Patent Grant
10000502
The present invention relates to thiohydantoin compounds that are androgen receptor antagonists and are useful for the treatment of disorders that are affected by the modulation of the androgen receptor (AR). The invention also relates to pharmaceutical compositions that include one or more of such compounds, to processes to prepare such compounds and compositions, and to the use of such compounds or pharmaceutical compositions for the treatment of prostate cancer and other diseases, syndromes, disorders, or conditions associated with androgen-resistant ARs or an AR mutant associated with “castration-resistant” prostate cancer.
Prostate cancer is the most common non-cutaneous malignancy in men and the second leading cause of death in men from cancer in the western world (Jemal A, Siegel R, Xu J, Ward E. Cancer Statistics. Cancer J Clin 2010; 60:277-300). As a male sexual organ, development of the prostate is highly regulated by androgens, the AR and by the products of androgen dependent genes. During all stages of prostate cancer progression, the disease remains dependent upon androgens. Anti-androgens, including AR antagonists, are used therapeutically to reverse the dependence of the tumor upon the actions of androgen (Scher H, Sawyers C. Biology of progressive, castration-resistant prostate cancer: directed therapies targeting the androgen-receptor signaling axis. J Clin Oncol 2005; 23:8253-8261; Tran C, Ouk S, Clegg N, Chen Y, Watson P, Arora V, et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science 2009; 324:787-790; Scher H, Fizazi K, Saad F, Taplin M, Sternberg C, Miller K, et al. Increased survival with enzalutamide in prostate cancer after chemotherapy. N Engl J Med 2012; 367:1187-1197). Unfortunately, the efficacy of even second-generation, highly potent AR antagonists, such as MDV-3100 (enzalutamide, Xtandi®), is short-lived in many patients.
AR antagonists have transformed patient care by targeting a key nodal point in tumor cell signaling. However, as with other molecularly targeted cancer therapies across different oncology indications, the emergence of acquired resistance via mutation of the therapeutic target is not uncommon. This is best exemplified by imatinib-treated patients with chronic myeloid leukemia in whom ABL kinase mutations render leukemia cells resistant to imatinib. Multiple next-generation ABL inhibitors have since been developed to circumvent the mutation and with activity in this setting (Gorre M, Mohammed M, Ellwood K, Hsu N, Paquette R, Rao P, Sawyers C. Clinical resistance to STI-571 cancer therapy caused by BCRABL gene mutation or amplification. Science 2001; 293:876-80; O'Hare T, Deininger M W, Eide C A, Clackson T, Druker B J. Targeting the BCR-ABL signaling pathway in therapy-resistant Philadelphia chromosome-positive leukemia. Clin Cancer Res 2011. 17: 212-21).
Importantly, the activity of second- and third-generation AR inhibitors indicates that the disease remains “addicted” to a deregulated driver. This has led to the paradigm of sequential therapy targeting the same driver oncogene in distinct resistant states and is applicable herein to targeting of AR and the lineage dependence of AR signaling.
AR mutations that result in receptor promiscuity and the ability of these anti-androgens to exhibit agonist activity might at least partially account for this phenomenon. For example, hydroxyflutamide and bicalutamide act as AR agonists in T877A and W741L/W741C AR mutants, respectively.
In the setting of prostate cancer cells that were rendered castration resistant via overexpression of AR, it has been demonstrated that certain anti-androgen compounds, such as bicalutamide, have a mixed antagonist/agonist profile (Tran C, Ouk S, Clegg N, Chen Y, Watson P, Arora V, et al. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science 2009; 324:787-790). This agonist activity helps to explain a clinical observation, called the anti-androgen withdrawal syndrome, whereby about 30% of men who progress on AR antagonists experience a decrease in serum PSA when therapy is discontinued (Scher, H. I. and Kelly, W. K., J Urol 1993 March; 149(3): 607-9). Prostate specific antigen decline after antiandrogen withdrawal: the flutamide withdrawal syndrome.
Accumulating evidence indicates that castration-resistant prostate cancer (CRPC) remains dependent upon AR signaling through reactivation of AR signaling (Yuan X, Balk S. Mechanisms mediating androgen receptor reactivation after castration. Urol Oncol 2009; 27: 36-41; Linja M, Savinainen K, Saramaki O, Tammela T, Vessella R, Visakorpi T. Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer. Cancer Res 2001; 61:3550-; Chen C, Welsbie D, Tran C, Baek S, Chen R, Vessella R, Rosenfeld M, Sawyers C. Molecular determinants of resistance to antiandrogen therapy. Nat Med 2004; 10(1): 33-9.555). Point mutation in the ligand-binding domain (LBD) of AR accounts for 10-20% of resistance and is characterized by receptor activation, rather than inhibition, by anti-androgen drugs (Beltran H, Yelensky R, Frampton G, Park K, Downing S, MacDonald T, et al. Targeted next-generation sequencing of advanced prostate cancer identifies potential therapeutic targets and disease heterogeneity. Eur Urol 2013; 63(5): 920-6; Bergerat J, Ceraline J. Pleiotropic functional properties of androgen receptor mutants in prostate cancer. Hum Mutat 2009; 30(2): 145-57). Many of these mutations broaden ligand specificity, and some confer resistance by converting the AR antagonist into an agonist of the mutant receptor (Veldscholte J, Ris-Stalpers C, Kuiper G G, Jenster G, Berrevoets C, Claassen E, van Rooij H C, Trapman J, Brinkmann A O, Mulder E. A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochem Biophys Res Commun. 1990; 173: 534-40; Haapala K, Hyytinen E, Roiha M, Laurila M, Rantala I, Helin H, Koivisto P. Androgen receptor alterations in prostate cancer relapsed during a combined androgen blockade by orchiectomy and bicalutamide. Lab Invest 2001; 81(12):1647-1651; Hara T, Miyazaki J, Araki H, Yamaoka M, Kanzaki N, Kusaka M, Miyamoto M. Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome. Cancer Res 2003; 63(1):149-153).
One mutation, phenylalanine to leucine at position 876 (F876L) of AR, was recently shown to arise in response to MDV-3100 and ARN-509 in preclinical models and in patients undergoing therapy with ARN-509 (Clegg N, Wongvipat J, Joseph J, Tran C, Ouk S, Dilhas A, et al. ARN-509: a novel antiandrogen for prostate cancer treatment. Cancer Res 2012; 72(6): 1494-503; Balbas M, Evans M, Hosfield D, Wongvipat J, Arora V, Watson P, et al. Overcoming mutation-based resistance to antiandrogens with rational drug design. Elife 2013. 2: e00499; Korpal M, Korn J, Gao X, Rakiec D, Ruddy D, Doshi S, et al. An F876L mutation in androgen receptor confers genetic and phenotypic resistance to MDV3100 (enzalutamide). Cancer Discov 2013; 39:1030-1043; Joseph J D, Lu N, Qian J, Sensintaffar J, Shao G, Brigham D, Moon M, Maneval E C, Chen I, Darimont B, Hager J H. A clinically relevant androgen receptor mutation confers resistance to second-generation antiandrogens enzalutamide and ARN-509. Cancer Discov 2013; 3:1020-1029).
AR F876L confers resistance to MDV-3100 and ARN-509. Comprehensive biological studies have demonstrated that prostate cancer cells harboring this mutation continued to grow when treated with either compound. In vitro reporter assays confirmed resistance and demonstrate agonist conversion of both compounds and in tumors engineered to express AR F876L, neither compound controlled tumor growth. Furthermore, the AR F876L mutant is detected in ARN-509-treated patients with progressive CRPC. The mutation was detected in the plasma DNA of patients undergoing longitudinal analysis in 3 of 29 patients eligible for assessment. All 3 of the patients were amongst the 18 patients with an increase in prostate specific antigen (PSA) whilst on drug, indicative of disease progression (Joseph 2013).
Structural modeling of wild-type (WT) and F876L mutated AR bound with MDV-3100, indicated that helices 11 and 12 were differentially displaced. Within the LBD of AR in the F876L mutant, helix 12 is not displaced by MDV-3100 as it is in WT AR, and this allows MDV 3100 to function as an agonist. The compounds described herein are designed to act as antagonists (third-generation), where second-generation compounds are not active.
Thus, androgen receptor antagonists of the present invention may provide therapeutic benefit for the treatment of prostate cancer and other diseases, syndromes, disorders, or conditions associated with androgen-resistant ARs or an AR mutant associated with castration-resistant prostate cancer.
The present invention is directed to compounds of Formula (I)
wherein
The present invention also provides a pharmaceutical composition comprising, consisting of and/or consisting essentially of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, a pharmaceutically acceptable diluent and a compound of Formula (I) or a pharmaceutically acceptable salt form thereof.
Also provided are processes for making a pharmaceutical composition comprising, consisting of, and/or consisting essentially of admixing a compound of Formula (I), and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable diluent.
The present invention further provides methods for treating or ameliorating a disease, syndrome, condition, or disorder in a subject, including a mammal and/or human, in which the disease, syndrome, condition, or disorder is affected by the antagonism of one or more androgen receptor types, such as prostate cancer, castration-resistant prostate cancer, and metastatic castration-resistant prostate cancer, using a compound of Formula (I).
The present invention also is directed to the use of any of the compounds described herein in the preparation of a medicament wherein the medicament is prepared for treating a disease, syndrome, condition, or disorder that is affected by the antagonism of one or more androgen receptor types, such as prostate cancer, castration-resistant prostate cancer, and metastatic castration-resistant prostate cancer.
The present invention is also directed to the preparation of substituted thiohydantoin derivatives that act as antagonists of one or more androgen receptors.
Exemplifying the invention are methods of treating a disease, syndrome, condition, or disorder mediated by one or more androgen receptors, selected from the group consisting of prostate cancer, castration-resistant prostate cancer, and metastatic castration-resistant prostate cancer, comprising, consisting of, and/or consisting essentially of, administering to a subject in need thereof a therapeutically effective amount of any of the compounds or pharmaceutical compositions described in the present invention.
In another embodiment, the present invention is directed to a compound of Formula (I) for use in the treatment of a disease, syndrome, condition, or disorder affected by the antagonism of one or more androgen receptor types, selected from the group consisting of prostate cancer, castration-resistant prostate cancer, and metastatic castration-resistant prostate cancer.
In another embodiment, the present invention is directed to a composition comprising a compound of Formula (I) for the treatment of a disease, syndrome, condition, or disorder affected by the antagonism of one or more androgen receptors, selected from the group consisting of prostate cancer, castration-resistant prostate cancer, and metastatic castration-resistant prostate cancer.
Another embodiment of the present invention is directed to a pharmaceutical composition comprising a compound of Formula (I)
With reference to substituents, the term “independently” refers to the situation where when more than one substituent is possible, the substituents may be the same or different from each other.
The term “alkyl” whether used alone or as part of a substituent group, refers to straight and branched carbon chains having 1 to 8 carbon atoms. Therefore, designated numbers of carbon atoms (e.g., C1-8) refer independently to the number of carbon atoms in an alkyl moiety or to the alkyl portion of a larger alkyl-containing substituent. In substituent groups with multiple alkyl groups such as, (C1-6alkyl)2amino-, the C1-6alkyl groups of the dialkylamino may be the same or different.
The term “alkoxy” refers to an —O-alkyl group, wherein the term “alkyl” is as defined above.
The terms “alkenyl” and “alkynyl” refer to straight and branched carbon chains having 2 to 8 carbon atoms, wherein an alkenyl chain contains at least one double bond and an alkynyl chain contains at least one triple bond.
The term “cycloalkyl” refers to saturated or partially saturated, monocyclic or polycyclic hydrocarbon rings of 3 to 14 carbon atoms. Examples of such rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and adamantyl.
The term “heterocyclyl” refers to a nonaromatic monocyclic or bicyclic ring system having 3 to 10 ring members that include at least 1 carbon atom and from 1 to 4 heteroatoms independently selected from N, O, and S. Included within the term heterocyclyl is a nonaromatic cyclic ring of 5 to 7 members in which 1 to 2 members are N, or a nonaromatic cyclic ring of 5 to 7 members in which 0, 1 or 2 members are N and up to 2 members are O or S and at least one member must be either N, O, or S; wherein, optionally, the ring contains 0 to 1 unsaturated bonds, and, optionally, when the ring is of 6 or 7 members, it contains up to 2 unsaturated bonds. The carbon atom ring members that form a heterocycle ring may be fully saturated or partially saturated. The term “heterocyclyl” also includes two 5 membered monocyclic heterocycloalkyl groups bridged to form a bicyclic ring. Such groups are not considered to be fully aromatic and are not referred to as heteroaryl groups. When a heterocycle is bicyclic, both rings of the heterocycle are non-aromatic and at least one of the rings contains a heteroatom ring member. Examples of heterocycle groups include, and are not limited to, pyrrolinyl (including 2H-pyrrole, 2-pyrrolinyl or 3-pyrrolinyl), pyrrolidinyl, imidazolinyl, imidazolidinyl, pyrazolinyl, pyrazolidinyl, piperidinyl, morpholinyl, thiomorpholinyl, and piperazinyl. Unless otherwise noted, the heterocycle is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.
The term “aryl” refers to an unsaturated, aromatic monocyclic or bicyclic ring of 6 to 10 carbon members. Examples of aryl rings include phenyl and naphthalenyl. The term “heteroaryl” refers to an aromatic monocyclic or bicyclic aromatic ring system having 5 to 10 ring members and which contains carbon atoms and from 1 to 4 heteroatoms independently selected from the group consisting of N, O, and S. Included within the term heteroaryl are aromatic rings of 5 or 6 members wherein the ring consists of carbon atoms and has at least one heteroatom member. Suitable heteroatoms include nitrogen, oxygen, and sulfur. In the case of 5 membered rings, the heteroaryl ring preferably contains one member of nitrogen, oxygen or sulfur and, in addition, up to 3 additional nitrogens. In the case of 6 membered rings, the heteroaryl ring preferably contains from 1 to 3 nitrogen atoms. For the case wherein the 6 membered ring has 3 nitrogens, at most 2 nitrogen atoms are adjacent. Examples of heteroaryl groups include furyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolyl, isoindolyl, benzofuryl, benzothienyl, indazolyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzothiadiazolyl, benzotriazolyl, quinolinyl, isoquinolinyl and quinazolinyl. Unless otherwise noted, the heteroaryl is attached to its pendant group at any heteroatom or carbon atom that results in a stable structure.
The term “halogen” or “halo” refers to fluorine, chlorine, bromine and iodine atoms.
The term “carboxy” refers to the group —C(═O)OH.
The term “formyl” refers to the group —C(═O)H.
The term “oxo” or “oxido” refers to the group (═O).
Whenever the term “alkyl” or “aryl” or either of their prefix roots appear in a name of a substituent (e.g., arylalkyl, alkylamino) the name is to be interpreted as including those limitations given above for “alkyl” and “aryl.” Designated numbers of carbon atoms (e.g., C1-C6) refer independently to the number of carbon atoms in an alkyl moiety, an aryl moiety, or in the alkyl portion of a larger substituent in which alkyl appears as its prefix root. For alkyl and alkoxy substituents, the designated number of carbon atoms includes all of the independent members included within a given range specified. For example C1-6 alkyl would include methyl, ethyl, propyl, butyl, pentyl and hexyl individually as well as sub-combinations thereof (e.g., C1-2, C1-3, C1-4, C1-5, C2-6, C3-6, C4-6, C5-6, C2-5, etc.).
In general, under standard nomenclature rules used throughout this disclosure, the terminal portion of the designated side chain is described first followed by the adjacent functionality toward the point of attachment. Thus, for example, a “C1-C6 alkylcarbonyl” substituent refers to a group of the formula:
In the present invention, as a non-limiting example, when R4 is
and V is “—OCH2—”, the substituent is oriented such that the oxygen atom of —OCH2— is covalently bound to the (R3)(R4)-substituted phenyl ring as shown:
The label “R” at a stereocenter designates that the stereocenter is purely of the R-configuration as defined in the art; likewise, the label “S” means that the stereocenter is purely of the S-configuration. As used herein, the labels “*R” or “*S” at a stereocenter are used to designate that the stereocenter is of pure but unknown absolute configuration. As used herein, the label “RS” refers to a stereocenter that exists as a mixture of the R- and S-configurations.
A compound containing one stereocenter drawn without a stereo bond designation is a mixture of two enantiomers. A compound containing two stereocenters both drawn without stereo bond designations is a mixture of four diastereomers. A compound with two stereocenters both labeled “RS” and drawn with stereo bond designations is a mixture of two enantiomers with relative stereochemistry as drawn. A compound with two stereocenters both labeled “*RS” and drawn with stereo bond designations is a mixture of two enantiomers with a single, but unknown, relative stereochemistry.
Unlabeled stereocenters drawn without stereo bond designations are mixtures of the R- and S-configurations. For unlabeled stereocenters drawn with stereo bond designations, the relative and absolute stereochemistry is as depicted.
Unless otherwise noted, it is intended that the definition of any substituent or variable at a particular location in a molecule be independent of its definitions elsewhere in that molecule. It is understood that substituents and substitution patterns on the compounds of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth herein.
The term “subject” refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment.
The term “therapeutically effective amount” refers to an amount of an active compound or pharmaceutical agent, including a compound of the present invention, which elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition, or disorder being treated.
The term “composition” refers to a pharmaceutical product that includes the specified ingredients sometimes in therapeutically effective amounts, as well as any product that results, directly, or indirectly, from combinations of the specified ingredients in the specified amounts.
The term “androgen receptor” as used herein is intended to include the wild-type androgen receptor as well as androgen-resistant ARs and/or AR mutants associated with castration-resistant prostate cancer.
The term “AR-mediated” refers to any disease, syndrome, condition, or disorder that might occur in the absence of androgen receptors but can occur in the presence of androgen receptors. Suitable examples of include, but are not limited to, prostate cancer, castration-resistant prostate cancer, and metastatic castration-resistant prostate cancer.
The term “Androgen-dependent disorder” refers to any disorder that can benefit from a decrease in androgen stimulation and includes pathological conditions that depend on androgen stimulation. An “androgen-dependent disorder” can result from an excessive accumulation of testosterone or other androgenic hormone, increased sensitivity of androgen receptors to androgen, or an increase in androgen-stimulated transcription.
Examples of “androgen-dependent disorders” include prostate cancer and disorders such as, for example, acne, seborrhea, hirsutism, alopecia, and hidradenitis suppurativa.
As used herein, the term “anti-androgen” refers to a group of hormone receptor antagonist compounds that are capable of preventing or inhibiting the biologic effects of androgens on normally responsive tissues in the body. In some embodiments, an anti-androgen is a small molecule. In some embodiments, an anti-androgen is an AR antagonist. In some embodiments, an anti-androgen is an AR full antagonist. In some embodiments, an anti-androgen is a first-generation anti-androgen. In some embodiments, an anti-androgen is a second-generation anti-androgen. In some embodiments, an anti-androgen is a third-generation anti-androgen.
As used herein, the term “AR antagonist” or “AR inhibitor” are used interchangeably and refer to an agent that inhibits or reduces at least one activity of an AR polypeptide. Exemplary AR activities include, but are not limited to, co-activator binding, DNA binding, ligand binding, or nuclear translocation.
As used herein, a “full antagonist” refers to an antagonist which, at an effective concentration, essentially completely inhibits an activity of an AR polypeptide. As used herein, a “partial antagonist” refers an antagonist that is capable of partially inhibiting an activity of an AR polypeptide, but that, even at a highest concentration is not a full antagonist. By ‘essentially completely’ is meant at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% at least about 99%, or greater inhibition of the activity of an AR polypeptide.
As used herein, the term “first-generation anti-androgen” refers to an agent that exhibits antagonist activity against a wild-type AR polypeptide. However, first-generation anti-androgens differ from second-generation anti-androgens in that first-generation anti-androgens can potentially act as agonists in castration resistant prostate cancers (CRPC). Exemplary first-generation anti-androgens include, but are not limited to, flutamide, nilutamide and bicalutamide.
As used herein, the term “second-generation anti-androgen” refers to an agent that exhibits full antagonist activity against a wild-type AR polypeptide. Second-generation anti-androgens differ from first-generation anti-androgens in that second-generation anti-androgens act as full antagonists in cells expressing elevated levels of AR, such as for example, in castration resistant prostate cancers (CRPC). Exemplary second-generation anti-androgens include 4-[7-(6-cyano-5-trifluoromethylpyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl]-2-fluoro-N methylbenzamide (also known as ARN-509; CAS No. 956104-40-8); 4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluoro-N-methylbenzamide (also known as MDV3100 or enzalutamide; CAS No: 915087-33-1) and RD162 (CAS No. 915087-27-3). In some embodiments, a second-generation anti-androgen binds to an AR polypeptide at or near the ligand binding site of the AR polypeptide.
