (EDC) (3.43 g, 18 mmol). The resulting reaction mixture was stirred at room temperature for 5 h then diluted with aq. sat. NaHCO3. The resulting mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated.
Crude A1.4 was stirred with NaOH (1.7 g, 42 mmol) in MeOH/water (40 mL/20 mL) at room temperature for 10 h. The solution was concentrated and the solid was dissolved in water (40 mL) and acidified with conc. HCl. The precipitated product was filtered and dried.
A1.5 (150 mg, 0.39 mmol) was mixed with 3,5-dimethylbenzylamine (62 mg, 0.45 mmol) and DIEA (0.5 mL) in THF (3 mL). To this solution was added 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronoium hexafluorphosphate (HATU) (217 mg, 0.58 mmol). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The mixture was extracted with CH2Cl2, dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by preparative HPLC to give Compound A1. 1H-NMR (400 MHz, DMSO-d6): δ 9.13 (t, 1H), 8.64 (s, 1H), 7.36 (m, 1H), 7.08 (t, 2H), 6.96 (s, 2H), 6.91 (s, 1H), 4.40 (m, 3H), 4.16 (d, 1H), 3.80 (d, 1H), 3.76 (d, 1H), 3.26 (m, 1H), 3.06 (m, 1H), 2.63 (s, 3H), 2.56 (m, 1H), 2.26 (s, 6H), 1.72 (m, 4H). MS (EI) for C28H30F2N4O2. found: 493.0 (MH+). Analytical HPLC, ret. time=13.5 min, 93% purity.
Compounds A2 to A79 were prepared from methods analogous to those used to prepare Compound A1 utilizing appropriate reagent replacements at various steps.
tert-Butyl-4-(3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate (6.0 g, 20.0 mmol), AcOH (57 μL, 1.0 mmol), pyridine (80 μL, 1.0 mmol), and toluene (100 mL) were charged into a 250 mL flask equipped with fractional distillation apparatus. The reaction mixture was stirred at 130° C. (bath temperature) and the solvent distilled at about the boiling point of the MeOH/toluene azeotrope for 48 h. The reaction mixture was cooled to room temperature and concentrated in vacuo to give tert-butyl 4-(2-(ethoxy(or methoxy)carbonyl)-3-methoxybut-2-enoyl)piperidine-1-carboxylate which was used directly in the next step. To a stirred suspension of acetamidine HCl (2.84 g, 30.0 mmol) in EtOH (60 mL) was added NaOEt (2.05 g, 30.0 mmol) and the reaction mixture was stirred at room temperature for 30 min. To this suspension was added the crude tert-butyl 4-(2-(ethoxy(or methoxy)carbonyl)-3-methoxybut-2-enoyl)piperidine-1-carboxylate in EtOH (20 mL) slowly at room temperature and the resulting mixture was stirred at reflux for 5 h. The reaction mixture was cooled down to room temperature, concentrated under reduced pressure, and the residue was partitioned between water and CH2Cl2. The separated aq. layer was extracted with CH2Cl2 (×2) and the combined organic layers were dried over sodium sulfate, concentrated in vacuo, and purified by flash chromatography to give a mixture of the methyl and ethyl esters of A80.1 (2.51 g, ca. 35%).
To a stirred solution of A80.1 (2.51 g, ca. 6.91 mmol) in 1,4-dioxane (20 mL) was added HCl (20 mL, 4M in 1,4-dioxane, 80 mmol) and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and the residue was used for the next step without further purification.
A mixture of crude A80.2, 2,6-difluorophenylacetic acid (1.43 g, 8.31 mmol), Et3N (4.82 mL, 34.6 mmol) and DMF (20 mL) was treated with HATU (3.16 g, 8.31 mmol) and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with EtOAc, washed with aq. sodium bicarbonate, brine, dried over sodium sulfate, concentrated in vacuo and the residue purified by flash chromatography to afford a mixture of the methyl and ethyl esters of A80.3 (2.34 g, ca. 81%).
To a stirred solution of A80.3 (2.1 g, ca 5.0 mmol) in MeOH (30 mL) was added aq. IN NaOH (20 mL) and the reaction mixture was stirred at room temperature overnight. MeOH was removed in vacuo and the residual aq. layer was acidified with IN HCl to pH 4-5, and extracted with CH2Cl2 (×5). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give acid A80.4 (1.88 g, ca. 97%).
Compound A80 was synthesized from A80.4 and 3,5-dimethylbenzylamine in an identical manner to Compound A1. 1H-NMR (400 MHz, DMSO-d6): δ 9.07 (t, 1H), 7.36 (m, 1H), 7.07 (m, 2H), 6.97 (s, 2H), 6.92 (s, 1H), 4.41 (m, 3H), 4.12 (d, 1H), 3.82 (d, 1H), 3.73 (d, 1H), 2.93 (m, 1H), 2.81 (m, 1H), 2.56 (s, 3H), 2.44 (m, 1H), 2.34 (s, 3H), 2.27 (s, 6H), 1.70 (m, 4H). MS (EI) for C29H32F2N4O2. found: 507.0 (MH+). Analytical HPLC, ret. time=3.46 min, 90% purity.
Compounds A81 to A84 were prepared from methods analogous to those used to prepare Compound A80 utilizing appropriate reagent replacements at various steps.
To a suspension of S-methyl thiourea sulfate (5.5 g, 39 mmol) in EtOH (50 mL) was added EtONa (2.7 g, 39 mmol). The resulting mixture was stirred for 15 min at room temperature. Crude enamine A1.1 in EtOH (30 mL) was added. The mixture was heated at 70° C. with stirring for 2 h. The solvent was removed under reduced pressure. The residue was stirred in EtOAc (500 mL) and filtered. Concentration of the filtrate gave the crude product, which was further purified by flash column chromatography.
To a solution of A85.1 (4.23 g, 11 mmol) in MeOH/water (50 mL/20 mL) was added NaOH (2.5 g, 55 mmol). The resulting mixture was stirred at room temperature for 10 h. MeOH was removed under reduced pressure. The aq. phase was acidified with conc. HCl. The precepited product was filtered and dried under vacuum. The resulting carboxylic acid (800 mg, 2.25 mmol) was mixed with 2,4-dichlorobenzylamine (475 mg, 2.7 mmol) and DIEA (0.5 mL) in CH2Cl2 (4 mL). To this solution was added HATU (1.0 g, 2.7 mmol). The resulting mixture was stirred at room temperature for 12 h then diluted with aq. sat. NaHCO3. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by flash column chromatography.
A85.2 (200 mg, 0.39 mmol) was treated with 4N HCl/dioxane (0.5 mL) in MeOH (3 mL) for 2 h. Ether (10 mL) was added. The precipitated HCl salt was filtered, dried and then mixed with 2,6-difluorophenylacetic acid (78 mg, 0.456 mmol), HOBt (62 mg, 0.456 mmol) and DIEA (193 mg, 1.5 mmol) in CH2Cl2 (3 mL). To this solution was added EDC (88 g, 0.456 mmol). The resulting reaction mixture was stirred at room temperature for 5 h then diluted with aq. sat. NaHCO3. The resulting mixture was extracted with EtOAc and the organic phase was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography. 1H-NMR (400 MHz, DMSO-d6): δ 9.21 (s, 1H), 8.64 (s, 1H), 7.63 (m, 2H), 7.36 (m, 2H), 7.07 (m, 2H), 4.47 (m, 3H), 4.15 (d, 1H), 3.83 (d, 1H), 3.75 (d, 1H), 3.25 (m, 1H), 3.05 (m, 1H), 2.56 (m, 1H), 2.54 (s, 3H), 1.70 (m, 4H). MS (EI) for C26H24Cl2F2N4O2S. found: 564.9 (MH+). Analytical HPLC, ret. time=5.22 min, 95% purity
To a 0° C. solution of A85.3 (50 mg, 0.088 mmol) in CH2Cl2 (3 mL) was added meta-chloroperoxybenzoic acid (mCPBA) (55 mg, 0.22 mmol, ˜70%). The resulting mixture was stirred for 2 h while slowly warming to room temperature. Aq. sat. NaHCO3 was added. The mixture was extracted with CH2Cl2 and the organic phase was washed with aq. Na2SO3 solution, dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography.
A86.1 was treated with MeONa, which was freshly prepared from Na (23 mg, 1 mmol) in MeOH (2 mL). The mixture was stirred at room temperature for 2 h. Water was added to quench the reaction. The mixture was extracted with EtOAc, and the organic phase was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by HPLC to give Compound A86. 1H-NMR (400 MHz, DMSO-d6): δ 9.18 (s, 1H), 8.64 (s, 1H), 7.63 (m, 2H), 7.37 (m, 2H), 7.07 (m, 2H), 4.47 (m, 3H), 4.15 (d, 1H), 3.95 (s, 3H), 3.83 (d, 1H), 3.75 (d, 1H), 3.28 (m, 1H), 3.10 (m, 1H), 2.56 (m, 1H), 1.70 (m, 4H). MS (EI) for C26H24Cl2F2N4O3. found: 549.1 (MH+). Analytical HPLC, ret. time=3.95 min, 95% purity.
Compounds A87 to A98 were prepared from methods analogous to those used to prepare Compounds A85 and A86 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: Phenomenex, Gemini, 50×4.6 mm, 5μ column, gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 5 min gradient time, 6 min total run time at 3.0 mL/min flow rate, λ=254 nM
To a stirred solution of 2-(2,6-difluorophenyl)acetic acid (150 mg, 0.871 mmol) in THF (2 mL) at room temperature was added borane dimethyl sulfide complex (1.30 mL, 2M in THF, 2.61 mmol). After stirring for 16 h at 60° C., the mixture was cooled down to room temperature and quenched with MeOH (1 mL). After stirring for 10 min, volatile materials were removed under vacuum and the resulting mixture was purified by silica gel chromatography (Hexane/EtOAc=6:1) to give A99.1 (105 mg, 76%) as a clear oil.
To a stirred solution of A99.1 (40.0 mg, 0.253 mmol) in CH2Cl2 (2 mL) at room temperature was added Dess-Martin periodinane (161 mg, 0.379 mmol). After stirring for 70 min, water (2 mL) was added and the resulting solution was extracted with EtOAc (4 mL and 2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=5:1) to give A99.2 (22.0 mg, 56%) as a clear oil.
A1.2 was hydrolyzed to A99.3 with the same technique used to hydrolyze A80.3 to A80.4.
A99.4 was synthesized from A99.3 and 3,5-dimethylbenzylamine in an identical manner to A1.6.
A99.5 was synthesized from A99.4 in a manner identical to that used to convert A1.2 to A1.3.
To a stirred solution of A99.2 (22.0 mg, 0.141 mmol), A99.5 (63.4 mg, 0.169 mmol) and CH2Cl2 (2 mL) at room temperature was added NaBH(OAc)3 (60.0 mg, 0.282 mmol). After stirring for 4.5 h at 40° C., aq. sat. NaHCO3 (5 mL) was added and the resulting solution was extracted with EtOAc (5 mL and 2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (EtOAc) to give Compound A99 (31.0 mg, 46%) as a white powder. 1H-NMR (400 MHz, CDCl3): δ 8.54 (s, 1H), 7.15 (m, 1H), 6.97 (s, 3H), 6.85 (t, 2H), 6.02 (t, 1H), 4.56 (d, 2H), 3.10 (m, 3H), 2.89 (m, 2H), 2.69 (s, 3H), 2.58 (m, 2H), 2.32 (s, 6H), 2.08 (m, 4H), 1.76 (m, 2H). MS (EI) for C28H32F2N4O. found 479.2 (MH+). Analytical HPLC, ret. time=13.060 min, 95% purity.
Compounds A100 to A101 were prepared from methods analogous to those used to prepare Compound A99 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: YMC C18, 5μ 150×4.6 mm column, gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 25 min gradient time, 27 min total run time at 1.5 mL/min flow rate, λ=254 nM
To a stirred solution of ethyl N-Boc-4-methylpiperidine-4-carboxylate (1 g, 3.69 mmol) in MeOH (30 mL) was added aq. IN NaOH (10 mL) and the resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and the aq. layer was acidified with aq. IN HCl to pH 4-5 and extracted with CH2Cl2 (×4). The combined organic layer was dried over sodium sulfate and concentrated in vacuo to give 1-(tert-butoxycarbonyl)-4-methylpiperidine-4-carboxylic acid (905 mg, quant.). Reaction mixture A: to a mixture of N,N′-dimethyldiimidazole (430 mg, 2.41 mmol) in THF (5 mL) was added the 1-(tert-butoxycarbonyl)-4-methylpiperidine-4-carboxylic acid (451 mg, 1.85 mmol) in THF (5 mL) dropwise. The reaction mixture was protected from light and stirred overnight at room temperature. Reaction mixture B: to a stirred mixture of potassium ethyl malonate (504 mg, 2.96 mmol) in EtOAc (15 mL) cooled to 0° C. was added Et3N (1.05 mL, 7.53 mmol) followed by MgCl2 (342 mg, 3.59 mmol). The reaction mixture was slowly heated to 35° C. and then maintained at 35° C. overnight. To reaction mixture B was added reaction mixture A slowly at room temperature and the resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with EtOAc, washed with aq. 0.5N HCl, aq. NaHCO3, dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to afford A102.1 (423 mg, 73%).
A mixture of A102.1 (423 mg, 1.35 mmol) and DMF/DMA (250 μL, 1.76 mmol) was stirred at 110° C. for 2 h. An additional amount of DMF/DMA (500 μL, 3.52 mmol) was added to the reaction mixture and the resulting mixture was stirred at 110° C. for 2 h. The reaction mixture was concentrated in vacuo to give A102.2, which was used directly for the next step without further purification.
To a stirred solution of acetamide-HCl (191 mg, 2.02 mmol) in EtOH (10 mL) was added NaOEt (136 mg, 2.0 mmol) and the resulting mixture was stirred at room temperature for 20 min. To this suspension was added A102.2 (crude material) in EtOH (5 mL) and the reaction mixture was stirred at reflux for 3 h. The reaction mixture was cooled to room temperature, concentrated in vacuo, and the residue was diluted with water, extracted with CH2Cl2 (×2), dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to afford A102.3 (369 mg, 75% from A102.1).
To a stirred solution of A102.3 (369 mg, 1.02 mmol) in 1,4-dioxane (6 mL) was added HCl (4 mL, 4M in 1,4-dioxane, 16 mmol) and the resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and the crude amine residue was used directly for the next step. To a stirred suspension of the crude amine, Et3N (711 μL, 5.10 mmol), and 2,6-difluorophenylacetic acid (211 mg, 1.23 mmol) was added HATU (465 mg, 1.22 mmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was partitioned between aq. IN NaOH and EtOAc and the separated organic layer was washed with brine, dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to give A102.4 (362 mg, 85% from A102.3).
To a stirred solution of A102.4 (362 mg, 0.87 mmol) in THF (5 mL)/MeOH (10 mL) was added aq. 1N NaOH (4 mL) and the reaction mixture was stirred at 50° C. for 5 h. The reaction mixture was cooled to room temperature, concentrated in vacuo, and the residue was diluted with water, acidified with aq. IN HCl to pH 3-4, and extracted with CH2Cl2 (×4). The combined extracts were dried over sodium sulfate and concentrated in vacuo to afford A102.5 (317 mg, 94%).
To a stirred solution of A102.5 (117 mg, 0.30 mmol), 3-chlorobenzylamine (44 μL, 0.36 mmol), and Et3N (209 μL, 1.50 mmol) in DMF (3 mL) was added HATU (137 mg, 0.36 mmol) and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was directly purified by preparative reverse phase HPLC to give Compound A102 (84 mg, 55%) as a white powder after lyophilization. Analytical HPLC: retention time=14.852 min, 98%. 1H-NMR (400 MHz, CDCl3): δ 8.45 (s, 1H), 7.31-7.17 (m, 5H), 6.89-6.85 (m, 2H), 6.49 (s, 1H), 4.59 (dd, 1H), 4.49 (dd, 1H), 3.76-3.71 (m, 1H), 3.68 (d, 1H), 3.58 (d, 1H), 3.59-3.55 (m, 1H), 3.35-3.29 (m, 1H), 3.18-3.11 (m, 1H), 2.71 (s, 3H), 2.48-2.42 (m, 1H), 2.33-2.27 (m, 1H), 1.75-1.65 (m, 2H), 1.43 (s, 3H). MS (EI) for C27H27ClF2N4O2. found 513.0 (MH+).
A stirred solution of lithium diisopropyl amide (LDA) (2.6 mL, 2.0 M in THF/heptane/ethylbenzene, 5.2 mmol) was added ethyl N-Boc-piperidine-4-carboxylate (1.03 g, 4.0 mmol) in THF (5 mL) dropwise over 10 min at 0° C. The reaction mixture was stirred at 0° C. for 30 min and then slowly added to a solution of N-fluorobenzenesulfonimide (1.77 g, 5.6 mmol) in THF (15 mL) at 0° C. The reaction mixture was stirred at 0° C. for 30 min and then at room temperature overnight. The reaction mixture was diluted with aq. NaHCO3 and extracted with CH2Cl2 (×3). The combined organic layer was dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to afford A103.1 (658 mg, 60%).
Compound A103 was synthesized from A103.1 in the same manner that Compound A102 was synthesized from ethyl N-Boc-4-methylpiperidine-4-carboxylate. Analytical HPLC: retention time=13.124 min, 98%. 1H-NMR (400 MHz, CDCl3): δ 8.62 (s, 1H), 7.40 (s, 1H), 7.31-7.20 (m, 4H), 6.94-6.88 (m, 2H), 6.09 (t, 1H), 4.66-4.58 (m, 3H), 4.00-3.96 (m, 1H), 3.77 (s, 2H), 3.61-3.53 (m, 1H), 3.07-2.99 (m, 1H), 2.73 (s, 3H), 2.43-2.19 (m, 3H), 2.12-2.06 (m, 1H). MS (EI) for C26H24ClF3N4O2. found 517.0 (MH+).
Compounds A104 to A105 were prepared from methods analogous to those used to prepare Compound A103 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: YMC C18, 5μ 150×4.6 mm column, gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 25 min gradient time, 27 min total run time at 1.5 mL/min flow rate, λ=254 nM
To a stirred solution of A99.5 (100 mg, 0.267 mmol), DIEA (90 μL, 0.53 mmol) and CH2Cl2 (2 mL) at room temperature was slowly added a solution of 2-fluorophenyl isocyanate (32.9 mg, 0.240 mmol) in CH2Cl2 (1 mL). After stirring for 1 h, the resulting mixture was filtered through cotton and the collected solid was purified by silica gel column chromatography (CH2Cl2/MeOH=19/1) to give Compound A106 (72.0 mg, 57%) as a white solid. 1H-NMR (400 MHz, CDCl3): δ 8.58 (s, 1H), 8.11 (td, 1H), 7.10 (q, 1H), 7.05 (dd, 1H), 6.97 (s, 3H), 6.96 (m, 1H), 6.64 (d, 1H), 6.16 (t, 1H), 4.56 (d, 2H), 4.19 (d, 2H), 3.42 (tt, 1H), 3.00 (td, 2H), 2.70 (s, 3H), 2.33 (s, 6H), 2.00 (qd, 2H), 1.86 (m, 2H). MS (EI) for C27H30FN5O2. found 476.2 (MH+). Analytical HPLC, ret. time=16.084 min, 99% purity.
Compound A107 was synthesized from A99.5 in the same manner as Compound A106. MS (EI) for C27H29F2N5O2. found 494.0 (MH+). Analytical HPLC, ret. time=15.368 min, 99% purity.
Diethyl malonate (3.75 g, 23 mmol) and N-Boc piperidine-4-carboxaldehyde (5 g, 23 mmol) were dissolved in iPrOH (30 mL). To this solution were added catalytic amounts of piperidine (70 mg) and AcOH (70 mg). The resulting solution was stirred at room temperature for 48 h. The mixture was then concentrated, and the crude product was used in the next step without further purification.
To a flask containing a suspension of acetamidine hydrochloride (2.2 g, 23 mmol) in EtOH (25 mL) was added EtONa (1.6 g, 23 mmol). The resulting mixture was stirred for 15 min at room temperature. To this mixture was added A108.1 as a solution in EtOH (15 mL). The mixture was heated to 70° C. with stirring for 2 h. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2 and washed with brine. Concentration gave crude dihydropyrimidinone which was used as is in the next step by mixing with N-bromosuccinimide (NBS) (1.2 g, 6.6 mmol), freshly ground K2CO3 (7.6 g, 55 mmol) and benzoyl peroxide (65 mg, 0.27 mmol) in CCl4 (25 mL). The resulting mixture was stirred for 1 h at 85° C. It was then cooled to room temperature. Water was added to dissolve the inorganic salts. The mixture was extracted with EtOAc. The organic layer was washed with brine and dried over sodium sulfate. Concentration gave the crude product, which was further purified by flash column chromatography to yield A108.2.
To a solution of A108.2 (1.1 g, 3 mmol) in EtOH (15 mL) was added 4N HCl in dioxane (2.5 mL). The resulting solution was stirred for 12 h at room temperature. Ether (100 mL) was added. The resulting solid was filtered and dried under vacuum. The crude amine HCl salt was used as is without further purification.
The ethyl 2-methyl-6-oxo-4-(piperidin-4-yl)-1,6-dihydropyrimidine-5-carboxylate hydrochloride obtained in the previous step from the Boc deprotection of A108.2 was mixed with 2,6-difluorophenylacetic acid (619 mg, 3.6 mmol), HOBt (607 mg, 4.5 mmol) and DIEA (3 mL) in CH2Cl2 (15 mL). To this solution was added EDC (860 g, 4.5 mmol). The resulting reaction mixture was stirred at room temperature for 5 h then diluted with aq. sat. NaHCO3. The resulting mixture was extracted with EtOAc and the organic phase was concentrated. The product was precipitated in ether. Filtration and drying under vacuum gave the desired product.
To a solution of A108.3 (200 mg, 0.476 mmol), 1,8-diazabicyclo[5.4.0.]undec-7-ene (DBU) (109 mg, 0.71 mmol) and MeNH2 (0.5 mL, 2.0 M in THF) in CH3CN (2 mL) was added (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP) (273 mg, 0.62 mmol). The resulting solution was stirred at room temperature for 3 h. It was then diluted with CH2Cl2, washed with Na2CO3, brine, and concentrated. The crude product was purified by flash column chromatography. The resulting pure ethyl 4-(1-(2-(2,6-difluorophenyl)acetyl)piperidin-4-yl)-2-methyl-6-(methylamino)pyrimidine-5-carboxylate was stirred with NaOH (72 mg, 1.8 mmol) in MeOH/water (5 mL/5 mL) at 80° C. for 2 h. The reaction solution was concentrated and the solid was dissolved in water (5 mL) and acidified with conc. HCl. The precipitated product was filtered and dried.
A108.4 (0.35 mmol) was mixed with 3,5-dimethylbenzylamine (60 mg, 0.45 mmol) and DIEA (0.5 mL) in DMF (3 mL). To this solution was added HATU (170 mg, 0.45 mmol). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The resulting mixture was extracted with CH2Cl2 and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by prep HPLC to give Compound A108. 1H-NMR (400 MHz, DMSO-d6): δ 8.91 (t, 1H), 7.35 (m, 1H), 7.07 (m, 2H), 6.96 (s, 2H), 6.91 (s, 1H), 6.61 (m, 1H), 4.38 (m, 3H), 4.10 (d, 1H), 3.82 (d, 1H), 3.73 (d, 1H), 3.38 (m, 1H), 2.81 (m, 1H), 2.80 (d, 3H), 2.56 (m, 1H), 2.35 (s, 3H), 2.26 (s, 6H), 1.70 (m, 4H). MS (EI) for C29H33F2N5O2. found: 522.1 (MH+). Analytical HPLC, ret. time=18.42 min, 96% purity.
A109 was synthesized from A108.4 in the same manner as Compound A108. 1H-NMR (400 MHz, DMSO-d6): δ 9.04 (t, 1H), 7.36 (m, 5H), 7.07 (m, 2H), 6.68 (m, 1H), 4.45 (m, 3H), 4.10 (d, 1H), 3.82 (d, 1H), 3.73 (d, 1H), 2.81 (m, 1H), 2.80 (d, 3H), 2.65 (m, 1H), 2.35 (s, 3H), 2.30 (m, 1H), 1.70 (m, 4H). MS (EI) for C27H28ClF2N5O2. found: 528.1 (MH+). Analytical HPLC, ret. time=3.23 min, 95% purity.
A1.5 (200 mg, 0.53 mmol) was mixed with 3-chlorophenylglycine methyl ester hydrochloride (150 mg, 0.63 mmol) and DIEA (0.5 mL) in CH2Cl2 (3 mL). To this solution was added HATU (240 mg, 0.63 mmol). The resulting mixture was stirred at room temperature for 3 h then diluted with aq. sat. NaHCO3. The mixture was extracted with EtOAc and the organic phase was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by flash column chromatography.
To a 0° C. solution of A110.1 (156 mg, 0.26 mmol) in THF (3 mL) was added litium aluminum hydride (LAH) (0.3 mL, 1.0 M in THF). The resulting solution was stirred for 1 h, and quenched with 2 N NaOH solution. The mixture was extracted with EtOAc and the organic phase was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography to give A110.2. 1H-NMR (400 MHz, DMSO-d6): δ 9.09 (d, 1H), 8.66 (s, 1H), 7.46 (m, 1H), 7.37 (m, 4H), 7.07 (m, 2H), 5.08 (m, 2H), 4.42 (m, 1H), 4.13 (m, 1H), 3.83 (m, 2H), 3.68 (t, 2H), 3.41 (m, 1H), 3.10 (m, 1H), 2.64 (s, 3H), 2.56 (m, 1H), 1.70 (m, 4H). MS (EI) for C27H27ClF2N4O3. found: 529.0 (MH+). Analytical HPLC, ret. time=3.70 min, 94% purity.
To a solution of A108.3 (100 mg, 0.23 mmol) and DBU (70 mg, 0.46 mmol) in THF (2 mL) was added BOP (140 mg, 0.31 mmol). The resulting solution was stirred at room temperature for 10 min, then Cs2CO3 (150 mg, 0.46 mmol) and MeOH (1.5 mL) were added. The mixture was stirred at room temperature for an additional 2 h. The crude product was extracted with EtOAc, washed with Na2CO3, brine, and concentrated. The resulting crude ethyl ester was stirred with NaOH (25 mg, 0.62 mmol) in MeOH/water (2 mL/2 mL) at 80° C. for 2 h. The reaction solution was concentrated and the resulting solid was dissolved in water (5 mL) and acidified with conc. HCl. The precipitated product was filtered and dried to give A111.1.
Crude A111.1 (0.2 mmol) was mixed with 3-chlorobenzylamine (42 mg, 0.3 mmol) and DIEA (0.2 mL) in DMF (2 mL). To this solution was added HATU (114 mg, 0.3 mmol). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The resulting mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by prep HPLC to give Compound A111. 1H-NMR (400 MHz, DMSO-d6): δ 9.08 (t, 1H), 7.36 (m, 5H), 7.07 (m, 2H), 4.45 (m, 3H), 4.12 (d, 1H), 3.95 (s, 3H), 3.82 (d, 1H), 3.73 (d, 1H), 3.02 (m, 1H), 2.84 (m, 1H), 2.55 (s, 3H), 2.45 (m, 1H), 1.70 (m, 4H). MS (EI) for C27H27ClF2N4O3. found: 529.0 (MH+). Analytical HPLC, ret. time=22.49 min, 92% purity.
Ethyl 4,4,4-trifluoroacetoacetate (4.3 g, 23 mmol) and N-Boc piperidine-4-carboxaldehyde (5 g, 23 mmol) were dissolved in iPrOH (30 mL). To this solution were added catalytic amounts of piperidine (70 mg) and AcOH (70 mg). The resulting solution was stirred at room temperature for 48 h. The mixture was then concentrated, and the crude product was used in the next step without further purification.
To a flask containing a suspension of acetamidine hydrochloride (2.5 g, 27 mmol) in EtOH (25 mL) was added EtONa (1.9 g, 27 mmol). The resulting mixture was stirred for 15 min at room temperature. To this mixture was added A112.1 as a solution in EtOH (15 mL). The mixture was stirred at room temperature for 12 h and then was concentrated. The residue was dissolved in DMF (20 mL), reacted with pTsOH (8.7 g, 45 mmol) at 110° C. for 2 h. The reaction mixture was cooled to room temperature, basified with 20% NaOH aq. solution until pH 10. The crude product was extracted with CH2Cl2, dried over sodium sulfate, and concentrated. The residue (4.2 g, 10 mmol) was dissolved in CH2Cl2 (20 mL), to which were added NaHCO3 (3.1 g, 30 mmol), Boc2O (3.2 g, 15 mmol) and water (20 mL). The mixture was stirred at room temperature for 12 h, then the organic layer was washed with brine, and concentrated. Flash column chromatography gave the desired dihydropyrimidine which was dissolved in DCE (30 mL), to which was added MnO2 (1.8 g, 21 mmol). The resulting mixture was stirred for 1 h at 85° C., cooled to room temperature and filtered through Celite. The filtrate was washed with brine and dried over sodium sulfate. Concentration gave crude A112.2 which was used in the next step without further purification.
To a solution of A112.2 (290 mg, 0.69 mmol) in MeOH (5 mL) was added 4N HCl in dioxane (1 mL). The resulting solution was stirred for 12 h at room temperature. EtOAc (25 mL) was added. The solid was filtered and dried under vacuum. The crude product was used without further purification by mixing with 2,6-difluorophenylacetic acid (180 mg, 1.1 mmol), HATU (418 mg, 1.1 mmol) and DIEA (645 mg, 5 mmol) in THF (5 mL). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The resulting mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude product was stirred with NaOH (200 mg, 5 mmol) in MeOH/water (2 mL/1 mL) at 80° C. for 3 h. The solution was concentrated and the solid was dissolved in water (5 mL) and acidified with conc. HCl. The precipitated product was filtered and dried.
A112.3 (80 mg, 0.18 mmol) was mixed with DIEA (0.5 mL) and HATU (100 mg, 0.27 mmol) in THF (3 mL). The formation of the HOAT ester was complete in 3 h. The reaction mixture was diluted with EtOAc, washed with NaHCO3, and concentrated. The crude HOAT ester was reacted with neat 3,5-dimethylbenzylamine (0.5 mL) at room temperature for 12 h. The resulting mixture was extracted with EtOAc and washed with 2 N HCl aq. solution. The organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by prep HPLC to give Compound A112. MS (EI) for C29H29F5N4O2. found: 561.0 (MH+). Analytical HPLC, ret. time=4.50 min, 86% purity.
To a stirred solution of A1 (90.0 mg, 0.183 mmol) in DMF (2.0 mL) at room temperature was added NaH (10.9 mg, 60% dispersion in mineral oil, 0.274 mmol). After stirring for 5 min at room temperature, iodomethane (40 μmol, 0.64 mmol) was added. The reaction mixture was stirred for 2 h at room temperature, quenched with aq. sat. NaHCO3 (3 mL) and then extracted with EtOAc (2×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=2:5) to give Compound A113 (45.0 mg, 49%) as a white powder after lyophilization. 1H-NMR (400 MHz, CDCl3, rotameric mixture): δ 8.47 and 8.45 (two of s, 1H), 7.23 (m, 1H), 6.99 (s, 2H), 6.90 (m, 2H), 6.67 (s, 1H), 4.73 (m, 2H), 4.35 (s, 1H), 4.09 (d, 1H), 3.75 (s, 2H), 3.25-3.09 (m, 1H), 3.15 and 2.79 (two of s, 3H), 3.02-2.92 (m, 1H), 2.73 and 2.70 (two of s, 3H), 2.68-2.53 (m, 1H), 2.33 and 2.28 (two of s, 6H), 2.05-1.77 (m, 4H). MS (EI) for C29H32F2N4O2. found 507.2 (MH+). Analytical HPLC, ret. time=18.180 min, 93% purity.
A mixture of ethyl 2-methyl-6-oxo-1,6-dihydropyrimidine-5-carboxylate (1.00 g, 5.49 mmol) and phosphorous oxychloride (12 mL) was stirred for 2 h at 90° C. The reaction mixture was concentrated under vacuum, dissolved in MeCN (10 mL), and concentrated again. The resulting material was dissolved in EtOAc (10 mL) and treated with aq. sat. NaHCO3 (10 mL). The aq. solution was separated and further extracted with EtOAc (2×5 mL). The organic layers were immediately washed with aq. sat. NaHCO3 (5 mL), dried over anhydrous magnesium sulfate, concentrated, and purified by silica gel chromatography (hexane/EtOAc=7:1) to provide A114.1 (670 mg, 61%) as a clear oil.
To a stirred mixture of A114.1 (670 mg, 3.34 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylate (1.34 g, 4.34 mmol) and PdCl2(dppf)2.CH2Cl2 (273 mg, 0.334 mmol) in DMF (10 mL) was added Et3N (1.16 mL, 8.35 mmol) and water (2 mL) in sequence. After stirring for 110 min at 90° C., water (3 mL) and aq. sat. NH4Cl (6 mL) were added and the resulting solution was extracted with EtOAc (4×8 mL). The combined organic layers were concentrated and purified by silica gel chromatography (Hexane/EtOAc=5:1→4:1) to provide A114.2 (980 mg, 85%) as a clear oil. 1H-NMR (400 MHz, CDCl3, rotameric mixture): δ 9.02 (s, 1H), 7.05 and 6.95 (two of d, J=8.0 Hz, 1H), 4.93 and 4.84 (two of d, J=8.0 Hz, 1H), 4.50-4.43 (m, 1H), 4.40 (q, J=7.2 Hz, 2H), 4.07-3.93 (m, 1H), 3.57-3.50 (m, 1H), 2.75 (s, 3H), 2.19-2.02 (m, 2H), 1.50 (s, 9H), 1.41 (t, J=7.2 Hz, 3H). MS (EI) for C18H25N3O4. found 348.1 (MH+).
A mixture of A114.2 (248 mg, 0.714 mmol), LiOH (25.7 mg, 1.07 mmol), THF (4 mL) and water (1 mL) was stirred for 80 min at 50° C. The resulting solution was concentrated under vacuum to give the corresponding acid; MS (EI) for C16H21N3O4. found 320.1 (MH+). To a stirred solution of the resulting carboxylic acid, (3,5-dimethylphenyl)methanamine (106 mg, 0.785 mmol), HATU (407 mg, 1.07 mmol) and DMF (4 mL) at room temperature was added DIEA (0.37 mL, 2.14 mmol). After stirring for 2.5 h, water (4 mL) was added and the resulting solution was extracted with EtOAc (3×4 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=3:2→4:3) to give A114.3 (210 mg, 67%) as a pale yellow foam. MS (EI) for C25H32N4O3. found 437.1 (MH+).
A mixture of A114.3 (210 mg, 0.481 mmol) and 4N HCl in 1,4-dioxane (3 mL) was stirred for 1 h at room temperature, and concentrated under vacuum. EtOAc (2 mL) was added and the mixture was concentrated again. To a stirred solution of the resulting residue, 2-(2,6-difluorophenyl)acetic acid (108 mg, 0.625 mmol), HATU (274 mg, 0.722 mmol) and DMF (4 mL) was added DIEA (0.33 mL, 1.9 mmol). After stirring for 4.5 h at room temperature, aq. sat. NaHCO3 (4 mL) and water (1 mL) were added and the resulting solution was extracted with EtOAc (3×4 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=2:1). The isolated material was further purified by prep HPLC to give Compound A114 (25.0 mg, 11%) as a white powder after lyophilization. 1H-NMR (400 MHz, CDCl3, rotameric mixture): δ 8.60 and 8.59 (two of s, 1H), 7.24 (m, 1H), 6.96 (s, 3H), 6.93 (dd, 1H), 4.56 (dd, 2H), 4.26-4.07 (m, 2H), 3.85 and 3.82 (two of s, 2H), 3.76 and 3.56 (two of ddd, 1H), 2.74 and 2.73 (two of s, 3H), 2.32 (s, 6H), 2.31-2.04 (m, 2H). MS (EI) for C28H28F2N4O2. found 491.0 (MH+). Analytical HPLC, ret. time=18.332 min, 99% purity.
To a stirred solution of A99.5 (100 mg, 0.267 mmol), 2-bromo-1-(2-chlorophenyl)ethanone (68.6 mg, 0.294 mmol) and CH2Cl2 (2 mL) at room temperature was added DIEA (0.14 mL, 0.80 mmol). After stirring for 70 min, aq. sat. NaHCO3 (3 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=1:3→1:4) to give Compound A115 (47 mg, 36%) as a pale yellow powder after lyophilization. 1H-NMR (400 MHz, CDCl3): δ 8.53 (s, 1H), 7.38 (m, 4H), 6.96 (s, 3H), 6.05 (t, 1H), 4.55 (d, 2H), 3.76 (s, 2H), 3.13 (tt, 1H), 3.05 (d, 2H), 2.68 (s, 3H), 2.32 (s, 6H), 2.26 (m, 2H), 2.08 (qd, 2H), 1.73 (m, 2H). MS (EI) for C28H31ClN4O2. found 491.1 (MH+). Analytical HPLC, ret. time=12.464 min, 97% purity.
To a stirred solution of the methyl/ethyl ester A80.3 (2.1 g, ca 5.0 mmol) in MeOH (30 mL) was added aq. IN NaOH (20 mL) and the reaction mixture was stirred at room temperature overnight. MeOH was removed in vacuo and the residual aq. layer was acidified with IN HCl to pH 4-5, and extracted with CH2Cl2 (×5). The combined organic layer was dried over sodium sulfate and concentrated in vacuo to give acid B1.1 (1.88 g, ca. 97%).
To a stirred solution of acid B1.1 (195 mg, 0.50 mmol) and Et3N (77 μL, 0.55 mmol) in THF (5 mL) was added ethyl chloroformate (53 μL, 0.55 mmol) at 0° C. After stirring for 1 h at 0° C., the precipitates were filtered and an aq. solution of NaBH4 was added dropwise to the filtrate at 0° C. After stirring for 1.5 h at 0° C., acetone (1 mL) was added to destroy the excess NaBH4 and the resulting mixture was stirred for 30 min. The reaction mixture was diluted with water and extracted with CH2Cl2 (×2). The combined organic layers were dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to afford the corresponding alcohol (104 mg, 55%, 0.28 mmol) which was then dissolved in CH2Cl2 (6 mL) and treated with Dess-Martin periodinane (235 mg, 0.55 mmol) with stirring at room temperature for 2 h. The reaction mixture was diluted with water and the separated aq. layer was extracted with CH2Cl2 (×2). The combined organic layers were dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to give B1.2 (101 mg, 98%).
To a stirred solution of aldehyde B1.2 (100 mg, 0.27 mmol) in THF (5 mL) was added MeMgBr (0.4 mL, 1.4 M in THF/toluene, 0.56 mmol) and the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was quenched with aq. NH4Cl and extracted with CH2Cl2 (×2). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give 2-(2,6-difluorophenyl)-1-(4-(5-(1-hydroxyethyl)-2,6-dimethylpyrimidin-4-yl)piperidin-1-yl)ethanone (110 mg, quant., 0.27 mmol), which was directly used for the next step by dissolving it in CH2Cl2 (6 mL) and then adding Dess-Martin periodinane (172 mg, 0.41 mmol). The reaction mixture was stirred at room temperature for 2 h, then diluted with aq. sodium bicarbonate and extracted with CH2Cl2 (×2). The combined organic layers were dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to give ketone B1.3 (91 mg, 88%).
A mixture of B1.3 (91 mg, 0.23 mmol) and O-(3-(trifluoromethyl)phenyl)hydroxylamine (82 mg, 0.46 mmol) in EtOH (8 mL) was treated with 2 drops of concentrated HCl and the reaction mixture was stirred at 50° C. for 4 days. 30-40% conversion was observed on LC-MS. The reaction mixture was diluted with aq. sodium bicarbonate and extracted with CH2Cl2 (×3). The combined organic layer was dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to afford B1.4 as an inseparable 3:1 mixture of E/Z isomers as a white powder after lyophilization (19 mg, 15%). Data for major isomer (E): Analytical HPLC: retention time=22.368 min, 98%. 1H-NMR (400 MHz, CDCl3): δ 7.48-7.44 (m, 2H), 7.36-7.32 (m, 2H), 7.26-7.19 (m, 1H), 6.92-6.87 (m, 2H), 4.75 (d, 1H), 4.11 (d, 1H), 3.74 (d, 2H), 3.21-3.14 (m, 1H), 2.96-2.87 (m, 1H), 2.80 (s, 3H), 2.68-2.61 (m, 1H), 2.57 (s, 3H), 2.42 (s, 3H), 2.10-1.95 (m, 2H), 1.87-1.75 (m, 2H). MS (EI) for C28H27F5N4O2. found 547.1 (MH+).
Compound B2 was synthesized from B1.3 in the same manner as Compound B1. In the case of Compound B2, a 10:1 mixture of Z/E isomers was recovered. Data for major isomer (Z): Analytical HPLC: retention time=19.692 min, 99%. 1H-NMR (400 MHz, CDCl3): δ 8.57 (t, 1H), 7.47 (d, 1H), 7.24-7.13 (m, 2H), 6.92-6.87 (m, 2H), 4.80-4.66 (m, 1H), 4.16-4.03 (m, 1H), 3.75 (s, 1H), 3.72 (d, 1H), 3.24-3.17 (m, 1H), 3.12-3.05 (m, 1H), 2.80 (s, 3H), 2.69-2.63 (m, 1H), 2.45 (s, 3H), 2.38 (s, 3H), 2.18-1.84 (m, 2H), 1.78-1.69 (m, 2H). MS (EI) for C27H26F5N5O2. found 548.0 (MH+).
B3.1 was synthesized from A1.5 in the same manner that B1.3 was synthesized from B1.1.
To a stirred solution of B3.1 (112 mg, 0.30 mmol) and O-(3-(trifluoromethyl)phenylhydroxylamine (54 mg, 0.30 mmol) in EtOH (5 mL) was added 2 drops of cone. HCl and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with EtOAc, washed with aq. sodium bicarbonate, concentrated in vacuo, and the residue was purified by flash chromatography (hexanes/methyl t-butylether/CH2Cl2=4/3/3) to give Compound B3 (49 mg, 36%) as a white powder after lyophilization. Analytical HPLC: retention time=23.664 min, 98%. 1H-NMR (400 MHz, CDCl3): δ 8.57 (s, 1H), 7.49 (s, 1H), 7.45 (t, 1H), 7.36 (dd, 1H), 7.32 (d, 1H), 7.24-7.19 (m, 1H), 6.93-6.86 (m, 2H), 4.75 (d, 1H), 4.12 (d, 1H), 3.75 (d, 2H), 3.31-3.23 (m, 1H), 3.22-3.15 (m, 1H), 2.75 (s, 3H), 2.69-2.61 (m, 1H), 2.48 (s, 3H), 2.14-1.95 (m, 2H), 1.92-1.79 (m, 2H). MS (EI) for C27H25F5N4O2. found 533.0 (MH+).
Compound B4 is the Z isomer of Compound B3, recovered as a second compound from the same reaction that generated Compound B3 during the purification process by flash chromatography. Analytical HPLC: retention time=22.856 min, 95%. 1H-NMR (400 MHz, CDCl3): δ 8.38 (s, 1H), 7.41-7.38 (m, 2H), 7.29 (d, 1H), 7.24-7.18 (m, 2H), 6.92-6.86 (m, 2H), 4.70 (d, 1H), 4.06 (d, 1H), 3.72*d, 2H), 3.11-3.04 (m, 1H), 2.77 (s, 3H), 2.76-2.70 (m, 1H), 2.60-2.54 (m, 1H), 2.37 (s, 3H), 2.10-1.90 (m, 2H), 1.77-1.67 (m, 2H). MS (EI) for C27H25F5N4O2. found 533.0 (MH+).
Compounds B5 to B22 were prepared from methods analogous to those used to prepare Compounds B3 and B4 utilizing appropriate reagent replacements.
To a stirred mixture of acid A106.1 (3.07 g, 9.55 mmol), N,O-dimethylhydroxylamine hydrogenchloride (1.12 g, 11.5 mmol), and Et3N (5.32 mL, 38.2 mmol) in dimethylacetamide (DMA) (30 mL) was added HATU (4.36 g, 11.5 mmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with EtOAc, washed with aq. sodium bicarbonate, brine, dried over sodium sulfate and concentrated in vacuo to give the corresponding Weinreb amide, which was directly used for the next step. The crude Weinreb amide was dissolved in THF (30 mL) and the resulting solution was treated with MeMgBr (27.3 mL, 1.4 M in THF/toluene, 38.2 mmol) at room temperature. After stirring for 2 h, the reaction mixture was cooled to 0° C., quenched with aq. NH4Cl and extracted with EtOAc (×2). The combined organic layers were dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to give ketone B23.1 (2.76 g, 90% in 2 steps).
To a stirred solution of B23.1 (639 mg, 2.0 mmol) in THF (20 mL) was added lithium bis(trimethylsilyl)amide (LHMDS) (3.0 mL, 1.0 M in THF, 3.0 mmol) at 0° C. and the reaction mixture was stirred for 30 min. To this resulting solution was added TMSCl (383 μL, 3.0 mmol). After stirring for 3 h, the reaction mixture was diluted with aq. sodium bicarbonate and extracted with EtOAc (×2). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give the corresponding trimethylsilylenol ether, which was directly used for the next step. The crude trimethylsilylenol ether was dissolved in THF (20 mL) and the solution was treated with NaHCO3 (252 mg, 3.0 mmol) followed by NBS (356 mg, 2.0 mmol) at 0° C. The reaction mixture was warmed to room temperature and stirred for 30 min. The reaction mixture was diluted with aq. sodium bicarbonate and extracted with EtOAc (×2). The combined organic layers were dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to afford the corresponding α-bromoketone (661 mg, 83% in 2 steps). To a stirred solution of this α-bromoketone (398 mg, 1.0 mmol) in DMA (5 mL) was added K2CO3 (294 mg, 3.0 mmol) and the reaction mixture was stirred for 1 h. The reaction mixture was diluted with water and extracted with EtOAc (×2). The combined organic layers were washed with brine, dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to give acetate B23.2 (354 mg, 94%).
A solution of B23.2 (354 mg, 0.94 mmol) in 1,4-dioxane (10 mL) was treated with HCl (5 mL, 4 M in 1,4-dioxane, 20 mmol) and the reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo to give the corresponding amine HCl salt, which was directly used for the next step. The crude amine HCl salt was dissolved in DMA (10 mL) and treated with Et3N (655 μL, 4.7 mmol) and 2,6-difluorophenylacetic acid (194 mg, 1.13 mmol) followed by HATU (430 mg, 1.13 mmol). After stirring for 30 min at room temperature, the reaction mixture was diluted with EtOAc, washed with aq. sodium bicarbonate, brine, dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to give B23.3 (338 mg, 83%).
The mixture of B23.3 (338 mg, 0.78 mmol) and hydroxylamine hydrochloride (542 mg, 7.8 mmol) in EtOH (10 mL) was treated with 5 drops of concentrated HCl and the resulting mixture was stirred at room temperature overnight. The reaction mixture was diluted with aq. sodium bicarbonate and extracted with CH2Cl2 (×5). The combined organic layers were dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to afford oxime B23.4 (244 mg, 70%) along with acetylated oxime (44 mg, 14%). The resulting mixture (89 mg, 0.55 mmol) was dissolved in MeOH (5 mL) and treated with K2CO3 to hydrolyze the acetylated oxime (138 mg, 1.0 mmol). After stirring for 1 h at room temperature, the reaction mixture was concentrated in vacuo and the residue was directly purified by flash chromatography to give B23.4 (77 mg, 95%).
A mixture of B23.3 (77 mg, 0.19 mmol) and 4-chloro-2-trifluoromethylpyridine (34 mg, 0.19 mmol) in DMA (4 mL) was treated with K2CO3 (131 mg, 0.95 mmol). After stirring for 24 h at room temperature, additional 4-chloro-2-trifluoromethylpyridine (34 mg, 0.19 mmol) was added to the reaction mixture. After stirring for 48 h at room temperature, the reaction mixture was diluted with water and extracted with EtOAc (×2). The combined organic layer was washed with brine, dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to give Compound B23 (37 mg, 35%) as a white powder after lyophilization. Analytical HPLC: retention time=16.904 min, 99%. 1H-NMR (400 MHz, CDCl3): δ 8.69 (br s, 1H), 8.63 (d, 1H), 7.53 (d, 1H), 7.29 (dd, 1H), 7.26-7.19 (m, 1H), 6.93-6.87 (m, 2H), 5.03 (s, 2H), 4.76 (d, 1H), 4.13 (d, 1H), 3.76 (d, 2H), 3.24-3.17 (m, 1H), 3.12-3.05 (m, 1H), 2.84 (s, 3H), 2.70-2.64 (m, 1H), 2.13-1.84 (m, 4H). MS (EI) for C26H24F5N5O3. found 549.9 (MH+).
To a stirred solution of B23 (32 mg, 0.058 mmol) and Et3N (81 μL, 0.58 mmol) in CH2Cl2 (4 mL) was added diethylaminosulfur trifluoride (DAST) dropwise at room temperature. After stirring for 4 h, the reaction mixture was diluted with aq. sodium bicarbonate and extracted with CH2Cl2 (×2). The combined organic layers were dried over sodium sulfate, concentrated in vacuo, and the residue was purified by reverse phase HPLC to afford Compound B24 (8.6 mg, 27%) as a white powder after lyophilization. Analytical HPLC: retention time=19.804 min, 99%. 1H-NMR (400 MHz, CDCl3): δ 8.65 (d, 1H), 8.58 (s, 1H), 7.53 (d, 1H), 7.30 (dd, 1H), 7.26-7.20 (m, 1H), 6.94-6.87 (m, 2H), 5.77 (d, 2H), 4.77 (d, 1H), 4.14 (d, 1H), 3.76 (d, 2H), 3.24-3.17 (m, 1H), 2.98-2.93 (m, 1H), 2.80 (s, 3H), 2.70-2.63 (m, 1H), 2.16-1.96 (m, 2H), 1.90-1.79 (m, 2H). MS (EI) for C26H23F6N5O2. found 552.1 (MH+).
Compounds B25 to B28 were prepared from methods analogous to those used to prepare Compounds B23 and B24 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: YMC C18, 5μ 150×4.6 mm column, gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 25 min gradient time, 27 min total run time at 1.5 mL/min flow rate, λ=254 nM
To a stirred solution of A1.4 (530 mg, 1.31 mmol) and trimethyl(trifluoromethyl)silane (280 mg, 1.97 mmol) in toluene (5 mL) at −78° C. was added Bu4NF (65 μL, 1M in THF, 0.065 mmol). The reaction mixture was warmed slowly to −5° C. for 40 min and then 2N HCl (1.2 mL) was added. After stirring for 1 h at room temperature, EtOAc (5 mL) was added. The organic layer was separated, concentrated, dissolved in THF (5 mL), and transferred to the aq. layer. After stirring for 2 h, water (2 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=1:1→1:2) to give B29.1 (205 mg, 37%) as a white foam. MS (EI) for C20H18F5N3O2. found 428.0 (MH+).
To a stirred solution of B29.1 (80.0 mg, 0.187 mmol), O-(3-fluorophenyl)hydroxylamine (48.0 mg, 0.374 mmol) and EtOH (1.5 mL) at room temperature was added a catalytic amount of cone HCl. After stirring for 41 h at 80° C., aq. sat. NaHCO3 (2 mL) and water (1 mL) were added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=3:1→5:2) to give Compound B29 (8.0 mg, a mixture of E/Z=5:2, 8%) as a pale red powder after lyophilization. 1H-NMR (400 MHz, CDCl3, geometric mixture): δ 8.56 and 8.50 (two of s, 1H, 1:2.8), 7.32 (m, 1H), 7.22 (m, 1H), 7.04-6.84 (m, 5H), 4.75 (m, 1H), 4.10 (m, 1H), 3.74 (m, 2H), 3.23-2.47 (m, 3H), 2.81 and 2.79 (two of s, 3H, 2.1:1), 2.12-1.75 (m, 4H). MS (EI) for C26H22F6N4O2. found 537.0 (MH+). Analytical HPLC, ret. time=21.632 min (major) and 21.808 min (minor), incomplete separation (gradient 10% to 100% MeCN/H2O), 97% purity.
B30.1 was synthesized from A1.5 in the same manner that B1.2 was synthesized from B1.1
To a stirred solution of B30.1 (40.0 mg, 0.111 mmol), O-(3-(trifluoromethyl)phenyl)hydroxylamine (29.5 mg, 0.167 mmol) and EtOH (1 mL) at room temperature was added a catalytic amount of conc HCl. After stirring for 3 h, aq. sat. NaHCO3 (3 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=4:3→2:3) to give Compound B30 (39.6 mg, 69%) as a white powder after lyophilization. 1H-NMR (400 MHz, CDCl3): δ 8.85 (s, 1H), 8.63 (s, 1H), 7.49 (m, 2H), 7.37 (m, 2H), 7.23 (m, 1H), 6.91 (t, 2H), 4.79 (d, 1H), 4.18 (d, 1H), 3.78 (s, 2H), 3.48 (tt, 1H), 3.29 (td, 1H), 2.78 (m, 1H), 2.77 (s, 3H), 2.10 (qd, 1H), 1.94 (m, 3H). MS (EI) for C26H23F5N4O2. found 519.0 (MH+). Analytical HPLC, ret. time=23.248 min, 98% purity.
To a stirred mixture of aldehyde B1.2 (46 mg, 0.123 mmol) and 3-trifluoromethylphenylhydroxylamine (33 mg, 0.186 mmol) in EtOH (6 mL) was added 2 drops of concentrated HCl and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with aq. sodium bicarbonate and extracted with CH2Cl2 (×3). The combined organic layers were dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to give Compound B31 (41 mg, 63%) as a white powder after lyophilization. Analytical HPLC: retention time=22.624 min, 99%. 1H-NMR (400 MHz, CDCl3): δ 8.77 (s, 1H), 7.50-7.46 (m, 2H), 7.36-7.34 (m, 2H), 7.26-7.19 (m, 1H), 6.93-6.87 (m, 2H), 4.77 (d, 1H), 4.15 (d, 1H), 3.76 (d, 2H), 3.46-3.39 (m, 1H), 3.27-3.21 (m, 1H), 2.75-2.68 (m, 1H), 2.71 (s, 3H), 2.66 (s, 3H), 2.15-1.82 (m, 4H). MS (EI) for C27H25F5N4O2. found 533.0 (MH+).
To a stirred solution of B23 (99 mg, 0.18 mmol) in CH2Cl2 (5 mL) was added Dess-Martin periodinane (153 mg, 0.36 mmol) and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with aq. sodium bicarbonate and extracted with CH2Cl2 (×3). The combined organic layers were dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to give the corresponding aldehyde, (Z)-2-(4-(1-(2-(2,6-difluorophenyl)acetyl)piperidin-4-yl)-2-methylpyrimidin-5-yl)-2-(2-(trifluoromethyl)pyridin-4-yloxyimino)acetaldehyde (79 mg, 80%). A solution of the aldehyde (44 mg, 0.080 mmol) in CH2Cl2 (5 mL) was treated with DAST (39 mg, 0.24 mmol) and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with aq. sodium bicarbonate and extracted with CH2Cl2 (×2). The combined organic layer was dried over sodium sulfate, concentrated in vacuo, and the residue was purified by reverse phase HPLC to afford Compound B32 (21 mg, 46%) as a white powder after lyophilization. Analytical HPLC: retention time=16.772 min, 98%. 1H-NMR (400 MHz, CDCl3): δ 8.69 (d, 1H), 8.60 (s, 1H), 7.57 (d, 1H), 7.34 (dd, 1H), 7.26-7.20 (m, 1H), 7.21 (t, 1H), 6.94-6.87 (m, 2H), 4.77 (d, 1H), 4.13 (d, 1H), 3.76 (d, 2H), 3.22-3.15 (m, 1H), 3.01-2.94 (m, 1H), 2.81 (s, 3H), 2.68-2.61 (m, 1H), 2.15-1.96 (m, 2H), 1.88-1.78 (m, 2H). MS (EI) for C26H22F7N5O2. found 570.1 (MH+).
A mixture of methyl triphenylphosphonium bromide (16.6 g, 46.6 mmol), tert-BuOK (4.48 g, 39.9 mmol) and THF (120 mL) was stirred for 30 min at 40° C. The reaction mixture was cooled down to room temperature and 1-benzyl-3,3-difluoropiperidin-4-one (3.00 g, 13.3 mmol, Bioorganic Med. Chem. Lett. 2011, 21, 6409-6413) in THF (15 mL) was added. After stirring for 16 h at room temperature, hexane (75 mL) and water (30 mL) were added. The organic layer was separated, concentrated and dissolved in EtOAc/Hexane (1:10, 75 mL). The resulting solution was washed with water (6 mL). Both aq. layers were combined and extracted with EtOAc/Hexane (1:10, 2×30 mL). All organic layers were combined, concentrated and purified by silica gel column chromatography (hexane/EtOAc=25:1) to give B33.1 (2.54 g, 86%) as a clear oil. MS (EI) for C13H15F2N. found 224.1 (MH+).
To a stirred solution of B33.1 (3.04 g, 13.6 mmol) in THF (50 mL) at room temperature was added BH3.THF (27 mL, 1M in THF, 27 mmol). After stirring for 130 min at 70° C., the reaction mixture was cooled to 0° C. and 2N NaOH (41 mL) and 30% H2O2 (41 mL) were added in sequence. The resulting mixture was stirred for 15 min at room temperature, stirred for 16 h at 40° C., and cooled to room temperature. Water (30 mL) was added and the resulting solution was extracted with EtOAc (4×30 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=3:1→7:4) to give B33.2 (2.0 g, 61%) as a clear oil. MS (EI) for C13H17F2NO. found 242.1 (MH+).
To a stirred solution of B33.2 (2.0 g, 8.29 mmol) in acetone (30 mL) at 0° C. was added Jones reagent (5.1 mL, 8N, 40.8 mmol). After stirring for 30 min at room temperature, iPrOH (3 mL) and water (24 mL) were added in sequence. The resulting mixture was washed with hexane (2×10 mL). The resulting aq. layer was neutralized with 6N NaOH and extracted with EtOAc (4×15 mL). The combined organic layers were dried over magnesium sulfate and concentrated to give B33.3 (1.57 g, 45%) as a brown oil. MS (EI) for C13H15F2NO2. found 256.1 (MH+).
To a stirred solution of B33.3 (750 mg, 2.94 mmol) in THF (6 mL) at 0° C. was slowly added 1,1′-carbonyldiimidazole (CDI) (715 mg, 4.41 mmol) in THF (12 mL) over 30 min. After stirring for 3 h at room temperature, the resulting solution was divided to three equal portions (three portions of the reaction A).
Three same reactions were carried out by the following procedures: To a stirred solution of ethyl potassium malonate (417 mg, 2.45 mmol) in THF (8 mL) at 0° C. were added Et3N (0.51 mL, 3.7 mmol) and MgCl2 (257 mg, 2.70 mmol). After stirring for 3 h at room temperature, one portion of the reaction A was slowly added for 10 min at 0° C. The resulting mixture was stirred for 30 min at room temperature, stirred for additional 64 h at 30° C., and cooled down to 0° C. Saturated aq. citric acid (8 mL) was added and the resulting solution was extracted with EtOAc (3×10 mL).
The combined organic layers from three reactions were washed with water (10 ml). The aq. layer was extracted with EtOAc (3×5 mL). The combined organic layers were dried over magnesium sulfate, concentrated, and purified by silica gel column chromatography (hexane/EtOAc=4:1→3:1) to give B33.4 (320 mg, crude) as a clear oil. MS (EI) for C17H21F2NO3. found 326.1 (MH+).
A mixture of the B33.4 and DMF/DMA (4 mL) was stirred for 5 h at room temperature and then concentrated under vacuum. MS (EI) for C20H26F2N2O3. found 381.1 (MH+). To a stirred solution of the resulting residue, acetamidine.HCl (186 mg, 1.97 mmol) and EtOH (8 mL) at 0° C. was added EtONa (0.55 mL, 2.68M in EtOH, 1.48 mmol). After stirring for 1 h at room temperature, aq. sat. NH4Cl (8 mL) was added at 0° C. and the resulting solution was extracted with EtOAc (3×10 mL). The combined organic layers were concentrated, dissolved in EtOAc (10 mL), and washed with water (1 mL). The aq. layer was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=7:1→5:1) to give B33.5 (180 mg, 16%) as a clear oil. MS (EI) for C20H23F2N3O2. found 376.0 (MH+).
A mixture of B33.5 (30.0 mg, 0.0799 mmol), Pd/C (17 mg, 10 wt. %, 0.016 mmol) and MeOH (2 mL) was stirred for 1 h at room temperature under a balloon of hydrogen. The mixture was filtered through Celite and the filtrate was concentrated. The residue was dissolved in EtOAc (2 mL) and the resulting solution was filtered and concentrated. MS (EI) for C13H17F2N3O2. found 286.0 (MH+). To a stirred solution of the resulting residue, 2-(2,6-difluorophenyl)acetic acid (20.7 mg, 0.120 mmol), HATU (61.0 mg, 0.160 mmol) and DMF (0.8 mL) was added DIEA (40 μL, 0.24 mmol). After stirring for 3.5 h at room temperature, aq. sat. NH4Cl (3 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=2:1→3:2) to give B33.6 (34 mg, 97%) as a yellow oil. MS (EI) for C21H21F4N3O3. found 440.2 (MH+).
To a stirred solution of B33.6 (30.0 mg, 0.0683 mmol) in THF (1 mL) at 0° C. was added LiAlH4 (50 μL, 1M in THF, 0.055 mmol). After stirring for 1.5 h at 0° C., water (0.01 mL), 15% NaOH (0.01 mL) and water (0.02 mL) were added in sequence at room temperature with vigorous stirring. The resulting mixture was stirred for additional 1.5 h at room temperature, concentrated and purified by silica gel column chromatography (EtOAc/7N NH3 in MeOH=40:1→25:1) to give 1-(3,3-difluoro-4-(5-(hydroxymethyl)-2-methylpyrimidin-4-yl)piperidin-1-yl)-2-(2,6-difluorophenyl)ethanone (11 mg, 41%) as a clear oil. MS (EI) for C19H19F4N3O2. found 398.0 (MH+).
To a stirred solution of 1-(3,3-difluoro-4-(5-(hydroxymethyl)-2-methylpyrimidin-4-yl)piperidin-1-yl)-2-(2,6-difluorophenyl)ethanone (11.0 mg, 0.0277 mmol) in CH2Cl2 (0.5 mL) at room temperature was added Dess-Martin periodinane (23.0 mg, 0.0544 mmol).
After stirring for 2 h, water (2 mL) was added and the resulting solution was extracted with EtOAc (3×1.5 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=2:3) to give B33.7 (11 mg, quant) as a clear oil. MS (EI) for C19H17F4N3O2. found 396.0 (MH+).
To a stirred solution of B33.7 (11.0 mg, 0.0277 mmol), O-(3-(trifluoromethyl)phenyl)hydroxylamine (9.8 mg, 0.055 mmol) and EtOH (1 mL) at room temperature was added a catalytic amount of cone HCl. After stirring for 17 h, aq. sat. NaHCO3 (1 mL) was added and the resulting solution was extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=2:1) to give Compound B33 (12 mg, 78%) as a white solid after lyophilization. 1H-NMR (400 MHz, CDCl3 8.98 (d, 1H), 8.65 (d, 1H), 7.48 (m, 2H), 7.38 (m, 2H), 7.26 (m, 1H), 6.92 (t, 2H), 4.65-4.37 (m, 1H), 4.24 (m, 1H), 4.04 (m, 1H), 3.86-3.64 (m, 1H), 3.81 (s, 2H), 3.53 (m, 1H), 2.81 (s, 3H), 2.65-2.39 (m, 1H), 2.08 (m, 1H). MS (EI) for C26H21F7N4O2. found 554.9 (MH+). Analytical HPLC, ret. time=21.500 min (gradient 10% to 100% MeCN/H2O), 95% purity.
To a stirred solution of B33.5 (36.0 mg, 0.0959 mmol) in THF (1 mL) at 0° C. was added LiAlH4 (50 μL, 1M in THF, 0.055 mmol). After stirring for 80 min at 0° C., water (0.01 mL) and 15% NaOH (0.01 mL) were added in sequence at room temperature with vigorous stirring. The resulting mixture was stirred for additional 30 min and EtOAc (2 mL) was added. The resulting mixture was filtered through Celite and concentrated to give the alcohol (4-(1-benzyl-3,3-difluoropiperidin-4-yl)-2-methylpyrimidin-5-yl)methanol. MS (EI) for C18H21F2N3O. found 334.0 (MH+). To a stirred solution of the alcohol in CH2Cl2 (2 mL) at room temperature was added Dess-Martin periodinane (81.4 mg, 0.192 mmol). After stirring for 1.5 h, water (2 mL) was added and the resulting solution was extracted with EtOAc (4 mL, 3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=7:3→2:1) to give B34.1 (19 mg, 60%) as a clear oil. MS (EI) for C18H19F2N3O. found 332.0 (MH+).
To a stirred solution of B34.1 (36.0 mg, 0.109 mmol) in THF (1 mL) at 0° C. was added methylmagnesium bromide (0.16 mL, 1.4M in Toluene/THF (3:1), 0.22 mmol). After stirring for 50 min at room temperature, aq. sat. NH4Cl (3 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were dried over magnesium sulfate and concentrated to give the alcohol 1-(4-(1-benzyl-3,3-difluoropiperidin-4-yl)-2-methylpyrimidin-5-yl)ethanol. MS (EI) for C19H23F2N3O. found 348.1 (MH+). To a stirred solution of the alcohol in CH2Cl2 (2 mL) at room temperature was added Dess-Martin periodinane (92.5 mg, 0.218 mmol). After stirring for 70 min, water (3 mL) was added and the resulting solution was extracted with EtOAc (5 mL, 3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=2:1) to give B34.2 (28 mg, 74%) as a clear oil. MS (EI) for C19H21F2N3O. found 346.1 (MH+).
A mixture of B34.2 (28.0 mg, 0.0811 mmol), Pd/C (43 mg, 10 wt. %, 0.041 mmol) and MeOH (2 mL) was stirred for 20 h at room temperature under a balloon of hydrogen. The mixture was filtered through Celite and the filtrate was concentrated. The residue was dissolved in MeOH (0.4 mL) and EtOAc (2 mL), and the resulting solution was filtered and concentrated to give the free amine 1-(4-(3,3-difluoropiperidin-4-yl)-2-methylpyrimidin-5-yl)ethanone. MS (EI) for C12H15F2N3O. found 256.0 (MH+). To a stirred solution of the free amine, 2-(2,6-difluorophenyl)acetic acid (18.1 mg, 0.105 mmol), HATU (46.3 mg, 0.122 mmol) and DMF (0.8 mL) was added DIEA (40 μL, 0.24 mmol). After stirring for 1.5 h at room temperature, water (3 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=3:5) to give B34.3 (17.5 mg, 53%) as a clear oil. MS (EI) for C20H19F4N3O2. found 410.0 (MH+).
To a stirred solution of B34.3 (17.0 mg, 0.0415 mmol) in EtOH (0.5 mL) were added hydroxylamine.HCl (8.7 mg, 0.13 mmol) and a catalytic amount of cone HCl. After stirring for 15 h at 50° C., aq. sat. NaHCO3 (2 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=3:5) to give B34.4 (16 mg, a mixture of E/Z=3.8:1, 91%) as a clear oil. MS (EI) for C20H20F4N4O2. found 425.0 (MH+).
To a stirred solution of B34.4 (15.0 mg, a mixture of E/Z=3.8:1, 0.0353 mmol), 4-chloro-2-(trifluoromethyl)pyridine (12.8 mg, 0.0707 mmol) and DMA (0.5 mL) at room temperature was added potassium carbonate (9.8 mg, 0.071 mmol). After stirring for 26 h, water (3 mL) was added and the resulting solution was extracted with EtOAc (4×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography twice (tBuOMe/CH2Cl2/Hexane=4:4:3 and Hexane/EtOAc=3:4) to give Compound B34 (5.2 mg, 26%) as a clear oil. 1H-NMR (400 MHz, CDCl3): δ 8.65 (d, 1H), 8.62 (d, 1H), 7.53 (d, 1H), 7.28 (m, 1H), 7.24 (m, 1H), 6.91 (t, 2H), 4.46 (m, 1H), 4.17 (m, 1H), 4.03 (m, 1H), 3.86-3.52 (m, 2H), 3.79 (s, 2H), 2.82 (s, 3H), 2.52 (d, 3H), 2.49-2.27 (m, 1H), 2.12 (m, 1H). MS (EI) for C26H22F7N5O2. found 569.9 (MH+). Analytical HPLC, ret. time=20.636 min, 95% purity.
A mixture of B33.5 (150 mg, 0.399 mmol), Pd/C (128 mg, 10 wt. %, 0.120 mmol) and MeOH (6 mL) was stirred for 70 min at room temperature under a balloon of hydrogen. The mixture was filtered through Celite and the filtrate was concentrated. The residue was dissolved in EtOAc (5 mL), and the resulting solution was filtered and concentrated to give the free amine ethyl 4-(3,3-difluoropiperidin-4-yl)-2-methylpyrimidine-5-carboxylate. MS (EI) for C13H17F2N3O2. found 286.0 (MH+). To a stirred solution of the free amine, 1,4-dioxane (3 mL) and water (0.7 mL) at room temperature were added DIEA (0.20 mL, 1.2 mmol) and Boc2O (130 mg, 0.600 mmol) in sequence. After stirring for 1 h, aq. sat. NH4Cl (5 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=6:1→4:1) to give B35.1 (134 mg, 77%) as a clear oil. 1H-NMR (400 MHz, CDCl3): δ 9.05 (s, 1H), 4.67 (m, 1H), 4.40 (q, 2H), 4.29 (m, 1H), 4.11 (m, 1H), 3.38 (m, 1H), 3.17 (m, 1H), 2.78 (s, 3H), 2.47 (m, 1H), 1.95 (m, 1H), 1.48 (s, 9H), 1.41 (t, 3H). MS (EI) for C18H25F2N3O4. found 386.0 (MH+).
To a stirred solution of B35.1 (100 mg, 0.259 mmol) in THF (3 mL) at 0° C. was added LiAlH4 (0.21 mL, 1M in THF, 0.208 mmol). After stirring for 1 h at 0° C., water (0.02 mL), 15% NaOH (0.02 mL) and water (0.04 mL) were added in sequence at room temperature with vigorous stirring. The resulting mixture was stirred for additional 1 h and EtOAc (3 mL) was added. The resulting mixture was filtered through Celite and concentrated to give the alcohol tert-butyl 3,3-difluoro-4-(5-(hydroxymethyl)-2-methylpyrimidin-4-yl)piperidine-1-carboxylate. MS (EI) for C16H23F2N3O3. found 344.1 (MH+). To a stirred solution of the alcohol in CH2Cl2 (3 mL) at room temperature was added Dess-Martin periodinane (220 mg, 0.518 mmol). After stirring for 70 min, water (5 mL) was added and the resulting solution was extracted with EtOAc (10 mL, 2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=3:1→4:3) to give B35.2 (40 mg, 45%) as a white solid.
To a stirred solution of B35.2 (40.0 mg, 0.117 mmol) in THF (1 mL) at 0° C. was added methylmagnesium bromide (0.17 mL, 1.4M in Toluene/THF (3:1), 0.23 mmol). After stirring for 1.5 h at room temperature, aq. sat. NH4Cl (3 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were dried over magnesium sulfate and concentrated to give the alcohol tert-butyl 3,3-difluoro-4-(5-(1-hydroxyethyl)-2-methylpyrimidin-4-yl)piperidine-1-carboxylate. MS (EI) for C17H25F2N3O3. found 358.1 (MH+). To a stirred solution of the alcohol in CH2Cl2 (2 mL) at room temperature was added Dess-Martin periodinane (99.0 mg, 0.234 mmol). After stirring for 1.5 h, water (3 mL) was added and the resulting solution was extracted with EtOAc (5 mL, 2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=3:2) to give B35.3 (37 mg, 89%) as a clear oil. MS (EI) for C17H23F2N3O3. found 356.0 (MH+).
To a stirred solution of B35.3 (37.0 mg, 0.104 mmol) in THF (1 mL) at 0° C. was added lithium bis(trimethylsilyl)amide (LiHMDS) (0.16 mL, 1M in THF, 0.16 mmol). After stirring for 15 min at 0° C., chlorotrimethylsilane (20 μL, 0.16 mmol) was added. After stirring for 4 h at 0° C., aq. sat. NaHCO3 (3 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were dried over magnesium sulfate and concentrated. To a stirred solution of the resulting residue in THF (1 mL) at 0° C. were added NaHCO3 (13.1 mg, 0.156 mmol) and NBS (18.5 mg, 0.104 mmol) in sequence. After stirring for 40 min at room temperature, aq. sat. NaHCO3 (3 mL) was added at 0° C. and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=7:3) to give B35.4 (30 mg, 66%) as a clear oil. MS (EI) for C17H22BrF2N3O3. found 434.0 (MH+).
A mixture of B35.4 (52.0 mg, 0.120 mmol), potassium acetate (35.3 mg, 0.360 mmol) and DMA (0.8 mL) was stirred for 30 min at room temperature and then purified by silica gel column chromatography (hexane/EtOAc=2:1) to give B35.5 (54 mg, crude) as a pale yellow oil. MS (EI) for C19H25F2N3O5. found 414.0 (MH+).
A mixture of B35.5 (27 mg) and 4N HCl in 1,4-dioxane (1 mL) was stirred for 1 h at room temperature and concentrated under vacuum. EtOAc (2 mL) was added and the mixture was concentrated again to give the amine HCl salt, 2-(4-(3,3-difluoropiperidin-4-yl)-2-methylpyrimidin-5-yl)-2-oxoethyl acetate hydrochloride. MS (EI) for C14H17F2N3O3. found 314.0 (MH+). To a stirred solution of the amine salt, 2-(2,6-difluorophenyl)acetic acid (13.4 mg, 0.0780 mmol), HATU (34.2 mg, 0.0900 mmol) and DMF (0.6 mL) at room temperature was added DIEA (30 μL, 0.18 mmol). After stirring for 40 min, water (3 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=1:1→2:3) to give B35.6 (25 mg, 89%) as a clear oil. MS (EI) for C21H21F4N3O4. found 468.0 (MH+).
To a stirred solution of B35.6 (18.0 mg, 0.0385 mmol) in EtOH (1 mL) were added hydroxylamine.HCl (8.0 mg, 0.12 mmol) and a catalytic amount of cone HCl. After stirring for 75 h at 60° C., aq. sat. NaHCO3 (1 mL) and water (1 mL) were added at 0° C., and the resulting solution was extracted with EtOAc (4 mL, 2×2 mL). The combined organic layers were concentrated, dissolved in EtOAc (3 mL) and washed with water (0.5 mL). The aq. layer was extracted with EtOAc (2×1 mL). The combined organic layers were purified by silica gel column chromatography (hexane/EtOAc=1:3→EtOAc) to give B35.7 (10 mg, 59%) as a clear oil. MS (EI) for C20H20F4N4O3. found 441.0 (MH+).
To a stirred solution of B35.7 (16.5 mg, 0.0375 mmol), 4-chloro-2-(trifluoromethyl)pyrimidine (20.5 mg, 0.112 mmol) and DMA (1.5 mL) at room temperature was added potassium carbonate (20.7 mg, 0.150 mmol). After stirring for 1 h, the resulting mixture was purified by silica gel column chromatography (hexane/EtOAc=1:1→2:5) to give B35.8 (10 mg, 46%) as a white foam. MS (EI) for C25H21F7N6O3. found 587.2 (MH+).
To a stirred solution of B35.8 (10.0 mg, 0.0171 mmol) in CH2Cl2 (1 mL) at 0° C. was added DAST (8.2 mg, 0.051 mmol). After stirring for 50 min at 0° C., water (2 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=4:3) to give Compound B35 (6.5 mg, 65%) as a white solid after lyophilization. 1H-NMR (400 MHz, CDCl3): δ 8.82 (d, 1H), 8.66 (s, 1H), 7.40 (d, 1H), 7.24 (m, 1H), 6.90 (t, 2H), 5.78 (m, 2H), 4.34 (m, 2H), 3.78 (s, 2H), 3.69 (m, 3H), 2.83 (s, 3H), 2.58-2.34 (m, 1H), 2.11 (m, 1H). MS (EI) for C25H20F8N6O2. found 589.2 (MH+). Analytical HPLC, ret. time=20.084 min, 98% purity.
To a mixture of 3-(trifluoromethyl)benzonitrile (1.80 g, 10.5 mmol) and hydroxylamine hydrochloride (4.39 g, 63.1 mmol) in EtOH (15 mL) was added Et3N (7.45 g, 73.6 mmol). The resulting white slurry was heated in a sealed tube at 80° C. with stirring for 2.5 h. After cooling to room temperature, the reaction mixture was diluted with EtOAc, and washed with water (3×) and brine, dried (magnesium sulfate), and concentrated in vacuo to give C1.1 as a solid (2.276 g): MS (EI) for C8H7F3N2O. found 205.0 (MH+). The material was used directly for the subsequent reaction without further purification.
To a mixture of A80.4 (120 mg, 0.31 mmol), HATU (141 mg, 0.37 mmol), and DMA (2.1 mL) was added DIEA (80 mg, 0.62 mmol). The resulting mixture was stirred at 35° C. for 5 min, then C1.1 (82 mg, 0.40 mmol) was added. The resulting mixture was stirred at 35° C. for 1 h, followed by heating at 100° C. for an additional 20 h. After cooling to room temperature, the reaction mixture was subjected to a routine aq. workup, and purified by prep HPLC. The fractions containing the desired product were combined and lyophilized to afford Compound C1 as a white solid (21 mg). 1H-NMR (400 MHz, CDCl3): δ 8.45 (s, 1H), 8.37 (d, 1H), 7.84 (d, 1H), 7.70 (t, 1H), 7.22 (m, 1H), 6.70 (m, 2H), 4.73 (d, 1H), 4.11 (d, 1H), 3.74 (dd, 2H), 3.15 (t, 1H), 2.93 (m, 1H), 2.78 (s, 3H), 2.62 (m, 1H), 2.56 (s, 3H), 2.10-1.83 (m, 4H). MS (EI) for C28H24F5N5O2. found 558.0 (MH+).
Compounds C2 to C4 were prepared from methods analogous to those used to prepare Compound C1 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: YMC C18, 5μ 150×4.6 mm column, gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 25 min gradient time, 27 min total run time at 1.5 mL/min flow rate, λ=254 nM
To a solution of methyl 2-(trifluoromethyl)pyrimidine-4-carboxylate (1.50 g, 7.28 mmol) in 10 mL of anhydrous MeOH was added hydrazine monohydrate (1.09 g, 21.83 mmol). The resulting solution was heated at 50° C. with stirring for 5 min, at which time the reaction was completed. The reaction mixture, as a yellow solution, was reduced to ⅓ of the volume in vacuo. The precipitate thus formed was filtered, washed with MeOH, and dried to give 1.16 g of C5.1 as a yellow crystalline solid. The combined filtrate was concentrated under high vacuum to afford an additional 0.20 g of the hydrazide product as a yellow solid. 1H-NMR (400 MHz, CDCl3): δ9.14 (d, 1H), 8.94 (br. s, 1H), 8.27 (d, 1H), 4.16 (d, 2H).
A solution of A80.4 (100 mg, 0.26 mmol), EDC (59 mg, 0.31 mmol), and HOBt (42 mg, 0.31 mmol) in DMA (1.5 mL) was stirred at 35° C. for 1.5 h. C5.1 (69 mg, 33 mmol) was then added to the above solution. The resulting mixture was heated at 50° C. for 24 h. After cooling to room temperature, the mixture was diluted with EtOAc, and washed with water and brine, dried with magnesium sulfate and concentrated in vacuo. The residue was purified by prep HPLC to afford C5.2 (55 mg) as a white solid. 1H-NMR (400 MHz, CDCl3): δ 9.65 (br. s, 1H), 9.25 (d, 1H), 8.34 (d, 1H), 8.0 (br. s, 1H), 7.23 (m, 1H), 6.90 (t, 2H), 4.74 (d, 1H), 4.13 (d, 1H), 3.76 (s, 2H), 3.34-3.23 (m, 2H), 2.78 (t, 1H), 2.71 (s, 3H), 2.62 (s, 3H), 2.08-1.82 (m, 4H). MS (EI) for C26H24F5N7O3. found 578.1 (MH+).
To a solution of C5.2 (76 mg, 0.13 mmol) in anhydrous MeCN (1.3 mL) was added anhydrous K2CO3 (55 mg, 0.40 mmol), followed by p-toluenesulfonyl chloride (p-TsCl) (38 mg, 0.20 mmol). The mixture was stirred at 50° C. for 30 min when the reaction was completed. The reaction mixture as a slurry was diluted with water. The organic layer was separated and purified by prep HPLC to give Compound C5 as a white solid (52 mg). 1H-NMR (400 MHz, CDCl3): δ 9.22 (d, 1H), 8.48 (d, 1H), 7.23 (m, 1H), 6.90 (m, 2H), 4.73 (d, 1H), 4.11 (d, 1H), 3.75 (dd, 2H), 3.16 (t, 1H), 2.98 (m, 1H), 2.78 (s, 3H), 2.61 (m, 1H), 2.60 (s, 3H), 2.13-1.95 (m, 3H), 1.84 (d, 1H). Analytical HPLC, ret. time=18.83 min, 99% purity. MS (EI) for C26H22F5N7O2. found 559.9 (MH+).
Compounds C6 to C23 were prepared from methods analogous to those used to prepare Compound C5 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: YMC C18, 5μ 150×4.6 mm column, gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 25 min gradient time, 27 min total run time at 1.5 mL/min flow rate, λ=254 nM
The ester of A80.1 was hydrolyzed to C24.1 with the same technique used to hydrolyze A80.3 to A80.4.
A solution of C24.1 (500 mg, 1.49 mmol), EDC (329 mg, 1.71 mmol), and HOBt (232 mg, 1.71 mmol) in DMA (10 mL) was stirred at 25° C. for 1.5 h. To the above mixture was added C5.1 (344 mg, 1.67 mmol). The resulting mixture was stirred at 40° C. for 17 h. After cooling to room temperature, the reaction mixture was diluted with EtOAc, washed with water (3×) and brine, dried (magnesium sulfate), and concentrated in vacuo to dryness, yielding a solid (793 mg). LC/MS analysis of the resulting residue showed the presence of C24.2 as a major product. MS (EI) for C23H28F3N7O4. found 524.2 (MH+). The crude product was used directly for the next reaction as is.
The entire amount (793 mg) of crude product obtained from the previous step (C24.2) was dissolved in 30 mL of anhydrous MeCN. To this solution was added K2CO3 (850 mg, 4.45 mmol), followed by p-toluenesulfonyl chloride (1236 mg, 4.94 mmol). The mixture was stirred at 50° C. for 1 h. After removal of solvent in vacuo, the crude mixture was diluted with EtOAc, washed with water (3×) and brine, dried (magnesium sulfate), and concentrated in vacuo to dryness, affording C24.3 as a solid (761 mg). MS (EI) for C23H26F3N7O3. found 506.2 (MH+). The crude product was used directly for the subsequent reaction.
Crude C24.3 (760 mg) obtained above was dissolved in MeCN (4 mL). To this solution was added 4M HCl in dioxane (6 mL), which was allowed to stir at room temperature for 10 min. After removal of solvent in vacuo, the residue was dissolved in water and washed with tert-butyl methyl ether (2×). The aq. layer was cooled to 0° C., and basified to pH 11 by slow addition of 2N NaOH aq. solution, and then extracted with EtOAc (3×). The combined organic extract was washed with brine, dried with magnesium sulfate, and concentrated under high vacuum, to give C24.4 as a solid (310 mg). 1H-NMR (400 MHz, CDCl3): δ 9.22 (d, 1H), 8.47 (d, 1H), 3.36 (d, 2H), 2.89 (m, 1H), 2.77 (s, 3H), 2.76 (m, 2H), 2.58 (s, 3H), 2.46 (br. s, 1H), 2.22-2.07 (m, 2H), 1.94-1.91 (m, 2H). MS (EI) for C18H18F3N7O3. found 406.2 (MH+).