As used herein, the term “third-generation anti-androgen” refers to an agent that exhibits full antagonist activity against a wild-type AR polypeptide and against mutant forms of the AR polypeptide, with mutations arising in the ligand binding domain (LBD) of the AR polypeptide as set forth below. Third-generation anti-androgens retain the differentiation from first-generation anti-androgens in that third-generation anti-androgens act as full antagonists in cells expressing elevated levels of AR, such as for example, in castration resistant prostate cancers (CRPC).
As used herein, the term “mutant” refers to an altered (as compared with a reference) nucleic acid or polypeptide, or to a cell or organism containing or expressing such altered nucleic acid or polypeptide.
As used herein, unless otherwise noted, the term “affect” or “affected” (when referring to a disease, syndrome, condition or disorder that is affected by antagonism of AR) includes a reduction in the frequency and/or severity of one or more symptoms or manifestations of said disease, syndrome, condition or disorder; and/or include the prevention of the development of one or more symptoms or manifestations of said disease, syndrome, condition or disorder or the development of the disease, condition, syndrome or disorder.
The compounds of the instant invention are useful in methods for treating or ameliorating a disease, a syndrome, a condition or a disorder that is affected by the antagonism of one or more AR receptors. Such methods comprise, consist of and/or consist essentially of administering to a subject, including an animal, a mammal, and a human in need of such treatment, amelioration and/or prevention, a therapeutically effective amount of a compound of Formula (I), or an enantiomer, diastereomer, solvate or pharmaceutically acceptable salt thereof.
One embodiment of the present invention is directed to a method of treating an androgen receptor dependent or androgen receptor mediated disease or condition in a subject in need thereof, including an animal, a mammal, and a human in need of such treatment, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I).
In another embodiment, the androgen receptor dependent or androgen receptor mediated disease or condition is selected from benign prostate hyperplasia, hirsutism, acne, adenomas and neoplasies of the prostate, benign or malignant tumor cells containing the androgen receptor, hyperpilosity, seborrhea, endometriosis, polycystic ovary syndrome, androgenic alopecia, hypogonadism, osteroporosis, suppression of spermatogenesis, libido, cachexia, anorexia, androgen supplementation for age related decreased testosterone levels, prostate cancer, breast cancer, endometrial cancer, uterine cancer, hot flashes, and Kennedy's disease muscle atrophy and weakness, skin atrophy, bone loss, anemia, arteriosclerosis, cardiovasculasr disease, loss of energy, loss of well-being, type 2 diabetes, or abdominal fat accumulation.
In particular, the compounds of Formula (I), or an enantiomer, diastereomer, solvate or pharmaceutically acceptable salt form thereof are useful for treating or ameliorating diseases, syndromes, conditions, or disorders such as prostate cancer, castration-resistant prostate cancer, and metastatic castration-resistant prostate cancer.
More particularly, the compounds of Formula (I), or an enantiomer, diastereomer, solvate or pharmaceutically acceptable salt form thereof, are useful for treating or ameliorating prostate cancer, castration-resistant prostate cancer, and metastatic castration-resistant prostate cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of Formula (I), or an enantiomer, diastereomer, solvate or pharmaceutically acceptable salt form thereof as herein defined.
Embodiments of the present invention include a compound of Formula (I)
wherein
and R3 is hydrogen or fluoro, R1 is chloro, methoxy, or difluoromethyl;
and R3 is hydrogen, R1 is chloro, methoxy, or difluoromethyl;
and R3 is hydrogen, R1 is chloro, methoxy, difluoromethyl, or trifluoromethyl;
and R3 is hydrogen, R1 is chloro, methyl, methoxy, or difluoromethyl;
wherein R8 is selected from the group consisting of hydrogen, methyl, and 2-hydroxyethyl;
wherein R11 is 1-(t-butoxycarbonyl)-azetidin-3-ylmethyl;
wherein R4 is selected from the group consisting of morpholin-4-yl, morpholin-4-ylmethyl, 5-methylmorpholin-2-ylmethoxy, (1-t-butoxycarbonyl)-4,4-difluoropyrrolidin-2-ylmethoxy, (6,6-difluoro-2-methyl-2-azaspiro[3.3]heptan-6-yl)ethoxy, 1H-pyrazol-3-yl, methylaminocarbonylmethyl, 8-methyl-8-azabicyclo[3.2.1]octan-3-yl, 5,8-diazaspiro[2.5]octan-5-ylmethyl, 1-azaspiro[3.3]heptan-6-yloxy, (4-aminopiperidin-1-yl)methyl, (2S,3R,4S)-3,4-dihydroxy-2-methyl-3,4-dihydro-2H-pyran-6-yl, 3,4-dihydroxy-3,4-dihydro-2H-pyran-6-yl, 2-methyl-2-azaspiro[3.3]heptan-6-yl)oxy, (1-methyl-1-azaspiro[3.3]heptan-6-yl)oxy, (2-methyl-2-azabicyclo[2.2.1]heptan-5-yl)oxy, (3-methyl-3-azaspiro[3.3]heptan-6-yl)oxy, 3-azabicyclo[3.2.1]octan-8-yloxy, (1-methylazetidin-3-yl)methylaminocarbonyl, 1-methylpyrrolidin-3-ylaminocarbonyl, (1-methyl-1-oxido-piperidin-1-ium-4-yl)oxy, (3-methyl-3-azabicyclo[3.2.1]octan-8-yl)oxy, 9-azaspiro[3.5]nonan-6-yloxy, (9-methyl-9-azaspiro[3.5]nonan-6-yl)oxy, (1-methylpyrrolidin-3-yl)methylaminocarbonyl, (piperidin-3-yl)methylaminocarbonyl, (4-fluoro-8-azabicyclo[3.2.1]octan-3-yl)oxy, (4-fluoro-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)oxy, (1-methyl-piperidin-4-yl)methylaminocarbonyl, piperidin-4-ylmethylaminocarbonyl, (1-methyl-piperidin-3-yl)methylaminocarbonyl, 1-(ethoxycarbonyl)piperidin-4-ylmethylaminocarbonyl, 1-(t-butoxycarbonyl)piperidin-4-ylmethylaminocarbonyl, 3-(t-butoxycarbonylamino)-2,2,4,4-tetramethylcyclobut-1-yloxy, and a substituent selected from a) to e);
wherein V is absent or —OCH2—; and wherein Rb is dimethylamino or t-butoxycarbonyl(N-methyl)amino;
wherein Lc absent or selected from O, S, or —CH2—; and wherein W is selected from NH, N(methyl), N(cyclopropyl), N(2-fluoroethyl), N(cyanomethyl), N(3-fluoropropyl), N(3-amino-2-hydroxy-prop-1-yl), N(3,3-dimethyl-butyl), CH(amino), CH(methylamino), or CH(dimethylamino);
R1 is chloro, methoxy, or difluoromethyl;
and R3 is hydrogen or fluoro, R1 is chloro, methoxy, or difluoromethyl;
and R3 is hydrogen, R1 is chloro, methyl, methoxy, or difluoromethyl;
wherein R is selected from the group consisting of hydroxy, aminosulfonyl, 2-azabicyclo[2.2.1]heptan-5-yloxy, 3-azabicyclo[3.2.1]octan-8-yloxy, (3-methyl-3-azabicyclo[3.2.1]octan-8-yl)oxy, 9-azaspiro[3.5]nonan-6-yloxy, 3-(dimethylamino)propyloxy, 3-(t-butoxycarbonyl)-3-azaspiro[3.3]heptan-6-yloxy, and substituent f)
wherein R8 is selected from the group consisting of hydrogen, methyl, and 2-hydroxyethyl;
wherein R11 is 1-(t-butoxycarbonyl)-azetidin-3-ylmethyl;
wherein R7 is selected from the group consisting of (3-methyl-3-azabicyclo[3.2.1]octan-8-yl)oxy, 3-(t-butoxycarbonyl)-3-azaspiro[3.3]heptan-6-yloxy, and substituent f)
wherein Wf is NH or N(2-fluoroethyl);
Embodiments of the present invention include a compound of Formula (I)
wherein
wherein La is absent or —(CH2)r—, wherein r is an integer of 1 or 2; Ra is a substituent selected from methyl or prop-2-yn-1-yl;
wherein V is absent or —OCH2—; and wherein Rb is amino, dimethylamino, or t-butoxycarbonyl(N-methyl)amino;
wherein Lc absent or selected from O, S, or —CH2—; and wherein W is selected from NH, N(methyl), N(cyclopropyl), N(2-hydroxyethyl), N(2-fluoroethyl), N(2,2,2-trifluoroethyl), N(cyanomethyl), N(1-prop-2-ynyl), N(3-fluoropropyl), N(methoxycarbonylmethyl), N(3-amino-2-hydroxy-prop-1-yl), N(3,3-dimethyl-butyl), CH(amino), CH(methylamino), CH(dimethylamino), S, or SO2;
and R3 is hydrogen or fluoro, R1 is chloro, methoxy, or difluoromethyl;
and R3 is hydrogen, R1 is chloro, methoxy, or difluoromethyl;
and R3 is hydrogen, R1 is chloro, methoxy, difluoromethyl, or trifluoromethyl;
and R3 is hydrogen, R1 is chloro, methyl, methoxy, or difluoromethyl;
wherein R7 is selected from the group consisting of hydroxy, methoxy, aminosulfonyl, 2-azabicyclo[2.2.1]heptan-5-yloxy, 3-azabicyclo[3.2.1]octan-8-yloxy, (3-methyl-3-azabicyclo[3.2.1]octan-8-yl)oxy, 9-azaspiro[3.5]nonan-6-yloxy, (9-methyl-9-azaspiro[3.5]nonan-6-yl)oxy, 3-(dimethylamino)propyloxy, 3-(t-butoxycarbonyl)-3-azaspiro[3.3]heptan-6-yloxy, and substituent f)
wherein R8 is selected from the group consisting of hydrogen, methyl, and 2-hydroxyethyl;
wherein R11 is methyl or 1-(t-butoxycarbonyl)-azetidin-3-ylmethyl;
wherein R10 is selected from the group consisting of methoxycarbonyl, phenyloxy, and phenylcarbonylamino;
Embodiments of the present invention include a compound of Formula (I)
wherein
wherein La is absent or —(CH2)r—, wherein r is an integer of 1 or 2; Ra is methyl;
wherein V is absent or —OCH2—; and wherein Rb is dimethylamino or t-butoxycarbonyl(N-methyl)amino;
wherein Lc absent or selected from O, S, or —CH2—; and wherein W is selected from NH, N(methyl), N(cyclopropyl), N(2-fluoroethyl), N(cyanomethyl), N(3-fluoropropyl), N(3-amino-2-hydroxy-prop-1-yl), N(3,3-dimethyl-butyl), CH(amino), CH(methylamino), CH(dimethylamino), or S;
and R3 is hydrogen or fluoro, R1 is chloro, methoxy, or difluoromethyl;
and R3 is hydrogen, R1 is chloro, methoxy, or difluoromethyl;
and R3 is hydrogen, R1 is chloro, methoxy, difluoromethyl, or trifluoromethyl;
and R3 is hydrogen, R1 is chloro, methyl, methoxy, or difluoromethyl;
wherein R7 is selected from the group consisting of hydroxy, aminosulfonyl, 2-azabicyclo[2.2.1]heptan-5-yloxy, 3-azabicyclo[3.2.1]octan-8-yloxy, (3-methyl-3-azabicyclo[3.2.1]octan-8-yl)oxy, 9-azaspiro[3.5]nonan-6-yloxy, (9-methyl-9-azaspiro[3.5]nonan-6-yl)oxy, 3-(dimethylamino)propyloxy, 3-(t-butoxycarbonyl)-3-azaspiro[3.3]heptan-6-yloxy, and substituent f)
wherein R8 is selected from the group consisting of hydrogen, methyl, and 2-hydroxyethyl;
wherein R11 is 1-(t-butoxycarbonyl)-azetidin-3-ylmethyl;
wherein R10 is selected from the group consisting of methoxycarbonyl, phenyloxy, and phenylcarbonylamino;
Embodiments of the present invention include a compound of Formula (I)
wherein
wherein R4 is selected from the group consisting of morpholin-4-yl, morpholin-4-ylmethyl, 5-methylmorpholin-2-ylmethoxy, (1-t-butoxycarbonyl)-4,4-difluoropyrrolidin-2-ylmethoxy, (6,6-difluoro-2-methyl-2-azaspiro[3.3]heptan-6-yl)ethoxy, 1H-pyrazol-3-yl, methylaminocarbonylmethyl, 8-methyl-8-azabicyclo[3.2.1]octan-3-yl, 5,8-diazaspiro[2.5]octan-5-ylmethyl, 1-azaspiro[3.3]heptan-6-yloxy, (4-aminopiperidin-1-yl)methyl, (2S,3R,4S)-3,4-dihydroxy-2-methyl-3,4-dihydro-2H-pyran-6-yl, 3,4-dihydroxy-3,4-dihydro-2H-pyran-6-yl, 2-methyl-2-azaspiro[3.3]heptan-6-yl)oxy, (1-methyl-1-azaspiro[3.3]heptan-6-yl)oxy, (2-methyl-2-azabicyclo[2.2.1]heptan-5-yl)oxy, (3-methyl-3-azaspiro[3.3]heptan-6-yl)oxy, 3-azabicyclo[3.2.1]octan-8-yloxy, (1-methylazetidin-3-yl)methylaminocarbonyl, 1-methylpyrrolidin-3-ylaminocarbonyl, (1-methyl-1-oxido-piperidin-1-ium-4-yl)oxy, (3-methyl-3-azabicyclo[3.2.1]octan-8-yl)oxy, 9-azaspiro[3.5]nonan-6-yloxy, (9-methyl-9-azaspiro[3.5]nonan-6-yl)oxy, (1-methylpyrrolidin-3-yl)methylaminocarbonyl, (piperidin-3-yl)methylaminocarbonyl, (4-fluoro-8-azabicyclo[3.2.1]octan-3-yl)oxy, (4-fluoro-8-methyl-8-azabicyclo[3.2.1]octan-3-yl)oxy, (1-methyl-piperidin-4-yl)methylaminocarbonyl, piperidin-4-ylmethylaminocarbonyl, (1-methyl-piperidin-3-yl)methylaminocarbonyl, 1-(ethoxycarbonyl)piperidin-4-ylmethylaminocarbonyl, 1-(t-butoxycarbonyl)piperidin-4-ylmethylaminocarbonyl, 3-(t-butoxycarbonylamino)-2,2,4,4-tetramethylcyclobut-1-yloxy, and a substituent selected from a) to e);
wherein V is absent or —OCH2—; and wherein Rb is dimethylamino or t-butoxycarbonyl(N-methyl)amino;
wherein Lc absent or selected from O, S, or —CH2—; and wherein W is selected from NH, N(methyl), N(cyclopropyl), N(2-fluoroethyl), N(cyanomethyl), N(3-fluoropropyl), N(3-amino-2-hydroxy-prop-1-yl), N(3,3-dimethyl-butyl), CH(amino), CH(methylamino), or CH(dimethylamino);
R1 is chloro, methoxy, or difluoromethyl;
and R3 is hydrogen or fluoro, R1 is chloro, methoxy, or difluoromethyl;
and R3 is hydrogen, R1 is chloro, methyl, methoxy, or difluoromethyl;
wherein R7 is selected from the group consisting of hydroxy, aminosulfonyl, 2-azabicyclo[2.2.1]heptan-5-yloxy, 3-azabicyclo[3.2.1]octan-8-yloxy, (3-methyl-3-azabicyclo[3.2.1]octan-8-yl)oxy, 9-azaspiro[3.5]nonan-6-yloxy, 3-(dimethylamino)propyloxy, 3-(t-butoxycarbonyl)-3-azaspiro[3.3]heptan-6-yloxy, and substituent f)
wherein Wf is selected from NH, N(methyl), N(2-methoxyethyl), N(2-fluoroethyl), N(2,2,2-trifluoroethyl), N(allyl), or S;
wherein R8 is selected from the group consisting of hydrogen, methyl, and 2-hydroxyethyl;
wherein R11 is 1-(t-butoxycarbonyl)-azetidin-3-ylmethyl;
wherein R10 is phenylcarbonylamino;
Embodiments of the present invention include a compound of Formula (I)
wherein
wherein R4 is selected from the group consisting of methylaminocarbonylmethyl, (2S,3R,4S)-3,4-dihydroxy-2-methyl-3,4-dihydro-2H-pyran-6-yl, 9-azaspiro[3.5]nonan-6-yloxy, (piperidin-3-yl)methylaminocarbonyl, 4-methyl-piperidin-4-yloxy, 1-methyl-piperidin-3-yloxy, and a substituent a)
wherein W is selected from N(cyanomethyl), or CH(dimethylamino);
wherein R7 is selected from the group consisting of (3-methyl-3-azabicyclo[3.2.1]octan-8-yl)oxy, 3-(t-butoxycarbonyl)-3-azaspiro[3.3]heptan-6-yloxy, and substituent f)
wherein Wf is NH or N(2-fluoroethyl);
or
Embodiments of the present invention include a compound of Formula (I)
wherein
Embodiments of the present invention include a compound of Formula (I)
wherein
Additional embodiments of the present invention include a compound of Formula (I)
wherein
wherein Wg is selected from NH, N(methyl), N(2-methoxyethyl), N(2-fluoroethyl), or N(allyl);
R1 is chloro or methoxy;
and R3 is hydrogen or fluoro, R1 is chloro or methoxy;
A further embodiment of the present invention is directed to a compound of Formula (I) selected from the group consisting of
and
Additional embodiments of the present invention include compounds of Formula (I) as herein defined, or an enantiomer, diastereomer, solvate, or a pharmaceutically acceptable salt form thereof, as exemplified in the listing in Table 1, below.
In a further embodiment, the invention is directed to a compound of Formula (I)
selected from the group consisting of
Cpd 49, ethyl 4-[[[4-[6-[6-cyano-5-(trifluoromethyl)-3-pyridyl]-5-oxo-7-thioxo-6,8-diazaspiro[3.4]octan-8-yl]benzoyl]amino]methyl]piperidine-1-carboxylate;
A further embodiment of the present invention is directed to an isotopic derivative of a compound of Formula (I). More particularly, the present invention is directed to a deuterated analog of a compound of Formula (I).
An embodiment of the present invention is directed to 5-[5,7-dioxo-8-[6-(4-piperidyloxy)-3-pyridyl]-6, 8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile (compound H-1)
and pharmaceutically acceptable salt forms thereof.
An embodiment of the present invention is directed to 5-[8-[4-[(1-methyl-4-piperidyl)oxy]phenyl]-5,7-dioxo-6, 8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile (compound H-2)
and pharmaceutically acceptable salt forms thereof.
For use in medicine, salts of compounds of Formula (I) refer to non-toxic “pharmaceutically acceptable salts.” Other salts may, however, be useful in the preparation of compounds of Formula (I) or of their pharmaceutically acceptable salt forms thereof. Suitable pharmaceutically acceptable salts of compounds of Formula (I) include acid addition salts that can, for example, be formed by mixing a solution of the compound with a solution of a pharmaceutically acceptable acid such as, hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of Formula (I) carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts such as, sodium or potassium salts; alkaline earth metal salts such as, calcium or magnesium salts; and salts formed with suitable organic ligands such as, quaternary ammonium salts. Thus, representative pharmaceutically acceptable salts include acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, pamoate (embonate), palmitate, pantothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate.
Representative acids and bases that may be used in the preparation of pharmaceutically acceptable salts include acids including acetic acid, 2,2-dichloroacetic acid, acylated amino acids, adipic acid, alginic acid, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D-glucoronic acid, L-glutamic acid, α-oxo-glutaric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, (+)-L-lactic acid, (+)-DL-lactic acid, lactobionic acid, maleic acid, (−)-L-malic acid, malonic acid, (+)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebaic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid and undecylenic acid; and bases including ammonia, L-arginine, benethamine, benzathine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)-ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, L-lysine, magnesium hydroxide, 4-(2-hydroxyethyl)-morpholine, piperazine, potassium hydroxide, 1-(2-hydroxyethyl)-pyrrolidine, sodium hydroxide, triethanolamine, tromethamine, and zinc hydroxide.