A solution of 2,6-difluorobenzoic acid (11 mg, 0.07 mmol), EDC (11 mg, 0.06 mmol), and HOBt (8 mg, 0.06 mmol) in DMA (0.6 mL) was stirred at room temperature for 1.5 h. To the mixture was added C24.4 (18 mg, 0.04 mmol). The reaction mixture was stirred at room temperature for 17 h. To the resulting mixture was added 2N NaOH aq. solution (2 drops). The reaction mixture was stirred for 5 min, and neutralized with AcOH. The crude mixture was purified on prep HPLC to give Compound C24 as a solid (8.5 mg). 1H-NMR (400 MHz, CDCl3): δ 9.21 (d, 1H), 8.46 (d, 1H), 7.34 (m, 1H), 6.94 (m 2H), 4.89 (d, 1H), 3.63 (d, 1H), 3.12 (m, 1H), 2.99 (m, 1H), 2.81 (m, 1H), m2.77 (s, 3H), 2.59 (s, 3H), 2.15-1.99 (m, 2H), 1.94 (d, 1H), 1.83 (d, 1H). Analytical HPLC, ret. time=18.300 min, 98% purity. MS (EI) for C25H20F5N7O2. found: 546.2 (MH+).
Compounds C25 to C27 were prepared from methods analogous to those used to prepare Compounds C24 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: YMC C18, 5μ 150×4.6 mm column, gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 25 min gradient time, 27 min total run time at 1.5 mL/min flow rate, λ=254 nM
To a solution of C24.4 (16 mg, 0.04 mmol) in CH2Cl2 was added 1,3-difluoro-2-isocyanatobenzene (9 mg, 0.06 mmol) and a drop of DIEA. The solution was stirred at room temperature for 2 min at which time the reaction was complete. After removal of the solvent in vacuo, the residue was purified on prep. HPLC, affording Compound C28 as a white solid (10 mg). 1H-NMR (400 MHz, CDCl3): δ 9.21 (d, 1H), 8.47 (d, 1H), 7.10 (m, 1H), 6.93 (t, 2H), 5.87 (s, 1H), 4.22 (d, 2H), 2.99-2.91 (m, 3H), 2.78 (s, 3H), 2.59 (s, 3H), 2.12 (m, 2H), 1.91 (d, 2H).). Analytical HPLC, ret. time=16.62 min, 98% purity. MS (EI) for C25H21F5N8O2. found: 561.2 (NH+)
Compounds C29 to C32 were prepared from methods analogous to those used to prepare Compound C28 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: YMC C18, 5μ 150×4.6 mm column,
gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 25 min gradient time, 27 min total run time at 1.5 mL/min flow rate, λ=254 nM
To a solution of C24.4 (17 mg, 0.04 mmol) in CH2Cl2 (0.5 mL) was added 2,6-difluorobenzene-1-sulfonyl chloride (16 mg, 0.07 mmol), followed by DIEA (9 mg). After stirring at room temperature for 2 min, the reaction mixture was concentrated in vacuo to remove solvent. The residue was dissolved in MeCN and purified on prep HPLC to give Compound C33 as a white solid (9 mg). 1H-NMR (400 MHz, CDCl3): δ 9.19 (d, 1H), 8.42 (d, 1H), 7.50 (m, 1H), 7.02 (t, 2H), 4.08 (d, 2H), 2.86 (m, 1H), 2.76 (s, 3H), 2.65 (d, 2H), 2.60 (s, 3H), 2.26-2.16 (m, 2H), 1.92 (d, 2H). Analytical HPLC, ret. time=19.692 min, 99% purity. MS (EI) for C24H20F5N7O3S. found: 582.1 (MH+).
Compound C34 was synthesized in a manner analogous to C33. Analytical HPLC, ret. time=19.628 min. MS (EI) for C25H23F4N7O3S. found: 578.2 (MH+).
To a stirred solution of 2-(2,6-difluorophenyl)propanoic acid (186 mg, 1.00 mmol, J. Med. Chem. 2005, 48, 6776.), DCC (248 mg, 1.20 mmol) and CH2Cl2 (4 mL) at room temperature was slowly added a solution of pentafluorophenol (184 mg, 1.00 mmol) in CH2Cl2 (4 mL). After stirring for 18 h, the resulting mixture was filtered through Celite. Water (3 mL) was added to the filtrate and the aq. layer was extracted with EtOAc (2×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (CH2Cl2/hexane=1:20) to give C35.1 (300 mg, 85%) as a clear oil.
To a stirred solution of (S)—4-phenyloxazolidin-2-one (32.6 mg, 0.200 mmol) in THF (1 mL) at −78° C. was added nBuLi (90 μL, 2.5M in Hexane, 0.22 mmol). After stirring for 1 h, a solution of C35.1 (140 mg, 0.400 mmol) in THF (1.5 mL) was slowly added at −78° C. After stirring for 2 h, water (2 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers, which contained a diastereomeric mixture with 94% de, were concentrated and purified by silica gel column chromatography (CH2Cl2/hexane=1:5→5:1) to give C35.2 (57 mg, a single diastereomer, 43%) as a white solid. The stereochemistry of C35.2 was predicted by following a method described in Synlett 2009, 960. 1H-NMR (400 MHz, CDCl3): δ 7.36 (m, 3H), 7.24 (m, 2H), 7.18 (m, 1H), 6.80 (t, 2H), 5.49 (dd, 1H), 4.97 (q, 1H), 4.68 (t, 1H), 4.22 (d, 1H), 1.44 (d, 3H). MS (EI) for C18H15F2NO3. found 332.1 (MH+).
To a stirred solution of C35.2 (25.0 mg, 0.0755 mmol), THF (0.6 mL) and water (0.2 mL) at room temperature were added H2O2 (17 mg, 30 wt. % in H2O, 0.15 mmol) and LiOH (3.6 mg, 0.15 mmol) in sequence. After stirring for 8 h, water (2 mL) was added and the resulting solution was washed with EtOAc (2×2 mL). The aq. layer was acidified with IN HCl solution and extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (CH2Cl2/MeOH=40:1→20:1) to give C35.3 (13.5 mg, 96%) as a clear oil.
To a stirred solution of C35.3 (16.0 mg, 0.0860 mmol), C24.4 (24.9 mg, 0.0614 mmol), HATU (46.7 mg, 0.123 mmol) and DMF (0.6 mL) at room temperature was added DIEA (30 μL, 0.18 mmol). After stirring for 1.5 h, water (2 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=1:1→2:3) to give Compound C35 (15 mg, 43%) as a white solid after lyophilization. 1H-NMR (400 MHz, CDCl3, rotameric mixture): δ 9.20 (m, 1H), 8.44 (t, 1H), 7.21 (m, 1H), 6.93 and 6.87 (two of t, 2H), 4.78 (d, 1H), 4.14 (m, 1H), 3.73 (t, 1H), 2.96-2.50 (m, 3H), 2.76 and 2.71 (two of s, 3H), 2.58 and 2.54 (two of s, 3H), 1.98 (m, 1H), 1.81 (m, 2H), 1.51-1.02 (m, 1H), 1.50 (d, 3H). MS (EI) for C27H24F5N7O2. found 574.2 (MH+). Analytical HPLC, ret. time=19.296 min, 95% purity.
To a stirred solution of C28 (11.0 mg, 0.0196 mmol), iodomethane (5.6 mg, 0.039 mmol) and THF (0.5 mL) at 0° C. was added NaH (1.2 mg, 60% dispersion in mineral oil, 0.029 mmol). After stirring for 2 h, aq. sat. NH4Cl (1 mL) was added and the resulting solution was extracted with EtOAc (3×1 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=1:1→1:2) to give Compound C36 (9.0 mg, 80%) as a white solid after lyophilization. 1H-NMR (400 MHz, CDCl3): δ 9.20 (d, 1H), 8.44 (d, 1H), 7.19 (m, 1H), 6.97 (t, 2H), 3.81 (d, 2H), 3.12 (s, 3H), 2.74 (s, 3H), 2.73 (m, 1H), 2.60 (td, 2H), 2.54 (s, 3H), 1.71 (qd, 2H), 1.62 (m, 2H) MS (EI) for C26H23F5N8O2. found 575.2 (MH+). Analytical HPLC, ret. time=18.872 min, 99% purity.
To a solution of C5 (12 mg) in anhydrous THF (1.2 mL) under N2 was added NaH (4 mg, 60% in mineral oil) with stirring at room temperature. The mixture was stirred at room temperature for 20 min. To the mixture was added deuterium oxide (3 mL) with stirring. After stirring for 1 h, 2 drops of AcOH were added, the crude mixture was purified on prep HPLC to give Compound C37 (4 mg). 1H-NMR (400 MHz, CDCl3); δ 9.23 (d, 1H), 8.48 (d, 1H), 7.23 (m, 1H), 6.90 (t, 2H), 4.74 (d, 1H), 4.11 (d, 1H), 3.74 (m, 1H), 3.16 (t, 1), 2.98 (m, 1H), 2.78 (s, 3H), 2.60 (s, 1H), 2.56 (m, 1H), 2.10-1.95 (m, 3H), 1.84 (d, 1H). Analytical HPLC, ret. time=18.752 min. 97% purity. MS for C26H19D3F5N7O2. found: 563.0 (MH+).
A mixture of 2-(2,6-difluorophenyl)acetic acid (1.00 g, 5.81 mmol), anhydrous K2CO3 (3.21 g, 23.25 mmol) and deuterium oxide (5 mL) was heated at 100° C. with stirring for 24 h. The reaction was cooled to 0° C., acidified to pH 2 with 6N HCl, and then extracted with CH2Cl2. The organic layer was dried over sodium sulfate, and concentrated in vacuo. This process was repeated three more times to give C38.1 as a white solid (0.96 g). 1H-NMR (400 MHz, DMSO-d6): δ 12.55 (s, 1H), 7.38 (m. 1H), 7.08 (m, 2H).
A solution of C38.1 (4.5 mg, 0.03 mmol), EDC (4.3 mg, 0.02 mmol) and HOBt (3.0 mg, 0.02 mmol) in DMA (0.5 mL) was stirred at room temperature for 45 min. To the above mixture was added C24.4 (4.5 mg, 0.03 mmol). The resulting mixture was stirred at room temperature for 40 min. The reaction was quenched by adding 2 drops of 2N NaOH aq. solution. The resulting mixture was purified on HPLC to give Compound C38 as a white solid (4 mg). 1H-NMR (400 MHz, CDCl3); δ 9.20 (d, 1H), 8.45 (d, 1H), 7.21 (m, 1H), 6.89 (t, 2H), 4.72 (d, 1H), 4.10 (d, 1H), 3.16 (t, 1H), 2.99 (m, 1H), 2.77 (s, 3H), 2.61 (m, 1H), 2.59 (s, 3H), 2.08-1.82 (m, 4H). Analytical HPLC, ret. time=18.72 min, 98% purity. MS (EI) for C26H20D2F5N7O2. found: 562.2 (MH+).
To a solution of C5.2 (34 mg, 0.06 mmol) in anhydrous 2-methyltetrahydrofuran (1 mL) was added Lawesson's reagent (21 mg, 0.05 mmol). The mixture was heated in a sealed tube at 100° C. with stirring for 3 days. Upon cooling down to room temperature, the mixture was diluted with water, and extracted with EtOAc. The organic layer was washed with brine, dried (magnesium sulfate), and concentrated in vacuo. The crude product thus obtained was purified by prep. HPLC to give Compound C39 as a solid (22 mg). 1H-NMR (400 MHz, CDCl3): δ 9.19 (d, 1H), 8.56 (d, 1H), 7.25 (m, 1H), 6.90 (t, 2H), 5.68 (d, 1H), 4.52 (d, 1H), 4.04 (s, 2H), 3.26 (dt, 1H), 2.99 (dt, 1H), 2.84 (m, 1H), 2.78 (s, 3H), 2.41 (s, 3H), 2.22-2.11 (m, 2H), 1.95-1.86 (m, 2H). Analytical HPLC, ret. time=21.97 min, 99% purity. MS (EI) for C26H22F5N7S2. found 591.9 (MH+).
To a mixture of Me3SI (300 mg, 1.5 mmol) in THF (4 mL) was added tBuOK (2 mL, 1.0 M in THF). The resulting mixture was stirred at room temperature for 30 min and B23.1 (320 mg, 1 mmol) was added. Stirring was continued at room temperature for 2 h and then diluted with aq. sat. NaHCO3. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude product was used as is in the next step.
A solution of D1.1 (40 mg, 0.12 mmol), 3-chloroaniline (46 mg, 0.36 mmol) and LiCl (21 mg, 0.5 mmol) in EtOH (0.5 mL) was stirred at 80° C. for 5 days. The mixture was diluted with EtOAc, washed with brine, and concentrated. The crude product was purified by flash column chromatography.
To a solution of D1.2 (20 mg, 0.043 mmol) in MeOH (2 mL) was added 4N HCl in dioxane (0.3 mL). The resulting solution was stirred for 12 h at room temperature. EtOAc (25 mL) was added. The mixture was basified with 20% NaOH aq. solution to pH 10. The EtOAc layer was washed with brine, dried over sodium sulfate, and concentrated. The crude amine, 1-(3-chlorophenylamino)-2-(2-methyl-4-(piperidin-4-yl)pyrimidin-5-yl)propan-2-ol, was used in the next step without further purification.
1-(3-Chlorophenoxy)-2-(2-methyl-4-(piperidin-4-yl)pyrimidin-5-yl)propan-2-ol (10 mg, 0.028 mmol) obtained above was mixed with 2,6-difluorophenylacetic acid (10 mg, 0.056 mmol), HATU (15 mg, 0.039 mmol) and DIEA (38 mg, 0.3 mmol) in THF (2 mL). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by prep HPLC to give D1.3. 1H-NMR (400 MHz, DMSO-d6): δ 8.58 (d, 1H), 7.36 (m, 1H), 7.04 (m, 3H), 6.70 (d, 1H), 6.60 (d, 1H), 6.50 (d, 1H), 5.78 (m, 1H), 5.54 (s, 1H), 4.45 (d, 1H), 4.13 (d, 1H), 3.82 (m, 4H), 3.23 (m, 1H), 3.08 (m, 1H), 2.62 (m, 1H), 2.51 (s, 3H), 1.75 (m, 4H), 1.68 (s, 3H). MS (EI) for C27H29ClF2N4O2. found: 515.0 (MH+). Analytical HPLC, ret. time=3.64 min, 99% purity.
Compounds D2 to D10 were prepared from methods analogous to those used to Compounds Compound D1 utilizing appropriate reagent replacements.
To a solution of 3,5-dimethylphenol (244 mg, 2.0 mmol) in DMF (4 mL) was added NaH (120 mg, 3 mmol, 60% in mineral oil). The resulting mixture was stirred at room temperature for 30 min, then D1.1 (1 mmol) was added. The mixture was stirred at 75° C. for 8 h, cooled to room temperature and diluted with aq. sat. NaHCO3. The crude product was extracted with EtOAc, washed with brine, and concentrated. The crude product was purified by flash column chromatography.
To a solution of D11.1 (400 mg, 0.87 mmol) in MeOH (5 mL) was added 4N HCl in dioxane (1 mL). The resulting solution was stirred for 12 h at room temperature. EtOAc (25 mL) was added. The mixture was basified with 20% NaOH aq. solution to pH 10. The EtOAc layer was washed with brine, dried over sodium sulfate, and concentrated. The crude amine was used in the next step without further purification.
1-(3,5-Dimethylphenoxy)-2-(2-methyl-4-(piperidin-4-yl)pyrimidin-5-yl)propan-2-ol (220 mg, 0.61 mmol) obtained above was mixed with 2,6-difluorophenylacetic acid (123 mg, 0.72 mmol), HATU (300 mg, 0.78 mmol) and DIEA (387 mg, 3 mmol) in THF (5 mL). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by prep HPLC to give Compound D11. 1H-NMR (400 MHz, DMSO-d6): δ 8.56 (d, 1H), 7.36 (m, 1H), 7.07 (m, 2H), 6.55 (m, 3H), 5.70 (s, 1H), 4.45 (d, 1H), 4.18 (m, 3H), 3.78 (m, 3H), 3.17 (m, 1H), 2.62 (m, 1H), 2.55 (s, 3H), 2.21 (s, 6H), 1.75 (m, 4H), 1.65 (s, 3H). MS (EI) for C29H33F2N3O3. found: 510.0 (MH+). Analytical HPLC, ret. time=17.17 min, 97% purity.
Compounds D12 to D14 were prepared from methods analogous to those used to prepare Compound D11 utilizing appropriate reagent replacements.
D15.1 was synthesized from B23.1 in the same manner by which B35.4 was synthesized from B35.3.
To a stirred solution diethyl phosphite (22.2 mg, 0.161 mmol) in DMF (1 mL) at room temperature was added cesium carbonate (105 mg, 0.322 mmol). After stirring 30 min, D15.1 (53.4 mg, 0.134 mmol) in DMF (1 mL) was added. After stirring 70 min, water (2 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated to give D15.2 as a brown oil. MS (EI) for C21H34N3O6P. found 456.0 (MH+).
To a stirred solution of D15.2, LiCl (18.2 mg, 0.429 mmol) and MeCN (1 mL) at room temperature were added DIEA (50 μL, 0.27 mmol) and 3,5-dimethylbenzaldehyde (17.8 mg, 0.134 mmol) in MeCN (0.5 mL) in sequence. After stirring for 24 h at 95° C., water (2 mL) was added and the resulting solution was extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=3:1) to give D15.3 (10 mg, 17%) as a pale yellow oil. MS (EI) for C269H33N3O3. found 436.1 (MH+).
A mixture of D15.3 (15.0 mg, 0.0344 mmol), Pd/C (18.0 mg, 10 wt. %, 0.0172 mmol) and MeOH (2 mL) was stirred for 1 h at room temperature under a balloon of hydrogen. The mixture was filtered through Celite and the filtrate was concentrated. The residue was dissolved in EtOAc (1 mL) and the resulting solution was filtered and concentrated to give tert-butyl 4-(5-(3-(3,5-dimethylphenyl)propanoyl)-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (10 mg, 66%) as a clear oil.
To a stirred solution of tert-butyl 4-(5-(3-(3,5-dimethylphenyl)propanoyl)-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (9.0 mg, 0.021 mmol) in THF (0.4 mL) at room temperature was added methylmagnesium bromide (0.12 mL, 1.4M in Toluene/THF (3:1), 0.16 mmol). After stirring for 4 h at 50° C., aq. sat. NH4Cl (2 mL) was added and the resulting solution was extracted with EtOAc (3×1.5 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=3:1→3:4) to give D15.4 (6.0 mg, 64%) as a clear oil. MS (EI) for C27H39N3O3. found 454.1 (MH+).
A mixture of D15.4 (6.0 mg, 0.0132 mmol) and 4N HCl in 1,4-dioxane (0.3 mL) was stirred for 1 h at room temperature, and concentrated under vacuum. EtOAc (1 mL) was added and the mixture was concentrated again to give the amine HCl salt, 4-(3,5-dimethylphenyl)-2-(2-methyl-4-(piperidin-4-yl)pyrimidin-5-yl)butan-2-ol. MS (EI) for C22H31N3O. found 354.1 (MH+). To a stirred solution of the resulting amine HCl salt, 2-(2,6-difluorophenyl)acetic acid (3.4 mg, 0.020 mmol), HATU (15.0 mg, 0.0396 mmol) and DMF (0.3 mL) at room temperature was added DIEA (10 μL, 0.053 mmol). After stirring for 1 h, water (1 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=2:5→1:3) to give Compound D15 (4.7 mg, 69%) as a white powder after lyophilization. 1H-NMR (400 MHz, CDCl3): δ 8.60 (s, 1H), 7.22 (m, 1H), 6.90 (t, 2H), 6.83 (s, 1H), 6.72 (s, 2H), 4.76 (d, 1H), 4.11 (d, 1H), 3.77 (d, 2H), 3.68 (m, 1H), 3.21 (m, 1H), 2.68 (s, 3H), 2.67 (m, 1H), 2.52 (t, 2H), 2.31-1.97 (m, 4H), 2.27 (s, 6H), 1.72 (s, 3H), 1.71 (m, 2H). MS (EI) for C30H35F2N3O2. found 508.1 (MH+). Analytical HPLC, ret. time=16.556 min, 96% purity.
To a freshly prepared MeONa (5 mmol) solution in MeOH (5 mL) was added D1.1 (166 mg, 0.5 mmol). The resulting solution was stirred at 60° C. for 12 h and cooled to room temperature. Water was added to quench the reaction. The mixture was extracted with EtOAc and the organic phase was washed with brine, dried over sodium sulfate, and concentrated. The crude product was purified by flash column chromatography.
To a solution of E1.1 (150 mg, 0.42 mmol), 3,5-dimethylbenzyl bromide (109 mg, 0.55 mmol) and Bu4NI (30 mg, 0.08 mmol) in THF (5 mL) was added NaH (50 mg, 1.2 mmol, 60% in mineral oil). The resulting mixture was stirred at room temperature for 12 h. Water was added to quench the reaction. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by flash column chromatography.
To a solution of E1.2 (150 mg, 0.31 mmol) in MeOH (2 mL) was added 4N HCl in dioxane (0.5 mL). The resulting solution was stirred for 12 h at room temperature. EtOAc (25 mL) was added. The mixture was basified with 20% NaOH aq. solution to pH 10. The EtOAc layer was washed with brine, dried over sodium sulfate, and concentrated. The crude product was used in the next step without further purification.
5-(2-(3,5-Dimethylbenzyloxy)-1-methoxypropan-2-yl)-2-methyl-4-(piperidin-4-yl)pyrimidine (100 mg, 0.26 mmol) obtained above was mixed with 2,6-difluorophenylacetic acid (54 mg, 0.31 mmol), HATU (128 mg, 0.34 mmol) and DIEA (129 mg, 1 mmol) in THF (2 mL). The resulting reaction mixture was stirred at room temperature for 2 h, then diluted with aq. sat. NaHCO3. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by prep HPLC to give Compound E1. MS (EI) for C31H37F2N3O3. found: 538.1 (MH+). Analytical HPLC, ret. time=20.20 min, 97% purity.
Compounds E2 to E4 were prepared from methods analogous to those used to prepare Compound E1 utilizing appropriate reagent replacements.
To a 0° C. solution of B1.3 (280 mg, 0.76 mmol) in THF (1 mL) was added MeMgBr (1 mL, 1.52 mmol, 1.4 M in THF). The reaction was quenched by water after 15 min. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by flash column chromatography to give desired alcohol E5.1.
To a solution of E5.1 (69 mg, 0.18 mmol), 3,5-dimethylbenzyl bromide (46 mg, 0.23 mmol) and Bu4NI (13 mg, 0.036 mmol) in THF (2 mL) was added NaH (22 mg, 0.54 mmol, 60% in mineral oil). The resulting mixture was stirred at room temperature for 12 h. Water was added to quench the reaction. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by HPLC to give Compound E5. MS (EI) for C30H35F2N3O2. found: 508.1 (MH+). Analytical HPLC, ret. time=20.40 min, 90% purity.
Compounds E6 and E7 were prepared from methods analogous to those used to prepare Compound E5 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: YMC C18, 5μ 150×4.6 mm column, gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 25 min gradient time, 27 min total run time at 1.5 mL/min flow rate, λ=254 nM
Sodium (100 mg, 4.3 mmol) was added to ice-water cooled (R)-1,2-isopropylideneglycerol (1.5 mL). The mixture was stirred for 15 min. To the prepared sodium alkoxide solution was added D1.1 (143 mg, 0.43 mmol). The resulting solution was stirred at 65° C. for 12 h. Water was added to the cooled reaction mixture. It was then extracted with EtOAc, washed with NaHCO3, dried over sodium sulfate, and concentrated. The crude product was purified by flash column chromatography.
To a solution of E8.1 (210 mg, 0.45 mmol), 3,5-dimethylbenzyl bromide (115 mg, 0.57 mmol) and Bu4NI (25 mg, 0.07 mmol) in THF (5 mL) was added NaH (54 mg, 1.35 mmol, 60% in mineral oil). The resulting mixture was stirred at room temperature for 12 h. Water was added to quench the reaction. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by flash column chromatography.
To a solution of E8.2 (230 mg, 0.39 mmol) in MeOH (5 mL) was added 4N HCl in dioxane (1 mL). The resulting solution was stirred for 12 h at room temperature. EtOAc (25 mL) was added. The mixture was basified with 20% NaOH aq. solution to pH 10. The EtOAc layer was washed with brine, dried over sodium sulfate, and concentrated. The resulting crude amine (0.39 mmol) obtained was mixed with 2,6-difluorophenylacetic acid (67 mg, 0.39 mmol), HATU (177 mg, 0.45 mmol) and DIEA (129 mg, 1 mmol) in THF (5 mL). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by prep HPLC to give Compound E8 as a mixture of diastereomers. MS (EI) for C33H41F2N3O5. found: 598.0 (MH+). Analytical HPLC, ret. time=15.20 min, 95% purity.
Compound E9 (as a mixture of diastereomers) was synthesized from D1.1 in the same manner as Compound E8. MS (EI) for C33H41F2N3O5. found: 598.1 (MH+). Analytical HPLC, ret. time=15.14 min, 98% purity.
To a stirred solution of A1.4 (200 mg, 0.496 mmol) in THF (3 mL) at 0° C. was added LiAlH4 (0.40 mL, 1M in THF, 0.396 mmol). After stirring for 2.5 h at 0° C., Et2O (4 mL), water (0.02 mL), 5% NaOH (0.06 mL) and water (0.02 mL) were added in sequence at room temperature with vigorous stirring. The resulting mixture was stirred for additional 2 h at room temperature and then EtOAc (3 mL) was added. The resulting mixture was filtered through Celite, concentrated and purified by silica gel column chromatography (EtOAc/7N NH3 in MeOH=30:1→20:1) to give 2-(2,6-difluorophenyl)-1-(4-(5-(hydroxymethyl)-2-methylpyrimidin-4-yl)piperidin-1-yl)ethanone (95 mg, 53%) as a pale yellow solid. MS (EI) for C19H21F2N3O2. found 362.1 (MH+).
To a stirred solution of 2-(2,6-difluorophenyl)-1-(4-(5-(hydroxymethyl)-2-methylpyrimidin-4-yl)piperidin-1-yl)ethanone (20.0 mg, 0.0553 mmol) in CH2Cl2 (1 mL) at room temperature were added Et3N (20 μL, 0.17 mmol), TsCl (13.7 mg, 0.0719 mmol) and N,N-dimethylaminopyridine (DMAP) (0.7 mg, 0.006 mmol) in sequence. After stirring for 5 h, aq. sat. NH4Cl (2 mL) was added and the resulting solution was extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=1:2) to give E10.1 (10.0 mg, 48%) as a white solid. MS (EI) for C19H20ClF2N3O. found 380.0 (MH+).
A mixture of (3,5-dimethylphenyl)methanol (5.4 mg, 0.040 mmol), NaH (1.6 mg, 60% dispersion in mineral oil, 0.40 mmol) and DMF (0.3 mL) was stirred for 10 min at room temperature. A solution of E10.1 (10.0 mg, 0.0263 mmol) in DMF (1 mL) was added. After stirring for 1 h at 50° C., the reaction mixture was quenched with aq. sat. NH4C (1 mL) and then extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=2:3) to give Compound E10 (3.1 mg, 25%) as a white solid. 1H-NMR (400 MHz, CDCl3): δ 8.48 (s, 1H), 7.23 (m, 1H), 6.97 (m, 3H), 6.91 (t, 2H), 4.73 (d, 1H), 4.51 (s, 2H), 4.50 (d, 2H), 4.11 (d, 1H), 3.76 (s, 2H), 3.16 (td, 1H), 3.07 (tt, 1H), 2.69 (s, 3H), 2.63 (td, 1H), 2.33 (s, 6H), 2.04 (qd, 1H), 1.89 (qd, 1H), 1.75 (m, 2H). MS (EI) for C28H31F2N3O2. found 480.1 (MH+). Analytical HPLC, ret. time=18.704 min, 95% purity.
To a 0° C. solution of B3.1 (172 mg, 0.45 mmol) in MeOH (3 mL) was added NaBH4 (52 mg, 1.4 mmol). The reaction was quenched by water after 30 min. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was used in the next step without further purification.
To a solution of E11.1 (160 mg, 0.42 mmol), 3,5-dimethylbenzyl bromide (109 mg, 0.55 mmol) and Bu4NI (30 mg, 0.08 mmol) in THF (5 mL) was added NaH (42 mg, 1 mmol, 60% in mineral oil). The resulting mixture was stirred at room temperature for 12 h. Water was added to quench the reaction. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by HPLC to give Compound E11. 1H-NMR (400 MHz, DMSO-d6): δ 8.61 (s, 1H), 7.35 (m, 1H), 7.07 (m, 2H), 6.86 (m, 3H), 4.84 (m, 1H), 4.45 (d, 1H), 4.31 (m, 2H), 4.12 (d, 1H), 3.78 (m, 2H), 3.17 (m, 2H), 2.62 (m, 1H), 2.55 (s, 3H), 2.21 (s, 6H), 1.63 (m, 4H), 1.45 (d, 3H). MS (EI) for C29H33F2N3O2. found: 494.0 (MH+). Analytical HPLC, ret. time=22.52 min, 90% purity.
To a −78° C. solution of A1.2 (13 g, 37 mmol) in THF (125 mL) was added dropwise LAH (28 mL, 1 M in THF). The solution was stirred at −78° C. for additional 30 min after the addition. It was then warmed to 0° C. The stirring was continued for 10 min and the reaction was quenched by slow addition of water (1.5 mL), 4 N NaOH (2.5 mL) and water (4.5 mL). The mixture was filtered through Celite, which was washed with EtOAc. Concentration of the EtOAc solution gave the crude alcohol (tert-butyl 4-(5-(hydroxymethyl)-2-methylpyrimidin-4-yl)piperidine-1-carboxylate), which was purified by flash column chromatography.
To a 0° C. solution of tert-butyl 4-(5-(hydroxymethyl)-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (7.15 g, 23 mmol) and Et3N (4.7 g, 46 mmol) in THF (100 mL) was added methanesulfonylchloride (MsCl) (3.14 g, 28 mmol). The mixture was stirred for 30 min after the addition, then quenched with water and extracted with EtOAc. The organic layer was washed with NaHCO3, brine, and dried over sodium sulfate. Concentration gave the crude mesylate (tert-butyl 4-(2-methyl-5-((methylsulfonyloxy)methyl)pyrimidin-4-yl)piperidine-1-carboxylate), which was used in the next step.
A mixture of KCN (1.8 g, 28 mmol) and Bu4NBr (9.0 g, 28 mmol) in DMF (25 mL) was stirred at 40° C. for 3 h. The crude mesylate (˜22 mmol) was added as a solution in DMF (15 mL). The reaction mixture was stirred at 40° C. for 2 days, then cooled to room temperature. Water (50 mL) was added and the crude product was extracted with EtOAc. The organic layer was washed with NaHCO3, brine, dried over sodium sulfate, and concentrated. The crude product was purified by flash column chromatography to give F1.1.
To a 0° C. solution of F1.1 (670 mg, 2.1 mmol) and MeI (898 mg, 6.3 mmol) in DMF (10 mL) was added tBuOK (716 mg, 6.3 mmol). The reaction mixture was stirred for 30 min, and quenched with water (10 mL). The mixture was extracted with EtOAc. The organic layer was washed with NaHCO3, brine, dried over sodium sulfate, and concentrated. The crude product was purified by flash column chromatography to give tert-butyl 4-(5-(2-cyanopropan-2-yl)-2-methylpyrimidin-4-yl)piperidine-1-carboxylate.
To a 250 mL hydrogenation flask were added tert-butyl 4-(5-(2-cyanopropan-2-yl)-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (103 mg, 0.3 mmol), methanolic ammonia (3 N, 20 mL), and Raney nickel (100 mg, Raney 2800 Ni slurry in water). The flask was transferred to a Parr shaker type hydrogenation apparatus, charged with hydrogen (40 psi), and was shaken for 4 h. The catalyst was removed by filtration through Celite (washing with MeOH), and the solution was concentrated in vacuo to afford F1.2.
F1.2 (100 mg, 0.28 mmol), 4-chloro-2-(trifluoromethyl)pyrimidine (51 mg, 0.28 mmol) and DIEA (112 mg, 0.86 mmol) were mixed in MeCN (3 mL). The resulting solution was stirred at 50° C. for 12 h. The mixture was concentrated to dryness. The residue was dissolved in EtOAc, washed with NaHCO3 and brine. Concentration gave crude F1.3, which was used as is in the next step.
To a solution of the crude F1.3 in MeOH (2 mL) was added 4N HCl in dioxane (0.3 mL). The resulting solution was stirred for 12 h at room temperature. EtOAc (25 mL) was added. The mixture was basified with 20% NaOH aq. solution to pH 10. The EtOAc layer was washed with brine, dried over sodium sulfate, and concentrated to give F1.4. The crude product was used in the next step without further purification.
F1.4 (100 mg, 0.25 mmol) was mixed with 2,6-difluorophenylacetic acid (52 mg, 0.3 mmol), HATU (114 mg, 0.3 mmol) and DIEA (129 mg, 1 mmol) in THF (2 mL). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by prep HPLC to give Compound F1. MS (EI) for C27H29F5N6O. found: 549.0 (MH+). Analytical HPLC, ret. time=3.30 min, 98% purity.
Compounds F2 to F3 were prepared from methods analogous to those used to prepare Compound F1 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: YMC C18, 5μ 150×4.6 mm column, gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 25 min gradient time, 27 min total run time at 1.5 mL/min flow rate, λ=254 nM
To a solution of F1.1 (570 mg, 1.79 mmol) in dimethylcarbonate (3 mL) was added MeONa (194 mg, 3.6 mmol). The resulting solution was stirred at room temperature for 2 h. The reaction was quenched with water and extracted with EtOAc. The organic phase was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography to give F4.1.
To a solution of F4.1 (370 mg, 1 mmol) and MeI (283 mg, 2 mmol) in DMF (5 mL) was added tBuOK (224 mg, 2 mmol). The reaction mixture was stirred for 12 h, and quenched with water (10 mL). The mixture was extracted with EtOAc. The organic layer was washed with NaHCO3, brine, dried over sodium sulfate, and concentrated. The crude product was purified by flash column chromatography.
To a 250 mL hydrogenation flask were added F4.2 (200 mg, 0.5 mmol), methanolic ammonia (3 N, 10 mL), and Raney nickel (100 mg, Raney 2800 Ni slurry in water). The flask was transferred to a Parr shaker type hydrogenation apparatus, charged with hydrogen (40 psi), and was shaken for 4 h. The catalyst was removed by filtration through Celite (washing with MeOH), and the solution was concentrated in vacuo to afford tert-butyl 4-(5-(3-amino-1-methoxy-2-methyl-1-oxopropan-2-yl)-2-methylpyrimidin-4-yl)piperidine-1-carboxylate.
Tert-butyl 4-(5-(3-amino-1-methoxy-2-methyl-1-oxopropan-2-yl)-2-methylpyrimidin-4-yl)piperidine-1-carboxylate (130 mg, 0.33 mmol), 4-chloro-2-(trifluoromethyl)pyrimidine (73 mg, 0.33 mmol) and DIEA (129 mg, 1 mmol) were mixed in MeCN (3 mL). The resulting solution was stirred at 50° C. for 12 h. The mixture was concentrated to dryness. The residue was dissolved in EtOAc, washed with NaHCO3 and brine. Concentration gave the crude product, which was purified by flash column chromatography to give F4.3.
To a solution of F4.3 (100 mg, 0.18 mmol) in MeOH (2 mL) was added 4N HCl in dioxane (0.3 mL). The resulting solution was stirred for 12 h at room temperature. EtOAc (25 mL) was added. The mixture was basified with 20% NaOH aq. solution to pH 10. The EtOAc layer was washed with brine, dried over sodium sulfate, and concentrated. The crude product was used in the next step without further purification.
Methyl 2-methyl-2-(2-methyl-4-(piperidin-4-yl)pyrimidin-5-yl)-3-(2-(trifluoromethyl)pyrimidin-4-ylamino)propanoate (28 mg, 0.13 mmol) obtained above was mixed with 2,6-difluorophenylacetic acid (28 mg, 0.16 mmol), HATU (60 mg, 0.16 mmol) and DIEA (129 mg, 1 mmol) in THF (2 mL). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by prep HPLC to give Compound F4. MS (EI) for C28H29F5N6O3. found: 593.2 (MH+). Analytical HPLC, ret. time=12.8 min, 94% purity.
To a 0° C. solution of F4.4 (20 mg, 0.033 mmol) in THF (2 mL) was added LAH (0.033 mL, 2.0 M in THF). The resulting solution was stirred at room temperature for 30 min. The reaction mixture was quenched with aq. 10% NaOH solution, and filtered through Celite. The Celite pad was washed with EtOAc. The EtOAc solution was washed with brine, dried over sodium sulfate, and concentrated. The crude product was purified by prep HPLC to give Compound F5. MS (EI) for C27H29F5N6O2. found: 565.2 (MH+). Analytical HPLC, ret. time=10.56 min, 93% purity.
To a stirred solution of E10.1 (9.0 mg, 0.024 mmol), (3,5-dimethylphenyl)methanamine (6.4 mg, 0.047 mmol) and DMF (0.5 mL) at room temperature was added DIEA (10 μL, 0.047 mmol). After stirring for 2 h at 50° C., aq. sat. NaHCO3 (1 mL) and water (1 mL) were added and the resulting solution was extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (EtOAc→EtOAc/MeOH=30:1) to give Compound F6 (8.5 mg, 75%) as a beige powder after lyophilization. 1H-NMR (400 MHz, CDCl3): δ 8.45 (s, 1H), 7.23 (m, 1H), 6.92 (m, 5H), 4.74 (d, 1H), 4.11 (d, 1H), 3.77 (s, 4H), 3.74 (s, 2H), 3.16 (m, 2H), 2.68 (s, 3H), 2.63 (td, 1H), 2.32 (s, 6H), 2.05 (qd, 1H), 1.88 (qd, 1H), 1.75 (m 2H). MS (EI) for C28H32F2N4O. found 479.1 (MH+). Analytical HPLC, ret. time=12.208 min, 97% purity.
To a stirred solution of A1.4 (530 mg, 1.31 mmol) and trimethyl(trifluoromethyl)silane (280 mg, 1.97 mmol) in toluene (5 mL) at −78° C. was added Bu4NF (65 μL, 1M in THF, 0.065 mmol). The reaction mixture was warmed slowly to −5° C. for 40 min and then 2N HCl (1.2 mL) was added. After stirring for 1 h at room temperature, EtOAc (5 mL) was added. The organic layer was separated, concentrated, dissolved in THF (5 mL), and transferred to the aq. layer. After stirring for 2 h, water (2 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=1:1-1:2) to give F7.1 (205 mg, 37%) as a white foam. MS (EI) for C20H18F5N3O2. found 428.0 (MH+).
To a stirred solution of F7.1 (10.0 mg, 0.0234 mmol) in MeOH (0.5 mL) at room temperature was added NaBH4 (2.7 mg, 0.070 mmol). After stirring for 2 h, aq. sat. NH4Cl (2 mL) was added and the resulting solution was extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=3:4) to give F7.2 (9.0 mg, 90%) as a yellow oil. MS (EI) for C20H20F5N3O2. found 430.0 (MH+).
To a stirred solution of F7.2 (20.0 mg, 0.0466 mmol) in CH2Cl2 (1 mL) at room temperature were added Et3N (20 μL, 0.14 mmol) and methanesulfonyl chloride (7 μL, 0.09 mmol) in sequence. After stirring for 20 min, aq. sat. NH4Cl (2 mL) was added and the resulting solution was extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=4:3→1:1) to give F7.3 (22 mg, 93%) as a clear oil. MS (EI) for C21H22F5N3O4S. found 507.9 (MH+).
A mixture of F7.3 (19.0 mg, 0.0374 mmol) and (3,5-dimethylphenyl)methanamine (25.0 mg, 0.187 mmol) was stirred for 1 day at room temperature. Aq. sat. NH4Cl (2 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=2:1→3:2). The isolated material was further purified by prep HPLC to give Compound F7 (8.5 mg, 42%) as a white solid after lyophilization. 1H-NMR (400 MHz, CDCl3, rotameric mixture): δ 8.81 and 8.78 (two of s, 1H), 7.24 (m, 1H), 6.91 (m, 3H), 6.82 (m, 2H), 4.67 (two of d, 1H), 4.48 (m, 1H), 4.04 (two of d, 1H), 3.82 (dd, 1H), 3.74 (s, 2H), 3.60 (dd, 1H), 3.13-2.94 (m, 1H), 2.75 (m, 1H), 2.71 (s, 3H), 2.58-2.38 (m, 1H), 2.29 (two of s, 6H), 2.12 (m, 1H), 2.07-1.71 (m, 2H), 1.62 (m, 1H), 1.44-1.28 (m, 1H). MS (EI) for C29H31F5N4O. found 547.0 (MH+). Analytical HPLC, ret. time=19.952 min (gradient 10% to 100% MeCN/H2O), 98% purity.
F1.4 (80 mg, 0.2 mmol), 2,6-difluorophenyl isocyanate (63 mg, 0.4 mmol) and Et3N (109 mg, 1 mmol) were mixed in acetone (2 mL). The solution was stirred at room temperature for 30 min and diluted with EtOAc (25 mL). The organic phase was washed with NaHCO3, brine, dried over sodium sulfate and concentrated. The crude mixture was purified by prep HPLC to give Compound F8. MS (EI) for C26H28F5N7O. found: 550.0 (MH+). Analytical HPLC, ret. time=2.98 min, 98% purity.
B3.1 (278 mg, 0.77 mmol), 3,5-dimethylbenzylamine (105 mg, 0.77 mmol) and Ti(OiPr)4 (420 mg, 1.47 mmol) were mixed in THF (2 mL). The resulting mixture was stirred at room temperature for 8 h. NaBH4 (90 mg, 2.4 mmol) and EtOH (0.5 mL) were added. The stirring was continued for additional 4 h. Aq. sat. NaHCO3 was added to quench the reaction. The mixture was extracted with EtOAc and the organic phase was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by prep HPLC to give Compound F9. 1H-NMR (400 MHz, DMSO-d6): δ 8.73 (s, 1H), 7.35 (m, 1H), 7.07 (m, 2H), 6.86 (m, 3H), 4.43 (d, 1H), 4.13 (d, 1H), 4.01 (m, 1H), 3.78 (m, 2H), 3.50 (m, 3H), 3.11 (m, 2H), 2.58 (s, 3H), 2.55 (m, 1H), 2.23 (d, 6H), 1.63 (m, 4H), 1.31 (d, 3H). MS (EI) for C29H34F2N4O. found: 493.1 (MH+). Analytical HPLC, ret. time=2.98 min, 99% purity.