Embodiments of the present invention include prodrugs of compounds of Formula (I). In general, such prodrugs will be functional derivatives of the compounds that are readily convertible in vivo into the required compound. Thus, in the methods of treating or preventing embodiments of the present invention, the term “administering” encompasses the treatment or prevention of the various diseases, conditions, syndromes and disorders described with the compound specifically disclosed or with a compound that may not be specifically disclosed, but which converts to the specified compound in vivo after administration to a patient. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in “Design of Prodrugs”, ed. H. Bundgaard, Elsevier, 1985.
Where the compounds according to embodiments of this invention have at least one chiral center, they may accordingly exist as enantiomers. Where the compounds possess two or more chiral centers, they may additionally exist as diastereomers. It is to be understood that all such isomers and mixtures thereof are encompassed within the scope of the present invention. Furthermore, some of the crystalline forms for the compounds may exist as polymorphs and as such are intended to be included in the present invention. In addition, some of the compounds may form solvates with water (i.e., hydrates) or common organic solvents, and such solvates are also intended to be encompassed within the scope of this invention. The skilled artisan will understand that the term compound as used herein, is meant to include solvated compounds of Formula (I).
Where the processes for the preparation of the compounds according to certain embodiments of the invention give rise to mixture of stereoisomers, these isomers may be separated by conventional techniques such as, preparative chromatography. The compounds may be prepared in racemic form, or individual enantiomers may be prepared either by enantiospecific synthesis or by resolution. The compounds may, for example, be resolved into their component enantiomers by standard techniques such as, the formation of diastereomeric pairs by salt formation with an optically active acid such as, (−)-di-p-toluoyl-d-tartaric acid and/or (+)-di-p-toluoyl-1-tartaric acid followed by fractional crystallization and regeneration of the free base. The compounds may also be resolved by formation of diastereomeric esters or amides, followed by chromatographic separation and removal of the chiral auxiliary. Alternatively, the compounds may be resolved using a chiral HPLC column.
One embodiment of the present invention is directed to a composition, including a pharmaceutical composition, comprising, consisting of, and/or consisting essentially of the (+)-enantiomer of a compound of Formula (I) wherein said composition is substantially free from the (−)-isomer of said compound. In the present context, substantially free means less than about 25%, preferably less than about 10%, more preferably less than about 5%, even more preferably less than about 2% and even more preferably less than about 1% of the (−)-isomer calculated as
Another embodiment of the present invention is a composition, including a pharmaceutical composition, comprising, consisting of, and consisting essentially of the (−)-enantiomer of a compound of Formula (I) wherein said composition is substantially free from the (+)-isomer of said compound. In the present context, substantially free from means less than about 25%, preferably less than about 10%, more preferably less than about 5%, even more preferably less than about 2% and even more preferably less than about 1% of the (+)-isomer calculated as
During any of the processes for preparation of the compounds of the various embodiments of the present invention, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned. This may be achieved by means of conventional protecting groups such as those described in Protective Groups in Organic Chemistry, Second Edition, J. F. W. McOmie, Plenum Press, 1973; T. W. Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991; and T. W. Greene & P. G. M. Wuts, Protective Groups in Organic Synthesis, Third Edition, John Wiley & Sons, 1999. The protecting groups may be removed at a convenient subsequent stage using methods known from the art.
Even though the compounds of embodiments of the present invention (including their pharmaceutically acceptable salts and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient and/or a pharmaceutically acceptable diluent selected with regard to the intended route of administration and standard pharmaceutical or veterinary practice. Thus, particular embodiments of the present invention are directed to pharmaceutical and veterinary compositions comprising compounds of Formula (I) and at least one pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, and/or pharmaceutically acceptable diluent.
By way of example, in the pharmaceutical compositions of embodiments of the present invention, the compounds of Formula (I) may be admixed with any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilizing agent(s), and combinations thereof.
Solid oral dosage forms such as, tablets or capsules, containing the compounds of the present invention may be administered in at least one dosage form at a time, as appropriate. It is also possible to administer the compounds in sustained release formulations.
Additional oral forms in which the present inventive compounds may be administered include elixirs, solutions, syrups, and suspensions; each optionally containing flavoring agents and coloring agents.
Alternatively, compounds of Formula (I) can be administered by inhalation (intratracheal or intranasal) or in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder. For example, they can be incorporated into a cream comprising, consisting of, and/or consisting essentially of an aqueous emulsion of polyethylene glycols or liquid paraffin. They can also be incorporated, at a concentration of between about 1% and about 10% by weight of the cream, into an ointment comprising, consisting of, and/or consisting essentially of a wax or soft paraffin base together with any stabilizers and preservatives as may be required. An alternative means of administration includes transdermal administration by using a skin or transdermal patch.
The pharmaceutical compositions of the present invention (as well as the compounds of the present invention alone) can also be injected parenterally, for example, intracavernosally, intravenously, intramuscularly, subcutaneously, intradermally, or intrathecally. In this case, the compositions will also include at least one of a suitable carrier, a suitable excipient, and a suitable diluent.
For parenteral administration, the pharmaceutical compositions of the present invention are best used in the form of a sterile aqueous solution that may contain other substances, for example, enough salts and monosaccharides to make the solution isotonic with blood.
For buccal or sublingual administration, the pharmaceutical compositions of the present invention may be administered in the form of tablets or lozenges, which can be formulated in a conventional manner.
By way of further example, pharmaceutical compositions containing at least one of the compounds of Formula (I) as the active ingredient can be prepared by mixing the compound(s) with a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, and/or a pharmaceutically acceptable excipient according to conventional pharmaceutical compounding techniques. The carrier, excipient, and diluent may take a wide variety of forms depending upon the desired route of administration (e.g., oral, parenteral, etc.). Thus, for liquid oral preparations such as, suspensions, syrups, elixirs and solutions, suitable carriers, excipients and diluents include water, glycols, oils, alcohols, flavoring agents, preservatives, stabilizers, coloring agents and the like; for solid oral preparations such as, powders, capsules, and tablets, suitable carriers, excipients and diluents include starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like. Solid oral preparations also may be optionally coated with substances such as, sugars, or be enterically coated so as to modulate the major site of absorption and disintegration. For parenteral administration, the carrier, excipient and diluent will usually include sterile water, and other ingredients may be added to increase solubility and preservation of the composition. Injectable suspensions or solutions may also be prepared utilizing aqueous carriers along with appropriate additives such as, solubilizers and preservatives.
A therapeutically effective amount of a compound of Formula (I) or a pharmaceutical composition thereof includes a dose range from about 0.1 mg to about 3000 mg, or any particular amount or range therein, in particular from about 1 mg to about 1000 mg, or any particular amount or range therein, or, more particularly, from about 10 mg to about 500 mg, or any particular amount or range therein, of active ingredient in a regimen of about 1 to about 4 times per day for an average (70 kg) human; although, it is apparent to one skilled in the art that the therapeutically effective amount for a compound of Formula (I) will vary as will the diseases, syndromes, conditions, and disorders being treated.
For oral administration, a pharmaceutical composition is preferably provided in the form of tablets containing about 1.0, about 10, about 50, about 100, about 150, about 200, about 250, and about 500 milligrams of a compound of Formula (I).
An embodiment of the present invention is directed to a pharmaceutical composition for oral administration, comprising a compound of Formula (I) in an amount of from about 25 mg to about 500 mg.
In another embodiment, the pharmaceutical composition for oral administration comprises a compound of Formula (I) in an amount of from about 40 mg to about 95 mg.
Advantageously, a compound of Formula (I) may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three and four times daily.
Optimal dosages of a compound of Formula (I) to be administered may be readily determined and will vary with the particular compound used, the mode of administration, the strength of the preparation and the advancement of the disease, syndrome, condition or disorder. In addition, factors associated with the particular subject being treated, including subject gender, age, weight, diet and time of administration, will result in the need to adjust the dose to achieve an appropriate therapeutic level and desired therapeutic effect. The above dosages are thus exemplary of the average case. There can be, of course, individual instances wherein higher or lower dosage ranges are merited, and such are within the scope of this invention.
Compounds of Formula (I) may be administered in any of the foregoing compositions and dosage regimens or by means of those compositions and dosage regimens established in the art whenever use of a compound of Formula (I) is required for a subject in need thereof.
An embodiment of the present invention is directed to a pharmaceutical composition comprising, consisting of, and/or consisting essentially of, a compound selected from the group consisting of
and at least one of a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable diluent.
In another embodiment of the present invention, the compounds and compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for treating a cancer or another proliferative disease, disorder or condition. In some embodiments, the cancer or other proliferative disease, disorder or condition is a prostate cancer.
In some embodiments, the cancer or other proliferative disease, disorder or condition is a castration-resistant prostate cancer (CRPC). In some embodiments, the cancer or other proliferative disease, disorder or condition is a castration-resistant prostate cancer (CRPC) bearing a mutation in AR. In some embodiments, the mutation in AR is a mutation of Phenylalanine (Phe)876.
In some embodiments, the mutation in AR is a mutation of Phe876 to leucine. In some embodiments, the mutation in AR is a mutation of Phe876 to isoleucine. In some embodiments, the mutation in AR is a mutation of Phe876 to valine. In some embodiments, the mutation in AR is a mutation of Phe876 to serine. In some embodiments, the mutation in AR is a mutation of Phe876 to cysteine. In some embodiments, the mutation in AR is a mutation of Phe876 to tyrosine.
In some embodiments, the cancer or other proliferative disease, disorder or condition is a prostate cancer that is resistant to any AR therapy as a consequence of mutation.
In some embodiments, the cancer or other proliferative disease, disorder or condition is a prostate cancer that is resistant to treatment using second-generation AR antagonists, including, but not limited to, Enzalutamide or ARN-509.
The present invention encompasses the recognition that mutations in the AR polypeptide can render the AR polypeptide resistant to anti-androgens or convert anti-androgens to androgen agonists. In some embodiments, the present invention provides compounds that can be used to effect anti-androgenic effects despite the presence of such mutations.
The amino acid sequence of an AR polypeptide described herein can exist in a mutant AR containing, or can be modified to produce an mutant AR polypeptide variant at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) additions, substitutions, or deletions of a wild-type amino acid residue.
In some embodiments, the AR polypeptide variants described herein result in a loss of inhibition of AR activity by one or more antiandrogens of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 100%. In some embodiments, the AR polypeptide variants described herein convert antiandrogens to androgen receptor agonists.
Specific, nonlimiting amino acid residues that can be modified in an AR mutant include, e.g., E566, E589, E669, C687, A700, N772, H777, C785, F877, K911, of the AR polypeptide. These amino acid residues can be substituted with any amino acid or amino acid analog. For example, the substitutions at the recited positions can be made with any of the naturally-occurring amino acids (e.g., alanine, aspartic acid, asparagine, arginine, cysteine, glycine, glutamic acid, glutamine, histidine, leucine, valine, isoleucine, lysine, methionine, proline, threonine, serine, phenylalanine, tryptophan, or tyrosine). In particular instances, an amino acid substitution is E566K, E589K, E669K, C687Y, A700T, N772S, H777Y, C785R, F877C, F877I, F877L, F877S, F877V, F877Y and/or K911E.
In some embodiments, the AR mutants as described herein can include additional modifications of the AR polypeptide previously described in the art, including but not limited to, e.g., A597T, S648G, P683T, D696E, R727H, N728I, I738F, W741L, W741C, W741L, M743V, G751S, A871V, H874Y, T878A, T878S, and P914S.
In some embodiments, the compounds and compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for treating a bone disease, disorder or condition. In some embodiments, the bone disease, disorder or condition is osteoporosis.
In some embodiments, the present invention is directed to a compound of Formula (I) for use in the treatment of a disease, a syndrome, a condition or a disorder in a subject, including an animal, a mammal and a human in which the disease, the syndrome, the condition or the disorder is affected by the antagonism of the androgen receptor, selected from the group consisting of prostate cancer, castration-resistant prostate cancer, and metastatic castration-resistant prostate cancer.
In certain embodiments, a compound of Formula (I), or a composition thereof, may be administered in combination with another modulator, agonist or antagonist of AR. In some embodiments, the compound of Formula (I), or composition thereof, may be administered in combination with one or more other therapeutic agents.
In some embodiments the AR modulators, agonists or antagonists include, but are not limited to gonadotropin-releasing hormone agonists or antagonists (e.g. Lupron, Zoladex (Goserelin), Degarelix, Ozarelix, ABT-620 (Elagolix), TAK-385 (Relugolix), EP-100 or KLH-2109); non-steroidal antiandrogens, aminoglutethimide, enzalutamide, bicalutamide, nilutamide, flutamide, steroidal antiandrogens, finasteride, dutasteride, bexlosteride, izonsteride, turosteride, epristeride, other inhibitors of 5-alphareductase, 3,3′-diindolylmethane (DIM), N-butylbenzene-sulfonamide (NBBS); or a CYP17 inhibitor such as abiraterone acetate, TAK-700 (orteronel), TOK-001 (galeterone) or VT-464.
A further embodiment of the present invention is directed to a pharmaceutical composition comprising, consisting of, and/or consisting essentially of a compound of Formula (I) and a therapeutically effective amount of abiraterone acetate.
A further embodiment of the present invention is directed to a pharmaceutical composition comprising, consisting of, and/or consisting essentially of a compound of Formula (I) and abiraterone acetate and, optionally, prednisone or dexamethasone.
In certain embodiments, a compound of Formula (I), or a pharmaceutical composition thereof, may be administered in combination with a PI3K pathway inhibitor.
In some embodiments the PI3K pathway inhibitors (PI3K, TORC or dual PI3K/TORC inhibitor) include, but are not limited to, everolimus, BEZ-235, BKM120, BGT226, BYL-719, GDC0068, GDC-0980, GDC0941, GDC0032, MK-2206, OSI-027, CC-223, AZD8055, SAR245408, SAR245409, PF04691502, WYE125132, GSK2126458, GSK-2636771, BAY806946, PF-05212384, SF1126, PX866, AMG319, ZSTK474, CallOl, PWT33597, LY-317615 (enzastaurin hydrochloride), CU-906, or CUDC-907.
In certain embodiments, a compound of Formula (I), or a composition thereof, may be administered in combination with radiation therapy. The term “radiotherapy” or “ionizing radiation” include all forms of radiation, including but not limited to α, β, and γ radiation and ultraviolet light.
In some embodiments radiation therapy includes, but is not limited to, radioactive implants directly inserted in a tumor or body cavity (brachytherapy, interstitial irradiation, and intracavitary irradiation are types of internal radiotherapy), radiopharmaceuticals (e.g. Alpharadin (Radium-223 Chloride), 177Lu-J591 PSMA conjugate), or external beam radiation therapy (including Proton beam).
In certain embodiments, a compound of Formula (I), or a pharmaceutical composition thereof, may be administered in combination with immunotherapy.
In some embodiments the immunotherapy includes, but is not limited to Provenge, Prostvac, Ipilimumab, a CTLA-4 inhibitor or a PD-1 inhibitor.
Representative compounds of the present invention can be synthesized in accordance with the general synthetic methods described below and illustrated in the schemes and examples that follow. Since the schemes are an illustration, the invention should not be construed as being limited by the chemical reactions and conditions described in the schemes and examples. Compounds analogous to the target compounds of these examples can be made according to similar routes. The disclosed compounds are useful as pharmaceutical agents as described herein. The various starting materials used in the schemes and examples are commercially available or may be prepared by methods well within the skill of persons versed in the art.
Abbreviations used in the instant specification, particularly the schemes and examples, are as follows:
Compounds of Formula (I) may be prepared according to the process outlined in Scheme 1, below.
Accordingly, a suitably substituted compound of formula (II), a known compound or compound prepared by known methods, may be reacted with thiophosgene (III) and phenyl chlorothionocarbonate, in the presence of a suitably selected base such as DMAP, K2CO3, Cs2CO3, and the like, in a suitably selected solvent or mixture of solvents such as CHCl3, CH2Cl2, 1,2-dichloroethane, water, THF, toluene, and the like, at a temperature ranging from about 0 to about 130° C., to yield the corresponding compound of formula (IV). A suitably substituted compound of formula (V), a known compound or compound prepared by known methods, may be reacted with cyclobutanone (VI) in the presence of a suitably selected source of cyanide (VII), such as KCN, NaCN, TMS-CN, and the like; in a suitably selected solvent or mixture of solvents such as acetic acid, EtOH, MeOH, and the like, at temperature ranging from about 10 to about 130° C., to yield the corresponding compound of formula (VIII).
The compound of formula (IV) may then be reacted with the compound of formula (VIII) in a suitably selected solvent or mixture of solvents such as DMA, DMF, NMP, DSMO, and the like, at temperature ranging from about 15 to about 180° C., to yield the corresponding compound of formula (I).
Compounds of formula (I) may alternatively be prepared according to the process as outlined in Scheme 2, below.
Alternatively, a suitably substituted compound of formula (II), may be reacted with a compound of formula (VIII), a known compound or compound prepared by known methods, and thiophosgene, in the presence of a Lewis acid such as TMSOTf, AlCl3, ZnCl2, and the like, in a suitably selected solvent or mixture of solvents such as DMA, DMF, NMP, DSMO, and the like, at a temperature ranging from about 0 to about 180° C., to yield the corresponding compound of formula (I).
Compounds of formula (I) may alternatively be prepared according to the process as outlined in Scheme 3, below.
Alternatively, a suitably substituted compound of formula (IX), a known compound or compound prepared by known methods, wherein RA is H, alkyl, and the like, may be reacted with a compound of formula (X), wherein LG1 is a leaving group such as I, Br, Cl, triflate, and the like, in the presence of a copper catalyst such as CuI, and the like, in the presence of a suitably selected base such as DBU, t-BuOK, and the like; in a suitably selected solvent such as DMA, DMF, NMP, DMSO, and the like; at a temperature ranging from about 15 to about 170° C., under Ullman coupling conditions, to yield the corresponding compound of formula (XI). The compound of formula (XI) is then reacted with the compound of formula (IV) in a suitably selected solvent or mixture of solvents such as THF, 1,4-dioxane, toluene, DMSO, and the like, at temperature ranging from about 15 to about 180° C., to yield the corresponding compound of formula (I).
Compounds of formula (I) may alternatively be prepared according to the process as outlined in Scheme 4, below.
Alternatively, a suitably substituted compound of formula (II) may be reacted with a compound of formula (IX), a known compound or compound prepared by known methods, wherein RA is H, alkyl, and the like, to yield the corresponding compound of formula (XII). The compound of formula (XII) may then be reacted with the compound of formula (X), wherein LG1 is a leaving group such as I, Br, Cl, triflate, and the like, in the presence of a copper catalyst such as CuI, and the like, in the presence of a suitably selected base such as DBU, t-BuOK, and the like; in a suitably selected solvent such as DMA, DMF, NMP, DMSO, and the like; at temperature ranging from about 15 to about 170° C., under Ullman coupling conditions, to yield the corresponding compound of formula (XIII). The compound of formula (XIII) is then reacted with thiophosgene (III), phenyl chlorothionocarbonate in the presence of a suitably selected base such as DMAP, K2CO3, Cs2CO3, and the like, in a suitably selected solvent or mixture of solvents such as CHCl3, CH2Cl2, 1,2-dichloroethane, water, THF, toluene, and the like, at temperature ranging from about 0 to about 130° C., to yield the corresponding compound of formula (I).
Scheme 5 illustrates the preparation of certain compounds of the present invention wherein G is
and R4 is an amide.
Treatment of a suitably substituted compound of formula (XIV), wherein R, is C1-6 alkyl or the like, a known compound or compound prepared by known methods, may be reacted with a suitable base such as NaOH, LiOH, and the like, in a suitably selected solvent or mixture of solvents such as THF, MeOH, water, EtOH, and the like, at a temperature ranging from about 0 to about 60° C., to provide a carboxylic acid of formula (XV). A carboxylic acid of formula (XV) may then be coupled with a variety of agents to provide compounds within the scope of the invention. For example, the carboxylic acid of formula (XV) may be reacted with an appropriately substituted amine of formula H—NRA(RB) in the presence of a coupling reagent such as EDCI, HOBt, DCC, BOP, HATU, and the like, and a base such as trimethylamine, DIPEA, N-methylmorpholine, pyridine, and the like, in a suitably selected solvent or mixture of solvents such as DCM, DCE, THF, DMF, NMP, and the like, at a temperature ranging from about 0 to about 150° C.
Scheme 6 illustrates the preparation of certain compounds of the present invention wherein G is
and RD includes but is not limited to, a heterocyclyl, optionally partially unsaturated pyranyl, or bridged heterocyclyl substituent of the present invention.
The compound of formula (XVII), wherein LG1 is a suitably selected leaving group such as Cl, Br, OTf, F and the like, is a known compound or compound prepared by known methods. The compound of formula (XVII) may be reacted with a suitably substituted compound of formula (XVIII), wherein X is a suitably selected leaving group such as Cl, Br, OH, triflate, B(OH)2, B(OC1-2alkyl)2,
and the like, a known compound or compound prepared by known methods, under Suzuki coupling conditions. More particularly, such conditions may comprise reaction of a compound of formula (XVII) with a compound of formula (XVIII) in the presence of a suitably selected catalyst or catalyst system, such as Pd(PPh3)4, Pd2(dba)3, Pd(dppf), a mixture of Pd(OAc)2 and PPh3, and the like; in the presence of a suitably selected inorganic base such as K2CO3, Cs2CO3, Na2CO3, and the like; in a suitably selected solvent such as DME, 1,4-dioxane, and the like, preferably mixed with water; to yield the corresponding compound of formula (XIX).
One skilled in the art will recognize that the RD substituent group may alternatively be incorporated into the desired compound of formula (XIX) by reacting a compound of formula (XVIII), wherein the LG1 group is replaced with a group of the formula —B(ORD)2 (wherein the two RD groups are the same in each instance and are selected from hydrogen and C1-2alkyl; or the ORD groups are taken together with the atoms to which they are attached to form
with a suitably substituted compound of formula (XVIII), wherein the —X substituent may be replaced with a suitably selected leaving group such as Cl, Br, triflate, F, and the like; under Suzuki coupling conditions, more particularly, in the presence of a suitably selected catalysts or catalyst system, such as Pd(PPh3)4, Pd2(dba)3, Pd(dppf), a mixture of Pd(OAc)2 and PPh3, and the like; in the presence of a suitably selected inorganic base such as K2CO3, Cs2CO3, Na2CO3, and the like; in a suitably selected solvent such as DME, 1,4-dioxane, and the like, preferably mixed with water.
Alternatively, certain compounds of the present invention wherein G is
may be prepared according to the process outlined in Scheme 7.
A compound of formula (XX), wherein LG1 is a suitably selected leaving group such as Cl, Br, OTf, F, and the like, a known compound or compound prepared by known methods, may be reacted with a suitably substituted compound of formula (XVIII), wherein X is a suitable leaving group such as Cl, Br, OH, triflate, B(OH)2, B(OC1-2alkyl)2,
and the like under Suzuki coupling conditions, more particularly, in the presence of a suitably selected catalyst or catalyst system, such as Pd(PPh3)4, Pd2(dba)3, Pd(dppf), a mixture of Pd(OAc)2 and PPh3, and the like; in the presence of a suitably selected inorganic base such as K2CO3, Cs2CO3, Na2CO3, and the like; in a suitably selected solvent such as DME, 1,4-dioxane, and the like, preferably mixed with water; to yield the corresponding compound of formula (XXI).
The compound of formula (XXI) then may be reacted with a source of hydrogen, under hydrogenation conditions, in the presence of a suitably selected catalyst or catalyst system, such as Pd/C, Pt, and the like, in a suitably selected solvent such as MeOH, EtOAc, and the like, to yield the corresponding compound of formula (XXII). The compound of formula (XXII) then may be reacted with cyclobutanone (VI) in the presence of a suitably selected source of cyanide (VII), such as KCN, NaCN, TMS-CN, and the like; in a suitably selected solvent or mixture of solvents such as acetic acid, EtOH, MeO, and the like; at a temperature ranging from about 10 to about 130° C., to yield the corresponding compound of formula (XXIII).
The compound of formula (XXIII) may be reacted with the compound of formula (IV) in a suitably selected solvent or mixture of solvents such as DMA, DMF, NMP, DSMO, and the like, at a temperature ranging from about 15 to about 180° C., to yield the corresponding compound of formula (XXIV).
One skilled in the art will recognize that when the RD is heterocyclyl, compounds of formula (XVIII) may be prepared as exemplified in Chem. Eur. J. 2014, 20, 4414-4419 or Canadian Journal of Chemistry 1994,72(5),1262-72.
Certain compounds of the present invention wherein G is
and RE and RF may be taken together with the atoms to which they are attached to form a heterocyclyl including, but not limited to, piperidinyl, piperazinyl, thiomorpholinyl, and morpholinyl, may be prepared according to the process outlined in Scheme 8.
The compound of formula (XX), wherein LG1 is a suitably selected leaving group such as Cl, Br, OTf, F, and the like, a known compound or compound prepared by known methods, may be reacted with a suitable compound of formula (XXV), in the presence of a base such as CsF, Cs2CO2, K2CO3, tBuOK, NaH, and the like, in a suitably selected solvent or mixture of solvents such as THF, DMF, DMSO, DMA, DME, and the like; at a temperature ranging from about 10 to about 180° C., to yield the corresponding compound of formula (XXVI).
The compound of formula (XXVI) may then be reacted with a source of hydrogen, under hydrogenation conditions, in the presence of a suitably selected catalyst or catalyst system, such as Pd/C, Pt, and the like, in a solvent such as MeOH, EtOAc, and the like, to yield the corresponding compound of formula (XXVII). The compound of formula (XXVII) may be reacted with cyclobutanone (VI) in the presence of a source of cyanide of formula (VII), such as KCN, NaCN, TMS-CN, and the like; in a suitably selected solvent or mixture of solvents such as acetic acid, EtOH, MeO, and the like; at a temperature ranging from 10 to 130° C., to yield the corresponding compound of formula (XXVIII).
The compound of formula (XXVIII) may be reacted with the compound of formula (IV) in a suitably selected solvent or mixture of solvents such as DMA, DMF, NMP, DSMO, and the like, at temperature ranging from about 15 to about 180° C., to yield the corresponding compound of formula (XXIX).
Certain compounds of the present invention wherein G is
and RH includes, but is not limited to C1-6 alkyl, C3-7 cycloalkyl, and heterocyclic substituents of the present invention, may be prepared according to the process outlined in Scheme 9.
The compound of formula (XXX), wherein PG1 is a suitably selected amino protecting group such as —COCH3, -Cbz, and the like, a known compound or compound prepared by known methods, may be reacted with a compound of formula (XXXI), in the presence of a Lewis acid catalyst such as AlCl3, FeCl3, BF3, ZnCl2, and the like, under Friedel Craft acylation conditions, in a suitably selected solvent or mixture of solvents such as DCE, DCM, CH3NO2, and the like, to yield a compound of formula (XXXII). The compound of formula (XXXII) may be deprotected under various conventional reaction conditions, using reagents such as HCl if PG1 is acetyl, or hydrogenolysis if PG1 is carboxybenzyl, and the like, to afford a compound of formula (XXXIII). The compound of formula (XXXIII) may be reacted with cyclobutanone (VI) in the presence of a suitably selected source of cyanide (VII), such as KCN, NaCN, TMS-CN, and the like; in a suitably selected solvent or mixture of solvents such as acetic acid, EtOH, MeOH, and the like; at a temperature ranging from about 10 to about 130° C., to yield the corresponding compound of formula (XXXIV).
The compound of formula (XXXIV) may be reacted with the compound of formula (IV) in a suitably selected solvent or mixture of solvents such as DMA, DMF, NMP, DSMO, and the like, at a temperature ranging from about 15 to about 180° C., to yield the corresponding compound of formula (XXXV).
Certain compounds of the present invention wherein G is substituent i) and R4 is —Z-(L)n-RI; or wherein G is substituent ii) and R7 is —Z-(L)n-RI, may be prepared according to the process outlined in Scheme 10. In Scheme 10, Z may be O or S, L is C1-3 alkyl, and RI is an appropriate substituent of the present invention, including but not limited to, substituted and unsubstituted heterocyclyl substituents, and substituted and unsubstituted bridged heterocyclyl substituents.
Treatment of a suitably substituted compound of formula (XXXVI), (wherein A is C(R3) and B is CH; or, A is CH and B is N), a known compound or compound prepared by known methods, may be reacted with a suitably substituted compound of formula (XXXVII), a known compound or compound prepared by known methods, in the presence of DIAD, DEAD, and the like, and PPh3, under Mitsunobu conditions, in a suitably selected solvent or mixture of solvents such as THF, Et2O, and the like; at a temperature ranging from about 0 to about 130° C., to yield the corresponding compound of formula (XXXVIII).
An alternative route for the preparation of certain compounds of formula (XXXVIII), Scheme 10, is shown in Scheme 11. In Scheme 11, A may be CH(R3) when B is N, or A and B may be N.
A suitably substituted compound of formula (XXXIX), a known compound or compound prepared by known methods, may be reacted with a suitably substituted compound of formula (XXXVII), a known compound or compound prepared by known methods, in the presence of DIAD, DEAD, and the like, and PPh3, under conventional Mitsunobu conditions, in a suitably selected solvent or mixture of solvents such as THF, Et2O, and the like; at temperature ranging from about 0 to about 130° C., to yield the corresponding compound of formula (XL).
The compound of formula (XL) then may be reacted with a source of hydrogen, under hydrogenation conditions, in the presence of a suitably selected catalyst or catalyst system, such as Pd/C, Pt, and the like, in a suitably selected solvent such as MeOH, EtOAc, and the like, to yield the corresponding compound of formula (XLI). The compound of formula (XLI) may be reacted with cyclobutanone (VI) in the presence of a suitably selected source of cyanide (VII), such as KCN, NaCN, TMS-CN, and the like; in a suitably selected solvent or mixture of solvents such as acetic acid, EtOH, MeO, and the like; at temperature ranging from about 10 to about 130° C., to yield the corresponding compound of formula (XLII).
The compound of formula (XLII) then may be reacted with the compound of formula (IV) in a suitably selected solvent or mixture of solvents such as DMA, DMF, NMP, DSMO, and the like, at temperature ranging from about 15 to about 180° C., to yield the corresponding compound of formula (XXXVIII).
One skilled in the art will recognize that the nitro group in compound of formula (XXXIX) may be substituted with a suitable protecting group, then subsequently deprotected to its corresponding amine subsequent to the Mitsunobu reaction.
Certain compounds of the present invention wherein G is
(and RJ includes, but is not limited to, substituted and unsubstituted heterocyclyl, and substituted and unsubstituted cycloalkyl) may be prepared according to the process outlined in Scheme 12.
A suitably substituted compound of formula (XLIII), a known compound or compound prepared by known methods, may be treated with cyclobutanone (VI) in the presence of a suitably selected source of cyanide (VII), such as KCN, NaCN, TMS-CN, and the like; in a suitably selected solvent or mixture of solvents such as acetic acid, EtOH, MeO, and the like; at temperature ranging from about 10 to about 130° C., to yield the corresponding compound of formula (XLIV).
The compound of formula (XLIV) may be reacted with the compound of formula (IV) in a suitably selected solvent or mixture of solvents such as DMA, DMF, NMP, DSMO, and the like, at temperature ranging from about 15 to about 180° C., to yield the corresponding compound of formula (XLV).
Certain compounds of the present invention wherein G is
and RK is methyl or a Boc-protected azetidinylmethyl group, may be prepared according to the process outlined in Scheme 13.
A suitably substituted compound of formula (XLVI), a known compound or compound prepared by known methods, may be treated with a compound of formula (XLVII), a known compound or compound prepared by known methods, in the presence of DIAD, DEAD, and the like, and PPh3, under Mitsunobu conditions, in a suitably selected solvent or mixture of solvents such as THF, Et2O, and the like; at a temperature ranging from about 0 to about 130° C., to yield the corresponding compounds of formula (XLVIII) and (XLIX).
Certain compounds of the present invention wherein G is
and RB is amino or dimethylamino, may be prepared according to the process outlined in Scheme 14.
Treatment of a suitably substituted compound of formula (L), a known compound or compound prepared by known methods (wherein RL is hydrogen or methyl), may be treated with cyclobutanone (VI) in the presence of a suitably selected source of cyanide (VII), such as KCN, NaCN, TMS-CN, and the like; in a suitably selected solvent or mixture of solvents such as acetic acid, EtOH, MeO, and the like; at a temperature ranging from about 10 to about 130° C., to yield the corresponding compound of formula (LI).
The compound of formula (LI) may be reacted with the compound of formula (IV) in a suitably selected solvent or mixture of solvents such as DMA, DMF, NMP, DSMO, and the like, at a temperature ranging from about 15 to about 180° C., to yield the corresponding compound of formula (LII). A compound of formula (LII) may be reacted with an acid such as HCl, TFA, and the like, in a suitably selected solvent or mixture of solvents such as MeOH, EtOH, 1,4-dioxane, water, DCM, and the like; at a temperature ranging from about 0 to about 80° C., to yield the corresponding compound of formula (LIII). A compound of formula LIII may be reacted with RL-LG1, wherein is a leaving group such as I, Br, Cl, triflate, and the like, in the presence of a suitably selected base, such as K2CO3, Cs2CO3, Et3N, DIPEA, and the like, in the presence of a suitably selected solvent such as THF, DCM, MeCN, DMF, NMP, DMSO, and the like; at a temperature ranging from about 15 to about 170° C., to yield the corresponding compound of formula (LIV). One skilled in the art will recognize that the RL substituent group may alternatively be incorporated into the desired compound of formula (LIV) by reacting a compound of formula (LIII) with RL—CHO, in the presence of an acid or not such as AcOH, p-TsOH, in the presence of a reductive agent such as NaBH(AcO)3, NaCNBH3, and the like, under reductive amination conditions, in the presence of a suitably selected solvent such as DCM, DCE, THF, MeOH, and the like, at a temperature ranging from about 0 to about 80° C., to yield the corresponding compound of formula (LIV).
Cesium fluoride (5.71 g, 37.6 mmol) was added to a solution of 2-Chloro-5-nitro-pyrimidine (4.0 g, 25.0 mmol) and 4-Hydroxy-piperidine-1-carboxylic acid tert-butyl ester (5.0 g, 25.0 mmol) in DMF (120 mL). The resulting mixture was stirred for 24 h at room temperature. The insolubles were collected by filtration through a short pad of diatomaceous earth. The filtrate was concentrated under reduced pressure. The residue was taken in EA (40 mL) and washed successively with water (40 mL) and brine (20 mL). The organic layer was dried over MgSO4, filtered, and concentrated to dryness. Chromatography over silica gel (gradient of EA in heptane from 0 to 35%) gave the pure product as a white solid (2.43 g, 30%). 1H NMR (300 MHz, Chloroform-d) δ 1.47 (s, 9H), 1.76-1.92 (m, 2H), 1.98-2.13 (m, 2H), 3.21-3.44 (m, 2H), 3.63-4.00 (m, 2H), 5.26-5.50 (m, 1H), 9.29 (s, 2H). MS m/z 269 C14H20N4O5 (M+H-tBu)+.
4-(5-Nitro-pyrimidin-2-yloxy)-piperidine-1-carboxylic acid tert-butyl ester (3.0 g, 9.23 mmol) was dissolved in MeOH (40 mL) and cooled in ice/water bath under nitrogen stream. Dry 10% Pd/C (0.6 g) was added to the cold solution. The reaction vessel was connected to a balloon filled with hydrogen. The suspension was then stirred under a hydrogen atmosphere at room temperature during 1 h. The catalyst was removed by filtration through a pad of diatomaceous earth. Removal of solvent gave the crude product that was used without further treatment (2.71 g, 100%). 1H NMR (300 MHz, Chloroform-d) δ 1.46 (s, 9H), 1.69-1.86 (m, 2H), 1.89-2.08 (m, 2H), 3.20-3.34 (m, 2H), 3.38 (br s, 2H), 3.65-3.90 (m, 2H), 4.90-5.16 (m, 1H), 8.03 (s, 2H). C14H22N4O3 MS m/z 295 (M+H)+.
Cyclobutanone (1.38 mL, 18.5 mmol) and sodium cyanide (0.90 g, 18.5 mmol) were added successively to a solution of the previous intermediate (2.71 g, 9.23 mmol) in acetic acid (45 mL). The reaction was stirred overnight at room temperature. The solution was then concentrated under reduced pressure in a fume hood. The residue was taken in EA (50 mL) and washed with 1M Na2CO3 (100 mL) and brine (25 mL). The organic layer was dried over MgSO4, filtered and concentrated to a crude oily residue. Chromatography over silica gel (gradient of EA in heptane from 0 to 60%) gave the pure product (2.47 g, 71%). 1H NMR (300 MHz, Chloroform-d) δ 1.47 (s, 9H), 1.72-1.86 (m, 2H), 1.91-2.07 (m, 2H), 2.12-2.31 (m, 2H), 2.31-2.45 (m, 2H), 2.68-2.88 (m, 2H), 3.17-3.37 (m, 2H), 3.70-3.91 (m, 3H), 5.00-5.16 (m, 1H), 8.05 (s, 2H). C19H27N5O3 MS m/z 374 (M+H)+
4-[5-(1-Cyano-cyclobutylamino)-pyrimidin-2-yloxy]-piperidine-1-carboxylic acid tert-butyl ester (2.47 g, 6.61 mmol), and freshly prepared 5-Isothiocyanato-3-trifluoromethyl-pyridine-2-carbonitrile (2.73 g, 11.9 mmol) were heated at 60° C. in DMA (35 mL) during 4 h and then allowed to cool to room temperature. The mixture was diluted with MeOH (7 mL) and 1M HCl (7 mL) was added. The stirring was maintained at room temperature overnight. EA (50 mL) was added and solution washed with water (100 mL), saturated 1M Na2CO3 (30 mL) and brine (50 mL). The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 50%). The fractions with product were collected and concentrated under reduced pressure to yield an amorphous solid (3.40 g, 85%). 1H NMR (300 MHz, Chloroform-d) δ 1.48 (s, 10H), 1.73-1.98 (m, 3H), 2.00-2.18 (m, 2H), 2.23-2.40 (m, 1H), 2.42-2.60 (m, 2H), 2.69-2.87 (m, 2H), 3.25-3.44 (m, 2H), 3.76-3.96 (m, 2H), 5.21-5.40 (m, 1H), 8.34 (d, J=2.2 Hz, 1H), 8.50 (s, 2H), 9.08 (d, J=2.2 Hz, 1H). C27H28F3N7O4S MS m/z 548 (M+H-tBu)+.
The previous 4-{4-[7-(6-Cyano-5-methoxy-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-benzyl}-piperidine-1-carboxylic acid tert-butyl ester (3.40 g, 5.63 mmol) was taken in dioxane (25 mL). Dry 4N HCl in dioxane (14.0 mL, 56.0 mmol) was added with stirring. The mixture was stirred overnight at room temperature and diluted with diethyl ether (150 mL). Triturating during 2 hours gave a white powder that was collected by filtration and dried under high vacuum (2.86 g, 94%). 1H NMR (300 MHz, DMSO-d6) δ 1.52-1.75 (m, 1H), 1.92-2.11 (m, 3H), 2.17-2.32 (m, 2H), 2.42-2.59 (m, 2H), 2.58-2.73 (m, 2H), 3.05-3.21 (m, 2H), 3.21-3.35 (m, 2H), 5.17-5.47 (m, 1H), 8.74 (s, 2H), 8.75 (d, J=2.0 Hz, 1H), 9.08 (br s, 2H), 9.21 (d, J=2.0 Hz, 1H). C22H21ClF3N7O2S MS m/z 504 (M+H)+.
1H NMR (300 MHz, Chloroform-d) δ 1.71-2.30 (m, 7H), 2.42-2.58 (m, 1H), 2.66-2.83 (m, 2H), 2.85-3.01 (m, 2H), 3.18-3.34 (m, 2H), 5.06-5.36 (m, 1H), 8.34 (d, J=2.3 Hz, 1H), 8.50 (s, 2H), 9.08 (d, J=2.3 Hz, 1H). C22H20F3N7O2S MS m/z 504 (M+H)+.
Formaldehyde (37% wt in water, 2.8 mL, 37.2 mmol) was added to a solution of 5-{8-Oxo-5-[2-(piperidin-4-yloxy)-pyrimidin-5-yl]-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl}-3-trifluoromethyl-pyridine-2-carbonitrile (2.88 g, 5.31 mmol) and sodium acetate (0.436 g, 5.31 mmol) in DCE (15 mL). The mixture was stirred at room temperature for 40 min, before Sodium triacetoxyborohydride (1.78 g, 7.96 mmol) was added in 3 portions within 45 minutes. The reaction was continued 2 h and diluted with DCM (125 mL). The solution was washed successively with 1M Na2CO3 (100 mL) and water (50 mL). The organic layer was dried over MgSO4, filtered and concentrated to give the crude product. Chromatography over silica gel (gradient of MeOH in DCM from 0 to 15%) gave, upon removal of solvent, a white foam (2.27 g, 82%). 1H NMR (300 MHz, Chloroform-d) δ 1.69-1.90 (m, 1H), 1.94-2.09 (m, 2H), 2.09-2.23 (m, 2H), 2.25-2.42 (m, 3H), 2.35 (s, 3H), 2.42-2.59 (m, 2H), 2.69-2.94 (m, 4H), 5.00-5.27 (m, 1H), 8.35 (d, J=2.2 Hz, 1H), 8.50 (s, 2H), 9.09 (d, J=2.2 Hz, 1H). C23H22F3N7O2S MS m/z 518 (M+H)+.