A1.5 (500 mg, 1.32 mmol) was mixed with (3-chlorophenyl)hydrazine (285 mg, 1.59 mmol) and DIEA (1.2 mL) in CH2Cl2 (10 mL). To this solution was added O-(benzotriazol-1-yl)-N,N,N′,N-tetramethyluronium tetrafluoroborate (TBTU) (550 mg, 1.71 mmol). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The mixture was extracted with CH2Cl2 and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by flash column chromatography to give Compound G1. MS (EI) for C25H24ClF2N5O2. found: 500.1 (MH+). Analytical HPLC, ret. time=18.29 min, 97% purity.
Compounds G2 to G4 were prepared from methods analogous to those used to prepare Compound G1 utilizing appropriate reagent replacements.
HPLC conditions used to determine retention times: Phenomenex, Gemini, 50×4.6 mm, 5 column, gradient 10% to 90% MeCN/H2O, in the presence of 0.1% TFA, 5 min gradient time, 6 min total run time at 3.0 mL/min flow rate, λ=254 nM
To a stirred solution of A1.5 (150 mg, 0.40 mmol), 0-(3-(trifluoromethyl)phenylhydroxylamine (78 mg, 0.44 mmol) and Et3N (279 μL, 2.0 mmol) in DMF (4 mL) was added HATU (168 mg, 0.44 mmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with EtOAc, washed with aq. IN NaOH and brine, dried over sodium sulfate, and concentrated in vacuo. The residue was purified by flash chromatography to afford Compound H1 (52 mg, 24%) as a white powder after lyophilization. Analytical HPLC: retention time=14.312 min, 99%. 1H-NMR (400 MHz, CDCl3): δ 10.40 (br s, 1H), 8.68 (br s, 1H), 7.48-7.44 (m, 1H), 7.36-7.34 (m, 2H), 7.30-7.26 (m, 1H), 7.19-7.12 (m, 1H), 6.86-6.81 (m, 2H), 4.54 (d, 1H), 4.08 (d, 1H), 3.71 (s, 2H), 3.47 (br s, 1H), 3.20 (t, 1H), 2.74 (s, 3H), 2.63 (t, 1H), 2.03-1.90 (m, 2H), 1.84-1.74 (m, 2H). MS (EI) for C26H23F5N4O3. found 535.0 (MH+).
Compounds H2 to H7 were prepared from methods analogous to those used to prepare Compound H1 utilizing appropriate reagent replacements.
A108.3 (420 mg, 1 mmol) was mixed with NaOH (400 mg, 10 mmol) in MeOH/water (10 mL/5 mL). The resulting mixture was stirred at 85° C. for 2 h. The solution was concentrated, and acidified with conc. HCl. The product was precipitated, filtered, and dried.
To a mixture of I1.1 (85 mg, 0.22 mmol), HATU (107 mg, 0.28 mmol), and CH2Cl2 (1.5 mL) was added DIEA (112 mg, 0.87 mmol). The mixture was stirred at 35° C. for 30 min. To this mixture was added (6-(trifluoromethyl)pyridin-2-yl)methanamine hydrochloride (69 mg, 0.33 m mol). The resulting yellow solution was stirred at 35° C. for 14 h. After removal of solvent in vacuo, the residue was purified on prep HPLC to give 11.2 (64 mg). 1H-NMR (400 MHz, DMSO-d6): δ 12.5 (br. s. 1H), 9.22 (t, 1H), 8.10 (t, 1H), 7.82 (d, 1H), 7.78 (d, 1H), 7.33 (m, 1H), 7.05 (t, 2H), 4.57 (d, 2H), 4.40 (d, 1H), 4.10 (d, 1H), 3.74 (dd, 2H), 3.27 (m, 1H), 3.02 (t, 2H), 2.30 (s, 3H), 1.77 (m, 1H), 1.67-1.60 (m, 3H). Analytical HPLC, ret. time=13.72 min, 98% purity. MS (EI) for C26H24F5N5O3. found 550.0 (MH+).
Compound I2 was recovered as a second product from the reaction that generated Compound I1 (9 mg recovered). 1H-NMR (400 MHz, DMSO-d6): δ 9.29 (br. s. 1H), 8.13 (t, 1H), 8.02 (t, 1H), 7.81 (d, 1H), 7.76 (d, 1H), 7.74 (d, 1H), 7.62 (d, 1H), 7.42 (br. s, 1H), 7.33 (m, 1H), 7.05 (t, 2H), 4.74 (s, 2H), 4.66 (d, 2H), 4.40 (d, 1H), 4.11 (d, 1H), 3.74 (dd, 2H), 2.96 (t, 2H), 2.81 (m, 1H), 2.30 (s, 3H), 1.81 (m, 1H), 1.71-1.63 (m, 3H). Analytical HPLC, ret. time=14.38 min, 97% purity. MS (EI) for C33H29F8N7O2. found 707.9 (MH+).
I1.1 (120 mg, 0.31 mmol) was mixed with 3,5-dimethylbenzylamine (67 mg, 0.5 mmol), HATU (130 mg, 0.34 mmol), and DIEA (0.5 mL) in THF (5 mL). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The resulting mixture was extracted with CH2Cl2 and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by prep HPLC to give Compound I3. MS (EI) for C28H30ClF2N4O3. found: 509.0 (MH+). Analytical HPLC, ret. time=17.25 min, 94% purity.
Compounds 14 and 15 were prepared from methods analogous to those used to prepare Compound I3 utilizing appropriate reagent replacements.
I6.1 was synthesized from I1.1 in the same manner that B1.2 was synthesized from B1.1
Compound I6 was synthesized from I6.1 in the same manner that B30.2 was synthesized from B30.1. MS (EI) for C26H23F5N4O3. found: 534.9 (MH+). Analytical HPLC, ret. time=19.74 min, 92% purity.
To a solution of A108.2 (200 mg, 0.55 mmol) in anhydrous ethylene glycol dimethyl ether (3.6 mL) was added NaH (66 mg, 60% suspension in mineral oil, 1.65 mmol) with stirring at room temperature. The resulting suspension was stirred at 45° C. for 15 min. To this mixture was added iodomethane (311 mg, 2.19 mmol). The resulting mixture was stirred at 45° C. for 1 h. After cooling down to 0° C. in an ice bath, the reaction mixture was quenched by addition of sat. NH4Cl solution. The mixture was then diluted with EtOAc, washed with water (3×) and brine, dried over magnesium sulfate, and concentrated in vacuo, affording the crude product of I7.1 (200 mg). 1H-NMR (400 MHz, CDCl3): δ 4.39 (q, 2H), 4.19 (br. s 2H), 3.51 (s, 3H), 2.75-2.66 (m 3H), 2.51 (s, 3H), 1.84 (m, 2H), 1.64 (m, 2H), 1.49 (s, 9H), 1.35 (t, 3H). MS (EI) for C19H29N3O5. found: 380.1 (MH+).
To a mixture of the crude I7.1 (200 mg) in 1:1 MeOH/water (6 mL) was added NaOH (110 mg). The mixture was stirred at room temperature for 15 h, and then heated 50° C. for an additional 3 h. The mixture was concentrated in vacuo to remove MeOH, and then washed with hexanes (2×). The aq. layer was acidified to pH 2 with IN hydrochloric acid at 0° C., and then extracted with EtOAc (5×). The combined organic phase was washed with water and brine, dried over magnesium sulfate, and concentrated in vacuo to give the I7.2 as an off-white solid (135 mg). 1H-NMR (400 MHz, CDCl3): δ 4.35 (m, 1H), 4.21 (br.s, 2H), 3.63 (s, 3H), 2.86 (m, 2H), 2.64 (s, 3H), 1.80 (m, 2H), 1.80 (m, 2H), 1.48 (s, 9H).
To a mixture of I7.2 (135 mg, 0.38 mmol) and HATU (190 mg, 0.50 mmol) in CH2Cl2 (3.8 mL) was added DIEA (248 mg, 1.92 mmol). The resulting mixture was stirred at 30° C. for 20 min. To the above mixture was added (6-(trifluoromethyl)pyridin-2-yl)methanamine hydrochloride (114 mg, 0.54 mmol). The reaction mixture was stirred at 30° C. for 20 min. The resulting yellow solution was concentrated in vacuo to remove solvent. The residue was dissolved in EtOAc, washed sequentially with 0.2 N HCl aq. solution (2×), 0.2 N NaOH aq. solution (2×), and brine, dried over magnesium sulfate, and concentrated in vacuo to give a crude I7.3 (233 mg). 1H-NMR (400 MHz, CDCl3): δ 9.48 (t, 1H), 7.84 (t, 1H), 7.57 (d, 2H), 4.82 (d, 2H), 4.18 (br.s. 2H), 3.98 (m, 1H), 3.56 (s, 3H), 2.80 (m, 2H), 2.57 (s, 3H), 1.84 (m, 2H), 1.68 (m, 2H), 1.47 (s, 9H). The crude product was used directly for the next reaction without further purification.
To a solution of I7.3 (233 mg) in MeOH (3 mL), was added 4 M HCl in dioxane (5 mL). The mixture was stirred at room temperature for 2.5 h. After removal of solvents in vacuo, the reaction mixture was dissolved in MeCN and concentrated in vacuo. The residue was triturated with tert-butyl methyl ether and the solvent was decanted. The residue was dried under high vacuum to give I7.4 (209 mg), which was used directly for the subsequent reaction. MS (EI) for C19H22F3N5O2. found: 401.0 (MH+).
To a mixture of 2-(2,6-difluorophenyl)acetic acid (98 mg, 0.57 mmol) and HATU (188 mg, 0.49 mmol) in CH2Cl2 (2 mL) was added DIEA (100 mg). The solution was stirred at room temperature for 20 min. The resulting solution was added into the solution of I7.4 obtained in the previous step (209 mg), DIEA (130 mg) and CH2Cl2 (1 mL). The resulting solution was stirred at room temperature for 15 min. After removal of solvent in vacuo, the reaction mixture was diluted with EtOAc, washed with water (2×) and brine, and concentrated in vacuo. The residue was purified on prep. HPLC to yield Compound I7 as a solid (136 mg). 1H-NMR (400 MHz, DMSO-d6): δ 911 (t, 1H), 8.12 (t, 1H), 7.88 (d, 1H), 7.78 (d, 1H), 7.34 (m, 1H), 7.05 (t, 2H), 4.58 (d, 2H), 4.41 (d, 1H), 4.11 (d, 1H), 3.75 (dd, 2H), 3.45 (s, 3H), 3.22 (m, 1H), 3.04 (t, 1H), 2.91 (m, 1H), 2.52 (s, 3H), 1.79 (m, 1H), 1.67-1.60 (m, 3H). Analytical HPLC, ret. time=15.18 min, 98% purity. MS (EI) for C27H26F5N5O3. found: 564.0 (MH+).
Compound I8 was synthesized with the same method utilized for Compound I7. Analytical HPLC, ret. time=16.228 min. MS (EI) for C28H28F5N5O3. found: 578.0 (MH+).
A mixture of 1-(2-iodoethyl)-3,5-dimethylbenzene (300 mg, 1.15 mmol), triphenylphosphine (452 mg, 1.73 mmol) and MeCN (3 mL) was stirred for 18 h at 85° C. The resulting mixture was concentrated and purified by silica gel column chromatography (CH2Cl2/MeOH=15/1→12/1) to give J1.1 (610 mg, quant) as a white solid. MS (EI) for C28H28P+. found 395.0 (M+).
To a stirred solution of J1.1 (248 mg, 0.474 mmol) in THF (3 mL) at 0° C. was added nBuLi (0.34 mmol, 2.5M in Hexane, 0.853 mmol). After stirring for 30 min at 0° C., B30.1 (85.0 mg, 0.237 mmol) in THF (1 mL) was added at 0° C. After stirring for 80 min at room temperature, aq. sat. NH4Cl (3 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=2:1→1:1) to give J1.2 as a white solid. 1H-NMR (400 MHz, CDCl3, geometric mixture of E/Z=1.8:1, data for E reported): δ 8.54 (s, 1H), 7.23 (m, 1H), 6.91 (t, 2H), 6.90 (s, 1H), 6.86 (s, 2H), 6.52 (d, J=15.6 Hz, 1H), 6.26 (dt, J=15.6, 6.4 Hz, 1H), 4.73 (d, 1H), 4.15 (d, 1H), 3.77 (s, 2H), 3.53 (d, J=6.4 Hz, 2H), 3.25 (td, 1H), 3.10 (tt, 1H), 2.74 (m, 1H), 2.67 (s, 3H), 2.31 (s, 6H), 2.06 (m, 1H), 1.81 (m, 3H). MS (EI) for C29H31F2N3O. found 476.1 (MH+). Analytical HPLC, ret. time=21.196 min (E) and 21.468 min (Z), 99% purity.
Compound J2 was an additional isomer recovered from the reaction that generated Compound J1. 1H-NMR (400 MHz, CDCl3, geometric mixture of Z/E=9:1, data for Z selected): δ 8.42 (s, 1H), 7.23 (m, 1H), 6.90 (t, 2H), 6.85 (s, 1H), 6.75 (s, 2H), 6.50 (d, J=11.2 Hz, 1H), 6.13 (dt, J=11.2, 7.6 Hz, 1H), 4.73 (d, 1H), 4.12 (d, 1H), 3.76 (s, 2H), 3.36 (d, J=7.6 Hz, 2H), 3.20 (td, 1H), 3.07 (tt, 1H), 2.71 (s, 3H), 2.70 (m, 1H), 2.28 (s, 6H), 2.05 (qd, 1H), 1.81 (m, 3H). MS (EI) for C29H31F2N3O. found 476.1 (MH+). Analytical HPLC, ret. time=21.248 min (E) and 21.540 min (Z), 90% purity.
A mixture of olefin Compounds J1 and J2 (25.0 mg, 0.0526 mmol), Pd/C (28 mg, 10 wt. %, 0.026 mmol) and MeOH (2 mL) was stirred for 1.5 h at room temperature under a balloon of hydrogen. The mixture was filtered through Celite and the filtrate was concentrated. The residue was dissolved in CH2Cl2 (1 mL) and the resulting solution was filtered and concentrated to give J3 (18.0 mg, 72%) as a pale yellow solid after lyophilization. 1H-NMR (400 MHz, CDCl3): δ 8.34 (s, 1H), 7.23 (m, 1H), 6.91 (t, 2H), 6.88 (s, 1H), 6.82 (s, 2H), 4.72 (d, 1H), 4.09 (d, 1H), 3.75 (s, 2H), 3.10 (td, 1H), 2.83 (tt, 1H), 2.65 (s, 3H), 2.60 (m, 5H), 2.30 (s, 6H), 2.03 (qd, 1H), 1.87 (m, 3H), 1.65 (m, 2H). MS (EI) for C29H33F2N3O. found 478.1 (MH+). Analytical HPLC, ret. time=19.088 min, 97% purity.
A mixture of A1.4 (300 mg, 0.744 mmol) and NH4OH (4 mL) was stirred for 21 h at 80° C. The resulting solution was cooled to room temperature and extracted with EtOAc (5 mL, 3×3 mL). The combined organic layers were concentrated under vacuum to give K1.1 (83 mg, 30%) as a white foam. MS (EI) for C19H20F2N4O2. found 375.0 (MH+).
To a stirred solution of K1.1 (75.0 mg, 0.200 mmol), Et3N (0.14 mL, 1.0 mmol) and THF (2 mL) at 0° C. was added trifluoroacetic anhydride (210 mg, 1.00 mmol). After stirring for 10 min at room temperature, the resulting mixture was concentrated, dissolved in water (3 mL), and extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=3:2) to give K1.2 (53.0 mg, 74%) as a white solid. MS (EI) for C19H18F2N4O. found 357.0 (MH+).
A mixture of K1.2 (53.0 mg, 0.149 mmol), Pd/C (158 mg, 10 wt. %, 0.149 mmol) and NH3 in MeOH (4 mL, 2N) was stirred for 1.5 h at room temperature under a balloon of hydrogen. The mixture was filtered through Celite and the filtrate was concentrated. The residue was dissolved in EtOAc (2.7 mL) and MeOH (0.3 mL), and the resulting solution was filtered and concentrated to give K1.3 (46 mg, 86%) as a white solid. MS (EI) for C19H22F2N4O. found 361.1 (MH+).
To a stirred solution of K1.3 (12.0 mg, 0.0333 mmol), 3,5-dimethylbenzoic acid (7.3 mg, 0.049 mmol), HATU (23.2 mg, 0.0609 mmol) and DMF (0.5 mL) was added DIEA (20 μL, 0.12 mmol). After stirring for 30 min at room temperature, aq. sat. NaHCO3 (2 mL) and water (1 mL) were added and the resulting solution was extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (hexane/EtOAc=1:5→1:10) to give Compound K1 (13 mg, 79%) as a white solid after lyophilization. 1H-NMR (400 MHz, CDCl3): δ 8.51 (s, 1H), 7.34 (s, 2H), 7.22 (m, 1H), 7.16 (s, 1H), 6.90 (t, 2H), 6.27 (t, 1H), 4.72 (m, 1H), 4.69 (d, 2H), 4.10 (d, 1H), 3.75 (s, 2H), 3.25 (m, 2H), 2.71 (m, 1H), 2.70 (s, 3H), 2.35 (s, 6H), 2.05 (qd, 1H), 1.91 (qd, 1H), 1.78 (m, 1H), 1.69 (m, 1H). MS (EI) for C28H30F2N4O2. found 493.1 (MH+). Analytical HPLC, ret. time=14.908 min, 95% purity.
L1.1 was synthesized from A99.3 in an identical manner to that from which A99.4 was synthesized from A99.3.
To a solution of L1.1 (525 mg, 1.1 mmol) in 1,4-dioxane (4 mL) was added Lawesson's reagent (547 mg, 1.35 mmol). The resulting mixture was stirred at 100° C. for 5 h, and then cooled to room temperature. To this mixture was added 4 N HCl/dioxane (2 mL) and MeOH (10 mL). The stirring was continued for 2 h. Water (25 mL) was added. The aq. phase was washed with EtOAc, basified with 20% NaOH solution, and extracted with EtOAc. The organic phase was dried over sodium sulfate, filtered, and concentrated. The crude L1.2 was used as is in the next step.
L1.2 (131 mg, 0.34 mmol) was mixed with 2,6-difluorophenylacetic acid (70 mg, 0.41 mmol), HOBt (69 mg, 0.51 mmol) and DIEA (258 mg, 2 mmol) in THF (5 mL). To this solution was added EDC (98 mg, 0.51 mmol). The resulting reaction mixture was stirred at room temperature for 5 h then diluted with aq. sat. NaHCO3. The resulting mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by prep HPLC to give Compound L. 1H-NMR (400 MHz, DMSO-d6): δ 11.23 (br s, 1H), 9.13 (s, 1H), 8.61 (s, 1H), 7.70 (m, 2H), 7.45 (m, 1H), 7.35 (m, 1H), 7.07 (m, 2H), 4.98 (m, 2H), 4.45 (d, 1H), 4.15 (d, 1H), 3.83 (d, 1H), 3.75 (d, 1H), 3.05 (m, 2H), 2.46 (m, 1H), 1.70 (m, 4H). MS (EI) for C25H22Cl2F2N4OS. found: 535.1 (MH+). Analytical HPLC, ret. time=25.0 min, 93% purity.
To a solution of B3.1 (100 mg, 0.26 mmol) in AcOH (1 mL) was added 0.3 mL of 33% HBr/AcOH, followed by the addition of Br2 (0.5 mL, 0.5 M in CHCl3). The mixture was stirred at room temperature for 30 min. Aq. sat. NaHCO3 was added to quench the reaction. The mixture was extracted with CH2Cl2 and the organic phase was dried over sodium sulfate, filtered, and concentrated. Crude M1.1 was used as is in the next step.
M1.1 (0.2 mmol) was mixed with 3-chlorophenol (51 mg, 0.4 mmol) and K2CO3 (60 mg, 0.43 mmol) in acetone (3 mL). The resulting mixture was stirred at 50° C. for 12 h and then water was added. The mixture was extracted with EtOAc and the organic phase was dried over sodium sulfate, filtered, and concentrated. The crude product was purified by flash column chromatography to give Compound M1. 1H-NMR (400 MHz, DMSO-d6): δ 9.18 (s, 1H), 7.32 (m, 2H), 7.11 (m, 5H), 5.55 (s, 2H), 4.46 (d, 1H), 4.18 (d, 1H), 3.82 (d, 1H), 3.73 (d, 1H), 3.45 (m, 1H), 3.18 (m, 1H), 2.68 (s, 3H), 2.62 (m, 1H), 1.70 (m, 4H). MS (EI) for C26H24ClF2N3O3. found: 500.0 (MH+). Analytical HPLC, ret. time=3.48 min, 90% purity.
tert-Butyl 4-(3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate (40 g, 133 mmol) and dimethylformamide dimethylacetal (DMF-DMA) (19 g, 160 mmol) were dissolved in toluene (40 mL). The solution was stirred under reflux for 5 h and cooled to room temperature. The solution was concentrated and used as is in the next reaction.
To a suspension of acetamidine hydrochloride (15.1 g, 160 mmol) in EtOH (100 mL) was added EtONa (11 g, 160 mmol). The resulting mixture was stirred for 15 min at room temperature. Crude enamine intermediate CI1.1 was dissolved in EtOH (100 mL) and added to the acetamidine hydrochloride mixture. The mixture was heated to 70° C. with stirring for 2 h. The solvent was removed under reduced pressure. The residue was stirred in EtOAc (500 mL) and filtered. Concentration of the filtrate gave the crude product, which was further purified by flash column chromatography.
tert-Butyl-4-(3-ethoxy-3-oxopropanoyl)piperidine-1-carboxylate (6.0 g, 20.0 mmol), AcOH (57 μL, 1.0 mmol), trimethyl orthoacetate, (3.0 mL), pyridine (80 μL, 1.0 mmol), and toluene (100 mL) were charged into a 250 mL flask equipped with fractional distillation apparatus. The reaction mixture was stirred at 130° C. (bath temperature) and the solvent distilled at about the boiling point of the MeOH/toluene azeotrope for 48 h. The reaction mixture was cooled to room temperature and concentrated in vacuo to give CI2.1 which was used directly in the next step.
To a stirred suspension of acetamidine HCl (2.84 g, 30.0 mmol) in EtOH (60 mL) was added NaOEt (2.05 g, 30.0 mmol) and the reaction mixture was stirred at room temperature for 30 min. To this suspension was added the crude intermediate CI2.1 in EtOH (20 mL) slowly at room temperature and the resulting mixture was stirred at reflux for 5 h.
The reaction mixture was cooled down to room temperature, concentrated under reduced pressure, and the residue was partitioned between water and CH2Cl2. The separated aq. layer was extracted with CH2Cl2 (×2) and the combined organic layers were dried over sodium sulfate, concentrated in vacuo, and purified by flash chromatography to give a mixture of the methyl and ethyl esters of CI2 (2.51 g, ca. 35%).
To a stirred solution of ester 1 (5.0 mmol) in MeOH (30 mL) was added aq. IN NaOH (20 mL) and the reaction mixture was stirred at room temperature overnight. MeOH was removed in vacuo and the residual aq. layer was acidified with IN HCl to pH 4-5, and extracted with CH2Cl2 (×5). The combined organic layers were dried over sodium sulfate and concentrated in vacuo to give acid 2 (˜97% yield). Alternatively, LiOH can be substituted for NaOH using THF as the solvent instead of MeOH. Mild heating can also be used to facilitate the ester hydrolysis.
To a solution of 3 (13.7 mmol) in an appropriate solvent such as EtOH, MeOH, DCE or dioxane (20 mL) was added 4N HCl in dioxane (12 mL). The resulting solution was stirred for 12 h at room temperature. Ether (100 mL) was added with stirring. The resulting solid was filtered and dried under vacuum. The crude solid HCl salt 4 was used in the next step without further purification. If no filterable solid formed, the reaction mixture was concentrated in vacuo and the residue was used for the next step without further purification. Alternatively, trifluoroacetic acid can be used instead of 4N HCl in dioxane. The reaction mixture is concentrated in vacuo once Boc deprotection is complete and the crude TFA salt is used for the next step without further purification.
Carboxylic acid 2 (0.39 mmol) was mixed with amine 5 (0.45 mmol) and DIEA (0.5 mL) in THF (3 mL). To this solution was added 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronoium hexafluorphosphate (HATU) (0.58 mmol). The resulting reaction mixture was stirred at room temperature for 2 h then diluted with aq. sat. NaHCO3. The mixture was extracted with CH2Cl2, dried over sodium sulfate, filtered, and concentrated. The crude mixture was purified by flash chromatography or preparative HPLC to give amide 6.
A mixture of amine 4, 2,6-difluorophenylacetic acid (8.31 mmol), Et3N (34.6 mmol) and DMF (20 mL) was treated with HATU (8.31 mmol) and the resulting mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with EtOAc, washed with aq. sodium bicarbonate, brine, dried over sodium sulfate, concentrated in vacuo and the residue purified by flash chromatography to afford phenylacetamide 7 (˜81% yield).
Amine 4 (0.2 mmol), 2,6-difluorophenyl isocyanate (0.4 mmol) and Et3N (1 mmol) were mixed in an appropriate solvent such as CH2Cl2 or acetone (2 mL). The solution was stirred at room temperature for 30 min and diluted with EtOAc (25 mL). The organic phase was washed with NaHCO3, brine, dried over sodium sulfate and concentrated. The crude mixture was purified by flash chromatography or prep HPLC to give phenylurea 8.
To a 0° C. solution of 9 (0.088 mmol) in CH2Cl2 (3 mL) was added meta-chloroperoxybenzoic acid (mCPBA) (55 mg, 0.22 mmol, ˜70%). The resulting mixture was stirred for 2 h while slowly warming to room temperature. Aq. sat. NaHCO3 was added. The mixture was extracted with CH2Cl2 and the organic phase was washed with aq. Na2SO3 solution, dried over sodium sulfate, filtered, and concentrated. The resulting product can be used crude or purified by flash column chromatography.
Compound 10 (0.17 mmol) was dissolved in dioxane (3.0 mL). To this mixture was added aqueous ammonium hydroxide (3.0 mL) and the resulting mixture was stirred at room temperature. Once the reaction was complete as monitored by LC-MS, the reaction mixture was diluted with EtOAc and washed with aq. NaHCO3 (2×), water (1×), dried (Na2SO4) and concentrated in vacuo. The resulting crude residue was purified by preparative HPLC to give compound 11 (32%).
Intermediate A116.1 was made from the ester hydrolysis of Common Intermediate CI1 using General Procedure 1.
Intermediate A116.2 was made from coupling intermediate A116.1 with 3,5-dimethylbenzylamine using General Procedure 3.
Intermediate A116.3 was made from the Boc deprotection of intermediate A116.2 using General Procedure 2.
To a solution of 2-(2,6-difluorophenyl)-2,2-difluoroacetic acid (23 mg, 0.13 mmol) and oxalyl chloride (17 mg, 0.13 mmol) in DCM (1 mL) was added 1 drop of DMF with stirring at room temperature for 10 min. The resulting crude reaction solution was used as is in the next reaction.
Intermediate A116.3 (50 mg, 0.12 mmol) was added to the above solution of intermediate A116.4, followed by DIEA (63 mg, 0.49 mmol). The resulting mixture was stirred at RT for 30 min, and then quenched with ice water. The crude mixture was partitioned between EtOAc and saturated aqueous KHCO3. The organic phase was washed with brine, dried over MgSO4, and concentrated. The crude product was purified on prep HPLC to give Compound A116 as a white solid (11 mg). 1H-NMR (400 MHz, CDCl3): δ 8.57 (s, 1H), 7.45 (m, 1H), 6.98 (t, 2H), 6.95 (s, 3H), 6.11 (t, 1H), 4.66 (d, 1H), 4.55 (m, 2H), 4.25 (d, 1H), 3.46 (m, 1H), 3.12 (t, 1H), 2.82 (t, 1H), 2.71 (s, 3H), 2.31 (s, 6H), 2.00-1.80 (m, 4H). MS (EI) for C28H28F4N4O2. found: 529.2 (MH+). Analytical HPLC, ret. time=15.20 min, 98% purity.
Compound A117 was made in a manner similar to Compound A116: 1H-NMR (400 MHz, CDCl3): δ 8.55 (s, 1H), 7.58-7.56 (m, 2H), 7.49-7.47 (m, 3H), 6.94-6.93 (m, 3H), 6.13 (t, 1H), 4.71 (d, 1H), 4.52 (d, 2H), 4.01 (d, 1H), 3.39 (m, 1H), 2.92 (m, 1H), 2.76 (t, 1H), 2.69 (s, 3H), 2.29 (s, 6H), 1.96-1.82 (m, 2H), 1.71-1.61 (m, 2H). MS (EI) for C28H30F2N4O2. found: 393.2 (MH+). Analytical HPLC, ret. time=14.96 min, 99% purity.
Compound A118 was made in a manner similar to Compound A116: 1H-NMR (400 MHz, CDCl3): δ 8.56 (s, 1H), 7.58 (t, 1H), 7.49 (dd, 1H), 7.25 (m, 1H), 7.16 (t, 1H), 6.95 (s, 3H), 6.12 (t, 1H), 4.67 (d, 1H), 4.54 (m, 2H), 4.25 (d, 1H), 3.44 (m, 1H), 3.09 (m, 1H), 2.80 (t, 1H), 2.71 (s, 3H), 2.31 (s, 6H), 2.00-1.77 (m, 4H). MS (EI) for C28H29F3N4O2. found: 511.2 (MH+). Analytical HPLC, ret. time=15.10 min, 99% purity.
To a mixture of intermediate A116.3 (50 mg, 0.12 mmol), diisopropylethylamine (DIEA) (63 mg, 0.49 mmol) and anhydrous THF (1.2 mL) was added 2-chlorophenyl chloroformate (26 mg, 0.13 mmol) at RT and the mixture was stirred at RT for 16 h. The resulting mixture was diluted with EtOAc and the mixture was partitioned between EtOAc and saturated aqueous sodium carbonate solution. The organic phase was separated, washed with brine, dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified on prep. HPLC to afford Compound A119 as a while solid (29 mg). 1H-NMR (400 MHz, CDCl3): δ 8.58 (s, 1H), 7.43 (d, 1H), 7.30-7.23 (m, 2H), 7.18-7.14 (t, 1H), 6.97 (s, 3H), 6.16 (t, 1H), 4.56 (m, 2H), 4.48 (d, 1H), 4.33 (d, 1H), 3.4 (m, 1H), 3.11 (t, 1H), 2.94 (t, 1H), 2.72 (s, 3H), 2.33 (s, 6H), 2.10-1.98 (m, 2H), 1.85 (t, 2H). MS (EI) for C27H29ClN4O3. found: 493.1 (MH+). Analytical HPLC, ret. time=15.77 min, 99% purity.
Compound A120 was made in a manner similar to Compound A119: 1H-NMR (400 MHz, CDCl3): δ 8.59 (s, 1H), 7.14 (m, 1H), 7.00-6.95 (m, 5H), 6.10 (t, 1H), 4.57 (m, 2H), 4.45 (d, 1H), 4.33 (d, 1H), 3.42 (m, 1H), 3.12 (t, 1H), 2.96 (t, 1H), 2.73 (s, 3H), 2.33 (s, 6H), 2.10-2.00 (m, 2H), 1.86 (t, 2H). MS (EI) for C27H28F2N4O3. found: 495.3 (MH+). Analytical HPLC, ret. time=15.88 min, 99% purity.
Intermediate A121.1 was made from the ester hydrolysis of Common Intermediate CI2 using General Procedure 1.
Intermediate A121.2 was made from coupling intermediate A121.1 with (2-(trifluoromethyl)pyrimidin-4-yl)methanamine using General Procedure 3.
Intermediate A121.3 was made from the Boc deprotection of intermediate A121.2 using General Procedure 2.
Compound A121 was made from intermediate A121.3 and 2,6-difluorophenylacetic acid using General Procedure 4. 1H-NMR (400 MHz, CDCl3): δ 8.92 (d, 1H), 7.59 (d, 1H), 7.22 (m, 1H), 6.89 (t, 2H), 6.86 (t, 1H), 4.92 (d, 1H), 4.86 (d, 1H), 4.68 (d, 1H), 4.07 (d, 1H), 3.73 (d, 2H), 3.10 (m, 1H), 2.91 (tt, 1H), 2.69 (s, 3H), 2.58 (td, 1H), 2.50 (s, 3H), 2.04 (qd, 1H), 1.93 (qd, 1H), 1.81 (m, 2H). MS (EI) for C26H25F5N6O2. found 549.2 (MH+). Analytical HPLC, ret. time=14.252 min, 96% purity.
Compound A122 was made from intermediate A121.3 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.93 (d, 1H), 7.59 (d, 1H), 7.11 (m, 1H), 6.93 (t, 2H), 6.89 (t, 1H), 5.88 (s, 1H), 4.89 (d, 2H), 4.18 (d, 2H), 2.90 (m, 3H), 2.69 (s, 3H), 2.51 (s, 3H), 2.07 (qd, 2H), 1.82 (m, 2H). MS (EI) for C25H24F5N7O2. found 550.2 (MH+). Analytical HPLC, ret. time=12.496 min, 97% purity.
Compound A123 was made from intermediate A121.3 and 2,6-difluorophenyl chloroformate (prepared using a general method according to the literature procedure as described in J. Med. Chem. 2006, 49, 4981) using a method analogous to that used to convert intermediate A116.3 to Compound A119. 1H-NMR (400 MHz, CDCl3): δ 8.94 (d, 1H), 7.60 (d, 1H), 7.14 (m, 1H), 7.00-6.95 (m, 2H), 6.84 (t, 1H), 4.92 (dd, 2H), 4.43 (d, 1H), 4.31 (d, 1H), 3.03 (t, 1H), 2.92-2.84 (m, 2H), 2.71 (s, 3H), 2.51 (s, 3H), 2.13-2.06 (m, 2H), 1.84-1.81 (m, 2H). 1MS (EI) for C25H23F5N6O3. found: 551.2 (MH+). Analytical HPLC, ret. time=13.21 min, 99% purity.
Compound A124 was made in a manner similar to Compound A123: 1H-NMR (400 MHz, CDCl3): δ 8.94 (d, 1H), 7.60 (d, 1H), 7.22-7.10 (m, 4H), 6.84 (t, 1H), 4.91 (t, 2H), 4.42 (d, 1H), 4.33 (d, 1H), 2.99 (t, 1H), 2.92-2.81 (m, 2H), 2.71 (s, 3H), 2.51 (s, 3H), 2.08 (m, 2H), 1.81 (d, 2H). MS (EI) for C25H24F4N6O3. found: 533.2 (MH+). Analytical HPLC, ret. time=12.66 min, 99% purity.
Compound A125 was made in a manner similar to Compound A123: 1H-NMR (400 MHz, CDCl3): δ 8.34 (d, 1H), 7.61 (d, 1H), 7.37 (t, 2H), 7.20 (t, 1H), 7.12 (d, 2H), 4.91 (m, 2H), 4.42-4.34 (m, 2H), 2.96-2.80 (m, 3H), 2.71 (s, 3H), 2.52 (s, 3H), 2.10-2.03 (m, 2H), 1.81 (d, 2H). MS (EI) for C25H25F3N6O3. found: 515.2 (MH+). Analytical HPLC, ret. time=12.34 min, 99% purity.
Compound A126 was made in five steps from intermediate C48.2 in the same manner that Compound A123 was made in three steps from intermediate A121.1. 2-Fluorophenyl chloroformate was substituted for 2,6-difluorophenyl chloroformate in the third step and the additional steps of methyl sulfide oxidation (as outlined in General Procedure 6) and conversion of the resulting 2-(methylsulfonyl)pyrimidine to the corresponding 2-aminopyrimidine (as outlined in General Procedure 7) were added to the reaction sequence. 1H-NMR (400 MHz, CDCl3): δ 8.92 (d, 1H), 7.59 (d, 1H), 7.15 (m, 4H), 8.86 (t, 1H), 5.13 (s, 2H), 4.86 (dd, 2H), 4.39 (d, 1H), 4.29 (d, 1H), 2.97 (t, 1H), 2.82 (m, 2H), 2.38 (s, 3H), 1.98 (m, 2H), 1.80 (m, 2H). MS (EI) for C24H23F4N7O3. found 534.2 (MH+). Analytical HPLC, ret. time=10.616 min, 99% purity.
To a stirred mixture of intermediate acid A116.1 (3.07 g, 9.55 mmol), N,O-dimethylhydroxylamine hydrogenchloride (1.12 g, 11.5 mmol), and Et3N (5.32 mL, 38.2 mmol) in dimethylacetamide (DMA) (30 mL) was added HATU (4.36 g, 11.5 mmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with EtOAc, washed with aq. sodium bicarbonate, brine, dried over sodium sulfate and concentrated in vacuo to give the corresponding Weinreb amide, which was directly used for the next step. The crude Weinreb amide was dissolved in THF (30 mL) and the resulting solution was treated with MeMgBr (27.3 mL, 1.4 M in THF/toluene, 38.2 mmol) at room temperature. After stirring for 2 h, the reaction mixture was cooled to 0° C., quenched with aq. NH4Cl and extracted with EtOAc (×2). The combined organic layers were dried over sodium sulfate, concentrated in vacuo, and the residue was purified by flash chromatography to give ketone intermediate B36.1 (2.76 g, 90% in 2 steps).
To a stirred solution of B36.1 (53.0 mg, 0.166 mmol) in ethanol (1 mL) were added hydroxylamine.HCl (34.6 mg, 0.498 mmol) and a catalytic amount of cone HCl. After stirring for 23 h at room temperature, aqueous saturated NaHCO3 (2 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=3:2→2:3) to give B36.2 (29 mg, 52%) as a white foam. MS (EI) for C17H26N4O3. found 335.2 (MH+).
To a stirred solution of B36.2 (36.6 mg, 0.109 mmol) and 4-chloro-2-(trifluoromethyl)pyrimidine (59.7 mg, 0.327 mmol) in DMA (0.6 mL) at room temperature was added potassium carbonate (60.3 mg, 0.436 mmol). After stirring for 5 h, the mixture was filtered through Celite. The filtrate was concentrated and purified by silica gel column chromatography (Hexane/EtOAc=3:2→4:3) to give B36.3 (50 mg, 95%) as a white foam. MS (EI) for C22H27F3N6O3. found 481.2 (MH+).
Intermediate B36.4 was made from the Boc deprotection of intermediate B36.3 using General Procedure 2.
Compound B36 was made from intermediate B36.4 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.79 (d, 1H), 8.57 (s, 1H), 7.41 (d, 1H), 7.12 (m, 1H), 6.94 (t, 2H), 5.90 (s, 1H), 4.26 (d, 2H), 3.13 (tt, 1H), 3.01 (td, 2H), 2.78 (s, 3H), 2.59 (s, 3H), 2.15 (qd, 2H), 1.85 (m, 2H). MS (EI) for C24H22F5N7O2. found 536.2 (MH+). Analytical HPLC, ret. time=17.128 min, 99% purity.
Compound B37 was made in a manner similar to Compound B36: 1H-NMR (400 MHz, CDCl3): δ 8.62 (d, 1H), 8.57 (s, 1H), 7.54 (d, 1H), 7.29 (dd, 1H), 7.12 (m, 1H), 6.94 (t, 2H), 5.89 (s, 1H), 4.26 (d, 2H), 3.15 (tt, 1H), 3.00 (td, 2H), 2.77 (s, 3H), 2.52 (s, 3H), 4.16 (qd, 2H), 1.85 (m, 2H). MS (EI) for C25H23F5N6O2. found 535.1 (MH+). Analytical HPLC, ret. time=17.632 min, 95% purity.
Compound B38 was made in a manner similar to Compound B36: 1H-NMR (400 MHz, CDCl3): δ 8.57 (s, 1H), 7.90 (t, 1H), 7.45 (t, 2H), 7.11 (m, 1H), 6.93 (t, 2H), 5.88 (s, 1H), 4.24 (d, 2H), 3.23 (tt, 1H), 3.00 (td, 2H), 2.76 (s, 3H), 2.56 (s, 3H), 2.13 (qd, 2H), 1.86 (m, 2H). MS (EI) for C25H23F5N6O2. found 535.1 (MH+). Analytical HPLC, ret. time=18.228 min, 97% purity.
Compound B39 was made in a manner similar to Compound B36: 1H-NMR (400 MHz, CDCl3): δ 8.79 (d, 1H), 8.57 (s, 1H), 7.45 (m, 1H), 7.40 (d, 1H), 7.33 (m, 2H), 6.08 (s, 1H), 4.20 (d, 2H), 3.15 (tt, 1H), 3.02 (td, 2H), 2.77 (s, 3H), 2.58 (s, 3H), 2.15 (qd, 2H), 1.85 (m, 2H). MS (EI) for C25H22F7N7O2. found 586.1 (MH+). Analytical HPLC, ret. time=18.352 min, 98% purity.
Compound B40 was made in a manner similar to Compound B36: 1H-NMR (400 MHz, CDCl3): δ 8.56 (s, 1H), 7.44 (d, 1H), 7.36 (d, 1H), 7.12 (m, 1H), 6.94 (t, 2H), 5.90 (s, 1H), 4.26 (d, 2H), 3.11 (m, 1H), 3.01 (t, 2H), 2.77 (s, 3H), 2.51 (s, 3H), 2.21-2.09 (m, 2H), 1.84 (d, 2H). MS (EI) for C25H22ClF5N6O2. found: 569.2 (MH+). Analytical HPLC, ret. time=16.3 min, 97% purity.
Z isomer isolated from the same reaction that formed Compound B40. 1H-NMR (400 MHz, CDCl3): δ 8.54 (s, 1H), 7.83 (d, 1H), 7.34 (d, 1H), 7.03 (m, 1H), 6.92-6.82 (m, 2H), 5.82 (s, 1H), 4.14 (d, 2H), 3.42 (m, 1H), 2.67 (s, 3H), 2.06-1.98 (m, 2H), 1.91-1.88 (m, 2H). MS (EI) for C25H22ClF5N6O2. found: 569.2 (MH+). Analytical HPLC, ret. time=15.5 min, 86% purity.
Compound B42 was made in a manner similar to Compound B36: 1H-NMR (400 MHz, CDCl3): δ 8.49 (s, 1H), 7.17 (s, 1H), 7.05 (m, 1H), 6.86 (t, 2H), 5.82 (s, 1H), 4.18 (d, 2H), 3.04 (m, 1H), 2.93 (dd, 2H), 2.69 (s, 3H), 2.54 (s, 3H), 2.49 (s, 3H), 2.12-2.02 (m, 2H), 1.77 (d, 2H). MS (EI) for C25H24F5N7O2. found: 550.2 (MH+). Analytical HPLC, ret. time=13.97 min, 98% purity.
To a stirred solution of B36.1 (63.9 mg, 0.200 mmol) and O-(3-(trifluoromethyl)phenyl)hydroxylamine (53.1 mg, 0.300 mmol) in ethanol (2 mL) at room temperature was added a catalytic amount of cone HCl. After stirring for 1.5 h at 50° C., aqueous saturated NaHCO3 (2 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/DCM/EtOAc=3:1:1) to give B8.1 (45 mg, a mixture of E/Z=8.2:1, 47%) as a clear oil.
Intermediate B43.2 was made from the Boc deprotection of intermediate B43.1 using General Procedure 2.
Compound B43 was made from intermediate B43.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.58 (s, 1H), 7.49 (m, 1H), 7.46 (m, 1H), 7.37 (m, 1H), 7.33 (m, 1H), 7.11 (m, 1H), 6.93 (t, 3H), 5.89 (s, 1H), 4.25 (d, 2H), 3.25 (tt, 1H), 3.00 (td, 2H), 2.76 (s, 3H), 2.49 (s, 3H), 2.14 (qd, 2H), 1.87 (m 2H). MS (EI) for C26H24F5N5O2. found 534.2 (MH+). Analytical HPLC, ret. time=20.780 min, 99% purity.
Z isomer isolated from the same reaction that formed Compound B43. 1H-NMR (400 MHz, CDCl3): δ 8.39 (s, 1H), 7.40 (m, 2H), 7.29 (d, 1H), 7.22 (d, 1H), 7.11 (m, 1H), 6.93 (t, 2H), 5.86 (s, 1H), 4.19 (d, 2H), 2.90 (t, 2H), 2.77 (s, 3H), 2.72 (tt, 1H), 2.38 (s, 3H), 2.10 (qd, 2H), 1.73 (m, 2H). MS (EI) for C26H24F5N5O2. found 534.2 (MH+). Analytical HPLC, ret. time=20.168 min, 79% purity (contaminated with B43, Analytical HPLC, ret. time=20.772 min, 21%).
Compound B45 was made in a manner similar to Compound B43: 1H-NMR (400 MHz, CDCl3): δ 8.58 (s, 1H), 7.46 (m, 3H), 7.34 (m, 4H), 6.09 (s, 1H), 4.20 (d, 2H), 3.26 (tt, 1H), 3.01 (td, 2H), 2.76 (s, 3H), 2.49 (s, 3H), 2.14 (qd, 2H), 1.87 (m, 2H). MS (EI) for C27H24F7N5O2. found 584.2 (MH+). Analytical HPLC, ret. time=21.900 min, 99% purity.
To a stirred solution of Common Intermediate CI2 (140 mg, 0.400 mmol) in THF (3 mL) at 0° C. was added LiAlH4 (0.32 mL, 1M in THF, 0.32 mmol). After stirring for 1 h at room temperature, 3% NaOH (60 mg) was added at 0° C. with vigorous stirring. The resulting mixture was stirred for 1 h at room temperature and EtOAc (2 mL) was added. The resulting mixture was filtered through Celite and concentrated to give crude B46.1 as a pale yellow foam.
To a stirred solution of crude B46.1 in dichloromethane (3 mL) at room temperature was added Dess-Martin periodinane (339 mg, 0.800 mmol). After stirring for 1 h, water (3 mL) was added and the resulting solution was filtered through Celite. Dichloromethane was removed in vacuo and the resulting mixture was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=2:1) to give B46.2 (80 mg, 63%, 2 steps) as a pale yellow oil.
To a stirred solution of B46.2 (31.9 mg, 0.100 mmol), O-(3-(trifluoromethyl)phenyl)hydroxylamine (35.4 mg, 0.200 mmol) and ethanol (1 mL) at room temperature was added a catalytic amount of cone HCl. After stirring for 1 h at room temperature, aqueous saturated NaHCO3 (3 mL) was added and the resulting solution was extracted with EtOAc (2×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=3:1→5:2) to give B46.3 (31 mg, 65%) as a clear oil.
Intermediate B46.4 was made from the Boc deprotection of intermediate B46.3 using General Procedure 2.
Compound B46 was made from intermediate B46.4 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.77 (s, 1H), 7.48 (m, 2H), 7.36 (m, 2H), 7.12 (m, 1H), 6.94 (t, 2H), 5.89 (s, 1H), 4.26 (d, 2H), 3.40 (tt, 1H), 3.06 (td, 2H), 2.71 (s, 3H), 2.66 (s, 3H), 2.12 (qd, 2H), 1.88 (m, 2H). MS (EI) for C26H24F5N5O2. found 534.1 (MH+). Analytical HPLC, ret. time=20.524 min, 90% purity.
To a stirred solution of ethyl 2-methyl-4-(2-methylpiperidin-4-yl)pyrimidine-5-carboxylate (100 mg, 0.380 mmol), 1,4-dioxane (2 mL) and H2O (0.5 mL) were added DIEA (0.20 mL, 1.1 mmol) and di-t-butyl dicarbonate (124 mg, 0.570 mmol) in sequence. After stirring for 1 h at room temperature, aqueous saturated NH4Cl (3 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=4:1→3:1) to give intermediate B47.1 (52 mg, one set of two enantiomers, 38%) as a clear oil.
Intermediate B47.2 was synthesized from intermediate B47.1 using a method analogous to the method used to synthesize intermediate B46.1 from Common Intermediate CI2.
Intermediate B47.3 was synthesized from intermediate B47.2 using a method analogous to the method used to synthesize intermediate B47.2 from intermediate B47.1.
To a stirred solution of intermediate B47.3 (34.0 mg, 0.106 mmol) in THF (1 mL) at 0° C. was added methylmagnesium bromide (0.15 mL, 1.4M in toluene/THF (3:1), 0.21 mmol). After stirring for 1 h at room temperature, aqueous saturated NH4Cl (2 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were dried over MgSO4 and concentrated to give crude intermediate B47.4.
Intermediate B47.5 was synthesized from intermediate B47.4 using a method analogous to the method used to synthesize intermediate B46.2 from intermediate B46.1.
Intermediate B47.6 was synthesized from intermediate B47.5 using a method analogous to the method used to synthesize intermediate B43.1 from intermediate B36.1.
Intermediate B47.7 was made from the Boc deprotection of intermediate B47.6 using General Procedure 2.
Compound B47 was synthesized from intermediate B47.7 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.55 (s, 1H), 7.48 (m, 1H), 7.41 (m, 1H), 7.35 (m, 1H), 7.27 (m, 1H), 7.09 (m, 1H), 6.89 (t, 2H), 5.74 (s, 1H), 3.99 (m, 2H), 3.40 (ddd, 1H), 3.27 (m, 1H), 2.76 (s, 3H), 2.46 (s, 3H), 2.13 (m, 2H), 2.01 (m, 2H), 1.34 (d, 3H). MS (EI) for C27H26F5N5O2. found 548.2 (MH+). Analytical HPLC, ret. time=21.292 min, 90% purity.
To a solution of intermediate B46.2 (1.03 g, 3.2 mmol) in dry THF (30 mL) was added vinyl magnesium chloride (5.0 mL, 1.6M in THF, 8 mmol). The resulting mixture was stirred at room temperature and monitored by LC-MS until complete. Upon completion, the reaction mixture was diluted with saturated aqueous NH4Cl and extracted with EtOAc (3×). The combined organic extractions were washed with saturated aqueous NaCl (1×), dried (Na2SO4) and concentrated in vacuo to give intermediate B48.1 which was used as is in subsequent reactions. 1H-NMR (400 MHz, CDCl3): δ 6.12 (m, 1H), 5.70 (m, 1H), 5.22 (m, 2H), 4.22 (m, 2H), 3.24 (m, 1H), 2.74 (m, 2H), 2.62 (s, 3H), 2.55 (s, 3H), 2.15 (br s, 1H), 1.90 (m, 2H), 1.60 (m, 2H), 1.47 (s, 9H). MS (EI) for C19H29N3O3. found: 348.3 (MH+).
Intermediate B48.2 was synthesized from intermediate B48.1 using a method analogous to the method used to synthesize intermediate B46.2 from intermediate B46.1. Crude material was purified by flash chromatography to give intermediate B48.2 (959 mg, 90% yield).
To a solution of intermediate B48.2 (959 mg, 2.8 mmol) in dry THF (30 mL) was added MeMgBr (5.5 mL, 1.4M in 1:3 THF:Toluene, 7.7 mmol). The resulting mixture was stirred at room temperature for 1 hour, at which time, it was diluted with saturated aqueous NH4Cl and extracted with EtOAc (3×). The combined organic extractions were washed with saturated aqueous NaCl (1×), dried (Na2SO4) and concentrated in vacuo. The resulting residue was purified by flash chromatography (18% EtOAc, 80% heptanes, 2% 7N NH3 in MeOH) to give intermediate B48.3 (643 mg, 64% yield). 1H-NMR (400 MHz, CDCl3): δ 4.22 (m, 2H), 2.71 (m, 4H), 2.66 (s, 3H), 2.50 (m, 1H), 2.38 (s, 3H), 1.92 (m, 2H), 1.78 (m, 2H), 1.65 (m, 2H), 1.47 (s, 9H) 1.02 (t, 3H). MS (EI) for C20H31N3O3. found: 362.3 (MH+).
Intermediate B48.4 was made from the Boc deprotection of intermediate B48.3 using General Procedure 2.
Intermediate B48.5 was made from intermediate B48.4 and 2,6-difluorophenylacetic acid using General Procedure 4.
Compound B48 was synthesized from intermediate B48.5 using a method analogous to the method used to synthesize intermediate B43.1 from intermediate B36.1. H-NMR (400 MHz, CDCl3): δ 7.43 (m, 2H), 7.37-7.17 (m, 3H), 6.88 (m, 2H), 4.75 (m, 1H), 4.10 (m, 1H), 3.73 (m, 2H), 3.14 (m, 1H), 2.85 (m, 2H), 2.70 (s, 3H), 2.68-2.48 (comp m, 2H), 2.47 (s, 3H), 1.75 (m, 2H), 1.50 (m, 4H), 1.03 (t, 3H). MS (EI) for C30H31F5N4O2. found: 575.3 (MH+). Analytical HPLC, ret. time=25.00 min, 80% purity.
Z isomer isolated from the same reaction that formed Compound B48. 1H-NMR (400 MHz, CDCl3): δ 7.37 (m, 2H), 7.30-7.15 (m, 3H), 6.88 (m, 2H), 4.70 (dd, 1H), 4.07 (dd, 1H), 3.72 (m, 2H), 3.12 (dt, 1H), 2.71 (s, 3H), 2.65 (m, 2H), 2.56 (m, 2H), 2.38 (s, 3H), 2.15 (m, (1H), 1.90 (m, 1H), 1.73 (m, 4H), 1.09 (t, 3H). MS (EI) for C30H31F5N4O2. found: 575.3 (MH+). Analytical HPLC, ret. time=24.74 min, 98% purity.
To a suspension of S-methyl thiourea sulfate (5.5 g, 39 mmol) in EtOH (50 mL) was added EtONa (2.7 g, 39 mmol). The resulting mixture was stirred for 15 min at room temperature. Common Intermediate CI1.1 in EtOH (30 mL) was added. The mixture was heated at 70° C. with stirring for 2 h. The solvent was removed under reduced pressure. The residue was stirred in EtOAc (500 mL) and filtered. Concentration of the filtrate gave the crude product, which was further purified by flash column chromatography.
Intermediate B50.2 was made from the ester hydrolysis of intermediate B50.1 using General Procedure 1.
Intermediate B50.3 was synthesized from intermediate B50.2 using a method analogous to the method used to synthesize intermediate B36.1 from intermediate A116.1.
Intermediate B50.4 was made from the Boc deprotection of intermediate B50.3 using General Procedure 2.
Intermediate B50.5 was made from intermediate B50.4 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Compound B50 was made from intermediate B50.5 using a method analogous to the method used to synthesize intermediate B43.1 from intermediate B116.1. 1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H), 7.51-7.43 (m, 2H), 7.39-7.30 (m, 2H), 7.17-7.06 (m, 1H), 6.94 (t, 2H), 5.89 (s, 1H), 4.24 (d, 2H), 3.29 (t, 1H), 2.99 (t, 2H), 2.61 (s, 3H), 2.48 (s, 3H), 2.12 (td, 2H), 1.90 (d, 2H). MS (EI) for C26H24F5N5O2S. found: 566.2 (MH+). Analytical HPLC, ret. time=23.54 min, 98% purity.
Intermediate B51.1 was synthesized from Compound B50 using General Procedure 6.
Compound B51 was synthesized from intermediate B51.1 using General Procedure 7. 1H NMR (400 MHz, CDCl3) δ 8.28 (s, 1H), 7.46 (dd, 2H), 7.33 (dd, 2H), 7.11 (t, 1H), 6.94 (t, 2H), 5.89 (s, 1H), 5.19 (s, 2H), 4.23 (d, 2H), 3.25 (s, 1H), 2.97 (t, 2H), 2.44 (s, 3H), 2.05 (dd, 2H), 1.86 (dd, 2H). MS (EI) for C25H23F5N6O2. found: 535.2 (MH+). Analytical HPLC, ret. time=17.42 min, 98% purity.
Intermediate B52.1 was made from intermediate B50.5 using a method analogous to the method used to synthesize intermediate B116.2 from intermediate B116.1.
To a solution of intermediate B52.1 in diemthylacetamide (2 mL) was added sodium hydride (40 mg, 1.20 mmol, 60% in mineral oil) at 0° C. The reaction mixture was allowed to warm up to room temperature for 15 min. To this heterogeneous mixture was added 4-chloro-2-(trifluoromethyl)pyridine (119 mg, 0.66 mmol) and stirred for 1 h. The reaction mixture was quenched with water at 0° C. and partitioned with EtOAc. The organic layer was washed with brine, dried over MgSO4 and concentrated. The resulting crude material was purified by flash column chromatography to give a (E/Z) mixture of Compound B52 (210 mg, 68%, E/Z=4.5:1). 1H NMR (400 MHz, CDCl3) δ 8.63 (d, 1H), 8.47 (s, 1H), 7.54 (t, 1H), 7.32-7.26 (m, 1H), 7.17-7.07 (m, 1H), 6.99-6.90 (m, 2H), 5.91 (s, 1H), 4.26 (d, 2H), 3.20 (dd, 1H), 3.06-2.94 (dt, 2H), 2.62 (s, 3H), 2.51 (s, 3H), 2.21-2.07 (m, 2H), 1.88 (m, 2H). MS (EI) for C25H23F5N6O2S. found: 567.2 (MH+). Analytical HPLC, ret. time=20.80 min, 99% purity.
Intermediate B53.1 was synthesized from Compound B52 using General Procedure 6.
Compound B53 was synthesized from intermediate B53.1 using General Procedure 7. 1H NMR (400 MHz, CDCl3) δ 8.61 (d, 1H), 8.27 (s, 1H), 7.54 (d, 1H), 7.28 (dd, 1H), 7.12 (tt, 1H), 6.99-6.87 (m, 2H), 5.95 (s, 1H), 5.37 (s, 2H), 4.24 (dd, 2H), 3.16 (tt, 1H), 3.04-2.89 (m, 2H), 2.47 (s, 3H), 2.06 (m, 2H), 1.84 (d, 2H). MS (EI) for C24H22F5N7O2. found: 373.2 (not shown MH+). Analytical HPLC, ret. time=11.66 min, 99% purity.
Compound B54 was made in a manner similar to Compounds B52 and B53: 1H NMR (400 MHz, CDCl3) δ 8.78 (d, 1H), 8.27 (s, 1H), 7.41 (d, 1H), 7.17-7.07 (m, 1H), 6.98-6.87 (m, 2H), 5.91 (s, 1H), 5.32 (d, 2H), 4.24 (d, 2H), 3.14 (m, 1H), 2.98 (dd, 2H), 2.54 (s, 3H), 2.10-1.97 (m, 2H), 1.93-1.77 (m, 2H). MS (EI) for C23H21F5N8O2. found: 537.2 (MH+). Analytical HPLC, ret. time=11.24 min, 99% purity.
Compound B55 was made in a manner similar to Compounds B52 and B53: 1H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 7.44 (d, 1H), 7.36 (d, 1H), 7.19-7.05 (m, 1H), 6.94 (t, 2H), 5.95 (s, 1H), 5.47 (d, 2H), 4.25 (d, 2H), 3.21-3.06 (m, 1H), 2.96 (dt, 2H), 2.48 (d, 3H), 2.14-1.97 (m, 2H), 1.83 (d, 2H). MS (EI) for C24H21ClF5N7O2. found: 570.1 (MH+). Analytical HPLC, ret. time=13.94 min, 99% purity.
Compound B56 was made in a manner similar to Compounds B52 and B53: 1H NMR (400 MHz, CDCl3) δ 8.26 (s, 1H), 7.18-7.07 (m, 1H), 6.94 (t, 2H), 6.63 (s, 2H), 5.91 (s, 1H), 5.25 (s, 2H), 4.25 (d, 2H), 3.12 (dd, 1H), 2.99 (t, 2H), 2.46 (d, 3H), 2.06 (ddd, 2H), 1.83 (d, 2H). MS (EI) for C23H21F4N7O2. found: 504.2 (MH+). Analytical HPLC, ret. time=11.12 min, 99% purity.
Z isomer isolated from the same reaction that formed Compound B56. 1H NMR (400 MHz, CDCl3) δ 8.28 (bs, 1H), 7.19-7.05 (m, 1H), 6.94 (t, 2H), 6.82 (dd, 1H), 6.40 (d, 1H), 5.90 (s, 1H), 5.23 (s, 2H), 4.24 (d, 2H), 3.20 (t, 1H), 3.00 (t, 2H), 2.47 (s, 3H), 2.11-1.96 (m, 2H), 1.84 (m, 2H). MS (EI) for C23H21F4N7O2. found: 504.2 (MH+). Analytical HPLC, ret. time=11.04 min, 99% purity.
To a solution of N-hydroxyphthalimide (1.34 g, 8.21 mmol), copper acetate (1.49 g, 8.21 mmol), 2-chloro-4-pyridnylboronic acid (2.58 g, 16.4 mmol) in dichloroethane (30 mL) was added pyridine (0.73 mL, 9.035 mmol) at room temperature. The resulting mixture was stirred for 18 h and diluted with DCM. The precipitate was filtered over Celite, and the filtrate concentrated. The residue was purified by flash column chromatography to give intermediate B58.1 (538 mg, 23.9%); 1H NMR (400 MHz, CDCl3-d) δ 8.34 (d, 1H), 8.02-7.94 (m, 2H), 7.93-7.85 (m, 2H), 7.07 (d, 1H), 7.03 (dd, 1H).
A mixture of intermediate B58.1 (520 mg, 1.89 mmol) and hydrazine hydrate (0.28 mL, 5.68 mmol) was stirred in MeOH-DCM (1:1) for 18 h. The reaction mixture was diluted with DCM and partitioned with saturated aqueous NaHCO3. The resulting solution was extracted with DCM (3×50 mL). The combined organic layers were washed with brine, dried over MgSO4, concentrated in vacuo and purified by flash column chromatography to give intermediate B58.2 (66 mg, 23.8%); MS (EI) for C5H5ClN2O. found 144.01 (MH+).
Intermediate B58.3 was synthesized from intermediate B50.3 and intermediate B58.2 using a method analogous to the method that was used to synthesize intermediate B43.1 from intermediate B36.1.
Intermediate B58.4 was made from the Boc deprotection of intermediate B58.3 using General Procedure 2.
Compound B58 was made from intermediate B58.4 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, CDCl3) δ 8.46 (s, 1H), 8.29 (d, 1H), 7.21 (d, 1H), 7.12 (d, 1H), 7.04 (dd, 1H), 6.94 (t, 2H), 5.91 (s, 1H), 4.27 (d, 2H), 3.21 (t, 1H), 3.02 (t, 2H), 2.62 (s, 3H), 2.48 (s, 3H), 2.21-2.06 (m, 2H), 1.88 (m, 2H). MS (EI) for C24H23ClF2N6O2S. found: 533.2 (MH+). Analytical HPLC, ret. time=19.62 min, 98% purity.
Intermediate B59.1 was synthesized from Compound B58 using General Procedure 6.
Compound B59 was synthesized from intermediate B59.1 using General Procedure 7. 1H NMR (400 MHz, CDCl3) δ 8.27 (d, 2H), 7.21 (d, 1H), 7.11 (dd, 1H), 7.03 (dd, 1H), 6.94 (t, 2H), 5.90 (s, 1H), 5.29 (s, 2H), 4.25 (d, 2H), 3.17 (t, 1H), 3.00 (t, 2H), 2.44 (s, 3H), 2.14-1.98 (m, 2H), 1.85 (dd, 2H). MS (EI) for C23H22ClF2N7O2. found: 502.2 (MH+). Analytical HPLC, ret. time=13.48 min, 99% purity.
To a solution of intermediate B53.1 (a mixture of E and Z isomers) (70 mg, 0.12 mmol) in dioxane (2 mL) was added ethylamine in THF (0.18 mL, 0.35 mmol) at room temperature and the resulting mixture was stirred for 2 h. The reaction mixture was then concentrated in vacuo and purified by preparative HPLC to give Compound B60 (20.7 mg, 31.4%). 1H NMR (400 MHz, CDCl3) δ 8.61 (d, 1H), 8.21 (bs, 1H), 7.54 (d, 1H), 7.31-7.27 (m, 1H), 7.17-7.06 (m, 1H), 6.99-6.87 (m, 2H), 6.34 (bs, 1H), 5.99 (s, 1H), 4.24 (d, 2H), 3.57-3.44 (m, 2H), 3.20 (t, 1H), 2.98 (t, 2H), 2.46 (s, 3H), 2.17-1.98 (m, 2H), 1.86 (d, 2H), 1.27 (t, 3H). MS (EI) for C26H26F5N7O2. found: 564.2 (MH+). Analytical HPLC, ret. time=18.62 min, 96% purity.
Compound B61 was made in a manner similar to Compound B60: 1H NMR (400 MHz, CDCl3) δ 8.60 (d, 1H), 8.31 (s, 1H), 7.54 (t, 1H), 7.30-7.26 (m, 1H), 7.17-7.07 (m, 1H), 6.94 (t, 2H), 5.91 (s, 1H), 4.23 (d, 2H), 3.23 (m, 2H, 6H), 3.00 (t, 2H), 2.46 (s, 3H), 2.10 (qd, 2H), 1.88 (d, 2H). MS (EI) for C26H26F5N7O2. found: 564.2 (MH+). Analytical HPLC, ret. time=19.33 min, 98% purity.
To a solution of methyl 2-(trifluoromethyl)pyrimidine-4-carboxylate (1.50 g, 7.28 mmol) in 10 mL of anhydrous MeOH was added hydrazine monohydrate (1.09 g, 21.83 mmol). The resulting solution was heated at 50° C. with stirring for 5 min, at which time the reaction was completed. The reaction mixture, as a yellow solution, was reduced to ⅓ of the volume in vacuo. The precipitate thus formed was filtered, washed with MeOH, and dried to give 1.16 g of intermediate C40.1 as a yellow crystalline solid. The combined filtrate was concentrated under high vacuum to afford an additional 0.20 g of the hydrazide product as a yellow solid. 1H-NMR (400 MHz, CDCl3): δ 9.14 (d, 1H), 8.94 (br. s, 1H), 8.27 (d, 1H), 4.16 (d, 2H).
A solution of intermediate A121.1 (500 mg, 1.49 mmol), EDC (329 mg, 1.71 mmol), and HOBt (232 mg, 1.71 mmol) in DMA (10 mL) was stirred at 25° C. for 1.5 h. To this mixture was added intermediate C40.1 (344 mg, 1.67 mmol). The resulting mixture was stirred at 40° C. for 17 h. After cooling to room temperature, the reaction mixture was diluted with EtOAc, washed with water (3×) and brine, dried (magnesium sulfate), and concentrated in vacuo to dryness, yielding a solid (793 mg). LC/MS analysis of the resulting residue showed the presence of intermediate C40.2 as the major product. MS (EI) for C23H28F3N7O4. found 524.2 (MH+). The crude product was used directly in the next reaction.
The entire amount (793 mg) of crude product obtained from the previous step (intermediate C40.2) was dissolved in 30 mL of anhydrous MeCN. To this solution was added K2CO3 (850 mg, 4.45 mmol), followed by p-toluenesulfonyl chloride (1236 mg, 4.94 mmol). The mixture was stirred at 50° C. for 1 h. After removal of solvent in vacuo, the crude mixture was diluted with EtOAc, washed with water (3×) and brine, dried (magnesium sulfate), and concentrated in vacuo to dryness, affording intermediate C40.3 as a solid (761 mg). MS (EI) for C23H26F3N7O3. found 506.2 (MH+). The crude product was used directly for the subsequent reaction.
Intermediate C40.4 was made from the Boc deprotection of intermediate C40.3 using General Procedure 2.
Compound C40 was made from intermediate C40.4 and 1-fluoro-2-isocyanato-3-(trifluoromethyl)benzene using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 9.22 (d, 1H), 8.48 (d, 1H), 7.44 (m, 1H), 7.34 (m, 2H), 6.08 (s, 1H), 4.18 (d, 2H), 2.97 (m, 3H), 2.79 (s, 3H), 2.60 (s, 3H), 2.12 (qd, 2H), 1.91 (m, 2H). MS (EI) for C26H21F7NO2. found 611.2 (MH+). Analytical HPLC, ret. time=17.144 min, 99% purity.
Compound C41 was made from intermediate C40.3 and 2-chlorophenylacetic acid using General Procedure 4. 1H-NMR (400 MHz, CDCl3): δ 9.21 (d, H), 8.46 (d, 1H), 7.38 (dd, 1H), 7.32 (dd, 1H), 7.26-7.19 (m, 2H), 4.77 (d, 1H), 3.85 (s, 2H), 3.04 (m, 1H), 2.94 (m, 1H), 2.76 (s, 3H), 2.60 (m, 1H), 2.58 (s, 3H), 2.02-1.91 (m, 2H), 1.86-1.83 (m, 2H). MS (EI) for C26H23ClF3N7O2. found: 558.2 (MH+). Analytical HPLC, ret. time=19.3 min, 98% purity.
Compound C42 was made in a manner similar to Compound C41: 1H-NMR (400 MHz, CDCl3): δ 9.21 (d, 1H), 8.47-8.45 (m, 2H), 7.68 (dd, 1H), 7.17 (dd, 1H), 4.76 (d, 1H), 4.08 (dd, 2H), 3.99 (d, 1H), 3.10 (m, H), 2.96 (m, 1H), 2.77 (s, 3H), 2.61 (m, 1H), 2.58 (s, 3H), 2.07-1.95 (m, 2H), 1.90-1.81 (m, 2H). MS (EI) for C25H22ClF3N8O2. found: 599.2 (MH+). Analytical HPLC, ret. time=14.6 min, 98% purity.
Compound C43 was made from intermediate C40.3 and intermediate A116.4 using a method analogous to the method that was used to synthesize Compound A116 from intermediate A116.3 and intermediate A116.4. 1H-NMR (400 MHz, CDCl3): δ 9.22 (d, 1H), 8.47 (d, 1H), 7.44 (m, 1H), 6.98 (t, 2H), 4.68 (d, 1H), 3.09 (t, 1H), 3.00 (m, 1H), 2.78 (s, 3H), 2.77 (t, 1H), 2.60 (s, 3H), 2.12-2.00 (m, 2H), 1.93-1.88 (m, 2H). MS (EI) for C26H20F7N7O2. found: 596.1 (MH+). Analytical HPLC, ret. time=16.10 min, 98% purity.
Compound C44 was made in a manner similar to Compound C43: 1H-NMR (400 MHz, CDCl3): δ 9.15 (d, 1H), 8.40 (d, 1H), 7.52 (t, 1H), 7.42 (m, 1H), 7.16 (m, 1H), 7.09 (t, 1H), 4.63 (d, 1H), 4.22 (d, 1H), 3.02-2.90 (m, 2H), 2.71 (s, 3H), 2.68 (t, 1H), 2.53 (s, 3H), 2.03-1.77 (m, 4H). MS (EI) for C26H21F6N7O2. found: 578.1 (MH+). Analytical HPLC, ret. time=16.02 min, 98% purity.
Compound C45 was made from intermediate C40.3 and 2-chlorophenyl chloroformate using a method analogous to the method that was used to synthesize Compound A119 from intermediate A116.3 and 2-chlorophenyl chloroformate. 1H-NMR (400 MHz, CDCl3): δ 9.22 (d, 1H), 8.48 (d, 1H), 7.42 (dd, 1H), 7.30-7.23 (m, 2H), 7.16 (m, 1H), 4.50 (d, 1H), 4.36 (d, 1H), 3.05 (t, 1H), 2.97 (m, 1H), 2.88 (t, 1H), 2.79 (s, 3H), 2.60 (s, 3H), 2.20-2.10 (m, 2H), 1.91 (m, 2H). MS (EI) for C25H21ClF3N7O3. found: 560.2 (MH+). Analytical HPLC, ret. time=16.70 min, 92% purity.
Compound C46 was made from intermediate C40.4 and 2,6-difluorophenyl isothiocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 9.23 (d, 1H), 8.48 (d, 1H), 7.24 (m, 1H), 6.98 (t, 2H), 6.50 (s, 1H), 4.83 (d, 2H), 3.22 (td, 2H), 3.08 (tt, 1H), 2.79 (s, 3H), 2.61 (s, 3H), 2.22 (qd, 2H), 1.98 (m, 2H). MS (EI) for C25H21F5N8OS. found 577.2 (MH+). Analytical HPLC, ret. time=18.948 min, 96% purity.
Compound C47 was made in a manner similar to Compound C46: 1H-NMR (400 MHz, CDCl3): δ 9.22 (d, 1H), 8.46 (d, 1H), 7.65 (d, 1H), 7.54 (m, 2H), 7.29 (m, 2H), 7.18 (s, 1H), 4.70 (d, 2H), 3.11 (td, 2H), 3.07 (tt, 1H), 2.78 (s, 3H), 2.61 (s, 3H), 2.18 (qd, 2H), 1.92 (m, 2H). MS (EI) for C26H22F6N8OS. found 609.2 (MH+). Analytical HPLC, ret. time=20.068 min, 91% purity.
To a suspension of S-methyl thiourea sulfate (1.15 g, 8.3 mmol) in EtOH (10 mL) was added EtONa (640 mg, 9.2 mmol). The resulting mixture was stirred for 15 min at room temperature. Common Intermediate CI2.1 (1.9 g, 4.9 mmol) in EtOH (15 mL) was added. The mixture was heated at 70° C. with stirring. The reaction was monitored by LC-MS. Once complete, the solvent was removed under reduced pressure. The resulting residue was stirred in EtOAc and any insoluble material was filtered. Concentration of the filtrate gave the crude product, which was further purified by flash column chromatography to give intermediate C48.1 (623 mg, 30%).
Intermediate C48.2 was made from the ester hydrolysis of intermediate C48.1 using General Procedure 1. 1H-NMR (400 MHz, DMSO-d6): δ 14.0 (br. s, 1H), 3.96 (m, 2H), 2.97 (m, 1H), 2.77 (m, 2H), 2.50 (s, 3H, overlap with DMSO-d6), 2.41 (s, 3H), 1.68-1.60 (m, 4H), 1.41 (s, 9H). MS (EI) for C17H25N3O4S. found: 368.2 (MH+).
Intermediate C48.3 was made from intermediate C48.2 and intermediate C40.1 using a method analogous to the method that was used to synthesize intermediate C40.2 from intermediate A121.1 and intermediate C40.1. MS (EI) for C23H28F3N7O4S. found: 556.2 (MH+).
Intermediate C48.4 was made from intermediate C48.3 using a method analogous to the method that was used to synthesize intermediate C40.3 from intermediate C40.2. 1H-NMR (400 MHz, CDCl3): δ 9.21 (d, 1H), 8.46 (d, 1H), 4.22 (m, 2H), 2.90 (m, 1H), 2.70 (m, 2H), 2.62 (s, 3H), 2.57 (s, 3H), 1.98-1.92 (m, 2H), 1.82 (d, 2H), 1.47 (s, 9H). MS (EI) for C23H26F3N7O3S. found: 538.2 (MH+).
Intermediate C48.5 was made from the Boc deprotection of intermediate C48.4 using General Procedure 2.
Intermediate C48.6 was made from intermediate C48.5 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Intermediate C48.7 was synthesized from intermediate C48.6 using General Procedure 6.
Compound C48 was synthesized from intermediate C48.7 using General Procedure 7. 1H-NMR (400 MHz, CDCl3): δ 9.22 (dd, 1H), 8.46 (dd, 1H), 7.12 (m, 1H), 6.92 (m, 3H), 5.93 (br s, 2H), 4.28 (d, 2H), 2.93 (m, 3H), 2.48 (s, 3H), 2.02 (m, 2H), 1.88 (m, 2H). MS (EI) for C24H20F5N9O2. found: 562.2 (MH+). Analytical HPLC, ret. time=14.58 min, 99% purity.
Compound C49 was made in a manner similar to Compound C48: 1H-NMR (400 MHz, CDCl3): δ 9.20 (d, 1H), 8.46 (d, 1H), 7.44 (m, 1H), 7.32 (m, 2H), 6.08 (s, 1H), 5.41 (s, 2H), 4.17 (m, 2H), 2.96 (m, 3H), 2.50 (s, 3H), 2.03 (m, 2H), 1.91 (m, 2H). MS (EI) for C25H20F7N9O2. found: 612.1 (MH+). Analytical HPLC, ret. time=15.87 min, 99% purity.
Intermediate C50.1 was made from intermediate C48.4 using General Procedure 6. MS (EI, Neg.) for C23H26F3N7O5S. found: 567.7 [(M-H)−].
Intermediate C50.2 was made from intermediate C50.1 using General Procedure 7. 1H-NMR (400 MHz, CDCl3): δ 9.19 (d, 1H), 8.44 (d, 1H), 5.29 (br. s, 2H), 4.20 (m, 2H), 2.83 (m, 1H), 2.69 (m, 2H), 2.47 (s, 3H), 1.90-1.77 (m, 4H), 1.47 (s, 9H). MS (EI) for C22H25F3N8O3. found: 507.2 (MH+).
Intermediate C50.3 was made from the Boc deprotection of intermediate C50.2 using General Procedure 2, and obtained as a light yellow solid. 1H-NMR (400 MHz, DMSO-d6): δ 9.38 (d, 1H), 8.78 (br. s, 1H), 8.56 (d, 1H), 8.53 (br. s, 1H), 7.34 (br. s, 2H), 4.82 (br. s, 1H), 3.31 (d, 2H), 3.02 (m, 1H), 2.87 (m, 2H), 2.38 (s, 3H), 2.03-1.91 (m, 4H). MS (EI) for C17H17F3N8O. found: 407.2 (MH+).
Compound C50 was made from intermediate C50.3 and 2,6-difluorophenyl chloroformate (prepared using a general method according to the literature procedure as described in J. Med. Chem. 2006, 49, 4981) using a method analogous to the method that was used to synthesize Compound A119 from intermediate A116.3. 1H-NMR (400 MHz, CDCl3): δ 9.20 (d, 1H), 8.45 (d, 1H), 7.14 (m, 1H), 6.99-6.94 (m, 2), 5.71 (br s, 2H), 4.44 (d, 1H), 4.32 (d, 1H), 3.06 (t, 1H), 2.97 (m, 1H), 2.89 (t, 1H), 2.49 (s, 3H), 2.03 (m, 2H), 1.90 (t, 2H). MS (EI) for C24H19F5N8O3. found: 563.2 (MH+). Analytical HPLC, ret. time=14.56 min, 98% purity.
Compounds C51-C57 were made from intermediate C50.3 and the corresponding substituted phenyl chloroformates (either purchased from commercial sources, or prepared using a general method according to the literature procedure as described in. J. Med. Chem. 2006, 49, 4981) in a manner similar to Compound C50:
1H-NMR (400 MHz, CDCl3): δ 9.20 (d, 1H), 8.45 (d, 1H), 7.42 (d, 1H), 7.30-7.22 (m, 2H), 7.16 (t, 1H), 5.34 (s, 2H), 4.49 (d, 1H), 4.35 (d, 1H), 3.08-2.95 (m, 2H), 2.87 (t, 1H), 2.50 (s, 3H), 2.10-1.98 (m, 2H), 1.95-1.85 (m, 2H). MS (EI) for C24H20ClF3NO3. found: 561.2 (MH+). Analytical HPLC, ret. time=14.72 min, 99% purity.
1H-NMR (400 MHz, CDCl3): δ 9.20 (d, 1H), 8.45 (d, 1H), 7.21-7.10 (m, 4H), 5.37 (s, 2H), 4.44 (d, 1H), 4.34 (d, 1H), 3.06-2.94 (m, 2H), 2.87 (t, 1H), 2.49 (s, 3H), 2.07-1.95 (m, 2H), 1.90 (t, 2H). MS (EI) for C24H20F4N8O3. found: 545.2 (MH+). Analytical HPLC, ret. time=14.18 min, 98% purity.
1H-NMR (400 MHz, CDCl3): δ 9.20 (d, 1H), 8.45 (d, 1H), 7.10-7.02 (m, 4H), 5.52 (s, 2H), 4.37 (dd, 2H), 3.02-2.94 (m, 2H), 2.84 (t, 1H), 2.49 (s, 3H), 2.05-1.85 (m, 4H). MS (EI) for C24H20F4N8O3. found: 545.2 (MH+). Analytical HPLC, ret. time=14.28 min, 97% purity.
1H-NMR (400 MHz, CDCl3): 9.20 (d, 1H), 8.46 (d, 1H), 7.09 (m, 1H), 6.98 (m, 1H), 6.89 (m, 1H), 5.39 (s, 2H), 4.37 (dd, 2H), 3.06-2.95 (m, 2H), 2.88 (t, 1H), 2.50 (s, 3H), 2.08-1.98 (m, 2H), 1.91 (t, 2H). MS (EI) for C24H19F5N8O3. found: 563.2 (MH+). Analytical HPLC, ret. time=14.92 min, 99% purity.
1H-NMR (400 MHz, CDCl3): δ 9.20 (d, 1H), 8.46 (d, 1H), 7.32 (m, 1H), 6.95-6.89 (m, 3H), 5.46 (s, 2H), 4.37 (dd, 2H), 3.03-2.94 (m, 2H), 2.85 (t, 1H), 2.50 (s, 3H), 2.06-1.87 (m, 4H). MS (EI) for C24H20F4N8O3. found: 545.2 (MH+). Analytical HPLC, ret. time=14.54 min, 99% purity.
1H-NMR (400 MHz, CDCl3): δ 9.20 (d, 1H), 8.45 (d, 1H), 7.16 (m, 1H), 6.94-6.84 (m, 2H), 5.44 (s, 2H), 4.37 (dd, 2H), 3.06-2.93 (m, 2H), 2.89 (t, 1H), 2.50 (s, 3H), 2.07-1.97 (m, 2H), 1.90 (t, 2H). MS (EI) for C24H19F5N8O3. found: 563.2 (MH+). Analytical HPLC, ret. time=14.90 min, 99% purity.
1H-NMR (400 MHz, CDCl3): δ 9.20 (d, 1H), 8.46 (d, 1H), 7.18 (t, 1H), 7.09 (d, 1H), 6.97-6.92 (m, 2H), 5.34 (s, 2H), 4.45 (d, 1H), 4.34 (d, 1H), 3.86 (s, 3H), 3.05-2.93 (m, 2H), 2.85 (m, 1H), 2.49 (s, 3H), 2.09-1.98 (m, 2H), 1.90-1.85 (m, 2H). MS (EI) for C25H23F3NO4. found: 557.2 (MH+). Analytical HPLC, ret. time=13.96 min, 98% purity.
Intermediate C58.1 was made from methyl 6-(trifluoromethyl)picolinate using a method analogous to the method that was used to synthesize intermediate C40.1 from methyl 2-(trifluoromethyl)pyrimidine-4-carboxylate.
Intermediate C58.2 was made from intermediate C48.2 and intermediate C58.1 using a method analogous to the method that was used to synthesize intermediate C40.2 from intermediate A121.1 and intermediate C40.1.
Intermediate C58.3 was made from intermediate C58.2 using a method analogous to the method used to synthesize intermediate C40.3 from intermediate C40.2.
Intermediate C58.4 was made from the Boc deprotection of intermediate C58.3 using General Procedure 2.
Compound C58 was made from intermediate C58.4 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.54 (d, 1H), 8.16 (t, 1H), 7.90 (d, 1H), 7.11 (m, 1H), 6.93 (t, 2H), 5.88 (s, 1H), 4.22 (d, 1H), 3.06-2.94 (m, 3H), 2.64 (s, 3H), 2.60 (s, 3H), 2.16-2.06 (m, 2H), 1.95 (d, 2H). MS (EI) for C26H22F5N7O2S. found: 591.9 (MH+). Analytical HPLC, ret. time=20.76 min, 99% purity.
Intermediate C59.1 was synthesized from Compound C58 using General Procedure 6.
Intermediate C59.1 (25 mg, 0.04 mmol) was dissolved in 4 mL of 7 N ammonium in MeOH solution and the resulting mixture was stirred at 30° C. for 10 min. After removal of solvent in vacuo, the crude mixture was purified on prep HPLC to give Compound C59. 1H-NMR (400 MHz, CDCl3): δ 8.46 (d, 1H), 8.07 (t, 1H), 7.81 (d, 1H), 7.03 (m, 1H), 6.86 (t, 2H), 5.80 (s, 1H), 5.27 (s, 2H), 4.13 (d, 2H), 2.91-2.85 (m, 3H), 2.42 (s, 3H), 2.00-1.90 (m 2H), 1.85 (d, 2H). MS (EI) for C25H21F5N8O2. found: 560.9 (MH+). Analytical HPLC, ret. time=15.37 min, 98% purity.