The previous 5-[8-[2-[(1-methyl-4-piperidyl)oxy]pyrimidin-5-yl]-5-oxo-7-thioxo-6,8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile (2.27 g, 4.38 mmol) was taken in dioxane (15 mL) and treated with 4N HCl in dioxane (1.26 mL, 5.04 mmol) with stirring. After 1.5 h, diethyl ether (50 mL) was added and the resulting suspension was stirred for another 30 min. The solid was collected by filtration through a sintered funnel and subsequently washed with diethyl ether (2×15 mL). The solid was collected and dried under high vacuum at room temperature to yield the pure title hydrochloride salt (2.24 g, 89%). 1H NMR (300 MHz, DMSO-d6) δ 1.48-1.77 (m, 1H), 1.87-2.14 (m, 2H), 2.15-2.29 (m, 2H), 2.29-2.40 (m, 1H), 2.43-2.58 (m, 2H), 2.58-2.71 (m, 2H), 2.78 (dd, J=13.7, 4.2 Hz, 3H), 3.02-3.30 (m, 2H), 3.30-3.44 (m, 2H), 3.44-3.55 (m, 1H), 5.07-5.31 (m, 0.5H), 5.31-5.46 (m, 0.5H), 8.74 (s, 1H), 8.75 (s, 2H); 9.21 (s, 1H), 10.75 (br s, 1H). C23H23ClF3N7O2S MS m/z 518 (M+H)+.
A. To a solution of 4-(4-nitrophenoxy)piperidine (0.845 g, 3.8 mmol) in EtOH (20 mL) was added a solution of sodium methoxide in MeOH ˜25% (0.878 mL, 4.68 mmol). After stirring overnight at RT, epichlorohydrin (0.367 mL, 4.68 mmol) was added. After stirring at RT overnight, the mixture was diluted with EtOAc and the organic layer washed with water and brine. The aqueous portion was extracted with EtOAc. The combined organic extracts were dried over MgSO4, filtered and concentrated to give 4-(4-nitrophenoxy)-1-(oxiran-2-ylmethyl)piperidine, directly used in the next step. 1H NMR (300 MHz, Chloroform-d) δ 1.81-1.97 (m, 2H), 1.98-2.13 (m, 2H), 2.24-2.36 (m, 1H), 2.36-2.58 (m, 3H), 2.71-2.85 (m, 3H), 2.85-2.96 (m, 1H), 3.05-3.17 (m, 1H), 4.36-4.57 (m, 1H), 6.94 (d, J=9.2 Hz, 2H), 8.19 (d, J=9.2 Hz, 2H); C14H18N2O4 MS m/z 279 (M+H)+.
B. Aqueous ammonia (8.23 mL, 95.05 mmol) was added to a solution of 4-(4-nitrophenoxy)-1-(oxiran-2-ylmethyl)piperidine in 1,4-dioxane (20 mL). After stirring at 60° C. in a seal tube overnight, the mixture was allowed to cool down to RT and the solvent concentrated. The residue was purified by chromatography over silica gel (gradient of 2.0M ammonia in MeOH in DCM from 0 to 100%) to give 1-amino-3-(4-(4-nitrophenoxy)piperidin-1-yl)propan-2-ol as a yellow oil (1.13 g, 43%). C14H21N3O4 MS m/z 296 (M+H)+.
C. Di-tert-butyl dicarbonate (0.545 mL, 2.54 mmol) was added to a solution of 1-amino-3-(4-(4-nitrophenoxy)piperidin-1-yl)propan-2-ol (0.716 g, 2.42 mmol) in DCM (10 mL). After stirring overnight at RT, the solvent was concentrated and the residue purified by chromatography over silica gel (gradient of MeOH in DCM from 0 to 30%) to give tert-butyl (2-hydroxy-3-(4-(4-nitrophenoxy)piperidin-1-yl)propyl)carbamate (0.455 g, 75%) as a yellow oil. 1H NMR (300 MHz, Chloroform-d) δ 1.44 (s, 9H), 1.78-1.95 (m, 2H), 1.96-2.16 (m, 2H), 2.27-2.46 (m, 3H), 2.53-2.76 (m, 2H), 2.85-2.99 (m, 1H), 2.98-3.15 (m, 1H), 3.24-3.46 (m, 1H), 3.65-3.88 (m, 1H), 4.39-4.58 (m, 1H), 4.99 (br s, 1H), 6.94 (d, J=9.3 Hz, 2H), 8.19 (d, J=9.3 Hz, 2H); C19H29N3O6 MS m/z 396 (M+H)+.
D. A solution of tert-butyl (2-hydroxy-3-(4-(4-nitrophenoxy)piperidin-1-yl)propyl)carbamate (0.45 g, 1.138 mmol) in MeOH (10 mL) was purged using nitrogen and vacuum. Palladium on charcoal (10% wet, 0.12 g) was added and the mixture was hydrogenated (20 psi) for 16 h. The reaction mixture was filtered through diatomaceous earth, the cake washed with MeOH, and the solvent concentrated under reduced pressure to give tert-butyl (3-(4-(4-aminophenoxy)piperidin-1-yl)-2-hydroxypropyl)carbamate (0.416 g, 93%) as a brown foam used directly into the next step. 1H NMR (300 MHz, DMSO-d6) δ 1.37 (s, 9H), 1.52-1.76 (m, 2H), 1.87-2.05 (m, 2H), 2.15-2.39 (m, 4H), 2.63-2.79 (m, 2H), 2.79-2.95 (m, 1H), 2.95-3.12 (m, 1H), 3.52-3.80 (m, 1H), 4.42-4.69 (m, 2H), 6.65 (t, J=5.8 Hz, 1H), 7.16 (d, J=9.3 Hz, 2H), 8.17 (d, J=9.1 Hz, 2H); C19H31N3O4 MS m/z 0.366 (M+H)+.
E. Cyclobutanone (0.23 mL, 3.1 mmol) and sodium cyanide (0.152 g, 3.1 mmol) were added successively to a solution of the previous intermediate (0.613 g, 0.738 mmol) in acetic acid (5 mL). The reaction was stirred overnight at room temperature. The solution was then concentrated under reduced pressure. The residue was taken in EtOAc (100 mL) and washed with water (50 mL), aqueous saturated NaHCO3 (50 mL) and brine (50 mL). The organic layer was dried over MgSO4, filtered and concentrated to give a crude residue. Chromatography over silica gel (gradient of MeOH/DCM (1/20) in DCM from 0 to 50%) gave tert-butyl (3-(4-(4-((1-cyanocyclobutyl)amino)phenoxy)piperidin-1-yl)-2-hydroxypropyl)carbamate (0.204 g, 20%); C24H36N4O4 MS m/z 445 (M+H)+.
Following the procedure described in Example 1, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of Formula (I) of the invention were prepared.
A mixture of 5-(8-oxo-5-(2-(piperidin-4-yloxy)pyrimidin-5-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.15 g, 0.298 mmol), (2-Bromoethoxy)-tert-butyldimethylsilane (0.128 mL, 0.596 mmol) and Et3N (0.082 mL, 0.596 mmol) in DMF (5 mL) was stirred at RT overnight. Additional (2-Bromoethoxy)-tert-butyldimethylsilane (0.128 mL, 0.596 mmol) and Et3N (0.082 mL, 0.596 mmol) were added to the mixture and after stirring at 50° C. overnight, the mixture was diluted with EtOAc and aqueous 1.0M Na2CO3. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated. The crude material was purified by chromatography over silica gel (gradient of MeOH in DCM from 0 to 10%). The fractions with product were collected and concentrated under reduced pressure to yield 5-(5-(2-((1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)oxy)pyrimidin-5-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.06 g, 29%). C30H38F3N7O3SSi MS m/z 661.8 (M+H)+.
TBAF (0.043 g, 0.136 mmol) was added to a solution of 5-(5-(2-((1-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperidin-4-yl)oxy)pyrimidin-5-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.06, 0.09 mmol) in THF (5 mL). After stirring at RT for 5 days the solvent was removed under reduced pressure. The crude material was purified by chromatography over silica gel (gradient of MeOH in DCM from 10 to 50%). The residue was then purified by preparative reverse phase HPLC chromatography (from 75% aqueous 25 mM NH4CO3/25% ACN to 38% aqueous 25 mM NH4CO3/62% ACN-MeOH) to give 5-(8-oxo-5-(2-(piperidin-4-yloxy)pyrimidin-5-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.06 g, 12%) as a solid. 1H NMR (300 MHz, Chloroform-d) δ 1.70-1.88 (m, 1H), 1.89-2.07 (m, 2H), 2.06-2.20 (m, 2H), 2.22-2.41 (m, 1H), 2.41-2.55 (m, 4H), 2.61 (t, J=5.4 Hz, 2H), 2.68-2.83 (m, 2H), 2.83-2.98 (m, 2H), 3.64 (t, J=5.4 Hz, 2H), 5.05-5.31 (m, 1H), 8.34 (d, J=2.2 Hz, 1H), 8.49 (s, 2H), 9.08 (d, J=2.2 Hz, 1H). C24H24F3N7O3S MS m/z 548 (M+H)+.
2-Oxa-6-azaspiro[3.3]heptane hemioxalate (0.30 g, 208 mmol) and triethylamine (0.87 mL, 4.16 mmol) were stirred at room temperature in a mixture of DCE (50 mL) and MeOH (5 mL). To the resulting solution was added (tert-Butyl-dimethyl-silyloxy)-acetaldehyde (0.79 mL, 4.16 mmol) with stirring. Sodium triacetoxyborohydride (0.88 g, 4.16 mmol) was added portion wise over 45 min. Upon reaction completion (2 h), the mixture was diluted with DCM (75 mL) and washed with 1M Na2CO3 (50 mL). The aqueous layer was extracted with DCM (20 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to yield an oily residue that was used without further treatment (0.536 g, 100%).
The crude oil was taken in THF (30 mL) and treated with TBAF-trihydrate (0.985 g, 3.12 mmol) overnight at room temperature. The solution was then concentrated and product was isolated by preparative LC (gradient of ACN/MeOH 50/50 in 25 mM aqueous NH4HCO3 from 5 to 37%). The desired fractions were collected and concentrated to an oil (0.298 g, 100%). 1H NMR (300 MHz, DMSO-d6) δ 2.59 (t, J=5.8 Hz, 2H), 3.37 (t, J=5.7 Hz, 2H), 3.55 (s, 4H), 4.59 (s, 4H), 8.21 (s, 1H). C7H13NO2 MS m/z 144 (M+H)+.
Sodium cyanide (1.01 g, 20.6 mmol) was added to a solution of cyclobutanone (5.13 mL, 68.7 mmol) and 4-amino-phenol (5.0 g, 45.8 mmol) in acetic acid (50 mL). The resulting mixture was stirred for 6 h. The solution was concentrated under reduced pressure. The residue was partitioned between EA (500 mL) and 1M Na2CO3 (250 mL). The organic layer was further washed with saturated NaHCO3 (250 mL), brine (250 mL), dried over MgSO4, filtered and concentrated to dryness. Chromatography over silica gel (gradient of MeOH in DCM from 0 to 3%) gave the pure product as a beige solid (6.94 g, 78%). 1H NMR (300 MHz, Chloroform-d) δ 1.79-1.97 (m, 1H), 2.05-2.27 (m, 3H), 2.29-2.46 (m, 2H), 3.80 (br s, 1H), 4.82 (br s, 1H), 6.58 (d, J=8.8 Hz, 2H), 6.75 (d, J=8.6 Hz, 2H). C11H12N20 MS m/z 189 (M+H)+
1-(4-Hydroxy-phenylamino)-cyclobutanecarbonitrile (6.93 g, 29.1 mmol) and freshly prepared 5-Isothiocyanato-3-trifluoromethyl-pyridine-2-carbonitrile (6.67 g, 29.1 mmol) were heated overnight at 60° C. in DMA (116 mL) and then allowed to cool to room temperature. The mixture was diluted with MeOH (58 mL) and 1M HCl (58 mL) was added. The stirring was maintained at room temperature for 1 h. EA (500 mL) was added and the solution washed with water (250 mL), saturated NaHCO3 (250 mL) and brine (250 mL). The combined aqueous layers were back extracted with EA (500 mL). The organic layers were dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 65%). The fractions with product were collected and concentrated under reduced pressure. Crystallization from ACN (50 mL) gave a white solid (7.92 g, 63%). 1H NMR (300 MHz, DMSO-d6) δ 1.45-1.66 (m, 1H), 1.87-2.07 (m, 1H), 2.35-2.49 (m, 2H), 2.54-2.71 (m, 2H), 6.95 (d, J=8.7 Hz, 2H), 7.18 (d, J=8.7 Hz, 2H), 8.76 (d, J=2.0 Hz, 1H), 9.22 (d, J=2.0 Hz, 1H), 9.92 (s, 1H). C19H3F3N4O2S MS m/z 419 (M+H)+.
5-[5-(4-Hydroxy-phenyl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl]-3-trifluoromethyl-pyridine-2-carbonitrile (0.57 g, 1.36 mmol), 2-(2-Oxa-6-aza-spiro[3.3]hept-6-yl)-ethanol (0.195 g, 1.36 mmol) and triphenylphosphine (0.71 g, 2.72 mmol) were dissolved in dry THF (15 mL) under nitrogen atmosphere and heated at 65° C. A solution of Diisopropyl azodicarboxylate (DIAD, 0.54 mL, 2.72 mmol) in THF (5 mL) was added dropwise over 15-20 min. Upon completion of the addition, the reaction was continued for 2 h at the same temperature. The mixture was then allowed to cool and concentrate to dryness. The crude residue was chromatographed over silica gel (gradient of MeOH in DCM from 0 to 10%). The pure fractions were concentrated to an amorphous solid. Triturating in diethyl ether gave a white powder (0.0157 g, 21%). 1H NMR (300 MHz, Chloroform-d) δ 1.58-1.79 (m, 1H), 2.09-2.34 (m, 1H), 2.46-2.76 (m, 4H), 2.87 (t, J=5.2 Hz, 2H), 3.57 (s, 4H), 4.05 (t, J=5.2 Hz, 2H), 4.77 (s, 4H), 7.07 (d, J=9.0 Hz, 2H), 7.22 (d, J=9.0 Hz, 2H), 8.36 (d, J=2.3 Hz, 1H), 9.10 (d, J=2.3 Hz, 1H). C26H24F3N5O3S MS m/z 544 (M+H)+.
Following the procedure described in Example 3, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of formula (I) of the invention were prepared.
To a solution of 5-(8-oxo-5-(4-(piperidin-4-yloxy)phenyl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.3 g, 0.598 mmol) in Et3N (0.125 mL, 0.897 mmol) and MeCN (4 mL) was added propargyl bromide 80% in toluene (0.077 mL, 0.718 mmol). After stirring at RT for 2 h, the solvent was removed under reduced pressure and the residue was diluted with DCM (60 mL) and the organic layer was washed with water and brine. The organic layer was dried over MgSO4, filtered and concentrated. The crude was purified by chromatography over silica gel (gradient of MeOH/DCM (1/50) from 0 to 100%). The fractions with product were collected and concentrated under reduced pressure. The residue was then purified by preparative reverse phase HPLC chromatography (from 70% aqueous 25 mM NH4HCO3/30% ACN to 27% aqueous 25 mM NH4HCO3/73% ACN). Desired fractions were collected, concentrated and aqueous layer extracted with DCM. Combined organic layers were dried over MgSO4, filtered and concentrated to give a solid further triturated with Et2O, filtered and dried to give 5-[5-oxo-8-[4-[(1-prop-2-ynyl-4-piperidyl)oxy]phenyl]-7-thioxo-6, 8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile (0.138 g, 42%) as a yellow solid. 1H NMR (300 MHz, Chloroform-d) δ 1.59-1.85 (m, 2H), 1.85-2.02 (m, 2H), 2.00-2.16 (m, 2H), 2.28 (s, 1H), 2.45-2.76 (m, 6H), 2.76-2.98 (m, 2H), 3.36 (s, 2H), 4.29-4.50 (m, 1H), 7.08 (d, J=8.5 Hz, 2H), 7.21 (d, J=8.4 Hz, 2H), 8.37 (d, J=2.3 Hz, 1H), 9.10 (d, J=2.3 Hz, 1H). C27H24F3N5O2S MS m/z 540 (M+H)+
To a solution of 5-(5-(4-((1-methylpiperidin-4-yl)oxy)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (9.29 g, 18.033 mmol) in dry chloroform (250 mL) was added at 0° C. under a nitrogen atmosphere, 3-chloroperbenzoic acid (6.062 g, 27.05 mmol). Upon reaction completion (3 h), the mixture was partitioned between aqueous 1.0M Na2CO3 and DCM. The aqueous layer was extracted with DCM. The combined organic layers were dried over MgSO4, filtered and concentrated to dryness to give crude cis/trans 5-[8-[4-(1-methyl-1-oxido-piperidin-1-ium-4-yl)oxyphenyl]-5-oxo-7-thioxo-6,8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile. The reaction was repeated a second time to generate a total of 13.27 g of crude cis/trans 5-[8-[4-(1-methyl-1-oxido-piperidin-1-ium-4-yl)oxyphenyl]-5-oxo-7-thioxo-6,8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile. The crude was purified by reverse phase chromatography (from 81% aqueous 25 mM NH4HCO3/19% MeCN-MeOH to 45% aqueous 25 mM NH4HCO3/55% MeCN-MeOH). The isomeric mixture was separated by reverse phase chromatography (from 72% aqueous 0.1% HCOOH/28% ACN-MeOH to 36% aqueous 0.1% HCOOH/64% ACN-MeOH) to give first Cpd 191 (cis isomer, first product to elute) and then Cpd 190 (trans isomer, second product to elute).
For each set of collected fractions, the aqueous layer was neutralized with solid Na2CO3, extracted with DCM, dried over MgSO4, filtered, and concentrated to dryness to generate Cpd 191 and Cpd 190 as a foam, each further triturated to generate a yellow solid: Cpd 191, (cis isomer, 2.1 g, 30%) and Cpd 190 (trans isomer, 0.58 g, 8.7%).
Cpd 191: 1H NMR (300 MHz, DMSO-d6) δ 1.45-1.64 (m, 1H), 1.78-2.04 (m, 4H), 2.34-2.46 (m, 3H), 2.62 (m, 2H), 3.00-3.12 (m, 2H), 3.20 (s, 3H), 3.49-3.66 (m, 2H), 4.72-4.83 (m, 1H), 7.21 (d, J=8.7 Hz, 2H), 7.34 (d, J=8.6 Hz, 2H), 8.76 (d, J=2.1 Hz, 1H), 9.22 (d, J=2.0 Hz, 1H). C25H24F3N5O3S MS m/z 532 (M+H)+.
Cpd 190: 1H NMR (300 MHz, DMSO-d6) δ 1.47-1.64 (m, 1H), 1.88-2.11 (m, 4H), 2.22-2.46 (m, 3H), 2.55-2.72 (m, 2H), 3.17 (s, 3H), 3.19-3.31 (m, 2H), 3.44-3.57 (m, 2H), 4.49-4.66 (m, 1H), 7.19 (d, J=8.6 Hz, 2H), 7.32 (d, J=8.5 Hz, 2H), 8.75 (s, 1H), 9.21 (s, 1H). C25H24F3N5O3S MS m/z 532 (M+H)+.
To a solution of 2-Hydroxy-5-nitropyridine (10 g, 69.24 mmol) in Tetrahydrofuran (350 mL) at room temperature under nitrogen, 1-Boc-4-hydroxypiperidine (18.67 g, 90 mmol) and Triphenylphosphine (54.5 g, 207.7 mmol) were added. Finally DIAD (40.9 mL, 207.7 mmol) was added dropwise and the mixture was stirred at room temperature overnight. The crude material was poured onto water/NaHCO3 and extracted with Ethyl acetate. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The residue was purified by flash chromatography over silica gel (Ethyl acetate-heptane gradient from 5% to 30%). Pure fractions were combined, concentrated and dried under high vacuum to give the product (22.3 g, 99%). C15H22N3O5 MS m/z 224.2 (M-100+H)+.