Compound C60 was recovered as a side product from the reaction that generated Compound C59. 1H-NMR (400 MHz, CDCl3): δ 8.48 (d, 2H), 8.09 (t, 1H), 7.83 (d, 1H), 7.04 (m, 1H), 6.86 (t, 2H), 5.80 (s, 1H), 4.15 (d, 2H), 4.04 (s, 3H), 2.99-2.88 (m, 3H), 2.53 (s, 3H), 2.10-1.99 (m, 2H), 1.89 (d, 2H). MS (EI) for C26H22F5N7O3. found: 575.9 (MH+). Analytical HPLC, ret. time=18.80 min, 99% purity.
Compound C61 was made in a manner similar to Compound C60: 1H-NMR (400 MHz, CDCl3): δ 9.21 (d, 1H), 8.47 (d, 1H), 7.11 (m, 1H), 6.93 (m, 2H), 5.90 (br s, 1H), 4.23 (m, 2H), 4.11 (s, 3H) 3.00 (m, 3H), 2.60 (s, 3H), 2.12 (m, 2H), 1.94 (m, 2H). MS (EI) for C25H21F5N8O3. found: 577.2 (MH+). Analytical HPLC, ret. time=17.84 min, 99% purity.
Intermediate C62.1 was made from the Boc deprotection of Common Intermediate CI1 using General Procedure 2.
Intermediate C62.2 was synthesized from intermediate C62.1 and 2,6-difluorophenylacetic acid using General Procedure 4.
Intermediate C62.3 was made from intermediate C62.2 using General Procedure 1.
Intermediate C62.4 was made from intermediate C62.3 and intermediate C40.1 using a method analogous to the method that was used to synthesize intermediate C40.2 from intermediate A121.1 and intermediate C40.1.
Compound C62 was synthesized from intermediate C62.4 using a method analogous to the method used to synthesize intermediate C40.3 from intermediate C40.2. 1H-NMR (400 MHz, CDCl3): δ 9.31 (s, 1H), 9.21 (d, 1H), 8.45 (d, 1H), 7.24 (m, 1H), 6.91 (t, 2H), 4.82 (d, 1H), 4.18 (d, 1H), 4.00 (tt, 1H), 3.79 (s, 2H), 3.37 (td, 1H), 2.84 (s, 3H), 2.83 (m, 1H), 2.03 (m, 4H). MS (EI) for C25H20F5N7O2. found 546.1 (MH+). Analytical HPLC, ret. time=18.784 min, 99% purity.
To a mixture of 3-(trifluoromethyl)benzonitrile (1.00 g, 5.84 mmol) and hydroxylamine hydrochloride (2.44 g, 35 mmol) in anhydrous ethanol (8.3 mL) was added triethylamine (4.14 g, 41 mmol). The resulting mixture was heated at 80° C. with stirring in a sealed tube for 20 min. After cooling to RT, the mixture was diluted with EtOAc, washed with water (3×) and brine, dried over MgSO4, and concentrated in vacuo to give (Z)—N′-hydroxy-3-(trifluoromethyl)benzimidamide as an off-white solid (1.18 g): 1H-NMR (400 MHz, DMSO-d3): δ 9.86 (s, 1H), 7.98 (s, 1H), 7.96 (d, 1H), 7.72 (d, 1H), 7.60 (t, 1H), 6.01 (s, 2H). MS (EI) for C8H7F3N2O. found: 205.1 (MH+). A mixture of intermediate A121.1 (300 mg, 0.89 mmol), HATU (408 mg, 1.07 mmol), DIEA (230 mg, 1.79 mmol) and DCM (7 mL) was heated at 35° C. with stirring for 40 min. To the mixture was added (Z)—N′-hydroxy-3-(trifluoromethyl)benzimidamide (237 mg, 1.16 mmol) and the resulting mixture was heated at 35° C. for 30 min. After removal of DCM in vacuo, the resulting mixture was partitioned between EtOAc and water. The organic phase was separated, washed with water (3×) and brine, dried over MgSO4 and concentrated in vacuo to afford intermediate C63.1 (515 mg) as a light orange solid: MS (EI) for C25H30F3N5O4. found: 522.2 (MH+). The crude product was used directly in the next reaction without further purification.
To crude intermediate C63.1 obtained above was added glacial acetic acid (10 ml). The mixture was heated at 95° C. with stirring for 2.5 days. The resulting crude reaction solution was used as is in the next reaction.
TFA (2 mL) was added to the crude reaction solution of intermediate C63.2 generated above. The resulting mixture was heated at 80-90° C. for an additional 2 h. The crude mixture was cooled to RT, diluted with water, and washed with t-butylethyl ether. The aqueous phase was basified with 0.5 N NaOH, and then extracted with EtOAc (3×). The organic phase was washed with water and brine, dried over MgSO4 and concentrated to give intermediate C63.3 (46 mg). MS (EI) for C20H20F3N5O. found: 404.2 (MH+). The compound was used for the next step without further purification.
Compound C63 was made from intermediate C63.3 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.44 (s, 1H), 8.36 (d, 1H), 7.83 (d, 1H), 7.70 (t, 1H), 7.10 (m, 1H), 6.93 (m, 2H), 5.97 (s, 1H), 4.22 (d, 2H), 2.95 (m, 2H), 2.89 (m, 1H), 2.77 (s, 3H), 2.56 (s, 3H), 2.10 (m, 2H), 1.88 (d, 2H). MS (EI) for C27H23F5N6O2. found: 559.2 (MH+). Analytical HPLC, ret. time=21.5 min, 99% purity.
Compound C64 was made in a manner similar to Compound C63: 1H-NMR (400 MHz, DMSO-d6): δ 8.41 (d, 1H), 8.33 (s, 1H), 8.18 (s, 1H), 8.04 (d, 1H), 7.89 (t, 1H), 7.60-7.49 (m, 3H), 4.12 (d, 2H), 3.35 (s, 3H), 3.03 (m, 1H), 2.82 (t, 2H), 2.67 (s, 3H), 1.83-1.72 (m, 2H). MS (EI) for C28H23F7N6O2. found: 609.1 (MH+). Analytical HPLC, ret. time=4.57 min over a 6 min-run, 99% purity.
Compound C65 was synthesized from intermediate C63.3 and 2,6-difluorobenzoic acid using General Procedure 4. 1H-NMR (400 MHz, DMSO-d6): δ 8.42 (d, 1H), 8.34 (s, 1H), 8.05 (d, 1H), 7.90 (t, 1H), 7.55 (m, 1H), 7.26-7.18 (m, 2H), 4.20 (d, 1H), 3.43 (m, 1H), 3.38 (s, 3H), 3.16 (m, 2H), 2.86 (m, 1H), 2.69 (s, 3H), 1.92 (m, 1H), 1.83-1.76 (m, 3H). MS (EI) for C27H22F5N5O2. found: 544.1 (MH+). Analytical HPLC, ret. time=23.6 min, 98% purity.
Intermediate C66.1 was made from intermediate A121.1 and 2-amino-1-(3-chlorophenyl)ethanone.HCl using General Procedure 3.
Intermediate C66.2 was made by the Boc-deprotection of intermediate C66.1 using General Procedure 2.
Intermediate C66.3 was made from intermediate C66.2 and 2,6-difluorophenylacetic acid using General Procedure 4.
A mixture of C66.3 (14 mg, 0.026 mmol) and POCl3 (0.5 mL) was stirred for 6.5 h at 95° C. After evaporating POCl3 in vacuo, the resulting residue was dissolved in EtOAc (2 mL) and treated with aqueous saturated NaHCO3 (2 mL). The aqueous layer was further extracted with EtOAc (2×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=4:3→1:1) to give Compound C66 (4.3 mg, 32%) as a clear oil. 1H-NMR (400 MHz, CDCl3): δ 7.66 (s, 1H), 7.57 (s, 1H), 7.56 (d, 1H), 7.41 (t, 1H), 7.36 (m, 1H), 7.22 (m, 1H), 6.89 (t, 2H), 4.71 (d, 1H), 4.09 (d, 1H), 3.74 (d, 2H), 3.13 (t, 1H), 3.04 (tt, 1H), 2.75 (s, 3H), 2.60 (td, 1H), 2.53 (s, 3H), 2.06 (qd, 1H), 1.89 (m, 3H). MS (EI) for C28H25ClF2N4O2. found 523.2 (MH+). Analytical HPLC, ret. time=22.116 min, 96% purity.
Intermediate C67.1 was made from intermediate C67.2 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Compound C67 was made from intermediate C67.1 using a method analogous to the cyclization of intermediate C66.3 to make Compound C66. 1H-NMR (400 MHz, CDCl3): δ 7.66 (t, 1H), 7.58 (s, 1H), 7.56 (m, 1H), 7.41 (t, 1H), 7.36 (m, 1H), 7.10 (m, 1H), 6.93 (t, 2H), 5.86 (s, 1H), 4.21 (d, 2H), 3.01 (tt, 1H), 2.95 (td, 2H), 2.75 (s, 3H), 2.53 (s, 3H), 2.08 (qd, 2H), 1.86 (m, 2H). MS (EI) for C27H24ClF2N5O2. found 524.1 (MH+). Analytical HPLC, ret. time=19.588 min, 99% purity.
2-Bromo-1-(2-(trifluoromethyl)pyrimidin-4-yl)ethanone was prepared from 4-chloro-2-(trifluoromethyl)pyrimidine by the same method used in the synthesis of 2-bromo-1-(2-chloropyrimidin-4-yl)ethanone (Bioorganic Med. Chem. Lett. 2011, 21, 3818-3822). To a stirred solution of 2-bromo-1-(2-(trifluoromethyl)pyrimidin-4-yl)ethanone (270 mg, 1.00 mmol) in THF (4 mL) at 0° C. were added sodium azide (98.0 mg, 1.50 mmol) and H2O (0.2 mL) in sequence and the mixture was stirred for 20 min at 0° C. The resulting crude reaction solution was used as is in the next reaction.
To the crude reaction solution of intermediate C68.1 was added NaBH4 (57 mg, 1.5 mmol). After stirring for 20 min at room temperature, aqueous saturated NH4Cl (4 mL) was added and the resulting solution was extracted with EtOAc (3×2 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=5:2) to give intermediate C68.2 (116 mg, 50%).
A mixture of intermediate C68.2 (116 mg, 0.498 mmol), palladium on activated carbon (53 mg, 10 wt. %, 0.050 mmol) and MeOH (5 mL) was stirred for 1 h at room temperature under hydrogen atmosphere using a balloon. The mixture was filtered through Celite and the filtrate was concentrated. The residue was dissolved in EtOAc (3 mL), and the resulting solution was filtered and concentrated to give a crude intermediate C68.3. MS (EI) for C7H8F3N3O. found 207.1 (MH+).
Intermediate C68.4 was made from the Boc-deprotection of Common Intermediate CI2 using General Procedure 2.
Intermediate C68.5 was made from intermediate C68.4 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Intermediate C68.6 was made from the ester hydrolysis of intermediate C68.5 using General Procedure 1.
Intermediate C68.7 was made from the coupling of intermediate C68.6 and intermediate C68.3 using General Procedure 3.
Intermediate C68.8 was made from intermediate C68.7 using a method analogous to the oxidation of intermediate B46.1 to intermediate B46.2.
Compound C68 was made from intermediate C68.8 using a method analogous to the cyclization of intermediate C66.3 to form Compound C66. 1H-NMR (400 MHz, CDCl3): δ 8.99 (d, 1H), 8.24 (s, 1H), 7.72 (d, 1H), 7.12 (m, 1H), 6.93 (t, 2H), 5.87 (s, 1H), 4.21 (d, 2H), 2.97 (m, 3H), 2.77 (s, 3H), 2.53 (s, 3H), 2.09 (qd, 2H), 1.86 (m, 2H). MS (EI) for C26H22F5N7O2. found 560.2 (MH+). Analytical HPLC, ret. time=17.628 min, 99% purity.
Intermediate C69.1 was made by coupling intermediate C48.2 with C68.3 using General Procedure 3.
Intermediate C69.2 was made from the Boc deprotection of intermediate C69.1 using General Procedure 2.
Intermediate C69.3 was made from intermediate C69.2 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Intermediate C69.4 was made from intermediate C69.3 using a method analogous to the oxidation of intermediate B46.1 to intermediate B46.2.
Intermediate C69.5 was made from intermediate C69.4 using a method analogous to the cyclization of intermediate C66.3 to form Compound C66.
Intermediate C69.6 was made from intermediate C69.5 using General Procedure 6.
To a stirred solution of intermediate C69.6 (4.6 mg, 0.0074 mmol) in 2-propanol (1.5 mL) at −78° C. was bubbled ammonia gas for 5 min. The mixture was sealed and stirred for 1 h at room temperature. The resulting solution was concentrated in vacuo and purified by preparative HPLC to give Compound C69 (3.0 mg, 72%) as a white solid after lyophilization. 1H-NMR (400 MHz, CDCl3, DMSO-d6): δ 9.00 (d, 1H), 8.21 (s, 1H), 7.80 (d, 1H), 7.59 (s, 1H), 7.14 (m, 1H), 6.92 (t, 2H), 6.27 (br s, 2H), 4.30 (d, 2H), 2.85 (m, 3H), 2.42 (s, 3H), 1.96 (qd, 2H), 1.82 (m, 2H). MS (EI) for C25H21F5N8O2. found 561.1 (MH+). Analytical HPLC, ret. time=11.360 min, 99% purity.
To a mixture of acetylacetone (2 g, 20 mmol) and N-boc-4-piperidinecarboxaldehyde (4.3 g, 20 mmol) in isopropanol (30 mL) were added acetic acid (60 mg) and piperidine (60 mg). The mixture was stirred at rt for 48 h. Isopropanol was removed. The residue was dissolved in EtOAc, washed with saturated NaHCO3, brine, and dried over anhydrous Na2SO4. Removal of EtOAc gave the crude intermediate C70.1. It was used in the next step without further purification.
To a solution of intermediate C70.1 (460 mg, 1.56 mmol) in DMF (3 mL) were added S-methylisothiourea hemisulfate (260 mg, 1.87 mmol) and NaHCO3 (524 mg, 6.2 mmol). The resulting mixture was stirred at 70° C. for 4 h. The reaction mixture was cooled to rt, extracted with EtOAc, and dried over anhydrous Na2SO4. EtOAc was removed, and the crude dihydropyrimidine product was dissolved in DCE (10 mL). To this solution was added MnO2 (1.1 g, 12.5 mmol). The mixture was stirred at 85° C. for 30 min. It was then cooled to rt, filtered through Celite, and concentrated. Purification by flash column chromatography gave intermediate C70.2.
To a stirred solution of C70.2 (365 mg, 1.00 mmol) in THF (4 mL) at 0° C. was added LiHMDS (1.5 mL, 1M in THF, 1.5 mmol). After stirring for 15 min at 0° C., chlorotrimethylsilane (0.19 mL, 1.5 mmol) was added. After stirring for 3 h at 0° C., aqueous saturated NaHCO3 (5 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were dried over MgSO4 and concentrated. To a stirred solution of the residue in THF (4 mL) at 0° C. were added NaHCO3 (126 mg, 1.50 mmol) and NBS (106 mg, 0.596 mmol) in sequence. After stirring for 1 h at room temperature, aqueous saturated NaHCO3 (5 mL) was added and the resulting solution was extracted with EtOAc (3×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=6:1→4:1→5:2) to give intermediate C70.3 (240 mg, 54%) as a pale brown oil. MS (EI) for C18H26BrN3O3S. found 446.1 (MH+).
A mixture of intermediate C70.3 (200 mg, 0.451 mmol), sodium azide (58.6 mg, 0.902 mmol) and acetone (4 mL) was stirred for 3.5 h at room temperature. Aqueous saturated NH4Cl (3 mL) was added and the resulting solution was extracted with EtOAc (2×3 mL). The combined organic layers were concentrated and purified by silica gel column chromatography (Hexane/EtOAc=3:1) to give intermediate C70.4 (157 mg, 86%) as a pale brown solid. MS (EI) for C18H26N6O3S. found 407.2 (MH+).
Intermediate C70.5 was made from intermediate C70.4 in EtOAc using a method analogous to the conversion of intermediate C68.2 to intermediate C68.3.
Intermediate C70.6 was made from intermediate C70.5 and 2-(trifluoromethyl)pyrimidine-4-carboxylic acid using standard amide coupling techniques analogous to those used in General Procedure 3.
Intermediate C70.7 was made from the Boc deprotection of intermediate C70.6 using General Procedure 2.
Intermediate C70.8 was made from intermediate C70.7 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Intermediate C70.9 was made from intermediate C70.8 using a method analogous to the cyclization of intermediate C66.3 to form Compound C66.
Intermediate C70.10 was made from intermediate C70.9 using General Procedure 6.
Compound C70 was made from intermediate C70.10 using a method analogous to that used to convert intermediate C69.6 to Compound C69. 1H-NMR (400 MHz, CDCl3): δ 9.09 (d, 1H), 8.27 (d, 1H), 7.38 (s, 1H), 7.11 (m, 1H), 6.93 (t, 2H), 5.87 (s, 1H), 5.43 (br s, 2H), 4.20 (d, 2H), 2.89 (td, 2H), 2.71 (tt, 1H), 2.36 (s, 3H), 2.02 (qd, 2H), 1.84 (m, 2H). MS (EI) for C25H21F5NO2. found 561.1 (MH+). Analytical HPLC, ret. time=10.088 min, 95% purity.
Compound C71 was made in three steps from intermediate C48.5 in the same manner that Compound C48 was made in three steps from intermediate C48.5, substituting 2-chlorophenyl isocyanate for 2,6-difluorophenyl isocyanate in the first step. 1H-NMR (400 MHz, CDCl3): δ 9.20 (d, 1H), 8.45 (d, 1H), 8.21 (dd, 1H), 7.34 (dd, 1H), 7.25 (m, 1H), 7.07 (s, 1H), 6.95 (dt, 1H), 5.34 (s, 2H), 4.22 (d, 2H), 3.01-2.93 (m, 3H), 2.50 (s, 3H), 2.06-1.91 (m, 4H). MS (EI) for C24H21ClF3N9O2. found: 560.2 (MH+). Analytical HPLC, ret. time=16.54 min, 98% purity.
To a 0° C. solution of vinyl magnesium chloride in THF (1 mL, 1.6 M in THF) was added dropwise a solution of intermediate B36.1 (346 mg, 1.1 mmol) in PhMe (2 mL). The reaction mixture was stirred at 0° C. for 10 min, and then was quenched by slow addition of ice-water. The mixture was extracted with EtOAc, washed with brine, and dried over anhydrous Na2SO4. After removal of the solvents, the residue was purified by flash column chromatography to give intermediate D16.1 (180 mg, 48%).
Ozone was bubbled through a −78° C. solution of intermediate D16.1 (180 mg, 0.57 mmol) in DCM/MeOH (5 mL/11 mL) until the blue color persisted. Nitrogen was then bubbled through the mixture to remove excess ozone. To this solution was added dimethylsulfide (160 mg, 2.5 mmol). The mixture was allowed to warm to rt gradually. Removal of the solvents gave the crude aldehyde, which was used in the next step without further purification.
Crude intermediate D16.2 (140 mg, 0.4 mmol) was dissolved in AcOH (0.8 mL). The solution was stirred for 1 h at rt. Then NaBH4 (10 mg) was added. The stirring was continued for 1 h. EtOAc was added. The organic phase was washed with saturated NaHCO3, brine, and dried over anhydrous Na2SO4. After removal of the solvents, the residue was purified by flash column chromatography to give intermediate D16.3 (118 mg, 59%).
Intermediate D16.4 was made from the Boc deprotection of intermediate D16.3 using General Procedure 2.
Compound D16 was made from intermediate D16.4 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.22 (s, 1H), 7.51 (t, 1H), 7.24 (m, 1H), 7.06 (m, 3H), 6.86 (d, 1H), 6.81 (d, 1H), 5.71 (s, 1H), 4.41 (m, 2H), 3.55 (m, 3H), 2.85 (m, 2H), 2.51 (s, 3H), 1.70 (m, 4H). MS (EI) for C26H27F5N6O2. found: 551.2 (MH+). Analytical HPLC, ret. time=11.08 min, 98% purity.
To a mixture of Me3SI (300 mg, 1.5 mmol) in THF (4 mL) was added tBuOK (2 mL, 1.0 M in THF). The resulting mixture was stirred at room temperature for 30 min and B1.1 (320 mg, 1 mmol) was added. Stirring was continued at room temperature for 2 h and then the reaction mixture was diluted with aq. sat. NaHCO3. The mixture was extracted with EtOAc and the organic mixture was dried over sodium sulfate, filtered, and concentrated. The crude product was used as is in the next step.
Intermediate D17.1 (668 mg, 2.0 mmol) and ammonium hydroxide (28%, 7 mL) were dissolved in 1,4-dioxane (7 mL). The mixture was warmed to 60° C. with stirring in a sealed tube for 72 h. After the mixture was cooled to RT, EtOAc was added to extract the product. The organic layer was washed with water and dried over anhydrous Na2SO4. Removal of the solvents gave crude intermediate D17.2, which was used in the next step without further purification.
To a solution of the crude intermediate D17.2 (133 mg, 0.38 mmol) prepared above in acetonitrile (2 mL) were added 2-chloro-6-trifluoromethylpyrimidine (69 mg, 0.38 mmol), and DIEA (147 mg, 1.14 mmol). The mixture was stirred at 50° C. for 12 h. After the mixture was cooled to rt, EtOAc was added to extract the product. The organic layer was washed with water and dried over anhydrous Na2SO4. Removal of the solvents gave crude intermediate D17.3, which was used in the next step without further purification.
Intermediate D17.4 was made from the Boc deprotection of intermediate D17.3 using General Procedure 2.
Compound D17 was made from intermediate D17.4 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.61 (s, 1H), 8.24 (s, 1H), 8.09 (d, 1H), 7.97 (m, 1H), 7.24 (m, 1H), 7.09 (m, 2H), 6.74 (d, 1H), 5.76 (s, 1H), 4.18 (m, 2H), 3.55 (m, 3H), 2.85 (m, 2H), 2.51 (s, 3H), 1.70 (m, 4H). MS (EI) for C25H26F5N7O2. found: 552.2 (MH+). Analytical HPLC, ret. time=11.50 min, 96% purity.
Compound D18 was made in a manner similar to Compound D17: 1H-NMR (400 MHz, CDCl3): δ 8.64 (s, 1H), 8.23 (d, 1H), 7.45 (m, 1H), 7.44-7.31 (m, 2H), 6.45 (d, 1H), 6.14 (s, 1H), 5.86 (t, 1H), 4.18 (t, 2H), 3.92 (br. s, 1H), 3.88 (m, 2H), 3.62 (m, 1H), 3.14-2.30 (m, 2H), 2.67 (s, 3H), 2.22-2.13 (m, 2H), 1.79-1.71 (m, 2H), 1.67 (s, 3H). MS (EI) for C26H26F7N7O2. found: 602.2 (MH+). Analytical HPLC, ret. time=11.4 min, 95% purity.
Intermediate D19.1 was made from intermediate D17.1 using a method analogous to the method that was used to convert intermediate D17.1 to intermediate D17.2.
Intermediate D19.2 was made from intermediate D19.1 using a method analogous to the method that was used to convert intermediate D17.2 to intermediate D17.3.
Intermediate D19.3 was made from the Boc deprotection of intermediate D19.2 using General Procedure 2.
Compound D19 was made from intermediate D19.3 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (m, 1H), 8.33 (m, 1H), 8.03 (m, 1H), 7.26 (m, 1H), 7.11 (m, 2H), 6.74 (m, 1H), 5.76 (s, 1H), 4.16 (m, 2H), 3.63 (s, 3H), 3.55 (m, 3H), 2.85 (m, 2H), 2.53 (s, 3H), 1.70 (m, 4H), 1.54 (s, 3H). MS (EI) for C26H28F5N7O2. found: 566.2 (MH+). Analytical HPLC, ret. time=12.44 min, 91% purity.
Compound D20 was made from intermediate D19.3 and 2,6-difluorophenylacetic acid using General Procedure 4. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (m, 1H), 8.33 (m, 1H), 7.34 (m, 1H), 7.05 (m, 2H), 6.90 (m, 1H), 5.73 (s, 1H), 4.47 (m, 2H), 4.16 (m, 2H), 3.63 (s, 3H), 3.55 (m, 3H), 2.85 (m, 2H), 2.53 (s, 3H), 1.70 (m, 4H), 1.54 (s, 3H). MS (EI) for C27H29F5N6O2. found: 565.2 (MH+). Analytical HPLC, ret. time=14.42 min, 95% purity.
Intermediate D21.1 was made from intermediate D17.2 and 2-chloro-4-(trifluoromethyl)pyrimidine using a method analogous to that used to convert intermediate D17.2 to intermediate D17.3.
Intermediate D21.2 was made from the Boc deprotection of intermediate D21.1 using General Procedure 2.
Compound D21 was made from intermediate D21.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.50 (m, 2H), 8.23 (s, 1H), 7.58 (m, 1H), 7.24 (m, 1H), 7.09 (m, 2H), 6.92 (d, 1H), 5.64 (s, 1H), 4.16 (m, 2H), 3.55 (m, 3H), 2.85 (m, 2H), 2.53 (s, 3H), 1.70 (m, 4H), 1.54 (s, 3H). MS (EI) for C25H26F5N7O2. found: 552.1 (MH+). Analytical HPLC, ret. time=12.47 min, 94% purity.
Compound D22 was made in a manner similar to Compound D21: 1H-NMR (400 MHz, CDCl3): δ 8.66 (s, 1H), 8.51 (d, 1H), 7.45 (d, 1H), 7.38-7.31 (m, 2H), 6.93 (d, 1H), 6.12 (s, 1H), 5.85 (br. s, 1H), 4.20 (m, 2H), 3.91 (d, 2H), 3.66 (m, 1H), 3.11-2.98 (m, 2H), 2.67 (s, 3H), 2.23-2.10 (m, 2H), 1.81-1.73 (m, 2H), 1.70 s, 3H). MS (EI) for C26H26F7N7O2. found: 602.2 (MH+). Analytical HPLC, ret. time=13.7 min, 99% purity.
Compound D23 was made from intermediate D21.2 and 2,6-difluorophenylacetic acid using General Procedure 4. 1H NMR (400 MHz, DMSO-d6) δ 8.50 (m, 2H), 7.58 (m, 1H), 7.34 (m, 1H), 7.03 (m, 2H), 6.91 (m, 1H), 5.64 (s, 1H), 4.47 (m, 2H), 4.16 (m, 2H), 3.55 (m, 3H), 2.85 (m, 2H), 2.53 (s, 3H), 1.70 (m, 4H), 1.54 (s, 3H). MS (EI) for C26H27F5N6O2. found: 551.2 (MH+). Analytical HPLC, ret. time=14.19 min, 97% purity.
Intermediate D24.1 was made from intermediate B36.1 using a method analogous to that used to convert intermediate C70.2 to intermediate C70.3.
Crude intermediate D24.1 (1.4 g, 3.5 mmol) was mixed with 2-hydroxy-6-trifluoromethylpyridine (700 mg, 3.85 mmol) in acetone (15 mL). To this mixture was added K2CO3 (1.0 g, 7.2 mmol). The resulting mixture was stirred for 3 h. EtOAc was added to extract the product. The organic phase was washed with saturated NaHCO3, brine, and dried over anhydrous Na2SO4. After removal of the solvents, the residue was purified by flash column chromatography to give intermediate D24.2 (810 mg, 48%).
To a 0° C. solution of methyl magnesium bromide in THF (0.3 mL, 1.4 M in THF, 0.42 mmol) was added dropwise a solution of intermediate D24.2 (50 mg, 0.1 mmol) in PhMe (1 mL). The reaction mixture was stirred at 0° C. for 10 min, and then was quenched by slow addition of ice-water. The mixture was extracted with EtOAc, washed with brine, and dried over anhydrous Na2SO4. After removal of the solvents, the residue was purified by flash column chromatography to give intermediate D24.3 (30 mg, 60%).
Intermediate D24.4 was made from the Boc deprotection of intermediate D24.3 using General Procedure 2.
Compound D24 was made from intermediate D24.4 and 2,6-difluorophenylacetic acid using General Procedure 4. 1H NMR (400 MHz, DMSO-d6) δ 8.64 (s, 1H), 7.95 (t, 1H), 7.48 (d, 1H), 7.35 (m, 1H), 7.09 (m, 3H), 5.77 (s, 1H), 4.57 (m, 2H), 4.48 (m, 1H), 4.16 (m, 1H), 3.80 (m, 3H), 3.20 (m, 1H), 2.65 (m, 1H), 2.55 (s, 3H), 1.90 (m, 1H), 1.65 (m, 3H), 1.68 (s, 3H). MS (EI) for C27H27F5N4O3. found: 551.2 (MH+). Analytical HPLC, ret. time=16.67 min, 99% purity.
Compound D25 was made from intermediate C24.4 and 2,6-difluorophenyl isocyanate using General Procedure 5. H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.21 (s, 1H), 7.95 (t, 1H), 7.49 (d, 1H), 7.27 (m, 1H), 7.09 (m, 3H), 5.77 (s, 1H), 4.57 (m, 2H), 4.21 (m, 2H), 3.82 (m, 1H), 2.90 (m, 2H), 2.55 (s, 3H), 1.70 (m, 4H), 1.66 (s, 3H). MS (EI) for C26H26F5N5O3. found: 552.2 (MH+). Analytical HPLC, ret. time=14.26 min, 95% purity.
Intermediate D26.1 was synthesized from intermediate B50.3 using a method analogous to the method used to convert intermediate C70.2 to intermediate C70.3.
Intermediate D26.2 was synthesized from intermediate D26.1 and 6-(trifluoromethyl)pyridin-2-ol using a method analogous to that used to synthesize intermediate D24.2 from intermediate D24.1.
Intermediate D26.3 was synthesized from intermediate D26.2 using a method analogous to that used to convert intermediate D24.2 to intermediate D24.3.
Intermediate D26.4 was synthesized from the Boc deprotection of intermediate D26.3 using General Procedure 2.
Compound D26 was made from intermediate D26.4 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.52 (s, 1H), 7.78 (t, 1H), 7.35 (d, 1H), 7.12 (m, 1H), 6.94 (m, 3H), 5.92 (s, 1H), 4.80 (d, 1H), 4.60 (d, 1H), 4.24 (d, 2H), 3.89 (s, 1H), 3.70 (tt, 1H), 3.05 (qd, 2H), 2.14 (m, 2H), 1.81 (m, 2H), 1.71 (s, 3H). MS (EI) for C26H26F5N5O3S. found 584.2 (MH+). Analytical HPLC, ret. time=19.552 min, 95% purity.
Intermediate D27.1 was synthesized from Compound D26 using General Procedure 6.
Compound D27 was synthesized from intermediate D27.1 using a method analogous to that used to convert intermediate C69.6 to Compound C69. 1H-NMR (400 MHz, CDCl3): δ 8.29 (s, 1H), 7.77 (t, 1H), 7.33 (d, 1H), 7.11 (m, 1H), 6.95 (m, 3H), 5.91 (s, 1H), 4.93 (s, 2H), 4.77 (d, 1H), 4.52 (d, 1H), 4.23 (d, 2H), 3.64 (tt, 1H), 3.57 (s, 1H), 3.03 (qd, 2H), 2.07 (m, 2H), 1.78 (m, 2H), 1.69 (s, 3H). MS (EI) for C25H25F5N6O3. found 553.1 (MH+). Analytical HPLC, ret. time=12.596 min, 95% purity.
Intermediate D28.1 was synthesized from intermediate D26.1 and 2-(trifluoromethyl)pyrimidin-4-ol using a method analogous to that used to synthesize intermediate D24.2 from intermediate D24.1.
Intermediate D28.2 was synthesized from intermediate D28.1 using a method analogous to that used to convert intermediate D24.2 to intermediate D24.3.
Intermediate D28.3 was synthesized from the Boc deprotection of intermediate D28.2 using General Procedure 2.
Compound D28 was made from intermediate D28.3 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.64 (d, 1H), 8.47 (s, 1H), 7.13 (m, 1H), 6.95 (m, 3H), 5.93 (s, 1H), 4.84 (d, 1H), 4.66 (d, 1H), 4.25 (d, 2H), 3.71 (m, 1H), 3.06 (t, 2H), 2.95 (s, 1H), 2.57 (s, 3H), 2.15 (m, 2H), 1.80 (m, 2H), 1.76 (s, 3H). MS (EI) for C25H25F5N6O3S. found 585.2 (MH+). Analytical HPLC, ret. time=14.052 min, 95% purity.
Intermediate D29.1 was synthesized from Compound D28 using General Procedure 6.
Compound D29 was synthesized from intermediate D29.1 using a method analogous to that used to convert intermediate C69.6 to Compound C69. 1H NMR (400 MHz, CDCl3) δ 8.63 (d, 1H), 8.24 (s, 1H), 7.11 (m, 1H), 6.95 (m, 3H), 5.91 (s, 1H), 5.04 (s, 2H), 4.81 (d, 1H), 4.59 (d, 1H), 4.24 (d, 2H), 3.64 (m, 1H), 3.04 (t, 2H), 2.72 (bs, 1H), 2.21 (dd, 2H), 1.75 (s, 3H), 1.74 (m, 2H). MS (EI) for C24H24F5N7O3. found: 554.2 (MH+). Analytical HPLC, ret. time=11.54 min, 99% purity.
Compound D30.1 was made from intermediate D28.3 and 2,6-difluorophenylacetic acid using General Procedure 4.
Intermediate D30.2 was synthesized from intermediate D30.1 using General Procedure 6.
Compound D30 was synthesized from intermediate D30.2 using a method analogous to that used to convert intermediate C69.6 to Compound C69. 1H NMR (400 MHz, CDCl3) δ 8.63 (d, 1H), 8.24 (bs, 1H), 7.22 (td, 1H), 6.98-6.84 (m, 3H), 5.04 (s, 2H), 4.79 (dt, 2H), 4.59 (d, 1H), 4.11 (d, 1H), 3.77 (s, 2H), 3.66 (d, 1H), 3.23 (t, 1H), 2.79-2.60 (m, 2H), 2.01 (ddd, 4H), 1.76 (s, 3H), 1.72 (d, 4H). MS (EI) for C25H25F5N6O3. found: 553.1 (MH+). Analytical HPLC, ret. time=9.80 min, 98% purity.
To a solution of Compound D29 (33.1 mg, 0.06 mmol) in pyridine (2 mL) was slowly added acetyl chloride (23.4 mg, 0.30 mmol) at 0° C. The resulting white mixture was stirred for a half hour at 0° C. Upon completion of the reaction, the mixture was quenched with ice and extracted with ethyl acetate (3×10 mL). The combined organic extracts were dried over magnesium sulfate and concentrated in vacuo. The crude product was purified by preparative HPLC to give Compound D31; 1H NMR (400 MHz, CDCl3) δ 8.64 (d, 1H), 8.54 (s, 1H), 7.93 (s, 1H), 7.13 (t, 1H), 6.94 (dd, 3H), 5.91 (s, 1H), 4.83 (d, 1H), 4.68 (d, 1H), 4.25 (d, 2H), 3.75 (s, 1H), 3.06 (t, 2H), 2.88 (s, 1H), 2.54 (s, 3H), 2.15-1.99 (m, 2H), 1.81 (m, 2H), 1.77 (s, 3H). MS (EI) for C26H26F5N7O4. found: 596.2 (MH+). Analytical HPLC, ret. time=13.76 min, 99% purity.
Compound D32 was made in a manner similar to Compound D31: 1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 7.94 (s, 1H), 7.79 (t, 1H), 7.35 (d, 1H), 7.16-7.07 (m, 1H), 6.99-6.90 (m, 3H), 5.92 (s, 1H), 4.80 (d, 1H), 4.61 (d, 1H), 4.24 (d, 2H), 3.85 (s, 1H), 3.76 (dd, 1H), 3.13-2.98 (m, 2H), 2.54 (s, 3H), 2.12-1.97 (m, 2H), 1.89-1.75 (m, 2H), 1.73 (s, 3H). MS (EI) for C27H27F5N6O4. found: 595.1 (MH+). Analytical HPLC, ret. time=11.59 min, 99% purity.
Compound D33 was made in a manner similar to Compound D31: 1H-NMR (400 MHz, CDCl3): δ 8.64 (d, 1H), 8.56 (br s, 1H), 8.23 (br s, 1H), 7.23 (m, 1H), 6.92 (m, 3H), 4.80 (m, 2H), 4.68 (d, 1H), 4.13 (d, 1H), 3.77 (comp m, 3H), 3.27 (t, 1H), 3.12 (br s, 1H), 2.70 (t, 1H), 2.51 (s, 3H), 2065-1.68 (comp m, 7H). MS (EI) for C27H27F5N6O4. found: 595.1 (MH+). Analytical HPLC, ret. time=11.47 min, 96% purity.
Compound D34 was synthesized from intermediate D29.1 and cyclopropylamine using a method analogous to the conversion of intermediate B53.1 to Compound B60. 1H NMR (400 MHz, CDCl3) δ 8.63 (d, 1H), 8.28 (s, 1H), 7.17-7.06 (m, 1H), 6.94 (t, 3H), 5.91 (s, 1H), 5.38 (s, 1H), 4.81 (d, 1H), 4.57 (d, 1H), 4.23 (d, 2H), 3.64 (d, 1H), 3.05 (t, 2H), 2.72 (t, 2H), 2.69 (bs, 1H), 2.08 (m, 2H), 1.85-1.66 (m, 2H), 1.77 (s, 3H), 0.83 (m, 2H), 0.54 (m, 2H). MS (EI) for C27H28F5N7O3. found: 594.2 (MH+). Analytical HPLC, ret. time=12.48 min, 97% purity.
Compound D35 was made in a manner similar to Compound D34: 1H-NMR (400 MHz, CDCl3, DMSO-d6): δ 8.62 (d, 1H), 8.23 (br s, 1H), 7.32 (s, 1H), 6.97 (d, 1H), 6.94 (t, 2H), 5.61 (br s, 1H), 4.70 (d, 1H), 4.61 (d, 1H), 4.34 (d, 2H), 3.80 (m, 1H), 3.58 (m, 2H), 3.00 (m, 2H), 2.01 (m, 2H), 1.80 (m, 2H), 1.72 (s, 3H), 1.30 (t, 3H). MS (EI) for C26H28F5N7O3. found 582.2 (MH+). Analytical HPLC, ret. time=13.528 min, 95% purity.
Compound D36 was made in a manner similar to Compound D34: 1H-NMR (400 MHz, CDCl3): δ 8.62 (d, 1H), 8.23 (s, 1H), 7.12 (m, 1H), 6.94 (t, 2H), 6.95 (d, 1H), 5.92 (s, 1H), 5.03 (m, 1H), 4.81 (d, 1H), 4.56 (d, 1H), 4.23 (d, 2H), 3.64 (tt, 1H), 3.05 (tt, 2H), 3.00 (d, 3H), 2.66 (s, 1H), 2.11 (m, 2H), 1.78 (m, 2H), 1.72 (s, 3H). MS (EI) for C25H26F5N7O3. found 568.2 (MH+). Analytical HPLC, ret. time=12.940 min, 97% purity.
Compound D37 was made in a manner similar to Compound D34: 1H NMR (400 MHz, CDCl3) δ 8.62 (d, 1H), 8.19 (s, 1H), 7.25 (m, 1H), 7.10 (m, 1H), 6.99-6.88 (m, 3H), 6.50-6.40 (m, 2H), 5.93 (s, 1H), 5.69-5.51 (m, 1H), 4.80 (d, 1H), 4.60-4.48 (m, 3H), 4.25 (d, 2H), 3.85 (s, 3H), 3.79 (s, 3H), 3.62 (m, 1H), 3.05 (m, 2H), 2.12 (m, 2H), 1.76 (m, 2H), 1.70 (s, 3H). MS (EI) for C33H34F5N7O5. found: 704.2 (MH+). Analytical HPLC, ret. time=14.58 min, 95% purity.
Intermediate M2.1 was made from the Boc deprotection of intermediate D28.1 using General Procedure 2.
Compound M2 was made from intermediate M2.1 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, CDCl3) δ 8.85 (s, 1H), 8.69 (d, 1H), 7.17-7.05 (m, 2H), 6.93 (t, 2H), 5.88 (s, 1H), 5.52 (s, 2H), 4.21 (d, 2H), 3.50 (t, 1H), 3.02 (t, 2H), 2.63 (s, 3H), 2.09-1.93 (m, 2H), 1.85 (d, 2H). MS (EI) for C24H21F5N6O3S. found: 569.1 (MH+). Analytical HPLC, ret. time=19.42 min, 99% purity.
Intermediate D38.1 was made from Compound M2 using General Procedure 6.
Intermediate D38.2 was made from intermediate D38.1 and dimethylamine using a method analogous to the conversion of intermediate B53.1 to Compound B60.
Compound D38 was made from intermediate D38.2 using a method analogous to the synthesis of intermediate D24.3 from D24.2. 1H NMR (400 MHz, CDCl3) δ 8.61 (d, 1H), 8.26 (s, 1H), 7.16-7.07 (m, 1H), 6.93 (dd, 3H), 5.91 (s, 1H), 4.81 (d, 1H), 4.55 (d, 1H), 4.23 (d, 2H), 3.64 (t, 1H), 3.18 (s, 6H), 3.06 (t, 2H), 2.55 (s, 1H), 2.22-1.98 (m, 3H), 1.77 (m, 2H), 1.71 (s, 3H). MS (EI) for C26H28F5N7O3. found: 582.2 (MH+). Analytical HPLC, ret. time=12.53 min, 97% purity.
Compound D39 was made in a manner similar to Compound D39: 1H NMR (400 MHz, CDCl3) δ 8.63 (d, 1H), 8.26 (s, 1H), 7.12 (tt, 1H), 6.99-6.88 (m, 3H), 6.08-5.80 (tt, 1H), 5.93 (s, 1H), 5.45 (bs, 1H), 4.79 (d, 1H), 4.60 (d, 1H), 4.23 (d, 2H), 3.92-3.76 (m, 2H), 3.73-3.60 (m, 1H), 3.04 (t, 2H), 2.70 (bs, 1H), 2.17-1.97 (m, 2H), 1.83-1.74 (m, 2H), 1.73 (s, 3H). MS (EI) for C26H26F7N7O3. found: 617.9 (MH+). Analytical HPLC, ret. time=14.76 min, 99% purity.
A solution of intermediate D29.1 (45.3 mg, 0.07 mmol) in isopropyl alcohol (3 mL) was treated with sodium (˜7 mg). The resulting mixture was monitored by LC/MS and quenched with an ice upon completion in 15 min at room temperature. The mixture was diluted with water (5 mL) and extracted with ethyl acetate (3×10 mL). The combined organic layers were dried over magnesium sulfate and concentrated in vacuo. The crude product was purified by prep HPLC to give Compound D40; 1H NMR (400 MHz, CDCl3) δ 8.63 (d, 1H), 8.46 (s, 1H), 7.17-7.06 (m, 1H), 7.01-6.88 (m, 3H), 5.89 (s, 1H), 5.39-5.31 (m, 1H), 5.28 (dt, 1H), 4.82 (d, 1H), 4.65 (d, 1H), 4.26 (d, 2H), 3.71 (dd, 1H), 3.06 (t, 2H), 2.80 (bs, 1H), 2.30-2.09 (m, 2H), 2.06-1.97 (m, 2H), 1.82 (d, 2H), 1.76 (s, 3H), 1.40 (d, 6H). MS (EI) for C27H29F5N6O4. found: 597.2 (MH+). Analytical HPLC, ret. time=17.84 min, 99% purity.
Compound D41 was made from intermediate D29.1 in a manner similar to Compound D40: 1H NMR (400 MHz, CDCl3) δ 8.64 (d, 1H), 8.52 (s, 1H), 7.16-7.07 (m, 1H), 6.99-6.90 (m, 3H), 6.29-6.01 (tt, 1H), 5.91 (s, 1H), 4.82 (d, 1H), 4.68 (d, 1H), 4.60 (td, 2H), 4.26 (d, 2H), 3.75 (t, 1H), 3.06 (t, 2H), 2.24-1.95 (m, 2H), 1.87-1.78 (m, 2H), 1.77 (s, 3H). MS (EI) for C26H25F7N6O4. found: 619.2 (MH+). Analytical HPLC, ret. time=17.78 min, 99% purity.
Compound D41 was made from intermediate D29.1 in a manner similar to Compound D40: 1H NMR (400 MHz, CDCl3) δ 8.65 (d, 1H), 8.52 (s, 1H), 7.12 (t, 1H), 7.00-6.90 (m, 3H), 5.89 (s, 1H), 5.58 (t, 1H), 4.87-4.80 (m, 3H), 4.70 (dd, 2H), 4.26 (d, 2H), 3.74 (s, 1H), 3.06 (t, 2H), 2.09 (d, 2H), 2.04 (s, 1H), 1.81 (m, 2H), 1.77 (s, 3H). MS (EI) for C27H27F7N6O4. found: 633.2 (MH+). Analytical HPLC, ret. time=17.62 min, 99% purity.
Compound D41 was made from intermediate D29.1 in a manner similar to Compound D40: 1H NMR (400 MHz, CDCl3) δ 8.64 (d, 1H), 8.50 (s, 1H), 7.17-7.08 (m 1H), 6.99-6.88 (m, 3H), 5.90 (s, 1H), 4.84 (dd, 2H), 4.75-4.70 (m, 1H), 4.70-4.65 (m, 2H), 4.63-4.59 (m, 1H), 4.26 (d, 2H), 3.73 (t, 1H), 3.06 (t, 2H), 2.22-2.06 (m, 3H), 1.79 (d, 2H), 1.77 (s, 3H). MS (EI) for C26H26F6N6O4. found: 601.2 (MH+). Analytical HPLC, ret. time=16.38 min, 99% purity.
Compound D41 was made from intermediate D29.1 in a manner similar to Compound D40: 1H NMR (400 MHz, CDCl3) δ 8.64 (d, 1H), 8.48 (s, 1H), 7.17-7.07 (m, 1H), 6.94 (dd, 3H), 5.91 (s, 1H), 4.84 (d, 1H), 4.66 (d, 1H), 4.26 (d, 2H), 4.01 (s, 3H), 3.72 (t, 1H), 3.06 (t, 2H), 2.25-2.07 (m, 2H), 1.76 (s, 3H). MS (EI) for C25H25F5N6O4. found: 569.2 (MH+). Analytical HPLC, ret. time=17.16 min, 99% purity.
Compound D41 was made from intermediate D12.1 in a manner similar to Compound D40: 1H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H), 7.78 (t, 1H), 7.34 (d, 1H), 7.17-7.06 (m, 1H), 6.98-6.89 (m, 3H), 5.89 (s, 1H), 4.79 (d, 1H), 4.59 (d, 1H), 4.25 (d, 2H), 4.00 (s, 3H), 3.83-3.62 (m, 2H), 3.04 (tt, 2H), 2.22-2.08 (m, 3H), 1.82 (dd, 2H), 1.72 (s, 3H). MS (EI) for C26H26F5N5O4. found: 568.2 (MH+). Analytical HPLC, ret. time=14.05 min, 99% purity.
Intermediate M3.1 was synthesized from intermediate M2.1 and 2,6-difluorophenyl chloroformate using a method analogous to that used to make Compound A119 from intermediate A116.3 and 2-chlorophenyl chloroformate.
Intermediate M3.2 was made from intermediate M3.1 using General Procedure 6.
Compound M3 was made from intermediate M3.2 using General Procedure 7. 1H NMR (400 MHz, CDCl3) δ δ 8.75 (s, 1H), 8.67 (d, 1H), 7.19-7.07 (m, 2H), 6.96 (t, 2H), 5.52 (dd, 4H), 4.41 (d, 1H), 4.30 (d, 1H), 3.61 (ddd, 1H), 3.11 (t, 1H), 2.96 (t, 1H), 1.88 (m, 4H). MS (EI) for C23H19F5N6O4. found: 539.2 (MH+). Analytical HPLC, ret. time=14.79 min, 99% purity.
Compound D46 was made from Compound M3 using a method analogous to that used to convert intermediate D24.2 to intermediate D24.3. 1H NMR (400 MHz, CDCl3) δ 8.63 (d, 1H), 8.23 (s, 1H), 7.14 (dq, 1H), 6.97 (dd, 3H), 5.15 (s, 2H), 4.80 (d, 1H), 4.60 (d, 1H), 4.46 (d, 1H), 4.35 (d, 1H), 3.64 (t, 1H), 3.13 (t, 1H), 2.98 (t, 1H), 2.70 (bs, 1H), 2.10 (m, 2H), 1.85-1.71 (m, 2H), 1.76 (s, 3H). MS (EI) for C24H23F5N6O4. found: 555.2 (MH+). Analytical HPLC, ret. time=15.62 min, 97% purity.
To a −78° C. solution of 2-methyl-6-trifluoromethylpyridine (320 mg, 2.0 mmol) in THF (6 mL) was added nBuLi (0.8 mL, 2.5 M in hexanes, 2.0 mmol). The resulting solution was stirred at −78° C. for 1 h. A solution of intermediate B36.1 (128 mg, 0.4 mmol) in THF (1 mL) was added. The mixture was stirred for 2 h and was allowed to warm to rt gradually. Water was added slowly to quench the reaction. EtOAc was added to extract the product. The organic phase was washed with saturated NaHCO3, brine, and dried over anhydrous Na2SO4. After removal of the solvents, the residue was purified by flash column chromatography to give intermediate D47.1 (30 mg, 16%).
Intermediate D47.2 was made from the Boc deprotection of intermediate D47.1 using General Procedure 2.
Compound D47 was synthesized from intermediate D47.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.26 (s, 1H), 8.21 (st, 1H), 7.91 (t, 1H), 7.60 (d, 1H), 7.52 (d, 1H), 7.24 (m, 1H), 7.08 (m, 2H), 5.76 (s, 1H), 4.25 (m, 2H), 3.50 (m, 3H), 2.85 (m, 2H), 2.45 (s, 3H), 1.80 (m, 4H), 1.62 (s, 3H). MS (EI) for C26H26F5N5O2. found: 536.2 (MH+). Analytical HPLC, ret. time=13.30 min, 100% purity.
Compound D48 was made in five steps from intermediate D26.2 in the same manner that Compound D46 was made in five steps from intermediate D28.1. 1H NMR (400 MHz, CDCl3) δ 8.60 (d, 1H), 8.13 (s, 1H), 7.25 (d, 1H), 7.15 (m, 1H), 6.98 (m, 3H), 5.11 (s, 2H), 4.47 (dd, 1H), 4.37 (d, 1H), 4.36 (dd, 1H), 4.12 (d, 1H), 3.69 (t, 1H), 3.13 (t, 1H), 2.97 (t, 1H), 2.57 (bs, 1H), 3.22 (m, 2H), 1.90 (m, 2H), 1.89 (s, 3H). MS (EI) for C25H24F5N5O4. found: 554.2 (MH+). Analytical HPLC, ret. time=11.25 min, 94% purity.
Compound D49 was made in four steps from intermediate M2.1 in the same manner that Compound D46 was made in four steps from intermediate M2.1, substituting 2,6-difluorophenyl chloroformate with 2-fluorophenyl chloroformate in the first step. 1H NMR (400 MHz, CDCl3) δ 8.63 (d, 1H), 8.26 (bs, 1H), 7.18 (m, 4H), 6.95 (d, 1H), 5.01 (s, 2H), 4.81 (d, 1H), 4.60 (d, 1H), 4.42 (dd, 2H), 3.64 (m, 1H), 3.11 (s, 1H), 2.95 (s, 1H), 2.72 (s, 1H), 2.09 (m, 2H), 1.74 (s, 3H), 1.73 (m, 2H). MS (EI) for C24H24F4N6O4. found: 537.2 (MH+). Analytical HPLC, ret. time=13.66 min, 99% purity.
Compound D50 was made in four steps from intermediate M2.1 in the same manner that Compound D46 was made in four steps from intermediate M2.1, substituting 2,6-difluorophenyl chloroformate with 4-fluorophenyl chloroformate in the first step. 1H NMR (400 MHz, CDCl3) δ 8.64 (d, 1H), 8.24 (s, 1H), 7.14-7.01 (m, 4H), 6.95 (d, 1H), 5.17 (bs, 2H), 4.81 (d, 1H), 4.61 (d, 1H), 4.40 (t, 2H), 3.64 (t, 1H), 3.07 (t, 1H), 2.92 (t, 1H), 2.74 (bs, 1H), 2.15-1.94 (m, 2H), 1.81 (m, 2H), 1.73 (s, 3H). MS (EI) for C24H24F4N6O4. found: 537.2 (MH+). Analytical HPLC, ret. time=13.72 min, 98% purity.
To a solution of intermediate D24.3 (37 mg, 0.07) in DCM (1 mL) at 0° C. was added DAST (30 mg, 0.19 mmol) with stirring. The solution was warmed to RT. After 25 min, the reaction mixture was quenched with saturated aqueous NaHCO3 solution, and diluted with EtOAc. The organic phase was separated, washed with brine, dried (MgSO4), and concentrated. The crude product was purified on prep HPLC to give intermediate E12.1 (16 mg). 1H-NMR (400 MHz, CDCl3): δ 8.54 (d, 1H), 7.72 (t, 1H), 7.28 (d, 1H), 6.91 (d, 1H), 4.76 (dd, 2H), 4.23 (m, 2H), 3.29 (m, 1H), 2.79 (m, 2H), 2.67 (s, 3H), 2.04 (m, 1H), 1.92 (m, 1H), 1.86 (d, 3H), 1.65 (m, 2H), 1.47 (s, 9H). MS (EI) for C24H30F4N4O3. found: 499.3 (MH+).
Intermediate E12.2 was made from the Boc deprotection of intermediate E12.1 using General Procedure 2.
Compound E12 was made from intermediate E12.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.55 (d, 1H), 7.74 (dd, 1H), 7.30 (d, 1H), 7.13-7.10 (m, 1H), 6.95-6.92 (m, 3H), 5.92 (s, 1H), 4.76 (m, 2H), 4.25 (m, 2H), 3.43 (m, 1H), 3.06 (m, 2H), 2.69 (s, 3H), 2.21-2.09 (m, 2H), 1.89 (d, 3H), 1.81-1.75 (m, 2H). MS (EI) for C26H25F6N5O2. found: 554.2 (MH+). Analytical HPLC, ret. time=18.8 min, 91% purity.
Compound E13 was made in a manner similar to Compound E12: 1H-NMR (400 MHz, CDCl3): δ 8.55 (d, 1H), 7.74 (dd, 1H), 7.44 (d, 1H), 7.37-7.30 (m, 3H), 6.93 (d, 1H), 6.10 (s, 1H), 4.77 (m, 2H), 4.20 (m, 2H), 3.43 (m, 1H), 3.08 (m, 2H), 2.69 (s, 3H), 2.23 (m, 1H), 2.09 (m, 1H), 1.89 (d, 3H), 1.81 (m, 2H). MS (EI) for C27H25F8N5O2. found: 604.2 (MH+). Analytical HPLC, ret. time=19.9 min, 92% purity.
To a solution of 3,5-dimethylphenol (244 mg, 2.0 mmol) in DMF (4 mL) was added NaH (120 mg, 3 mmol, 60% in mineral oil). The resulting mixture was stirred at room temperature for 30 min, then D2.1 (1 mmol) was added. The mixture was stirred at 75° C. for 8 h, cooled to room temperature and diluted with aq. sat. NaHCO3. The crude product was extracted with EtOAc, washed with brine, and concentrated. The crude product was purified by flash column chromatography.
Intermediate E14.2 was made from the Boc deprotection of intermediate E14.1 using General Procedure 2.
Intermediate E14.3 was made from intermediate E14.2 and 2,6-difluorophenylacetic acid using General Procedure 4. 1H-NMR (400 MHz, DMSO-d6): δ 8.56 (d, 1H), 7.36 (m, 1H), 7.07 (m, 2H), 6.55 (m, 3H), 5.70 (s, 1H), 4.45 (d, 1H), 4.18 (m, 3H), 3.78 (m, 3H), 3.17 (m, 1H), 2.62 (m, 1H), 2.55 (s, 3H), 2.21 (s, 6H), 1.75 (m, 4H), 1.65 (s, 3H). MS (EI) for C29H33F2N3O3. found: 510.0 (MH+). Analytical HPLC, ret. time=17.17 min, 97% purity.
Compound E14 was made from intermediate E14.3 using a method analogous to that used to convert intermediate D24.3 to intermediate E12.1. 1H-NMR (400 MHz, CDCl3): δ 8.52 (s, 1H), 7.22 (m, 1H), 6.90 (dd, 2H), 6.04 (s, 1H), 6.50 (s, 2H), 4.77 (m, 1H), 4.25 (m, 1H), 4.11 (m, 1H), 3.77 (s, 2H), 3.47 (m, 1H), 3.21 (m, 1H), 2.69 (s, 3H), 2.27 (s, 6H), 2.17-2.00 (m, 2H), 1.90 (d, 3H), 1.80-1.72 (m, 2H). MS (EI) for C29H32F3N3O2. found: 512.3 (MH+). Analytical HPLC, ret. time=22.1 min, 85% purity.
The following compound was made from Compound D17 in a manner similar to the way Compound E14 was made from intermediate E14.3:
Compound E15 was made from intermediate D17 in a manner similar to the way Compound E14 was made from intermediate E14.3: 1H-NMR (400 MHz, CDCl3): δ 8.64 (br. s, 1H), 8.30 (s, 1H), 7.13 (m, 1H), 6.70-6.93 (m, 2H), 6.54 (br. s, 1H), 5.91 (s, 1H), 5.44 (br. s, 1H), 4.37-4.23 (m, 4H), 3.99 (m, 1H), 3.30 (m, 1H), 3.16-3.02 (m, 2H), 2.74 (s, 3H), 2.19 (m, 2H), 1.83-1.75 (m, 5H). MS (EI) for C25H25F6N7O. found: 554.2 (MH+). Analytical HPLC, ret. time=15.3 min, 98% purity.
Intermediate M4.1 was made from the Boc deprotection of intermediate D24.2 using General Procedure 2.
Compound M4 was made from intermediate M4.1 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.99 (s, 1H), 7.80 (dd, 1H), 7.32 (d, 1H), 7.12-7.05 (m, 2H), 6.93 (m, 2H), 5.89 (s, 1H), 5.41 (s, 2H), 4.19 (d, 1H), 3.39 (m, 1H), 3.02 (m, 1H), 2.07-1.97 (m, 2H), 1.81 (d, 2H). MS (EI) for C25H22F5N5O3. found: 536.2 (MH+). Analytical HPLC, ret. time=18.0 min, 98% purity.
To a solution of Compound M4 (60 mg, 0.12 mmol) in MeOH (1 mL) were added NH4OAc (270 mg, 3.5 mmol) and Na(CN)BH3 (20 mg, 0.32 mmol). The mixture was stirred at rt for 48 h. MeOH was removed. The residue was extracted with EtOAc. The organic phase was washed with saturated NaHCO3, brine, and dried over anhydrous Na2SO4. After removal of the solvents, the residue was purified by preparative HPLC to give Compound N1 (23 mg, 35%). 1H NMR (400 MHz, DMSO-d6) δ 8.72 (s, 1H), 8.23 (s, 1H), 7.97 (t, 1H), 7.50 (d, 1H), 7.25 (m, 1H), 7.11 (m, 3H), 5.90 (m, 1H), 5.34 (m, 1H), 4.47 (m, 2H), 4.20 (d, 1H), 3.22 (m, 1H), 2.82 (m, 2H), 2.57 (s, 3H), 1.62 (m, 4H). MS (EI) for C25H24F5N5O3. found: 538.2 (MH+). Analytical HPLC, ret. time=14.93 min, 97.8% purity.
Intermediate M5.1 was made from intermediate M2.1 and 2,6-difluorophenylacetic acid using General Procedure 4.
Intermediate M5.2 was made from intermediate M5.1 using General Procedure 6.
Compound M5 was synthesized from intermediate M5.2 using General Procedure 7. 1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 8.66 (d, 1H), 7.21 (td, 1H), 7.10 (d, 1H), 6.94-6.84 (m, 2H), 5.51 (q, 4H), 4.70 (d, 1H), 4.05 (d, 1H), 3.73 (s, 2H), 3.68-3.56 (m, 1H), 3.20 (t, 1H), 2.66 (dd, 1H), 2.11 (s, 1H), 1.89-1.72 (m, 4H). MS (EI) for C24H21F5N6O3. found: 537.1 (MH+). Analytical HPLC, ret. time=12.62 min, 99% purity.
Compound M6 was synthesized from intermediate D38.1 using General Procedure 7. 1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 8.67 (d, 1H), 7.14-7.05 (m, 2H), 6.97-6.89 (m, 2H), 5.87 (s, 1H), 5.52 (m, 4H), 4.19 (d, 2H), 3.67-3.53 (m, 1H), 3.00 (dd, 2H), 1.98-1.74 (m, 4H). MS (EI) for C23H20F5N7O3. found: 538.1 (MH+). Analytical HPLC, ret. time=15.76 min, 97% purity.
Compound M7 was synthesized from intermediate D38.1 using a method analogous to that used to convert intermediate B53.1 to Compound B60.1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 8.66 (d, 1H), 7.10 (d, 2H), 6.93 (t, 2H), 5.87 (s, 1H), 5.85 (s, 1H), 5.52 (s, 2H), 4.69 (t, 1H), 4.58 (t, 1H), 4.18 (d, 2H), 3.95-3.77 (m, 2H), 3.65 (bs, 1H), 3.02 (t, 2H), 1.83 (m, 4H). MS (EI) for C25H23F6N7O3. found: 584.1 (MH+). Analytical HPLC, ret. time=17.30 min, 98% purity.
To a solution of Compound M6 (55.9 mg, 0.104 mmol) in MeOH (2 mL) was added NaBH4 (12 mg, 0.312 mmol) at 0° C. The reaction mixture was stirred at 0° C. for 15 min. The mixture was monitored by LC/MS and quenched with water at 0° C. upon completion. The resulting mixture was diluted with EtOAc, washed with water, brine, dried over magnesium sulfate and concentrated in vacuo to give a crude product, which was purified by preparative HPLC to give Compound N2 (17 mg, 30.3%). 1H NMR (400 MHz, CDCl3) δ 8.64 (d, 1H), 8.41 (s, 1H), 7.12 (dt, 1H), 7.03-6.89 (m, 3H), 5.90 (s, 1H), 5.32 (dd, 1H), 5.15 (s, 2H), 4.69 (dd, 1H), 4.51 (dd, 1H), 4.23 (d, 2H), 3.07 (dt, 3H), 2.19-1.96 (m, 2H), 1.87-1.69 (m, 2H). MS (EI) for C23H22F5N7O3. found: 540.2 (MH+). Analytical HPLC, ret. time=11.30 min, 99% purity.
Intermediate N3.1 was made from the Boc deprotection of intermediate D26.2 using General Procedure 2.
Intermediate N3.2 was made from intermediate N3.1 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Intermediate N3.3 was made from intermediate N3.2 using General Procedure 6.
Intermediate N3.4 was made from intermediate N3.3 using General Procedure 7.
Compound N3 was made from intermediate N3.4 using a method analogous to that used to convert Compound M6 to compound N2. 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.80 (t, 1H), 7.35 (d, 1H), 7.15-7.06 (m, 1H), 7.01 (t, 1H), 6.98-6.89 (m, 2H), 5.89 (s, 1H), 5.32 (dd, 1H), 5.03 (s, 2H), 4.63 (dd, 1H), 4.42 (dd, 1H), 4.22 (d, 2H), 3.07 (m, 3H), 2.16-1.94 (m, 2H), 1.86-1.70 (m, 2H). MS (EI) for C24H23F5N6O3. found: 539.2 (MH+). Analytical HPLC, ret. time=9.76 min, 99% purity.
Intermediate N4.1 was made from intermediate N3.3 using a method analogous to that used to convert intermediate D29.1 to Compound D40.
Compound N4 was made from intermediate N4.1 using a method analogous to that used to convert Compound M6 to compound N2. 1H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 7.81 (t, 1H), 7.36 (d, 1H), 7.12 (s, 1H), 7.05-6.98 (m, 1H), 6.94 (t, 2H), 5.88 (s, 1H), 5.39 (dd, 1H), 4.67 (dd, 1H), 4.44 (dd, 1H), 4.23 (m, 2H), 4.03 (s, 3H), 3.26 (d, 1H), 3.20 (m, 1H), 3.06 (m, 2H), 2.05 (m, 2H), 1.82 (m, 2H). MS (EI) for C25H24F5N5O4. found: 554.1 (MH+). Analytical HPLC, ret. time=18.02 min, 99% purity.
To a solution of intermediate B46.2 (870 mg, 2.7 mmol) in dry THF (25 mL) was added MeMgBr (5.0 mL, 1.4M in 1:3 THF:Toluene, 7.0 mmol). The resulting mixture was stirred at room temperature and monitored by LC-MS until complete. Upon completion, the reaction mixture was diluted with saturated aqueous NH4Cl and extracted with EtOAc (3×). The combined organic extractions were washed with saturated aqueous NaCl (1×), dried (Na2SO4) and concentrated in vacuo to give intermediate M8.1 which was used as is in subsequent reactions. MS (EI) for C18H29N3O3. found: 336.3 (MH+).
Intermediate M8.2 was made from intermediate M8.1 using a method analogous to the oxidation of intermediate B46.1 to intermediate B46.2.
Intermediate M8.3 was made from intermediate M8.2 using a method analogous to the conversion of intermediate C70.2 to intermediate C70.3.
Intermediate M8.4 was made from intermediate M8.3 and 2-(trifluoromethyl)pyrimidin-4-ol using a method analogous to the synthesis of intermediate D24.2 from intermediate D24.1.
Intermediate M8.5 was made from the Boc deprotection of intermediate M8.4 using General Procedure 2.
Compound M8 was made from intermediate M8.5 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.71 (d, 1H), 7.12 (m, 2H), 6.94 (m, 2H), 5.90 (s, 1H), 5.42 (s, 2H), 4.23 (m, 2H), 3.05 (m, 2H), 2.74 (m, 1H), 2.71 (s, 3H), 2.58 (s, 3H), 2.13 (m, 2H), 1.80 (m, 2H). MS (EI) for C25H23F5N6O3. found: 551.2 (MH+). Analytical HPLC, ret. time=17.00 min, 98% purity.
Compound N5 was made from Compound M8 using a method analogous to that used to convert Compound M6 to compound N2. 1H-NMR (400 MHz, CDCl3): δ 8.64 (d, 1H), 7.11 (m, 1H), 6.98 (d, 1H) 6.93 (m, 2H), 5.95 (s, 1H), 5.28 (m, 1H), 4.70 (m, 2H), 4.20 (m, 2H), 3.53 (m, 1H), 3.28 (m, 1H), 3.03 (m, 2H), 2.64 (s, 3H), 2.63 (s, 3H), 2.18 (m, 1H), 2.03 (m, 1H), 1.85-1.65 (m, 2H). MS (EI) for C25H25F5N6O3. found: 553.2 (MH+). Analytical HPLC, ret. time=12.22 min, 99% purity.
Intermediate M9.1 was made from intermediate M8.3 using a method analogous to the synthesis of intermediate D24.2 from intermediate D24.1.
Intermediate M9.2 was made from the Boc deprotection of intermediate M9.1 using General Procedure 2.
Compound M9 was made from intermediate M9.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 7.83 (t, 1H), 7.37 (d, 1H), 7.11 (m, 2H), 6.94 (m, 2H), 5.90 (s, 1H), 5.38 (s, 2H), 4.21 (m, 2H), 3.03 (m, 2H), 2.77 (m, 1H), 2.70 (s, 3H), 2.57 (s, 3H), 2.11 (m, 2H), 1.78 (m, 2H). MS (EI) for C26H24F5N5O3. found: 550.2 (MH+). Analytical HPLC, ret. time=14.57 min, 99% purity.
Compound N6 was made from Compound M4 using a method analogous to that used to convert Compound M6 to compound N2. 1H-NMR (400 MHz, CDCl3): δ 7.83 (t, 1H), 7.36 (d, 1H), 7.11 (m, 1H), 7.05 (d, 1H), 6.94 (m, 2H), 5.93 (s, 1H), 5.60 (dd, 1H), 4.70 (dd, 1H), 4.60 (dd, 1H), 4.24 (m, 2H), 3.55 (m, 1H), 3.38 (m, 1H), 3.03 (m, 2H), 2.65 (s, 3H), 2.64 (s, 3H), 2.25-2.00 (m, 2H), 1.87-1.70 (m, 2H). MS (EI) for C26H26F5N5O3. found: 552.2 (MH+). Analytical HPLC, ret. time=10.43 min, 99% purity.
Intermediate M10.1 was made from intermediate C70.3 and 2-(trifluoromethyl)pyrimidin-4-ol using a method analogous to the synthesis of intermediate D24.2 from intermediate D24.1.
Intermediate M10.2 was made from the Boc deprotection of intermediate M10.1 using General Procedure 2.
Intermediate M10.3 was made from intermediate M10.2 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Intermediate M10.4 was made from intermediate M10.3 using General Procedure 6.
Compound M10 was made from intermediate M10.4 using General Procedure 7. 1H-NMR (400 MHz, CDCl3): δ 8.68 (d, 1H), 7.09 (m, 2H), 6.93 (m, 2H), 5.84 (s, 1H), 5.39 (s, 2H), 5.20 (br s, 2H), 4.20 (m, 2H), 3.01 (m, 2H), 2.69 (m, 1H), 2.46 (s, 3H), 2.05 (m, 2H), 1.89 (m, 2H). MS (EI) for C24H22F5N7O3. found: 552.2 (MH+). Analytical HPLC, ret. time=14.44 min, 97% purity.
Compound N7 was made from Compound M10 using a method analogous to that used to convert Compound M6 to compound N2. 1H NMR (400 MHz, CDCl3) δ 8.64 (d, 1H), 7.15-7.07 (m, 1H), 6.99 (d, 1H), 6.93 (t, 2H), 5.90 (s, 1H), 5.50 (dd, 1H), 5.01 (s, 2H), 4.73 (dd, 1H), 4.63 (dd, 1H), 4.22 (t, 2H), 3.41 (t, 1H), 3.03 (td, 2H), 2.59 (bs, 1H), 2.51 (s, 3H), 2.17-1.93 (m, 4H), 1.75 (m, 4H). MS (EI) for C24H24F5N7O3. found: 554.2 (MH+). Analytical HPLC, ret. time=12.37 min, 99% purity.
Intermediate N8.1 was made from intermediate M10.4 using a method analogous to that used to convert intermediate D29.1 to Compound D40.
Compound N8 was made from intermediate N8.1 using a method analogous to that used to convert Compound M6 to compound N2. 1H NMR (400 MHz, CDCl3) δ 8.65 (d, 1H), 7.15-7.07 (m, 1H), 6.99 (d, 1H), 6.98-6.89 (m, 2H), 5.89 (s, 1H), 5.58 (d, 1H), 4.75 (dd, 1H), 4.66 (dd, 1H), 4.24 (td, 2H), 4.00 (s, 3H), 3.51 (t, 1H), 3.15-2.96 (m, 2H), 2.66 (s, 1H), 2.62 (s, 3H), 2.20 (dt, 1H), 2.08 (dt, 1H), 1.84 (d, 1H), 1.76 (d, 1H). MS (EI) for C25H25F5N6O4. found: 569.2 (MH+). Analytical HPLC, ret. time=15.82 min, 97% purity.
Intermediate M11.1 was made from intermediate C70.3 using a method analogous to the synthesis of intermediate D24.2 from intermediate D24.1.
Intermediate M11.2 was made from the Boc deprotection of intermediate M11.1 using General Procedure 2.
Intermediate M11.3 was made from intermediate M11.2 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Intermediate M11.4 was made from intermediate M11.3 using General Procedure 6.
Compound M11 was made from intermediate M11.4 using General Procedure 7. 1H-NMR (400 MHz, CDCl3): δ 7.81 (t, 1H), 7.34 (d, 1H), 7.10 (m, 2H), 6.93 (m, 2H), 5.88 (s, 1H), 5.32 (s, 2H), 5.25 (m, 2H), 4.19 (m, 2H), 3.01 (m, 2H), 2.72 (m, 1H), 2.47 (s, 3H), 2.00 (m, 2H), 1.79 (m, 2H). MS (EI) for C25H23F5N6O3. found: 551.2 (MH+). Analytical HPLC, ret. time=15.71 min, 99% purity.
Compound M12 was made from Compound M11 using a method analogous to that used to convert Compound D30 to Compound D31. 1H NMR (400 MHz, CDCl3) δ 7.93 (s, 1H), 7.82 (t, 1H), 7.37 (d, 1H), 7.16-7.06 (m, 2H), 6.98-6.89 (m, 2H), 5.89 (s, 1H), 5.34 (s, 2H), 4.20 (d, 2H), 3.03 (t, 2H), 2.80 (dd, 1H), 2.58 (d, 6H), 2.01 (qd, 2H), 1.80 (d, 2H). MS (EI) for C27H25F5N6O4. found: 593.1 (MH+). Analytical HPLC, ret. time=13.72 min, 99% purity.
Compound N9 was made from Compound M11 using a method analogous to that used to convert Compound M6 to compound N2. 1H-NMR (400 MHz, CDCl3): δ 7.73 (t, 1H), 7.28 (d, 1H), 7.03 (m, 1H), 6.96 (d, 1H), 6.86 (t, 2H), 5.81 (s, 1H), 5.43 (dd, 1H), 4.86 (s, 2H), 4.61 (dd, 1H), 4.49 (dd, 1H), 4.15 (t, 2H), 3.37 (m, 1H), 3.00-2.90 (m, 2H), 2.45 (s, 3H), 2.05-1.88 (m, 2H), 1.76-1.64 (m, 2H). MS (EI) for C25H25F5N6O3. found: 553.2 (MH+). Analytical HPLC, ret. time=13.39 min, 98% purity.
Compound N10 was made from Compound N10 using a method analogous to that used to convert Compound D30 to Compound D31. 1H NMR (400 MHz, CDCl3) δ 7.82 (dd, 2H), 7.37 (d, 1H), 7.16-7.07 (m, 1H), 7.04 (d, 1H), 6.94 (t, 2H), 5.92 (s, 1H), 5.56 (d, 1H), 4.64 (ddd, 2H), 4.23 (t, 2H), 3.57 (t, 1H), 3.27 (s, 1H), 3.12-2.95 (m, 2H), 2.61 (d, 6H), 2.17-1.91 (m, 2H), 1.87 (d, 1H), 1.75 (d, 1H). MS (EI) for C27H27F5N6O4. found: 595.1 (MH+). Analytical HPLC, ret. time=11.56 min, 99% purity.
Compound N11 was isolated as a side product generated from the reaction that made Compound N10. 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.77 (t, 1H), 7.33 (d, 1H), 7.18-7.07 (m, 1H), 6.94 (t, 3H), 6.56 (dd, 1H), 5.92 (s, 1H), 4.77 (ddd, 2H), 4.26 (dd, 2H), 3.45 (m, 1H), 3.08 (dt, 2H), 2.69 (s, 3H), 2.59 (s, 3H), 2.02 (s, 3H), 2.13-1.94 (m, 2H), 1.87 (d, 1H), 1.72-1.63 (m, 1H). MS (EI) for C29H29F5N6O5. found: 637.1 (MH+). Analytical HPLC, ret. time=18.27 min, 95% purity.
Intermediate N12.1 was synthesized from intermediate D26.1 using a method analogous to the conversion of intermediate C70.3 to intermediate C70.4.
Intermediate N12.2 was synthesized from intermediate N12.1 using a method analogous to the conversion of Compound M6 to compound N2.
Intermediate N12.3 was synthesized from intermediate N12.2 using a method analogous to that used to convert intermediate C68.2 to intermediate C68.3.
Intermediate N12.4 was synthesized from intermediate N12.3 using a method analogous to that used to synthesize intermediate D17.3 from intermediate D17.2.
Intermediate N12.5 was made from intermediate N12.4 using General Procedure 6.
To a 48 mL sealed-tube were added intermediate N12.5 (80.0 mg, 0.146 mmol), dioxane (4 mL) and conc. ammonium hydroxide (2 mL) at room temperature. The tube was sealed and heated at 100° C. overnight. After completion, the reaction mixture was diluted with 10 mL of water and extracted with EtOAc (3×10 mL). The combined organic layers were dried over anhydrous magnesium sulfate and concentrated in vacuo to give the desired crude intermediate N12.6 (50 mg, 71%); MS (EI) for C21H28F3N7O3. found 484.2 (MH+).
Intermediate N12.7 was synthesized from the Boc deprotection of intermediate N12.6 using General Procedure 2.
Compound N12 was synthesized from intermediate N12.7 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, CDCl3) δ 8.39 (s, 1H), 8.28 (d, 1H), 7.11 (dd, 1H), 6.96 (dd, 2H), 6.52 (d, 1H), 5.92 (s, 1H), 5.70 (bs, 1H), 5.17 (d, 1H), 5.04 (s, 2H), 4.21 (m, 2H), 4.04-3.86 (bs, 1H), 3.44 (s, 1H), 3.14-2.96 (m, 3H), 2.17-1.93 (m, 2H), 1.75 (d, 2H). MS (EI) for C23H23F5N8O2. found: 539.2 (MH+). Analytical HPLC, ret. time=7.82 min, 99% purity.
Compound M13 was made in three steps from intermediate M2.1 in the same manner that Compound M5 was made in three steps from intermediate M2.1, substituting 2,6-difluorobenzoic acid for 2-(2,6-difluorophenyl)acetic acid in the first step. 1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 8.66 (d, 1H), 7.33 (m, 2H), 7.10 (d, 1H), 6.95 (m, 2H), 5.50 (m, 4H), 4.88 (dd, 1H), 3.60 (m, 2H), 3.16 (dt, 1H), 2.87 (m, 1H), 1.88 (m, 2H), 1.73 (m, 2H). MS (EI) for C23H19F5N6O3. found: 523.2 (MH+). Analytical HPLC, ret. time=12.50 min, 98% purity.
Compound N13 was made in four steps from intermediate M2.1 in the same manner that Compound N2 was made in four steps from intermediate M2.1, substituting 2,6-difluorophenyl chloroformate for 2,6-difluorophenyl isocyanate in the first step and using standard reaction conditions typical with chloroformates such as those elaborated on in the synthesis of Compound A119 from intermediate A116.3. 1H NMR (400 MHz, CDCl3) δ 8.65 (d, 1H), 8.42 (s, 1H), 7.15 (dq, 1H), 7.03-6.92 (m, 3H), 5.33 (d, 1H), 5.08 (s, 2H), 4.69 (d, 1H), 4.56-4.41 (m, 2H), 4.36 (bs, 1H), 3.10 (m, 3H), 2.61 (s, 1H), 2.19-1.96 (m, 2H), 1.86-1.71 (m, 2H). MS (EI) for C23H21F5N6O4. found: 541.2 (MH+). Analytical HPLC, ret. time=13.72 min, 99% purity.
Compound M14 was made in three steps from intermediate N3.1 in the same manner that intermediate N3.4 was made in three steps from intermediate N3.1, substituting 2-fluorophenyl chloroformate for 2,6-difluorophenyl isocyanate in the first step and using standard reaction conditions typical with chloroformates such as those elaborated on in the synthesis of Compound A119 from intermediate A116.3. 1H NMR (400 MHz, CDCl3) δ 8.74 (s, 1H), 8.59 (d, 1H), 7.24-7.09 (m, 5H), 7.01-6.95 (m, 1H), 5.56 (s, 2H), 5.24 (s, 2H), 4.43 (d, 1H), 4.34 (d, 1H), 3.77-3.65 (m, 1H), 3.12 (t, 1H), 2.97 (t, 1H), 1.91 (dd, 2H), 1.80 (d, 2H). MS (EI) for C24H21F4N5O4. found: 520.2 (MH+). Analytical HPLC, ret. time=18.06 min, 95% purity.
To a stirred solution of tert-butyl intermediate D24.2 (50 mg, 0.10 mmol) in DCM (1 mL) was added bis(2-methoxyethyl)aminosulfur trifluoride (92 mg, 0.42 mmol). The reaction mixture was heated at 65° C. in a sealed tube with stirring for 21 h. The resulting mixture was cooled to 0° C. and partitioned between EtOAc and saturated aqueous NaHCO3. The aqueous layer was extracted with EtOAc. The combined organic portions were washed with brine, dried (MgSO4), filtered, and concentrated in vacuo. The residue was purified on prep. HPLC to give intermediate N14.1 (24 mg). 1H-NMR (400 MHz, CDCl3): δ 8.69 (s, 1H), 7.75 (t, 1H), 7.32 (d, 1H), 6.93 (d, 1H), 4.89 (t, 2H), 4.23 (m, 2H), 3.18 (m, 1H), 2.80 (m, 2H), 2.70 (s, 3H), 1.98 (m, 2H), 1.68 (m, 2H), 1.47 (s, 9H). MS (EI) for C23H27F5N4O3. found: 503.2 (MH+).
Intermediate N14.2 was synthesized from the Boc deprotection of intermediate N14.1 using General Procedure 2.
Compound N14 was synthesized from intermediate N14.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.72 (s, 1H), 7.77 (dd, 1H), 7.34 (d, 1H), 7.11 (m, 1H), 6.96-6.92 (m, 3H), 5.90 (s, 1H), 4.92 (t, 2H), 4.25 (d, 2H), 3.30 (m, 1H), 3.07 (t, 2H), 2.73 (s, 3H), 2.18-2.09 (m, 2H), 1.78 (d, 2H). MS (EI) for C25H22F7N5O2. found: 558.2 (MH+). Analytical HPLC, ret. time=19.9 min, 97% purity.
Compound N15 was made in a manner similar to Compound N14. 1H-NMR (400 MHz, CDCl3): δ 8.72 (s, 1H), 7.77 (dd, 1H), 7.44 (d, 1H), 7.37-7.30 (m, 3H), 6.95 (d, 1H), 6.10 (s, 1H), 4.92 (dd, 2H), 4.21 (d, 2H), 3.31 (m, 1H), 3.08 (m, 2H), 2.73 (s, 3H), 2.19-2.09 (m, 2H), 1.79 (d, 2H). MS (EI) for C26H22F9N5O2. found: 608.2 (MH+). Analytical HPLC, ret. time=21.2 min, 98% purity.
Intermediate N16.1 was synthesized from intermediate D26.2 using a method analogous to that used to synthesize intermediate N14.1 from intermediate D24.2.
Intermediate N16.2 was synthesized from the Boc deprotection of intermediate N16.1 using General Procedure 2.
Intermediate N16.3 was synthesized from intermediate N16.2 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Intermediate N16.4 was synthesized from intermediate N16.3 using General Procedure 6.
Compound N16 was synthesized from intermediate N16.4 using a method analogous to that used to convert intermediate C69.6 to Compound C69. 1H-NMR (400 MHz, CDCl3): δ 8.39 (s, 1H), 7.78 (t, 2H), 7.34 (d, 1H), 7.12 (m, 1H), 6.98 (d, 1H), 6.94 (t, 2H), 5.91 (s, 1H), 5.19 (s, 2H), 4.88 (t, 2H), 4.24 (d, 2H), 3.20 (tt, 1H), 3.05 (td, 2H), 2.04 (qd, 2H), 1.79 (m, 2H). MS (EI) for C24H21F7N6O2. found 559.0 (MH+). Analytical HPLC, ret. time=19.772 min, 95% purity.
Intermediate N17.1 was synthesized from intermediate D28.1 using a method analogous to that used to synthesize intermediate N14.1 from intermediate D24.2.
Intermediate N17.2 was synthesized from the Boc deprotection of intermediate N17.1 using General Procedure 2.
Intermediate N17.3 was synthesized from intermediate N17.2 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Intermediate N17.4 was synthesized from intermediate N17.3 using General Procedure 6.