A solution of 4-(5-Nitro-pyridin-2-yloxy)-piperidine-1-carboxylic acid tert-butyl ester (22.4 g, 69.24 mmol) in methanol (210 mL) was purged with nitrogen. Then Pd/C 10% wet catalyst (1.34 g) was added to the solution. The mixture was purged with hydrogen and stirred under a hydrogen atmosphere at room temperature for 14 h. The catalyst was removed by filtration through diatomaceous earth and the solvent evaporated under vacuum to afford the product (20.3 g, 100%). 1H NMR (300 MHz, Chloroform-d) δ 1.46 (s, 9H), 1.56-1.80 (m, 2H), 1.80-2.11 (m, 2H), 3.06-3.39 (m, 2H), 3.62-3.95 (m, 2H), 5.05 (tt, J=7.8, 3.7 Hz, 1H), 6.53-6.59 (m, 1H), 7.01 (dd, J=8.7, 3.0 Hz, 1H), 7.62 (d, J=2.9 Hz, 1H). C15H23N3O3 MS m/z 294.2 (M+H)+.
To a solution of 4-(5-Amino-pyridin-2-yloxy)-piperidine-1-carboxylic acid tert-butyl ester (20.3 g, 69.2 mmol) and Cyclobutanone (10.35 mL, 138.5 mmol) in Ethanol (56 mL) and Acetic acid (56 mL), sodium cyanide (13.57 g, 276.95 mmol) was added. The mixture was heated to 50° C. and stirred at this temperature for 15 h. The solution was then poured onto water followed by extraction with dichloromethane. The organic layer was separated and washed with brine, then dried over MgSO4, filtered and concentrated. The product was purified by flash chromatography over silica gel (Methanol-Dichloromethane gradient from 0% to 10%). Fractions were combined and concentrated to dryness. The residue was recrystallized from diisopropyl ether to afford the product as a beige solid (17.75 g, 70%). 1H NMR (300 MHz, Chloroform-d) δ 1.47 (s, 9H), 1.61-1.81 (m, 2H), 1.89-2.01 (m, 2H), 2.12-2.27 (m, 1H), 2.31-2.45 (m, 2H), 2.76 (ddd, J=11.8, 8.2, 5.7 Hz, 2H), 3.16-3.35 (m, 2H), 3.63-3.85 (m, 3H), 5.10 (dt, J=8.0, 4.1 Hz, 1H), 6.65 (d, J=8.8 Hz, 1H), 7.04 (dd, J=8.8, 2.9 Hz, 1H), 7.62 (d, J=2.9 Hz, 1H). C20H28N4O3 MS m/z 373.3 (M+H)+.
A solution of 4-[5-(1-Cyano-cyclobutylamino)-pyridin-2-yloxy]-piperidine-1-carboxylic acid tert-butyl ester (13.36 g, 35.86 mmol) and 3-Chloro-5-isothiocyanato-pyridine-2-carbonitrile (7.02 g, 35.86 mmol) in N,N-Dimethylacetamide was heated to 60° C. and stirred at that temperature for 15 h. The mixture was allowed to cool to room temperature. Methanol (50 mL) and 1M HCl (50 mL) were added. The mixture was stirred at room temperature for 30 min. The crude reaction mixture was quenched with a NaHCO3 saturated solution and extracted with ethyl acetate. The organic layer was separated and washed with brine, then dried over MgSO4, filtered and concentrated. The product was purified by flash chromatography over silica gel (Ethyl acetate-heptane gradient from 5% to 40%). Fractions were combined and concentrated to dryness to give the product as a foam (17.3 g, 84.9%). C27H29ClN6O4S MS m/z 513.0 (M−55)+.
To a solution of 4-{5-[7-(5-Chloro-6-cyano-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-pyridin-2-yloxy}-piperidine-1-carboxylic acid tert-butyl ester (17.3 g, 30.4 mmol) in Dichloromethane (90 mL), at 0° C. under Nitrogen, Trifluoroacetic acid (60 mL) was added. The mixture was then stirred at room temperature for 2 h, then evaporated to dryness. The residue was dissolved in dichloromethane, and washed with NaHCO3 saturated solution. The organic layer was dried over MgSO4, filtered and concentrated. The product was purified by flash chromatography over silica gel (Methanol-dichloromethane gradient from 0 to 10%). Product fractions were combined and concentrated to dryness to give the product as a foam (10.2 g, 72%). C22H21ClN6O2S MS m/z 468.9 (M+H)+.
To a solution of 3-chloro-5-[5-oxo-8-[6-(4-piperidyloxy)-3-pyridyl]-7-thioxo-6,8-diazaspiro[3.4]octan-6-yl]pyridine-2-carbonitrile in ethyl acetate, a 4N Hydrogen chloride solution in dioxane was added, followed by evaporation of solvents. The obtained beige solid was suspended in 40 mL of acetonitrile and stirred at 50° C. for 20 min, then cooled to room temperature and collected by filtration. The white solid was then dried under reduced pressure to constant weight (6.4 g, 88%). 1H NMR (300 MHz, DMSO-d6) δ 1.59 (d, J=10.7 Hz, 1H), 1.93-2.05 (m, 3H), 2.17-2.31 (m, 2H), 2.39-2.47 (m, 2H), 2.59-2.71 (m, 2H), 3.07-3.31 (m, 4H), 5.28-5.37 (m, 1H), 7.09 (d, J=8.8 Hz, 1H), 7.81 (dd, J=8.8, 2.6 Hz, 1H), 8.24 (d, J=2.6 Hz, 1H), 8.55 (d, J=2.1 Hz, 1H), 8.91 (d, J=2.0 Hz, 1H), 9.23 (s, 2H). C22H21ClN6O2S MS m/z 468.9 (M+H)+.
Formaldehyde (37% wt in water, 0.143 mL, 1.92 mmol) was added to a solution of 3-chloro-5-[5-oxo-8-[6-(4-piperidyloxy)-3-pyridyl]-7-thioxo-6,8-diazaspiro[3.4]octan-6-yl]pyridine-2-carbonitrile (0.300 g, 0.64 mmol) in DCE (8 mL). The mixture was stirred at room temperature for 10 min, then Sodium triacetoxyborohydride (0.407 g, 1.92 mmol) was added. The reaction was stirred for 15 h and diluted with ethyl acetate. The solution was washed successively with saturated NaHCO3 solution, water and brine. The organic layer was dried over MgSO4, filtered and concentrated to give the crude product. The product was purified by flash chromatography over silica gel (Methanol-dichloromethane gradient from 0 to 10%). Pure product fractions were combined and concentrated to dryness. The hydrochloride salt was prepared by addition of 4N Hydrogen chloride solution in dioxane to a solution of product in ethyl acetate followed by evaporation of solvents. The white solid Cpd 26 was then dried under reduced pressure to constant weight (0.176 g, 35%). 1H NMR (300 MHz, DMSO-d6) δ 1.43-1.60 (m, 1H), 1.84-2.00 (m, 3H), 2.04-2.16 (m, 2H), 2.19-2.39 (m, 1H), 2.44 (t, J=1.9 Hz, 3H), 2.51-2.61 (m, 1H), 2.64-2.83 (m, 3H), 3.02-3.17 (m, 2H), 3.37-3.46 (m, 1H), 5.04-5.41 (m, 1H), 7.01 (dd, J=8.8, 4.1 Hz, 1H), 7.75 (ddd, J=8.9, 6.4, 2.7 Hz, 1H), 8.17 (dd, J=4.5, 2.6 Hz, 1H), 8.47 (s, OH), 8.83 (d, J=1.9 Hz, 1H), 10.33-10.84 (m, 1H). C23H24Cl2N6O2S MS m/z 483.0 (M+H)+.
Following the procedure described in Example 6, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of formula (I) of the invention were prepared.
To a solution of 5-amino-3-(trifluoromethyl)picolinonitrile (14.97 g, 80 mmol) in chloroform (150 mL) was added water (90 mL) and the mixture was stirred vigorously. DMA (10 mL) and thiophosgene (12.2 mL, 160 mmol) were then added dropwise. After 20 min, the layers were separated, the organic layer was dried over MgSO4, filtered and concentrated to give 5-isothiocyanato-3-(trifluoromethyl)picolinonitrile (18.335 g, 100%) used directly into the next step.
A solution of 5-nitropyridin-2-ol (150 g, 1.07 mol) in MeOH (2 L) was purged using nitrogen and vacuum. Palladium on charcoal (10% wet) was added the mixture was hydrogenated (40 psi) for 16 hours. The reaction mixture was filtered through Diatomaceous earth and concentrated under reduced pressure to give 5-aminopyridin-2-ol as a dark oil used directly into the next step.
To a solution of 5-aminopyridin-2-ol (60 g, 490.4 mmol) and cyclobutanone (47.65 mL, 637.75 mmol) in MeOH (700 mL) was added zinc iodide (7.8 g, 24.43 mmol) at RT. Trimethylsilyl cyanide (73 g, 735.8 mmol) was then added in several portions and the mixture was stirred at 50° C. for 16 h, allowed to cool to RT, and then concentrated under reduced pressure. The residue was purified by chromatography over silica gel (gradient of MeOH in DCM from 0 to 8%). The fractions with product were collected and concentrated under reduced pressure to yield 1-((6-hydroxypyridin-3-yl)amino) cyclobutanecarbonitrile as a dark solid (45 g, 48%). 1H NMR (300 MHz, Chloroform-d) δ 1.93-2.10 (m, 2H) 2.18-2.32 (m, 2H) 2.55 (br. s., 2H) 5.77-5.92 (m, 1H) 6.26-6.39 (m, 1H) 6.48-6.67 (m, 1H) 6.99-7.19 (m, 1H) 10.81-11.19 (m, 1H) C10H11N3O MS m/z 190.1 (M+H)+.
A solution of 5-isothiocyanato-3-(trifluoromethyl)picolinonitrile (13 g, 45.38 mmol) in DMA (60 mL) was added to a solution of 1-((6-hydroxypyridin-3-yl)amino) cyclobutanecarbonitrile (10.416 g, 54.5 mmol) in DMA (60 mL). The mixture was heated at 60° C. for 2 h and then allowed to cool to room temperature. The mixture was treated with MeOH (100 mL) and 2M HCl (100 mL). The resulting suspension stirred at 60° C. for 1 h. The mixture was filtered and the filter cake was washed with water, MeOH, and then dried to give 5-(5-(6-hydroxypyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a grey solid (16.7 g, 86%). 1H NMR (400 MHz, DMSO-d6) δ 1.60-1.71 (m, 1H) 1.88-2.01 (m, 1H) 2.36-2.44 (m, 2H) 2.53-2.60 (m, 2H) 6.48 (d, J=9.54 Hz, 1H) 7.40 (dd, J=9.66, 2.32 Hz, 1H) 7.58 (br. s., 1H) 8.67 (s, 1H) 9.15 (s, 1H) 12.01 (br. s., 1H). C18H12F3N5O2S MS m/z 420 (M+H)+.
5-[5-(6-Hydroxy-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl]-3-trifluoromethyl-pyridine-2-carbonitrile (16.6 g, 39.6 mmol), 4-Hydroxy-piperidine-1-carboxylic acid tert-butyl ester (8.94 g, 43.5 mmol) and triphenylphosphine (22.8 g, 87.1 mmol) were dissolved in dry THF (150 mL) under a nitrogen atmosphere and heated at 50° C. A solution of Diisopropyl azodicarboxylate (DIAD, 15.6 mL, 79.1 mmol) in THF (50 mL) was added dropwise. Upon completion of the addition, the reaction was continued for 3 h at the same temperature. The mixture was then allowed to cool and concentrated to dryness. The crude residue was purified by column chromatography on silica gel (gradient of Ethyl acetate in heptane from 0 to 30%). The fractions with product were concentrated to an amorphous solid directly used in the next step. 1H NMR (300 MHz, Chloroform-d) δ 1.47 (s, 9H), 1.66-1.86 (m, 3H), 1.94-2.10 (m, 2H), 2.15-2.35 (m, 1H), 2.44-2.62 (m, 2H), 2.63-2.80 (m, 2H), 3.22-3.40 (m, 2H), 3.70-3.90 (m, 2H), 5.21-5.35 (m, 1H), 6.91 (d, J=8.8 Hz, 1H), 7.52 (dd, J=8.8, 2.6 Hz, 1H), 8.09 (d, J=2.6 Hz, 1H), 8.36 (d, J=2.2 Hz, 1H), 9.09 (d, J=2.2 Hz, 1H). C28H29F3N6O4S MS m/z 547 (M+H-tBu)+.
The previous 4-{5-[7-(6-Cyano-5-trifluoromethyl-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-pyridin-2-yloxy}-piperidine-1-carboxylic acid tert-butyl ester (43.4 g) was taken in DCM (300 mL). TFA (60 mL) was added with stirring. The mixture was stirred overnight at room temperature and concentrated under reduced pressure. The residue was taken in toluene (150 mL) and again concentrated (3 times). The crude residue was then purified by column chromatography on silica gel (gradient of MeOH in DCM from 0 to 10%) to afford a yellowish amorphous solid (19.8 g). Final purification was performed by preparative LC (gradient of a mixture ACN/MeOH (1/1, v/v) in 0.1% aqueous formic acid from 10 to 54%). The pure fractions were collected and pH brought to 8-9 by addition of solid Na2CO3. The product was extracted with EA (3×400 mL). The combined organic layers were washed with brine (300 mL), dried over MgSO4, filtered and concentrated to a white foam (9.57 g, 47% for two steps). 1H NMR (300 MHz, Chloroform-d) δ 1.45-1.57 (m, 1H), 1.87-2.15 (m, 5H), 2.20-2.41 (m, 2H), 2.42-2.60 (m, 2H), 2.94-3.09 (m, 2H), 3.10-3.30 (m, 2H), 5.15-5.27 (m, 1H), 6.74 (d, J=8.7 Hz, 1H), 7.39 (dd, J=8.7, 2.6 Hz, 1H), 7.90 (d, J=2.6 Hz, 1H), 8.24 (d, J=2.2 Hz, 1H), 8.91 (d, J=2.2 Hz, 1H). C23H21F3N6O2S MS m/z 503 (M+H)+.
The previous 5-{8-Oxo-5-[6-(piperidin-4-yloxy)-pyridin-3-yl]-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl}-3-trifluoromethyl-pyridine-2-carbonitrile (9.57 g, 19.0 mmol) was taken in dioxane (54 mL) and treated with 4N HCl in dioxane (5.24 mL, 20.9 mmol) with stirring. After 1 h, the mixture was concentrated to dryness under reduced pressure. Diethyl ether (50 mL) was added and the resulting suspension was stirred overnight. The solid was collected on a sintered funnel and washed with diethyl ether (2×15 mL). The solid was dried under high vacuum at room temperature to yield the pure title hydrochloride salt (9.85 g, 93%). 1H NMR (300 MHz, DMSO-d6) δ 1.48-1.69 (m, 1H), 1.87-2.04 (m, 3H), 2.12-2.29 (m, 2H), 2.34-2.48 (m, 2H), 2.58-2.73 (m, 2H), 3.04-3.20 (m, 2H), 3.21-3.35 (m, 2H), 5.26-5.40 (m, Hz, 1H), 7.09 (d, J=8.8 Hz, 1H), 7.80 (dd, J=8.8, 2.6 Hz, 1H), 8.22 (d, J=2.6 Hz, 1H), 8.75 (d, J=2.0 Hz, 1H), 8.92 (br s, 2H), 9.21 (d, J=2.0 Hz, 1H). C23H22ClF3N6O2S MS m/z 503 (M+H)+.
Formaldehyde (37% wt in water, 1.2 mL, 15.2 mmol) was added to a solution of 5-{8-Oxo-5-[6-(piperidin-4-yloxy)-pyridin-3-yl]-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl}-3-trifluoromethyl-pyridine-2-carbonitrile (4.68 g, 7.13 mmol) in THF (40 mL). The mixture was stirred at room temperature for 30 min, before Sodium triacetoxyborohydride (2.54 g, 11.38 mmol) was added. The reaction was continued overnight and diluted with EA (200 mL). The solution was washed with 1M Na2CO3 (100 mL). The aqueous layer was back extracted once with EA (100 mL). The combined organic layers were dried over MgSO4, filtered and concentrated to give the crude product. Purification by column chromatography on silica gel (gradient of MeOH in DCM from 0 to 10%) gave, after removal of solvent, a white foam. Preparative LC (gradient from 30 to 73% of a mixture ACN/MeOH (1/1, v/v) in 25 mM aqueous NH4HCO3) afforded title compound as a white solid (1.57 g, 41%). 1H NMR (300 MHz, Chloroform-d) δ 1.64-1.82 (m, 1H), 1.82-1.98 (m, 2H), 2.05-2.19 (m, 2H), 2.19-2.42 (m, 3H), 2.34 (s, 3H), 2.44-2.62 (m, 2H), 2.63-2.84 (m, 4H), 5.00-5.24 (m, 1H), 6.91 (d, J=8.8 Hz, 1H), 7.50 (dd, J=8.8, 2.7 Hz, 1H), 8.09 (d, J=2.6 Hz, 1H), 8.36 (d, J=2.2 Hz, 1H), 9.09 (d, J=2.2 Hz, 1H). C24H23F3N6O2S MS m/z 517 (M+H)+.
5-{5-[6-(1-Methyl-piperidin-4-yloxy)-pyridin-3-yl]-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl}-3-trifluoromethyl-pyridine-2-carbonitrile (1.57 g, 3.04 mmol) was taken in dioxane (10 mL) and treated with 4N HCl in dioxane (0.84 mL, 3.37 mmol) with stirring. After 1 h, the mixture was concentrated to dryness under reduced pressure. Diethyl ether (30 mL) was added and the resulting suspension was stirred until a powdered solid was obtained. The solid was collected by filtration on a sintered funnel and washed with diethyl ether (2×15 mL). The solid was dried under high vacuum at room temperature to yield the pure title hydrochloride salt (1.51 g, 90%). 1H NMR (300 MHz, DMSO-d6) δ 1.50-1.70 (m, 1H), 1.87-2.36 (m, 5H), 2.37-2.51 (m, 2H), 2.57-2.71 (m, 2H), 2.77 (br s, 3H), 3.04-3.26 (m, 2H), 3.32-3.54 (m, 2H), 5.10-5.51 (m, 1H), 7.08 (d, J=8.8 Hz, 1H), 7.81 (dd, J=8.7, 2.6 Hz, 1H), 8.23 (d, J=2.6 Hz, 1H), 8.75 (d, J=2.1 Hz, 1H), 9.22 (d, J=2.1 Hz, 1H), 10.72 (s, 1H). C24H24ClF3N6O2S MS m/z 517 (M+H)+.
A. To a solution of tert-butyl 3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (10 g, 44.38 mmol) and triethylamine (10.8 g, 106.73 mmol) in DMF (100 mL) was added dropwise trimethylsilyl chloride (5.8 g, 53.38 mmol). The mixture was stirred at 100° C. for 16 h, allowed to cool to room temperature, and concentrated under reduced pressure. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 100%). The fractions with product were collected and concentrated under reduced pressure to yield tert-butyl 3-((trimethylsilyl)oxy)-8-azabicyclo[3.2.1]oct-3-ene-8-carboxylate as a light yellow oil (8 g, 61%).
B. To a solution of tert-butyl 3-((trimethylsilyl)oxy)-8-azabicyclo[3.2.1]oct-3-ene-8-carboxylate (4 g, 13.44 mmol) in MeCN (50 mL) was added 1-chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate) (5.2 g, 14.67 mmol). The mixture was stirred at RT for 16 h and diluted with water and EtOAc. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 100%). The fractions with product were collected and concentrated under reduced pressure to yield tert-butyl 2-fluoro-3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate as a white solid (2.5 g, 76%). C12H18FNO3 MS m/z 266.12 (M+Na)+.
C. To a solution of tert-butyl 2-fluoro-3-oxo-8-azabicyclo[3.2.1]octane-8-carboxylate (2.5 g, 10.27 mmol) in MeOH (30 mL) was added sodium borohydride (1.17 g, 30.92 mmol) at 0° C. and stirred at RT for 16 h. The mixture was concentrated under reduced pressure and diluted with water and EtOAc. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 100%). The fractions with product were collected and concentrated under reduced pressure to yield tert-butyl 2-fluoro-3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylate as a white solid (0.25 g, 11%). C12H20FNO3 MS m/z 268.13 (M+Na)+.
Following the procedure described in Example 7, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of formula (I) of the invention were prepared.
To a solution of 5-(8-oxo-5-(6-(piperidin-4-yloxy)pyridin-3-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.246 g, 0.4 mmol), DIEA (0.206 g, 1.6 mmol) in THF (5 mL) was added 2,2,2-trifluoroethyl trifluoromethanesulfonate (0.185 g, 0.8 mmol). The mixture was heated at 80° C. for 12 h, cooled down to RT and concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC (Column: Gemini C18 150*25 mm*10 μm, Flow rate: 25 mL/min, Mobile Phase A: Purified water (containing 0.1% HCl), Mobile Phase B: acetonitrile, Gradient: 36-66(% B) from 0-15 min) to afford 5-[5-oxo-7-thioxo-8-[6-[[1-(2,2,2-trifluoroethyl)-4-piperidyl]oxy]-3-pyridyl]-6,8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile hydrochloride salt (0.0562 g, 23%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.53-1.64 (m, 1H) 1.83-2.02 (m, 3H) 2.16 (br. s., 2H) 2.38-2.47 (m, 2H) 2.64 (t, J=10.16 Hz, 2H) 2.97 (br. s., 2H) 3.18 (br. s., 2H) 3.65-3.81 (m, 2H) 5.19 (br. s., 1H) 7.06 (d, J=8.78 Hz, 1H) 7.78 (dd, J=8.53, 2.76 Hz, 1H) 8.22 (d, J=2.51 Hz, 1H) 8.75 (d, J=2.01 Hz, 1H) 9.21 (d, J=1.76 Hz, 1H) C25H22F6N6O2S. HCl MS m/z 585.1 (M+H)+.
To a solution of 5-(8-oxo-5-(6-(piperidin-4-yloxy)pyridin-3-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.154 g, 0.25 mmol), DIEA (0.129 g, 1 mmol) in DMF (3 mL) was added 1-fluoro-2-iodoethane (0.087 g, 0.5 mmol). The mixture was heated at 80° C. for 12 h, cooled down to RT and concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC ((Column: Gemini C18 150*25 mm*10 μm, Flow rate: 25 mL/min, Mobile Phase A: Purified water (containing 0.1% HCl), Mobile Phase B: acetonitrile, Gradient: 15-45(% B) from 0-15 min) to afford 5-[8-[6-[[1-(cyanomethyl)-4-piperidyl]oxy]-3-pyridyl]-5-oxo-7-thioxo-6,8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile hydrochloride salt (0.075 g, 50%). 1H NMR (400 MHz, DMSO-d6) d ppm 1.52-1.65 (m, 1H) 1.90-2.02 (m, 1H) 2.09 (br. s., 2H) 2.22-2.34 (m, 2H) 2.38-2.49 (m, 2H) 2.64 (t, J=9.79 Hz, 2H) 3.16 (br. s., 2H) 3.30 (br. s., 2H) 4.41 (br. s., 2H) 5.28 (br. s., 1H) 7.05-7.10 (m, 1H) 7.80 (dd, J=8.78, 2.51 Hz, 1H) 8.24 (d, J=2.51 Hz, 1H) 8.76 (d, J=2.01 Hz, 1H) 9.22 (d, J=1.76 Hz, 1H). C25H22F3N7O2S. HCl MS m/z 542.2 (M+H)+.
To a solution of 5-(8-oxo-5-(6-(piperidin-4-yloxy)pyridin-3-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.154 g, 0.25 mmol), DIEA (0.129 g, 1 mmol) in DMF (3 mL) was added 2-chloroacetonitrile (0.038 g, 0.5 mmol). The mixture was heated at 80° C. for 12 h, cooled down to RT and concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC ((Column: Gemini C18 150*25 mm*10 μm, Flow rate: 25 mL/min, Mobile Phase A: Purified water (containing 0.1% HCl), Mobile Phase B: acetonitrile, Gradient: 15-45(% B) from 0-15 min) to afford 5-[8-[6-[[1-(2-fluoroethyl)-4-piperidyl]oxy]-3-pyridyl]-5-oxo-7-thioxo-6,8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile hydrochloride salt (0.078 g, 53%). 1H NMR (400 MHz, DMSO-d6) d ppm 1.51-1.67 (m, 1H) 1.90-2.13 (m, 2H) 2.15-2.37 (m, 3H) 2.39-2.48 (m, 2H) 2.65 (t, J=9.79 Hz, 2H) 3.18-3.28 (m, 2H) 3.45-3.64 (m, 4H) 4.78-5.02 (m, 2H) 5.19-5.43 (m, 1H) 7.09 (d, J=8.78 Hz, 1H) 7.81 (d, J=6.78 Hz, 1H) 8.24 (br. s., 1H) 8.75 (d, J=2.01 Hz, 1H) 9.22 (d, J=2.01 Hz, 1H) 10.83 (br. s., 1H). C25H24F4N6O2S. HCl MS m/z 549.1 (M+H)+.
3,3-Dimethylbutanal (0.032 g, 0.32 mmol) was added to a solution of 5-(8-oxo-5-(4-(piperidin-4-yloxy)phenyl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl) picolinonitrile (0.1 g, 0.164 mmol), acetic acid (0.009 g, 0.162 mmol) in DCE (3 mL). The mixture was stirred at RT for 30 min, before Sodium triacetoxyborohydride (0.069 g, 0.324 mmol) was added. The reaction was stirred at RT overnight, washed with aqueous saturated NaHCO3 and extracted with EtOAc. The organic layer was washed successively with water, brine, dried over MgSO4, filtered and concentrated to give the crude product. The residue was purified by preparative reverse phase HPLC (Column: Gemini 150*25 mm*5 μm, Flow rate: 25 mL/min, Mobile Phase A: Purified water (containing 0.1% HCl), Mobile Phase B: acetonitrile, Gradient: 30-60(% B) from 0-10 min). Desired fractions were collected, concentrated under reduced pressure, neutralized with aqueous saturated NaHCO3 and extracted with DCM. The organic layer was dried over MgSO4, filtered and concentrated to afford 5-(5-(4-((1-(3,3-dimethylbutyl)piperidin-4-yl)oxy)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.048 g, 50%). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.88 (s, 9H) 1.34 (d, J=12.57 Hz, 2H) 1.49-1.71 (m, 3H) 1.97 (br. s., 2H) 2.19 (br. s., 2H) 2.24-2.35 (m, 2H) 2.42 (br. s., 2H) 2.56-2.67 (m, 3H) 2.72 (br. s., 2H) 4.44 (br. s., 1H) 7.14 (d, J=8.60 Hz, 2H) 7.28 (d, J=8.60 Hz, 2H) 8.75 (s, 1H) 9.21 (s, 1H). C30H34F3N5O2S MS m/z 586.1 (M+H)+.
A. To a solution of 5-(5-(4-hydroxyphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.21 g, 0.5 mmol), (R)-benzyl 3-hydroxypyrrolidine-1-carboxylate (0.124 g, 0.6 mmol) and triphenylphosphine (0.2 g, 0.75 mmol) in dry THF (6 mL) was added DIAD (0.15 g, 0.75 mmol) under a nitrogen atmosphere. After stirring at RT overnight the mixture was diluted with water and extracted with EtOAc. The organic layer was then washed with water, brine, dried over Na2SO4, filtered and concentrated to dryness. Chromatography over silica gel (gradient of EA in heptane from 0 to 50%) gave (R)-benzyl 3-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenoxy)pyrrolidine-1-carboxylate as a pale yellow solid (0.22 g, 71%).
To a solution of (R)-benzyl 3-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenoxy)pyrrolidine-1-carboxylate (0.2 g, 0.32 mmol) in DCM (6.4 mL) was added borane dimethyl sulfide complex (0.41 g, 3.2 mmol). The mixture was stirred at room temperature 4 h, then poured into water/aqueous saturated NaHCO3 and extracted with DCM. The organic layer was then washed with water, brine, dried over Na2SO4, filtered and concentrated to dryness. Chromatography over silica gel (gradient of MeOH in DCM from 0 to 10%) gave (R)-5-(8-oxo-5-(4-(pyrrolidin-3-yloxy)phenyl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl) picolinonitrile as an off-white solid (0.08 g, 82%).
Formaldehyde (37% wt in water, 36 μl, 0.49 mmol) was added at 0° C. to a solution of (R)-5-(8-oxo-5-(4-(pyrrolidin-3-yloxy)phenyl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.08 g, 0.16 mmol) and AcOH (46 μL, 0.8 mmol) in MeOH (3.2 mL). The mixture was stirred at room temperature overnight, then diluted with water and aqueous saturated NaHCO3 and extracted with EtOAc. The organic layer was washed with water, brine, dried over Na2SO4, filtered and concentrated to dryness. The resulting material was purified by preparative reverse phase HPLC (C18 column using gradient of a mixture MeCN/0/1% aqueous TFA from 30 to 70%). The pure fractions were collected, concentrated to dryness, redissolved in EtOAc, and treated with aqueous 2.0M HCl in Et2O at 0° C., then concentrated to give 5-[8-[4-[(3R)-1-methylpyrrolidin-3-yl]oxyphenyl]-5-oxo-7-thioxo-6, 8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile HCl as a pale yellow solid (0.036 g, 42%). 1H NMR (500 MHz, DMSO-d6) δ ppm 1.54-1.62 (m, 1H), 1.98-2.05 (m, 1H), 2.15-2.26 (m, 1H), 2.31 (dtd, 1H, J=14.2, 9.4, 4.9 Hz), 2.39-2.51 (m, 3H), 2.67 (br t, 3H, J=9.5 Hz), 2.93 (br s, 1H), 3.44-3.56 (m, 1H), 3.75-3.83 (m, 1H), 5.26 (br s, 1H), 7.22 (br d, 2H, J=8.8 Hz), 7.40 (d, 2H, J=7.9 Hz), 8.79 (d, 1H, J=1.9 Hz), 9.25 (d, 2H, J=1.6 Hz) C24H22F3N5O2S. HCl MS m/z 502.1 (M+H)+.
Cyanomethylenetributylphosphorane (0.181 g, 0.75 mmol) was added to a solution of 5-(5-(4-hydroxyphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl) picolinonitrile (0.209 g, 0.5 mmol) and tert-butyl (1S,5R)-3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylate (0.17 g, 0.75 mmol) in THF (10 mL). The solution was stirred at 80° C. for 10 h, allowed to cool to RT and concentrated under reduced pressure. The residue was purified by chromatography over silica gel (petroleum ether:EtOAc=2:1) to yield (1R,3r,5S)-tert-butyl 3-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenoxy)-8-azabicyclo[3.2.1]octane-8-carboxylate (0.2 g, 45%), directly used into the next step.
TFA (2 mL, 26 mmol) was added to a solution of (1R,3r,5S)-tert-butyl 3-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenoxy)-8-azabicyclo[3.2.1]octane-8-carboxylate (0.2 g, 0.223 mmol) in DCM (10 mL). The mixture was stirred for 2 h at RT and then concentrated under reduced pressure. The residue was purified by preparative reverse phase HPLC ((Column: Synergi 150*25 mm*10 μm, Flow rate: 30 mL/min, Mobile Phase A: Purified water (containing 0.1% HCl), Mobile Phase B: acetonitrile, Gradient: 40-70(% B) from 0-15 min) to afford 5-[8-[4-[[(1R,3r, 5S)-8-azabicyclo[3.2.1]octan-3-yl]oxy]phenyl]-5-oxo-7-thioxo-6,8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile (0.057 g, 46%) as a TFA salt. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.69 (br. s., 1H) 2.01 (br. s., 2H) 2.24 (d, J=9.54 Hz, 1H) 2.30-2.52 (m, 6H) 2.57 (d, J=10.79 Hz, 2H) 2.68 (br. s., 2H) 4.25 (br. s., 2H) 4.71 (br. s., 1H) 7.08 (d, J=7.03 Hz, 2H) 7.23 (d, J=8.03 Hz, 2H) 8.36 (d, J=1.76 Hz, 1H) 9.10 (s, 1H) 9.92 (s, 1H). C26H24F3N5O2S MS m/z 528.1 (M+H)+.
Cyclobutanone (1.54 mL, 20.6 mmol) was added to a solution of 4-(4-Amino-benzyl)-piperidine-1-carboxylic acid tert-butyl ester (3.0 g, 10.3 mmol) in Acetic acid (50 mL). The solution was stirred for 15 min at room temperature before Sodium cyanide (1.01 g, 20.6 mmol) was added and the reaction stirred for 4 h. The solution was concentrated under reduced pressure. The resultant residue was partitioned between EA (75 mL) and 1M Na2CO3 (125 mL). The organic layer was further washed with brine (50 mL), dried over MgSO4, filtered and concentrated to dryness. Purification by chromatography over silica gel (gradient of EA in heptane from 0 to 50%) gave the pure product as an oily residue (2.82 g, 74%). 1H NMR (300 MHz, Chloroform-d) δ 0.73-0.97 (m, 1H), 1.00-1.19 (m, 2H), 1.20-1.34 (m, 1H), 1.45 (s, 9H), 1.61 (d, J=11.4 Hz, 2H), 2.09-2.31 (m, 2H), 2.31-2.41 (m, 2H), 2.44 (d, J=6.8 Hz, 2H), 2.63 (t, J=12.7 Hz, 2H), 2.72-2.89 (m, 2H), 6.58 (d, J=8.3 Hz, 2H), 7.00 (d, J=8.3 Hz, 2H). C22H31N3O2 MS m/z 270 (M+H-Boc)+.
4-[4-(1-Cyano-cyclobutylamino)-benzyl]-piperidine-1-carboxylic acid tert-butyl ester (0.484 g, 1.24 mmol) and freshly prepared 5-Isothiocyanato-3-methoxy-pyridine-2-carbonitrile (0.59 g, 3.08 mmol) were mixed in DMA (12 mL). The resulting solution was stirred at 60° C. for 4 hours and then allowed to cool to room temperature. The mixture was diluted with MeOH (2.5 mL) and 1M HCl (2.5 mL) was added. The stirring was maintained overnight. EA (35 mL) was added and solution washed with 1M Na2CO3 (150 mL) and brine (25 mL). The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 60%) to yield pure product (0.744 g, 53%). 1H NMR (300 MHz, Chloroform-d) δ 1.08-1.30 (m, 3H), 1.45 (s, 9H), 1.62-1.84 (m, 4H), 2.12-2.34 (m, 1H), 2.48-2.77 (m, 9H), 4.00 (s, 3H), 7.23 (d, J=8.2 Hz, 2H), 7.35 (d, J=8.0 Hz, 2H), 7.57 (d, J=1.9 Hz, 1H), 8.43 (d, J=1.8 Hz, 1H). C30H35N5O4S MS m/z 506 (M+H-tBu)+.
The previous 4-{4-[7-(6-Cyano-5-methoxy-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-benzyl}-piperidine-1-carboxylic acid tert-butyl ester (0.744 g, 1.32 mmol) was taken in DCM (15 mL). Trifluoroacetic acid (2.65 mL, 35.6 mmol) was added with stirring. The mixture was concentrated to dryness after 30 min at room temperature. Preparative LC (gradient of ACN in 25 mM aqueous NH4HCO3 from 19 to 55%) afforded the desired pure product. Trituration in diethyl ether gave 3-Methoxy-5-[8-oxo-5-(4-piperidin-4-ylmethyl-phenyl)-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl]-pyridine-2-carbonitrile as a white powder (0.30 g, 96%). 1H NMR (300 MHz, Chloroform-d) δ 1.42-1.73 (m, 3H), 1.81 (d, J=12.4 Hz, 2H), 2.13-2.34 (m, 1H), 2.48-2.82 (m, 9H), 3.32 (d, J=12.3 Hz, 2H), 4.00 (s, 3H), 7.23 (d, J=7.8 Hz, 2H), 7.35 (d, J=8.0 Hz, 2H), 7.56 (d, J=1.9 Hz, 1H), 8.43 (d, J=1.9 Hz, 1H). C25H27N5O2S MS m/z 462 (M+H)+.
Formaldehyde (37% wt in water, 0.34 mL, 4.55 mmol) was added to a solution of 3-Methoxy-5-[8-oxo-5-(4-piperidin-4-ylmethyl-phenyl)-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl]-pyridine-2-carbonitrile (0.30 g, 0.65 mmol) in DCE (3 mL). The mixture was stirred at room temperature for 30 min, before Sodium triacetoxyborohydride (0.22 g, 0.97 mmol) was added in 2 portions within 30 min. The reaction was continued for 1 h and diluted with DCM (75 mL). The solution was washed successively with 1M Na2CO3 (40 mL) and water (15 mL). The organic layer was dried over MgSO4, filtered and concentrated to give the crude product. Preparative LC (gradient of ACN in 25 mM aqueous NH4HCO3 from 25 to 62%), upon removal of solvent, gave the pure product as a white solid (0.20 g, 64%). 1H NMR (300 MHz, Chloroform-d) δ 1.32-1.52 (m, 2H), 1.64-1.75 (m, 3H), 1.95 (t, J=11.6 Hz, 2H), 2.14-2.28 (m, 1H), 2.30 (s, 3H), 2.50-2.75 (m, 7H), 2.90 (d, J=11.3 Hz, 2H), 4.00 (s, 3H), 7.22 (d, J=8.2 Hz, 2H), 7.36 (d, J=8.0 Hz, 2H), 7.57 (d, J=1.9 Hz, 1H), 8.44 (d, J=1.9 Hz, 1H). C26H29N5O2S MS m/z 476 (M+H)+.
The previous intermediate (0.20 g, 0.421 mmol) was taken in dioxane (5 mL) and treated with 4N HCl in dioxane (0.126 mL, 0.505 mmol) with stirring. After 2.5 h, diethyl ether (40 mL) was added and the resulting suspension was stirred for another 30 min. The solid was collected on a sintered funnel and washed with diethyl ether (2×10 mL). The solid was dried under high vacuum at room temperature to yield the pure title hydrochloride salt (0.214 g, 97%). 1H NMR (300 MHz, DMSO-d6) δ 1.43-1.65 (m, 3H), 1.71-1.86 (m, 3H), 1.89-2.06 (m, 1H), 2.34-2.47 (m, 2H), 2.55-2.66 (m, 4H), 2.69 (s, 3H), 2.79-3.00 (m, 2H), 3.33-3.44 (m, 2H), 3.99 (s, 3H), 7.35 (d, J=8.0 Hz, 2H), 7.43 (d, J=8.1 Hz, 2H), 8.06 (d, J=1.8 Hz, 1H), 8.46 (d, J=1.8 Hz, 1H), 10.27 (br s, 1H). C26H29N5O2S. HCl MS m/z 476 (M+H)+.
A. Sodium hydride (60% in oil, 0.7 g, 17.51 mmol) was added to a solution of 1,4-dioxaspiro[4.5]decan-8-one (2 g, 12.81 mmol) and diethyl 4-aminobenzylphosphonate (3.5 g, 14.397 mmol) in dry DMF (15 mL). The mixture was stirred at RT overnight, diluted with water and the solution extracted with Et2O. The organic layer was dried over MgSO4, filtered and concentrated to give 4-(1,4-dioxaspiro[4.5]decan-8-ylidenemethyl)aniline directly used into the next step (3.01 g, 77%). 1H NMR (300 MHz, DMSO-d6) δ 1.50-1.73 (m, 4H), 2.23-2.31 (m, 2H), 2.36-2.46 (m, 2H), 3.89 (s, 4H), 5.03 (br s, 2H), 6.10 (s, 1H), 6.50 (d, J=8.1 Hz, 2H), 6.87 (d, J=8.1 Hz, 2H). C15H19NO2 MS m/z 246 (M+H)+.
B. Benzyl chloroformate (1.77 mL, 11.79 mmol) was added to a solution of 4-(1,4-dioxaspiro[4.5]decan-8-ylidenemethyl)aniline (3.01 g, 9.82 mmol) in DCM (35 mL) and Et3N (2.73 mL, 19.65 mmol). The mixture was stirred at RT overnight and concentrated under reduced pressure. The residue was diluted with DCM and aqueous 1.0M Na2CO3. The organic layer was washed with aqueous 1.0M Na2CO3, dried over MgSO4, filtered, concentrated and the crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 40%) to yield benzyl (4-(1,4-dioxaspiro[4.5]decan-8-ylidenemethyl)phenyl)carbamate as a white solid (1.05 g, 28%). 1H NMR (300 MHz, Chloroform-d) δ 1.61-1.73 (m, 2H), 1.72-1.84 (m, 2H), 2.35-2.46 (m, 2H), 2.46-2.60 (m, 2H), 3.98 (s, 4H), 5.20 (s, 2H), 6.25 (s, 1H), 6.64 (br s, 1H), 7.14 (d, J=8.5 Hz, 2H), 7.28-7.47 (m, 7H). C23H25NO4 MS m/z 380 (M+H)+.