Compound N17 was synthesized from intermediate N17.4 using a method analogous to that used to convert intermediate C69.6 to Compound C69. 1H-NMR (400 MHz, CDCl3): δ 8.67 (d, 1H), 8.38 (s, 1H), 7.12 (m, 1H), 7.00 (d, 1H), 6.95 (t, 2H), 5.91 (s, 1H), 5.23 (s, 2H), 4.94 (t, 2H), 4.25 (d, 2H), 3.15 (tt, 1H), 3.05 (td, 2H), 2.06 (qd, 2H), 1.79 (m, 2H). MS (EI) for C23H20F7N7O2. found 560.2 (MH+). Analytical HPLC, ret. time=16.280 min, 99% purity.
Intermediate N18.1 was synthesized from intermediate D24.1 using a method analogous to that used for the conversion of intermediate C70.3 to intermediate C70.4.
Intermediate N18.2 was made from intermediate N18.1 using a method analogous to that used to convert intermediate D24.2 to intermediate N14.1
To a solution of intermediate N18.2 (56 mg, 0.15 mmol) in THF-H2O (2 mL/0.1 mL) was added Ph3P (43 mg, 0.16 mmol). The mixture was stirred at 60° C. for 4 h, and then cooled to rt. The crude reaction solution was used as is in the next reaction.
To the crude reaction solution of intermediate N18.3 were added 4-chloro-2-trifluoromethylpyrimidine (55 mg, 0.30 mmol) and excess triethylamine. The reaction mixture was stirred at 60° C. for 24 h and cooled to rt. EtOAc was added to extract the product. The organic phase was washed with saturated NaHCO3, brine, and dried over anhydrous Na2SO4. After removal of the solvents, the residue was purified by flash column chromatography to give intermediate N18.4 (50 mg, 66%).
Intermediate N18.5 was made from the Boc deprotection of intermediate N18.4 using General Procedure 2.
Compound N18 was made from intermediate N18.5 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H), 8.59 (m, 1H), 8.29 (m, 2H), 7.28 (m, 1H), 7.11 (m, 2H), 6.81 (m, 1H), 4.24 (m, 2H), 3.60 (m, 2H), 3.45 (m, 1H), 2.80 (m, 1H), 2.62 (s, 3H), 1.62 (m, 5H). MS (EI) for C24H22F7N7O. found: 558.2 (MH+). Analytical HPLC, ret. time=16.06 min, 99.7% purity.
Compound N19 was made from intermediate N18.5 and 2,6-difluorophenylacetic acid using General Procedure 4. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.58 (m, 1H), 7.34 (m, 1H), 7.28 (m, 1H), 7.09 (m, 2H), 6.76 (m, 1H), 4.40 (m, 2H), 4.24 (m, 2H), 3.60 (m, 2H), 3.45 (m, 1H), 2.80 (m, 1H), 2.60 (s, 3H), 1.62 (m, 5H). MS (EI) for C25H23F7N6O. found: 557.2 (MH+). Analytical HPLC, ret. time=18.17 min, 99.7% purity.
Intermediate N20.1 was made from intermediate N18.3 and 2-chloro-4-(trifluoromethyl)pyrimidine using a method analogous to that used to synthesize intermediate N18.4 from intermediate N18.3
Intermediate N20.2 was made from the Boc deprotection of intermediate N20.1 using General Procedure 2.
Compound N20 was made from intermediate N20.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.67 (s, 1H), 8.59 (m, 1H), 8.29 (m, 2H), 7.26 (m, 1H), 7.08 (m, 2H), 7.02 (m, 1H), 4.24 (m, 2H), 3.60 (m, 2H), 3.45 (m, 1H), 2.80 (m, 1H), 2.58 (s, 3H), 1.62 (m, 5H). MS (EI) for C24H22F7N7O. found: 558.2 (MH+). Analytical HPLC, ret. time=16.75 min, 95% purity.
Compound N21 was made from intermediate N20.2 and 2,6-difluorophenylacetic acid using General Procedure 4. 1H NMR (400 MHz, DMSO-d6) δ 8.65 (s, 1H), 8.59 (m, 1H), 8.48 (m, 1H), 7.36 (m, 1H), 7.08 (m, 3H), 4.50 (m, 1H), 4.24 (m, 3H), 3.80 (m, 2H), 3.45 (m, 1H), 2.80 (m, 1H), 2.58 (s, 3H), 1.62 (m, 5H). MS (EI) for C25H23F7N6O. found: 557.2 (MH+). Analytical HPLC, ret. time=19.74 min, 98% purity.
Intermediate N22.1 was synthesized from intermediate D24.2 using a method analogous to the reduction of Compound M6 to Compound N2.
To a stirred solution of intermediate N22.1 (41 mg, 0.08 mmol) at 0° C. was added DAST (34 mg, 0.21 mmol). The mixture was stirred at 0° C. for 30 min and then partitioned between EtOAc and saturated aqueous NaHCO3. The aqueous layer was extracted with EtOAc. The combined organic portions were washed with brine, dried (MgSO4), filtered, and concentrated in vacuo to give a crude intermediate N22.2. The crude product was used directly in the next reaction without further purification. MS (EI) for C23H28F4N4O3. found: 485.2 (MH+).
Intermediate N22.3 was made from the Boc deprotection of intermediate N22.2 using General Procedure 2.
Compound N22 was synthesized from intermediate N22.3 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.55 (s, 1H), 7.65 (t, 1H), 7.20 (d, 1H), 6.98 (m, 1H), 6.88 (d, 1H), 6.83-6.78 (m 2H), 6.00 (ddd, 1H), 5.76 (s, 1H), 4.70-4.47 (m, 2H), 4.11 (d, 2H), 3.03-2.90 (m 3H), 2.58 (s, 3H), 2.13-1.88 (m, 2H), 1.65 (m, 2H). MS (EI) for C25H23F6N5O2. found: 540.2 (MH+). Analytical HPLC, ret. time=14.7 min, 98% purity.
Compound N23 was recovered from the same procedure that was used to generated Compound N. 1H NMR (400 MHz, DMSO-d6) δ 8.77 (s, 1H), 8.21 (s, 1H), 7.94 (t, 1H), 7.47 (d, 1H), 7.25 (m, 1H), 7.08 (m, 3H), 4.61 (m, 1H), 4.40 (m, 2H), 4.18 (m, 2H), 3.45 (m, 3H), 2.85 (m, 2H), 2.55 (s, 3H), 1.62 (m, 4H). MS (EI) for C25H25F5N6O2. found: 537.2 (MH+). Analytical HPLC, ret. time=12.47 min, 99% purity.
Compound N24 was made from intermediate N3.4 in a manner similar to the way Compound N23 was made from Compound M4. 1H-NMR (400 MHz, CDCl3): δ 8.50 (s, 1H), 7.76 (t, 1H), 7.31 (d, 1H), 7.12 (m, 1H), 6.95 (m, 3H), 5.93 (br s, 1H), 5.03 (br s, 2H), 4.69 (dd, 1H), 4.58 (dd, 1H), 4.22 (m, 3H), 3.18 (m, 1H), 3.03 (t, 2H), 2.18-1.82 (comp m, 4H), 1.76 (m, 2H). MS (EI) for C24H24F5N7O2. found: 538.1 (MH+). Analytical HPLC, ret. time=8.54 min, 98% purity.
Intermediate N25.1 was synthesized from intermediate D24.1 and 2-(trifluoromethyl)pyrimidin-4-ol using a method analogous to the method used to synthesize intermediate D24.2 from intermediate D24.1 and 6-(trifluoromethyl)pyridin-2-ol.
Intermediate N25.2 was synthesized from the Boc deprotection of intermediate N25.1 using General Procedure 2.
Intermediate N25.3 was made from intermediate N25.2 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Compound N25 was synthesized from intermediate N25.3 using a method analogous to that used to synthesize Compound N23 from Compound M4. 1H-NMR (400 MHz, DMSO-d6): δ 8.77 (d, 1H), 8.57 (s, 1H), 8.26 (s, 1H), 8.21 (d, 1H), 7.27 (m, 1H), 7.11 (m, 2H), 6.83 (d, 1H), 5.45 (m, 1H), 5.28 (m, 1H), 4.24 (m, 2H), 3.72 (m, 2H), 3.38 (m, 1H), 2.91 (m, 2H), 2.55 (s, 3H), 1.86 (m, 2H), 1.70 (m, 2H). MS (EI) for C24H24F5N7O2. found: 538.2 (MH+). Analytical HPLC, ret. time=9.30 min, 88% purity.
Compound M4 (32 mg, 0.06 mmol) was dissolved in EtOH (2 mL). Hydroxylamine hydrochloride (15 mg) was added. The reaction mixture was stirred at 40° C. for 12 h. EtOH was removed. The residue was purified by preparative HPLC to give a 5.6:1 mixture of the desired oximes. Data for the major isomer: 1H NMR (400 MHz, DMSO-d6) δ 12.04 (s, 1H), 8.45 (s, 1H), 8.24 (s, 1H), 7.88 (t, 1H), 7.47 (d, 1H), 7.25 (m, 1H), 7.11 (m, 2H), 6.96 (d, 1H), 5.60 (s, 1H), 4.16 (m, 2H), 3.45 (m, 2H), 3.08 (m, 1H), 2.82 (m, 2H), 2.52 (s, 3H), 1.72 (m, 2H), 1.50 (m, 2H). MS (EI) for C25H23F5N6O3. found: 551.2 (MH+). Analytical HPLC, ret. time=16.34 min, 5.6:1 mixture, 99% purity.
To a solution of intermediate N3.4 (60 mg, 0.11 mmol) in 2M methylamine in MeOH (1.5 mL, 3.0 mmol methylamine) was added AcOH (50 μL) and Na(CN)BH3 (21 mg, 0.33 mmol). The resulting mixture was heated with stirring to 45° C. overnight, followed by the addition of additional aliquots of Na(CN)BH3 (21 mg), 2M methylamine in MeOH (1.0 mL), and AcOH (50 μL) and heating in a sealed tube at 60° C. for several days. The reaction mixture was allowed to cool to room temperature, diluted with EtOAc and washed with saturated aqueous NaHCO3 (3×), saturated aqueous NaCl (1×) dried (Na2SO4) and concentrated in vacuo. The resulting residue was purified by preparative HPLC to give Compound N27 (10 mg, 16% yield). 1H-NMR (400 MHz, CDCl3): δ 8.53 (br s, 1H), 7.76 (t, 1H), 7.32 (d, 1H), 7.12 (m, 1H), 7.00 (d, 1H), 6.94 (t, 2H), 5.93 (br s, 1H), 5.21 (br s, 2H), 4.63 (m, 1H), 4.37 (m, 2H), 4.22 (m, 2H), 3.17 (m, 1H), 3.05 (m, 2H), 2.39 (s, 3H), 2.25-1.92 (m, 3H), 1.74 (m, 2H). MS (EI) for C25H26F5N7O2. found: 552.2 (MH+). Analytical HPLC, ret. time=8.65 min, 92% purity.
To a solution of intermediate N3.4 (106 mg, 0.20 mmol) in MeOH (3.0 mL) was added tert-butyl 4-aminopiperidine-1-carboxylate (1.16 g, 6.8 mmol), AcOH (150 μL) and Na(CN)BH3 (120 mg, 1.9 mmol). The resulting mixture was heated with stirring to 60° C. in a sealed tube for several days. The reaction mixture was allowed to cool to room temperature, diluted with EtOAc and washed with saturated aqueous NaHCO3 (3×), water (1×), aqueous 10% citric acid (3×), saturated aqueous NaCl (1×) dried (Na2SO4) and concentrated in vacuo to give crude intermediate N28.1 which was used as is in the next reaction. MS (EI) for C35H42F5N7O4. found: 720.3 (MH+).
Intermediate N28.2 was made from the Boc deprotection of intermediate N28.1 using General Procedure 2.
Compound N28 was made from intermediate N28.2 using a method analogous to that used to convert Compound D29 to Compound D31. 1H-NMR (400 MHz, CDCl3): δ 8.85 (br s, 1H), 7.78 (t, 1H), 7.35 (d, 1H), 7.12 (m, 1H), 6.94 (m, 3H), 5.93 (br s, 1H), 4.74 (m, 1H), 4.56 (m, 1H), 4.39 (m, 1H), 4.22 (m, 2H), 3.80-3.55 (m, 2H), 3.27 (m, 1H), 3.05 (m, 2H), 2.70 (s, 3H), 2.60 (m, 2H), 2.22 (m, 1H), 2.07 (s, 3H), 1.80-1.10 (comp m, 9H). MS (EI) for C32H36F5N7O3. found: 662.3 (MH+). Analytical HPLC, ret. time=9.85 min, 95% purity.
Compound N29 was made in four steps from intermediate N29.1 in the same manner that Compound N24 was made in four steps from intermediate N3.1, substituting 2-fluorophenyl chloroformate for 2,6-difluorophenyl isocyanate in the first step and using standard reaction conditions typical with chloroformates such as those elaborated on in the synthesis of Compound A119 from intermediate A116.3. 1H NMR (400 MHz, CDCl3) δ 8.22 (d, 1H), 8.20 (bs, 1H), 7.25-7.08 (m, 4H), 6.74 (d, 1H), 6.45 (s, 1H), 5.45 (s, 1H), 5.24 (s, 2H), 4.72 (d, 1H), 4.57-4.31 (td, 2H), 3.96 (d, 1H), 3.76 (d, 1H), 3.01 (ddd, 3H), 1.83 (s, 2H), 1.65 (d, 1H). MS (EI) for C24H24F4N6O3. found: 521.2 (MH+). Analytical HPLC, ret. time=10.70 min, 98% purity.
To a 0° C. solution of intermediate D24.2 (450 mg, 0.93 mmol) in THF (3 mL) was added dropwise allyl magnesium bromide (2.8 mL, 1.0 M in diethyl ether). The reaction mixture was stirred at 0° C. for 10 min, and then was quenched by slow addition of ice-water. The mixture was extracted with EtOAc, washed with brine, and dried over anhydrous Na2SO4. After removal of the solvents, the residue was purified by flash column chromatography to give intermediate N30.1 (290 mg, 60%).
Ozone was bubbled though a −78° C. solution of the intermediate N30.1 prepared above (33 mg, 0.063 mmol) in DCM/MeOH (5 mL/2 mL) until the blue color persisted. Nitrogen was then bubbled through the mixture to remove excess ozone. To this solution was added NaBH4 (10 mg, 0.26 mmol). The mixture was allowed to warm to rt gradually. Removal of the solvents gave crude intermediate N30.2, which was used in the next step without further purification.
Intermediate N30.3 was made from the Boc deprotection of intermediate N30.2 using General Procedure 2.
Compound N30 was made from intermediate N30.3 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.66 (s, 1H), 8.22 (s, 1H), 7.95 (t, 1H), 7.48 (m, 1H), 7.26 (m, 1H), 7.08 (m, 3H), 5.74 (s, 1H), 4.65 (m, 3H), 4.12 (m, 2H), 3.60 (m, 1H), 3.52 (m, 1H), 2.89 (m, 2H), 2.57 (s, 3H), 2.20 (m, 2H), 1.82 (m, 3H), 1.68 (d, 1H), 1.55 (d, 1H). MS (EI) for C27H28F5N5O4. found: 582.2 (MH+). Analytical HPLC, ret. time=14.88 min, 88% purity.
To a solution of intermediate N30.2 (80 mg, 0.15 mmol) in DCM (2 mL) were added pyridine (120 mg, 1.5 mmol) and tosyl chloride (43 mg, 0.23 mmol). The reaction mixture was stirred at rt for 12 h. Saturated NaHCO3 was added to quench the reaction. The mixture was diluted with DCM, washed with saturated NaHCO3, and dried over anhydrous Na2SO4. The solvents were removed under reduced pressure. The residue was dissolved in DMF (0.6 mL) to which was added NaN3 (65 mg, 1 mmol). The mixture was stirred at 55° C. for 24 h. The mixture was extracted with EtOAc, washed with brine and dried over anhydrous Na2SO4. Removal of the solvents gave the crude intermediate N31.1 which was used in the next step without further purification.
Intermediate N31.2 was made from the Boc deprotection of intermediate N31.1 using General Procedure 2.
Intermediate N31.3 was made from intermediate N31.2 and 2,6-difluorophenyl isocyanate using General Procedure 5.
Compound N31 was made from intermediate N31.3 using a method analogous to that used to convert intermediate N18.2 to intermediate N18.3. 1H NMR (400 MHz, DMSO-d6) δ 8.69 (s, 1H), 8.21 (s, 1H), 7.94 (t, 1H), 7.48 (d, 1H), 7.26 (m, 1H), 7.08 (m, 3H), 4.58 (s, 2H), 4.18 (m, 2H), 3.75 (m, 1H), 2.90 (m, 2H), 2.78 (m, 1H), 2.63 (m, 1H), 2.56 (s, 3H), 2.14 (m, 2H), 1.86 (m, 4H), 1.62 (m, 2H), 1.55 (d, 1H). MS (EI) for C27H29F5N6O3. found: 581.2 (MH+). Analytical HPLC, ret. time=11.91 min, 94% purity.
To a 0° C. suspension of methyltriphenylphosphonium bromide (1.1 g, 2.86 mmol) in THF (12 mL) was added KOtBu (2.86 mL, 2.86 mmol, 1.0 M in THF). The ice bath was removed and mixture was stirred at rt for 30 min. The mixture was cooled to 0° C. and a solution of intermediate D24.2 (920 mg, 1.91 mmol) in THF (1 mL) was added. Then the reaction mixture was stirred at rt for 12 h. Water was added to quench the reaction. The mixture was extracted with EtOAc, washed with brine, and dried over anhydrous Na2SO4. Further purification by flash column chromatography gave intermediate N32.1 (550 mg, 60%).
To a solution of intermediate N32.1 (100 mg, 0.21 mmol) in acetone/H2O/tBuOH (1 mL/0.2 mL/0.2 mL) were added NMO (50 mg, 0.42 mmol) and OsO4 (0.12 mL, 2.5% w/w solution in tBuOH). The reaction was stirred at rt for 48 h. The solvent was removed under reduced pressure, and the residue was extracted with EtOAc (10 mL) and washed with 5% aq HCl and saturated Na2S2O3 solution. The organic layer was dried over anhydrous Na2SO4. Further purification by flash column chromatography gave intermediate N32.2 (80 mg, 78%).
Intermediate N32.3 was made from the Boc deprotection of intermediate N32.2 using General Procedure 2.
Compound N32 was made from intermediate N32.3 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.58 (s, 1H), 8.24 (s, 1H), 7.95 (t, 1H), 7.49 (d, 1H), 7.27 (m, 1H), 7.10 (m, 3H), 5.73 (s, 1H), 5.19 (s, 1H), 4.84 (d, 1H), 4.57 (d, 1H), 4.20 (t, 1H), 3.82 (m, 3H), 2.83 (m, 2H), 2.57 (s, 3H), 1.80 (m, 4H). MS (EI) for C26H26F5N5O4. found: 568.2 (MH+). Analytical HPLC, ret. time=10.48 min, 98% purity.
Intermediate N33.1 was made from intermediate N32.2 using a method analogous to that used to make N31.1 from N30.2
Intermediate N33.2 was made from the Boc deprotection of intermediate N33.1 using General Procedure 2.
Compound N33 was made from intermediate N33.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 8.21 (s, 1H), 7.96 (t, 1H), 7.51 (d, 1H), 7.27 (m, 1H), 7.11 (m, 3H), 6.33 (s, 1H), 4.65 (q, 2H), 4.19 (m, 2H), 3.90 (m, 2H), 3.76 (m, 1H), 2.90 (m, 2H), 2.56 (s, 3H), 1.83 (m, 2H), 1.66 (m, 2H). MS (EI) for C26H25F5N8O3. found: 593.2 (MH+). Analytical HPLC, ret. time=17.09 min, 92% purity.
Compound N34 was made from Compound N33 using a method analogous to that used to convert intermediate N18.2 to intermediate N18.3. 1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 7.73 (t, 1H), 7.25 (m, 2H), 7.09 (m, 1H), 6.90 (m, 3H), 5.92 (s, 1H), 4.75 (m, 1H), 4.60 (m, 1H), 4.25 (m, 2H), 3.60 (m, 1H), 3.38 (m, 1H), 3.15 (m, 1H), 3.05 (m, 2H), 2.60 (s, 3H), 2.12 (m, 2H), 1.86 (m, 4H). MS (EI) for C26H27F5N6O3. found: 567.2 (MH+). Analytical HPLC, ret. time=12.21 min, 95% purity.
Intermediate N35.1 was made from intermediate N33.2 and 2,6-difluorophenylacetic acid using General Procedure 4.
Compound N35 was made from intermediate N35.1 using a method analogous to the conversion of intermediate C68.2 to intermediate C68.3. 1H-NMR (400 MHz, CDCl3, DMSO-d6): δ 8.66 (d, 1H), 7.79 (t, 1H), 7.34 (d, 1H), 7.24 (m, 1H), 6.99 (d, 1H), 6.91 (t, 2H), 4.74 (m, 1H), 4.70 (t, 2H), 4.12 (d, 1H), 3.78 (s, 2H), 3.66 (m, 1H), 3.48 (m, 1H), 3.29 (m, 2H), 2.73 (t, 2H), 2.66 (s, 3H), 2.04 (m, 2H), 1.79 (m, 2H). MS (EI) for C27H28F5N5O3. found 566.1 (MH+). Analytical HPLC, ret. time=10.216 min, 95% purity.
Intermediate N36.1 was made from intermediate N33.1 using a method analogous to that used to convert intermediate N35.1 to Compound N35.
Intermediate N36.2 was made from intermediate N36.1 and ethyl isocyanate using General Procedure 5. MS (EI) for C27H37F3N6O5. found 583.3 (MH+).
Intermediate N36.3 was made from the Boc deprotection of intermediate N36.2 using General Procedure 2.
Compound N36 was made from intermediate N36.3 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.58 (s, 1H), 7.74 (t, 1H), 7.31 (d, 1H), 7.11 (m, 1H), 6.94 (t, 2H), 6.89 (d, 1H), 6.24 (s, 1H), 5.93 (s, 1H), 5.21 (t, 1H), 4.91 (d, 1H), 4.65 (d, 1H), 4.50 (t, 1H), 4.23 (t, 1H), 3.76 (d, 2H), 3.75 (m, 1H), 3.21 (m, 2H), 3.09 (qd, 2H), 2.65 (s, 3H), 2.12 (m, 2H), 1.83 (m, 2H), 1.13 (t, 3H). MS (EI) for C29H32F5N7O4. found 638.3 (MH+). Analytical HPLC, ret. time=13.672 min, 99% purity.
To a stirred solution of N36.1 (6.5 mg, 0.013 mmol) in dichloromethane (0.3 mL) were added Et3N (3.9 mg, 0.039 mmol) and acetic anhydride (2.0 mg, 0.020 mmol) in sequence. After stirring for 40 min at room temperature, aqueous saturated NH4Cl (3 mL) was added and the resulting solution was extracted with EtOAc (2×1 mL). The combined organic layers were dried over MgSO4 and concentrated to give a crude intermediate N36.1. MS (EI) for C26H34F3N5O5. found 554.3 (MH+).
Intermediate N37.2 was made from the Boc deprotection of intermediate N37.1 using General Procedure 2.
Compound N37 was made from intermediate N37.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.62 (s, 1H), 7.76 (t, 1H), 7.34 (d, 1H), 7.11 (m, 1H), 6.94 (t, 2H), 6.90 (d, 1H), 6.33 (t, 1H), 5.92 (s, 1H), 5.74 (s, 1H), 4.93 (d, 1H), 4.70 (d, 1H), 4.26 (m, 2H), 3.86 (m, 1H), 3.76 (m, 1H), 3.63 (tt, 1H), 3.10 (qd, 2H), 2.65 (s, 3H), 2.15 (m, 2H), 2.03 (s, 3H), 1.85 (m, 2H). MS (EI) for C28H29F5N6O4. found 609.2 (MH+). Analytical HPLC, ret. time=10.628 min, 99% purity.
Intermediate N38.1 was synthesized by the oxidation of Compound N32 using a method analogous to that used to oxidize intermediate B46.1 to intermediate B46.2.
To a stirred solution of crude intermediate N38.1 (5.7 mg, 0.010 mmol), N-(2-aminoethyl)acetamide (1.7 mg, 90%, 0.015 mmol) and dichloromethane (0.5 mL) at room temperature was added NaBH(OAc)3 (6.4 mg, 0.030 mmol). After stirring for 40 min at 95° C., the resulting solution was purified by preparative HPLC to give Compound N38 (2.2 mg, 34%, 2 steps) as a white solid after lyophilization. 1H-NMR (400 MHz, CDCl3): δ 8.59 (s, 1H), 7.75 (t, 1H), 7.31 (d, 1H), 7.11 (m, 1H), 6.94 (t, 2H), 6.92 (d, 1H), 5.93 (s, 1H), 5.74 (t, 1H), 4.75 (d, 1H), 4.61 (d, 1H), 4.24 (d, 2H), 3.71 (tt, 1H), 3.35 (q, 2H), 3.27 (d, 1H), 3.07 (td, 2H), 3.06 (d, 1H), 2.80 (m, 2H), 2.67 (s, 3H), 2.12 (m, 2H), 1.98 (s, 3H), 1.78 (m, 2H). MS (EI) for C30H34F5N7O4. found 652.3 (MH+). Analytical HPLC, ret. time=11.496 min, 98% purity.
Compound N39 was made in a manner similar to Compound N38: 1H-NMR (400 MHz, CDCl3): δ 8.70 (s, 1H), 7.74 (t, 1H), 7.30 (d, 1H), 7.12 (m, 1H), 6.94 (t, 2H), 6.91 (d, 1H), 5.91 (s, 1H), 4.98 (br s, 1H), 4.74 (d, 1H), 4.59 (d, 1H), 4.25 (t, 2H), 3.60 (m, 3H), 3.43 (m, 2H), 3.13 (d, 1H), 3.04 (t, 2H), 2.92 (d, 1H), 2.67 (s, 3H), 2.54 (m, 4H), 2.14 (m, 2H), 2.06 (s, 3H), 1.75 (m, 2H). MS (EI) for C32H36F5N7O4. found 678.3 (MH+). Analytical HPLC, ret. time=11.540 min, 98% purity.
Compound N40 was made in a manner similar to Compound N38: 1H-NMR (400 MHz, CDCl3, a mixture of four stereomers): δ 8.55 and 8.53 (s, 1H), 7.76 (m, 1H), 7.32 (m, 1H), 7.12 (m, 1H), 6.93 (m, 3H), 5.98 (m, 1H), 4.77 (m, 1H), 4.55 (m, 1H), 4.25 (m, 2H), 3.76-3.55 (m, 3H), 3.52-3.18 (m, 4H), 3.12-2.93 (m, 3H), 2.68 (s, 3H), 2.23-2.06 (m, 3H), 2.05 and 2.03 (s, 3H), 1.85 (m, 2H), 1.70 (m, 2H). MS (EI) for C32H36F5N7O4. found 678.3 (MH+). Analytical HPLC, ret. time=11.120 min, 98% purity.
Compound N41 was made from Compound N34 and 3-acetamidopropanoic acid using standard amide coupling procedures such as those elaborated on in General Procedure 3. 1H-NMR (400 MHz, CDCl3): δ 8.62 (s, 1H), 7.77 (t, 1H), 7.35 (d, 1H), 7.12 (m, 1H), 6.95 (t, 2H), 6.92 (d, 1H), 6.67 (t, 1H), 6.13 (t, 1H), 5.93 (s, 1H), 5.59 (s, 1H), 4.85 (d, 1H), 4.70 (d, 1H), 4.27 (m, 2H), 3.85 (m, 2H), 3.65 (t, 1H), 3.50 (m, 2H), 3.11 (m, 2H), 2.66 (s, 3H), 2.45 (t, 2H), 2.16 (m, 2H), 1.92 (s, 3H), 1.85 (m, 2H). MS (EI) for C31H34F5N7O5. found 680.3 (MH+). Analytical HPLC, ret. time=10.416 min, 95% purity.
Compound N42 was made in a manner similar to Compound N41. 1H-NMR (400 MHz, CDCl3): δ 8.64 (s, 1H), 7.70 (t, 1H), 7.34 (d, 1H), 7.12 (m, 1H), 6.95 (t, 2H), 6.91 (d, 1H), 6.65 (t, 1H), 5.93 (s, 1H), 4.90 (d, 1H), 4.74 (d, 1H), 4.26 (m, 2H), 3.89 (d, 1H), 3.87 (t, 2H), 3.80 (d, 1H), 3.62 (tt, 1H), 3.11 (m, 2H), 2.65 (s, 3H), 2.47 (t, 2H), 2.15 (m, 2H), 1.85 (m, 2H). MS (EI) for C29H31F5N6O5. found 639.2 (MH+). Analytical HPLC, ret. time=10.520 min, 91% purity.
Compound N43 was made from intermediate N37.2 and 2-fluorophenyl chloroformate using a method analogous to that used to make Compound A119 from intermediate A116.3 and 2-chlorophenyl chloroformate. 1H-NMR (400 MHz, CDCl3, rotameric mixture): δ 8.64 and 8.61 (two of s, 1H), 7.77 (t, 1H), 7.35 (d, 1H), 7.18 (m, 4H), 6.90 (d, 1H), 6.34 (m, 1H), 5.84 and 5.72 (two of s, 1H), 4.95 (d, 1H), 4.71 and 4.68 (two of d, 1H), 4.48 (m, 1H), 4.40 (m, 1H), 3.88 (m, 1H), 3.75 (m, 1H), 3.65 (m, 1H), 3.18 (q, 1H), 3.02 (t, 1H), 2.67 (s, 3H), 2.16 (m, 2H), 2.04 (s, 3H), 1.89 (m, 1H), 1.80 (m, 1H). MS (EI) for C28H29F4N5O5. found 592.2 (MH+). Analytical HPLC, ret. time=13.408 min, 99% purity.
To a stirred solution of crude intermediate N38.1 (16.5 mg, 0.029 mmol) and 2-methyl-2-butene (50 μL) in t-BuOH (0.3 mL) at room temperature was added a mixture of NaH2PO4.2H2O (23 mg, 0.15 mmol), NaClO2 (13 mg, 0.15 mmol) and H2O (0.3 mL). After stirring for 15 min at room temperature, the resulting solution was purified by preparative HPLC to give Compound N44 (8.5 mg, 50%, 2 steps) as a white solid after lyophilization. 1H-NMR (400 MHz, MeOH-d4): δ 8.66 (s, 1H), 7.88 (t, 1H), 7.40 (d, 1H), 7.25 (m, 1H), 7.07 (d, 1H), 7.00 (t, 2H), 5.49 (s, 1H), 5.07 (d, 1H), 4.86 (d, 1H), 4.28 (t, 2H), 3.66 (t, 1H), 3.02 (q, 2H), 2.65 (s, 3H), 1.98 (m, 2H), 1.85 (m, 1H), 1.69 (m, 1H). MS (EI) for C26H24F5N5O5. found 582.2 (MH+). Analytical HPLC, ret. time=11.244 min, 95% purity.
Compound N45 was made from Compound N44 and methylamine using standard amide coupling procedures such as those elaborated on in General Procedure 3. 1H-NMR (400 MHz, CDCl3): δ 8.76 (s, 1H), 7.83 (t, 1H), 7.39 (d, 1H), 7.11 (m, 1H), 7.03 (m, 2H), 6.94 (t, 2H), 6.25 (s, 1H), 5.90 (s, 1H), 5.16 (d, 1H), 4.97 (d, 1H), 4.24 (m, 2H), 3.53 (m, 1H), 3.01 (m, 2H), 2.89 (d, 3H), 2.67 (s, 3H), 2.08 (m, 2H), 1.75 (m, 2H). MS (EI) for C27H27F5N6O4. found 595.2 (MH+). Analytical HPLC, ret. time=11.260 min, 95% purity.
Compound N46 was made from intermediate N37.2 in the same manner that Compound N43 was from intermediate N37.2, substituting 4-fluorophenyl chloroformate for 2-fluorophenyl chloroformate. 1H-NMR (400 MHz, CDCl3, rotameric mixture): δ 8.64 and 8.61 (two of s, 1H), 7.77 (t, 1H), 7.35 (d, 1H), 7.08 (m, 4H), 6.90 (d, 1H), 6.37 (m, 1H), 5.86 and 5.72 (two of s, 1H), 4.94 (d, 1H), 4.71 and 4.68 (two of d, 1H), 4.43 (m, 2H), 3.87 (m, 1H), 3.75 (m, 1H), 3.64 (m, 1H), 3.14 (q, 1H), 2.99 (t, 1H), 2.66 (s, 3H), 2.12 (m, 2H), 2.04 (s, 3H), 1.88 (m, 1H), 1.79 (m, 1H). MS (EI) for C28H29F4N5O5. found 592.2 (MH+). Analytical HPLC, ret. time=13.416 min, 96% purity.
Compound N47 was made from intermediate N37.2 in the same manner that Compound N43 was from intermediate N37.2, substituting 2,6-difluorophenyl chloroformate for 2-fluorophenyl chloroformate. 1H-NMR (400 MHz, CDCl3, rotameric mixture): δ 8.63 and 8.61 (two of s, 1H), 7.77 (t, 1H), 7.34 (d, 1H), 7.14 (m, 1H), 6.97 (t, 2H), 6.90 (d, 1H), 6.37 (m, 1H), 5.84 and 5.73 (two of s, 1H), 4.94 (d, 1H), 4.71 and 4.68 (two of d, 1H), 4.49 (m, 1H), 4.38 (m, 1H), 3.88 (m, 1H), 3.74 (m, 1H), 3.65 (m, 1H), 3.21 (q, 1H), 3.05 (t, 1H), 2.66 (s, 3H), 2.16 (m, 2H), 2.04 (s, 3H), 1.89 (m, 1H), 1.80 (m, 1H). MS (EI) for C28H28F5N5O5. found 610.2 (MH+). Analytical HPLC, ret. time=16.840 min, 98% purity.
To a mixture of intermediate D24.2 (115 mg, 0.24 mmol) and NH3/MeOH (0.4 mL, 7 N) was added Ti(OiPr)4 (68 mg, 0.24 mmol). The mixture was stirred at rt for 4 h, and then was added TMSCN (100 mg, 1.0 mmol). The mixture was stirred at rt for 72 h. The reaction was diluted with EtOAc, and filtered through the Celite. The filtrate was concentrated and purified by flash column chromatography to intermediate N48.1 (60 mg, 49%).
Intermediate N48.2 was made from the Boc deprotection of intermediate N48.1 using General Procedure 2.
Compound N48 was made from intermediate N48.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H NMR (400 MHz, DMSO-d6) δ 8.85 (s, 1H), 8.27 (s, 1H), 8.03 (t, 1H), 7.57 (d, 1H), 7.27 (m, 1H), 7.21 (d, 1H), 7.12 (m, 2H), 4.85 (d, 1H), 4.68 (d, 1H), 4.24 (m, 2H), 3.90 (m, 1H), 3.51 (s, 2H), 2.88 (m, 2H), 2.61 (s, 3H), 1.70 (m, 4H). MS (EI) for C26H24F5N7O2. found: 562.2 (MH+). Analytical HPLC, ret. time=20.08 min, 95% purity.
To a solution of Compound N48 (28 mg, 0.05 mmol) in THF/H2O (1 mL/0.5 mL) was added LiOH H2O (20 mg, 0.47 mmol). The mixture was stirred at rt for 72 h. The reaction progress was monitored by LC-MS. After the starting material was consumed, EtOAc was added to extract the product. The organic layer was washed with water, dried over anhydrous Na2SO4. Removal of the solvents gave the crude amide. Further purification by HPLC gave the desired product. 1H NMR (400 MHz, DMSO-d6) δ 8.59 (s, 1H), 8.24 (s, 1H), 7.99 (t, 1H), 7.54 (m, 2H), 7.41 (m, 1H), 7.26 (m, 1H), 7.13 (m, 3H), 4.72 (m, 2H), 4.20 (m, 2H), 3.53 (m, 1H), 2.65 (m, 1H), 2.55 (s, 3H), 1.65 (m, 4H). MS (EI) for C26H26F5N7O3. found: 580.2 (MH+). Analytical HPLC, ret. time=11.32 min, 99% purity.
To a stirred solution of intermediate D17.2 (30 mg, 0.086 mmol), DIEA (33 mg, 0.257 mmol) and DCM (1 mL) was added triphosgene (10 mg, 0.034 mmol). The mixture was stirred at RT for 10 min. Saturated NaHCO3 was added and the mixture was stirred for 15 min. The reaction mixture was diluted with DCM and worked up, and purified on prep HPLC to give intermediate N50.1 (23 mg): MS (EI) for C19H28N4O4. found: 377.2. Analytical HPLC, ret. time=12.32 min, 97% purity.
To solution of intermediate N50.1 (23 mg, 0.061 mmol) in DMAC was added NaH (60% in mineral oil, 24 mg) at RT with stirring. The resulting suspension was stirred at RT for 10 min, and cooled to 0° C., to which was added 4-chloro-2-(trifluoromethyl)pyrimidine (17 mg, 0.091 mmol). The reaction mixture was stirred at 0° C. for 30 min, and then saturated NH4Cl solution was added at 0° C. with stirring. The resulting mixture was diluted with MeOH and water, and then filtered. The filtrate was concentrated in vacuo. The residue was purified on prep HPLC to give intermediate N50.2 (23 mg) as a white solid. 1H-NMR (400 MHz, CDCl3): δ 8.78 (d, 1H), 8.74 (br. s, 1H), 8.38 (d, 1H), 4.89 (d, 1H), 4.45 (d, 1H), 4.33 (br. s, 2H), 2.88-2.72 (m, 3H), 2.71 (s, 3H), 2.12-2.01 (m 2H), 1.95 (s, 3H), 1.78-1.66 (m, 3H), 1.50 (s, 9H). MS (EI) for C24H29F3N6O4. found: 523.3 (MH+). Analytical HPLC, ret. time=20.76 min, 98% purity.
Intermediate N50.3 was made from the Boc deprotection of intermediate N50.2 using General Procedure 2.
Compound N50 was made from intermediate N50.3 and 2,6-difluorophenylacetic acid using General Procedure 4. 1H-NMR (400 MHz, CDCl3): δ 8.79 (d, 1H), 8.73 (br. s 1H), 8.38 (m, 1H), 7.24 (m, 1H), 6.96-6.90 (m, 2H), 4.84 (m, 1H), 4.60 (d, 2H), 4.48 (m, 1H), 4.20 (m, 1), 3.80 (m, 2H), 3.30 (t, 1H), 3.00 (m, 1H), 2.75 (m, 1H), 2.71 (s, 3H), 2.23-2.07 (m, 2H), 1.96 (s, 3H), 1.85-1.78 (m, 2H). MS (EI) for C27H25F5N6O3. found: 577.2 (MH+). Analytical HPLC, ret. time=18.5 min, 96% purity.
To a 0° C. solution of intermediate N51.2 (134 mg, 0.25 mmol) and Et3N (80 mg, 0.76 mmol) in DCM (5 mL) was added MsCl (34 mg, 0.3 mmol). The mixture was stirred at rt for 1 h. THF was added (8 mL), followed by the addition of KOtBu (1.5 mL, 1 M in THF). The resulting solution was stirred at rt for 1 h. The mixture was diluted with DCM, washed with saturated NaHCO3, brine, and dried over anhydrous Na2SO4. Further purification by flash column chromatography gave intermediate N51.1 (102 mg, 80%).
Intermediate N51.2 was made from the Boc deprotection of intermediate N51.1 using General Procedure 2.
Intermediate N51.3 was made from intermediate N51.2 and 2,6-difluorophenyl isocyanate using General Procedure 5.
To a solution intermediate N51.3 (40 mg, 0.07 mmol) in THF (5 mL) was added KOtBu (0.5 mL, 1 M in THF, 0.5 mmol). The mixture was stirred at rt for 2.5 h. EtOAc was added, the organic layer was washed with brine and dried over anhydrous Na2SO4. Further purification by flash column chromatography gave Compound N51 (7 mg, 17%). 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.26 (s, 1H), 7.99 (t, 1H), 7.51 (d, 1H), 7.26 (m, 1H), 7.18 (d, 1H), 7.11 (m, 2H), 4.74 (d, 1H), 4.69 (m, 1H), 4.61 (d, 1H), 4.46 (m, 1H), 4.18 (m, 2H), 3.27 (m, 2H), 2.88 (m, 3H), 2.59 (s, 3H), 1.90 (m, 1H), 1.72 (m, 2H), 1.62 (d, 1H). MS (EI) for C27H26F5N5O3. found: 564.1 (MH+). Analytical HPLC, ret. time=13.42 min, 95% purity.
Intermediate N52.1 was synthesized from intermediate N36.1 using a method analogous to that used to synthesize intermediate N50.1 from intermediate D17.2.
Intermediate N52.2 was made from the Boc Deprotection of intermediate N52.1 using General Procedure 2.
Compound N52 was synthesized from intermediate N52.2 and 2,6-difluorophenyl isocyanate using General Procedure 5. 1H-NMR (400 MHz, CDCl3): δ 8.8.74 (s, 1H), 7.79 (t, 1H), 7.35 (d, 1H), 7.13 (m, 1H), 7.00 (d, 1H), 6.95 (t, 2H), 5.92 (s, 1H), 5.09 (s, 1H), 4.96 (d, 1H), 4.59 (d, 1H), 4.33 (d, 1H), 4.32 (d, 1H), 4.22 (d, 1H), 3.82 (d, 1H), 3.14 (td, 1H), 3.02 (t, 2H), 2.72 (s, 3H), 2.28 (qd, 1H), 2.15 (qd, 1H), 1.84 (m, 1H), 1.74 (m, 1H). MS (EI) for C27H25F5N6O4. found 593.1 (MH+). Analytical HPLC, ret. time=12.308 min, 99% purity.
The binding of potential ligands to RORγ is measured by competition with [3H]25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. O1 nM of [3H]25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer). IC50 values are determined from the percentage inhibition of [3H]25-hydroxycholesterol binding.
IC50 values of the compounds of Tables 1-14 in the RORγ binding assay are provided in the Table, below.
This application claims the benefit of priority of U.S. Provisional Patent Application Ser. No. 61/716,244, filed Oct. 19, 2012; and U.S. Provisional Patent Application Ser. No. 61/716,233, filed Oct. 19, 2012, each of which is hereby incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/065480 | 10/17/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61716244 | Oct 2012 | US | |
| 61716233 | Oct 2012 | US |