C. Aqueous 1.0M HCl (8.35 mL, 8.35 mmol) was added to a solution of benzyl (4-(1,4-dioxaspiro[4.5]decan-8-ylidenemethyl)phenyl)carbamate (2.64 g, 6.96 mmol) in acetone (17 mL). After stirring at RT overnight, the mixture was filtered and the solid washed with water and dried to give benzyl (4-((4-oxocyclohexylidene)methyl) phenyl)carbamate as a white solid) (1.57 g, 67%). The mother waters were extracted with EtOAc. The organic layers were dried over MgSO4, filtered, concentrated to generate more benzyl (4-((4-oxocyclohexylidene)methyl)phenyl)carbamate (0.65 g, 24%). 1H NMR (300 MHz, DMSO-d6) δ 2.39 (m, 4H), 2.61 (t, J=6.9 Hz, 2H), 2.70 (t, J=7.1 Hz, 2H), 5.15 (s, 2H), 6.38 (s, 1H), 7.20 (d, J=8.2 Hz, 2H), 7.28-7.53 (m, 7H), 9.79 (s, 1H). C21H21NO3 MS m/z 336 (M+H)+.
D. Titanium (IV) isopropoxide (2.47 mL, 8.11 mmol) was added to a solution of benzyl (4-((4-oxocyclohexylidene)methyl)phenyl)carbamate (1.36 g, 4.06 mmol) in 7.0M ammonia in MeOH (34 mL). After stirring at RT for 6 h, sodium borohydride (0.232 g, 6.12 mmol) was added to the previous mixture. The reaction was stirred at RT overnight, diluted with aqueous 1.0M Na2CO3, and extracted with EtOAc. The organic layers were combined, dried over MgSO4, filtered, concentrated to give benzyl (4-((4-aminocyclohexylidene)methyl)phenyl)carbamate as a foam (1.23 g, 90%), used directly into the next step. 1H NMR (300 MHz, Chloroform-d) δ 1.06-1.41 (m, 2H), 1.59 (br s, 2H), 1.83-2.11 (m, 3H), 2.16-2.30 (m, 1H), 2.30-2.47 (m, 1H), 2.72-3.00 (m, 2H), 5.20 (s, 2H), 6.20 (s, 1H), 6.71 (s, 1H), 7.13 (d, J=8.2 Hz, 2H), 7.28-7.45 (m, 7H). C21H24N2O2 MS m/z 337 (M+H)+.
E. Di-tert-butyl dicarbonate (1.01 g, 4.63 mmol) was added to a solution of benzyl (4-((4-aminocyclohexylidene)methyl)phenyl)carbamate (1.41 g, 4.209 mmol) in DCM (20 mL). The mixture was stirred at RT for 1 h, concentrated and the residue purified by chromatography over silica gel (gradient of EA in heptane from 0 to 40%) to yield tert-butyl N-[4-[[4-(benzyloxycarbonylamino)phenyl]methylene]cyclohexyl]carbamate (1.024 g, 55%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ 1.07-1.34 (m, 2H), 1.38 (s, 9H), 1.70-1.90 (m, 2H), 1.90-2.08 (m, 1H), 2.09-2.24 (m, 1H), 2.24-2.40 (m, 1H), 2.62-2.82 (m, 1H), 3.35-3.57 (m, 1H), 5.14 (s, 2H), 6.16 (s, 1H), 6.76 (d, J=8.0 Hz, 1H), 7.10 (d, J=8.4 Hz, 2H), 7.27-7.53 (m, 7H), 9.75 (s, 1H). C26H32N2O4 MS m/z 459 (M+Na)+.
F. To a solution of tert-butyl N-[4-[[4-(benzyloxycarbonylamino)phenyl]methylene]cyclohexyl]carbamate (1.28 g, 2.93 mmol) in MeOH (15 mL) was added, at 0° C., Palladium on charcoal (10% wet, 0.15 g, 1.41 mmol) under a hydrogen atmosphere for 20 h at RT. The reaction mixture was filtered through diatomaceous earth and concentrated under reduced pressure to give tert-butyl (4-(4-aminobenzyl)cyclohexyl)carbamate (0.805 g, 90%), directly used into the next step. 1H NMR (300 MHz, Chloroform-d) δ 0.93-1.06 (m, 1H), 1.05-1.26 (m, 2H), 1.44 (s, 9H), 1.45-1.61 (m, 4H), 1.61-1.78 (m, 2H), 1.96 (m, 1H), 2.39 (dd, J=15.7, 6.9 Hz, 2H), 3.54 (br s, 2H), 4.16-4.82 (m, 1H), 6.62 (d, J=8.2 Hz, 2H), 6.91 (d, J=8.2 Hz, 2H). C18H28N2O2 MS m/z 0.305 (M+H)+.
G. Cyclobutanone (0.395 mL, 5.28 mmol) was added to a solution of tert-butyl (4-(4-aminobenzyl)cyclohexyl)carbamate (0.805 g, 2.64 mmol) in Acetic acid (15 mL). The solution was stirred for 15 min at room temperature before Sodium cyanide (0.267 g, 5.28 mmol) was added and the reaction stirred for 3 h. The solution was concentrated under reduced pressure, and the resulting material residue was partitioned between EA and 1M Na2CO3. The organic layer was further washed with brine, dried over MgSO4, filtered and concentrated to yield tert-butyl (4-(4-((1-cyanocyclobutyl)amino)benzyl)cyclohexyl) carbamate as a foam (1.014 g, 90%), used directly in the next step without further purification. 1H NMR (300 MHz, Chloroform-d) δ 0.92-1.08 (m, 1H), 1.08-1.23 (m, 2H), 1.44 (s, 9H), 1.48-1.62 (m, 4H), 1.61-1.80 (m, 2H), 1.88-2.03 (m, 1H), 2.09-2.29 (m, 2H), 2.29-2.50 (m, 4H), 2.69-2.88 (m, 2H), 3.72 (br s, 1H), 4.64 (br s, 1H), 6.59 (d, J=8.2 Hz, 2H), 7.00 (d, J=8.2 Hz, 2H). C23H33N3O2 MS m/z 406 (M+Na)+.
Following the procedure described in Example 14, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of Formula (I) of the invention were prepared.
Aluminium Chloride (4.23 g, 31.77 mmol) was added portionwise under a nitrogen atmosphere to a mixture of 1-methylpiperidine-4-carbonyl chloride (1.71 g, 10.58 mmol) and N-phenylacetamide (1.71 g, 12.71 mmol) in DCE (35 mL) at 0° C. The mixture was stirred at 85° C. overnight, allowed to cool to RT and poured into ice and aqueous 6.0M NaOH. The solution was basified to pH 10 to 11 and the aqueous layer extracted with DCM. The organic layers were combined, dried over MgSO4, filtered, concentrated and the residue purified by chromatography over silica gel (gradient of MeOH:DCM 1:9 in DCM from 0 to 100%) to give N-(4-(1-methylpiperidine-4-carbonyl)phenyl)acetamide (1.21 g, 47%) as a solid. 1H NMR (300 MHz, Chloroform-d) δ 1.78-1.90 (m, 4H), 2.00-2.13 (m, 2H), 2.20 (s, 3H), 2.29 (s, 3H), 2.83-2.98 (m, 2H), 3.08-3.28 (m, 1H), 7.62 (d, J=8.3 Hz, 2H), 7.81 (br s, 1H), 7.90 (d, J=8.5 Hz, 2H). C15H20N2O2 MS m/z 261 (M+H)+.
Aqueous 2.0N HCl (15.37 mL, 30.74 mmol) was added to a solution of N-(4-(1-methylpiperidine-4-carbonyl)phenyl)acetamide (1.2 g, 4.61 mmol) in MeOH (8 mL). The mixture was refluxed for 2 h, allowed to cool to RT, and the mixture was basified to pH 9 to 10 with aqueous 10.0M NaOH and extracted with DCM. The organic layers were combined, dried over MgSO4, filtered, and concentrated under reduced pressure to give (4-aminophenyl)(1-methylpiperidin-4-yl)methanone (0.848 g, 84%), directly used into the next step. 1H NMR (300 MHz, Chloroform-d) δ 1.77-1.92 (m, 4H), 2.01-2.17 (m, 2H), 2.31 (s, 3H), 2.85-2.98 (m, 2H), 3.05-3.24 (m, 1H), 4.11 (br s, 2H), 6.65 (d, J=8.6 Hz, 2H), 7.80 (d, J=8.7 Hz, 2H). C13H18N2O MS m/z 219 (M+H)+.
Cyclobutanone (0.435 mL, 5.77 mmol) was added to a solution of (4-aminophenyl)(1-methylpiperidin-4-yl)methanone (0.84 g, 3.84 mmol) in Acetic acid (2.5 mL) and MeOH (25 mL). The solution was stirred for 15 min at room temperature before trimethylsilyl cyanide (0.963 mL, 7.698 mmol) was added dropwise. After stirring at RT overnight, aqueous 1.0M Na2CO3 (100 mL) was added carefully and the solution was extracted with DCM. The organic layers were dried over MgSO4, filtered and concentrated to dryness. Chromatography over silica gel (gradient of MeOH:DCM 1:9 in DCM from 0 to 100%) gave 1-((4-(1-methylpiperidine-4-carbonyl)phenyl)amino)cyclobutanecarbonitrile (0.5 g, 44%) as a yellow solid. 1H NMR (300 MHz, Chloroform-d) δ 1.76-1.95 (m, 4H), 2.00-2.13 (m, 2H), 2.13-2.27 (m, 2H), 2.29 (s, 3H), 2.32-2.47 (m, 2H), 2.77-2.88 (m, 2H), 2.88-2.97 (m, 2H), 3.03-3.24 (m, 1H), 4.67 (br s, 1H), 6.64 (d, J=8.7 Hz, 2H), 7.87 (d, J=8.7 Hz, 2H). C18H23N3O MS m/z 298 (M+H)+.
A mixture of 5-amino-3-(trifluoromethyl)picolinonitrile (0.28 g, 1.496 mmol), 1-((4-(1-methylpiperidine-4-carbonyl)phenyl)amino)cyclobutanecarbonitrile (0.444 g, 1.493 mmol) and thiophosgene (0.172 mL, 2.244 mmol) in MeOH (15 mL) was stirred at 65° C. overnight. The mixture was allowed to cool down to RT, poured into water/ice and extracted with DCM. The organic layers were dried over MgSO4, filtered and concentrated to dryness. Chromatography over silica gel (gradient of MeOH:DCM 1:9 in DCM from 0 to 100%) gave a residue further triturated with Et2O to yield 5-(5-(4-(1-methylpiperidine-4-carbonyl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl) picolinonitrile (0.202 g, 26%) as a solid. 1H NMR (300 MHz, Chloroform-d) δ 1.57-1.85 (m, 1H), 2.04-2.22 (m, 3H), 2.21-2.39 (m, 3H), 2.62 (s, 3H), 2.69-3.08 (m, 4H), 3.07-3.39 (m, 2H), 3.32-3.81 (m, 2H), 7.48 (d, J=8.0 Hz, 2H), 8.16 (d, J=8.0 Hz, 2H), 8.37 (s, 1H), 9.11 (s, 1H). C26H24F3N5O2S MS m/z 528 (M+H)+.
A 250 mL round-bottom flask equipped with a stirring bar, addition funnel and a nitrogen inlet, was charged with 4-Hydroxy-piperidine-1-carboxylic acid tert-butyl ester (8 g, 39.7 mmol) and triethyl amine (6.08 mL, 43.7 mmol) in DCM (125 mL) and cooled in an ice bath under light nitrogen steam. Methanesulfonyl chloride (3.7 mL, 47.7 mmol) was added dropwise over 10-15 min. The mixture was allowed to come to room temperature and stirred for 1.5 h. The solution was washed with water (70 mL), dried over MgSO4, filtered and concentrated to yield the crude product as an oil (11.1 g, 100%). C11H21NO5S MS m/z 280 (M+H)+.
5-Nitro-1H-indole (2 g, 12.3 mmol) was dissolved in DMF (30 mL) and cooled in an ice bath under a nitrogen atmosphere. Sodium hydride (60% in mineral oil, 0.543 g, 13.5 mmol) was added in one portion. The mixture was stirred for 30 min while allowed to come to room temperature. The solution was then heated at 100° C. before a solution of 4-Methanesulfonyloxy-piperidine-1-carboxylic acid tert-butyl ester (4.14 g, 16.7 mmol) in DMF (40 mL) was added dropwise in two portions at 30 min intervals. The reaction was continued for 4 h at 100° C. and then allowed to cool to room temperature. The mixture was recharged with sodium hydride (60% in mineral oil, 0.543 g, 13.5 mmol) and after 30 min, a solution of 4-Methanesulfonyloxy-piperidine-1-carboxylic acid tert-butyl ester (4.14 g, 16.7 mmol) in DMF (40 mL) was also added dropwise. The reaction was continued for another 4 h at room temperature and quenched with water (200 mL). The organic mixture was extracted with EA (2×40 mL). The combined organic extracts were dried over MgSO4, filtered, and concentrated to dryness. Chromatography over silica gel (gradient of DCM in heptane from 0 to 60% and then of EA in heptane from 0 to 35%) afforded the title compound (1.58 g, 37%). 1H NMR (300 MHz, Chloroform-d) δ 1.50 (s, 9H), 1.93 (qd, J=12.4, 4.4 Hz, 2H), 2.03-2.18 (m, 2H), 2.94 (t, J=13.3 Hz, 2H), 4.26-4.51 (m, 3H), 6.72 (d, J=3.4 Hz, 1H), 7.33 (d, J=3.4 Hz, 1H), 7.40 (d, J=9.1 Hz, 1H), 8.11 (dd, J=9.1, 2.3 Hz, 1H), 8.59 (d, J=2.2 Hz, 1H). C18H23N3O4 MS m/z 290 (M+H-tBu)+.
4-(5-Nitro-indol-1-yl)-piperidine-1-carboxylic acid tert-butyl ester (1.58 g, 4.57 mmol) was dissolved in MeOH (50 mL) and cooled to 0-5° C. under nitrogen stream. 10% Pd/C (0.316 g) was added. The reaction vessel was connected to a balloon filled with hydrogen. The reaction was evacuated and placed under an atmosphere of hydrogen (3 times) and finally stirred at room temperature for 1 h under hydrogen. The catalyst was removed by filtration through a short pad of diatomaceous earth, then rinsed with MeOH (2×10 mL). The filtrate was concentrated to give the crude 4-(5-Amino-indol-1-yl)-piperidine-1-carboxylic acid tert-butyl ester (1.40 g, 97%). 1H NMR (300 MHz, Chloroform-d) δ 1.49 (s, 9H), 1.87 (qd, J=12.5, 4.4 Hz, 2H), 2.07 (d, J=10.5 Hz, 2H), 2.90 (t, J=13.0 Hz, 2H), 3.49 (br s, 2H), 4.18-4.42 (m, 3H), 6.33 (d, J=3.2 Hz, 1H), 6.68 (dd, J=8.7, 2.2 Hz, 1H), 6.94 (d, J=2.1 Hz, 1H), 7.09 (d, J=3.2 Hz, 1H), 7.17 (d, J=8.7 Hz, 1H). C18H25N3O2 MS m/z 316 (M+H)+.
Cyclobutanone (0.432 mL, 4.78 mmol) was added to a solution of the previous intermediate (1.40 g, 4.44 mmol) and Acetic acid (5 mL) in MeoH (50 mL). The solution was stirred 15 min at room temperature before Trimethylsilyl cyanide (1.11 mL, 8.89 mmol) was added dropwise. Upon completion of the addition, the reaction was continued overnight. The solution was diluted with EA (100 mL) and washed with 1M Na2CO3 (70 mL). The organic layer was dried over MgSO4, filtered and concentrated to a crude oily residue. Chromatography over silica gel (gradient of EA in heptane from 0 to 35%) gave the pure product (0.488 g, 28%). 1H NMR (300 MHz, Chloroform-d) δ 1.50 (s, 9H), 1.88 (qd, J=12.4, 4.4 Hz, 2H), 2.07 (d, J=13.4, 3.4 Hz, 2H), 2.13-2.33 (m, 2H), 2.42 (ddd, J=12.1, 9.4, 7.1 Hz, 2H), 2.73-3.02 (m, 4H), 3.87 (br s, 1H), 4.20-4.48 (m, 3H), 6.41 (d, J=3.1 Hz, 1H), 6.67 (dd, J=8.8, 2.3 Hz, 1H), 6.91 (d, J=2.2 Hz, 1H), 7.12 (d, J=3.2 Hz, 1H), 7.25 (d, J=8.8 Hz, 1H). C23H30N4O2 MS m/z 395 (M+H)+.
4-[5-(1-Cyano-cyclobutylamino)-indol-1-yl]-piperidine-1-carboxylic acid tert-butyl ester (0.488 g, 1.24 mmol) and freshly prepared 5-Isothiocyanato-3-trifluoromethyl-pyridine-2-carbonitrile (0.510 g, 2.23 mmol) were mixed in DMA (5 mL). The resulting solution was stirred at 60° C. for 4 h and then allowed to cool to room temperature. The mixture was diluted with MeOH (1.5 mL) and 1M HCl (1.5 mL) was added. The stirring was maintained overnight. EA (25 mL) was added and the solution washed with 1M Na2CO3 (10 mL) and brine (5 mL). The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 50%) to yield pure product (0.693 g, 89%). 1H NMR (300 MHz, Chloroform-d) δ 1.51 (s, 9H), 1.56-1.76 (m, 1H), 1.96 (qd, J=12.4, 4.4 Hz, 2H), 2.07-2.31 (m, 3H), 2.68 (dd, J=9.1, 7.0 Hz, 4H), 2.95 (t, J=12.8 Hz, 2H), 4.25-4.55 (m, 3H), 6.65 (d, J=3.2 Hz, 1H), 7.10 (dd, J=8.7, 2.0 Hz, 1H), 7.33 (d, J=3.3 Hz, 1H), 7.50-7.62 (m, 2H), 8.40 (d, J=2.2 Hz, 1H), 9.14 (d, J=2.2 Hz, 1H). C31H31F3N6O3S MS m/z 569 (M+H-tBu)+.
The previous 4-{5-[7-(6-Cyano-5-trifluoromethyl-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-indol-1-yl}-piperidine-1-carboxylic acid tert-butyl ester (0.693 g, 1.11 mmol) was taken in dioxane (5 mL). 4N HCl in dioxane (2.5 mL, 10 mmol) was added with stirring for 3 h at room temperature. Diethyl ether (40 mL) was added and the resulting precipitate was stirred for 15 min and collected by filtration on a sintered funnel. The solid was washed with diethyl ether (10 mL). The solid was re-dissolved in DCM (40 mL). The solution was washed with 1M Na2CO3 (10 mL), water (20 mL). The organic layer was dried over MgSO4, filtered and concentrated to dryness to yield 0.598 g (103%) of the crude product. Half of the yielded product was purified by preparative LC (gradient of ACN in 25 mM aqueous NH4HCO3 from 30 to 73%). The pure fractions were collected and concentrated to afford the desired product 0.100 g (17%). 1H NMR (300 MHz, Chloroform-d) δ 1.53-1.75 (m, 1H), 1.99-2.36 (m, 7H), 2.61-2.73 (m, 5H), 2.85-2.99 (m, 2H), 3.40 (d, J=12.6 Hz, 2H), 4.32-4.51 (m, 1H), 6.65 (d, J=3.2 Hz, 1H), 7.09 (dd, J=8.7, 2.0 Hz, 1H), 7.38 (d, J=3.4 Hz, 1H), 7.53-7.63 (m, 2H), 8.40 (d, J=2.2 Hz, 1H), 9.14 (d, J=2.1 Hz, 1H). C26H23F3N6OS MS m/z 525 (M+H)+.
Formaldehyde (37% wt in water, 0.30 mL, 4.00 mmol) was added to a solution of 5-[8-Oxo-5-(1-piperidin-4-yl-1H-indol-5-yl)-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl]-3-trifluoromethyl-pyridine-2-carbonitrile (0.30 g, 0.572 mmol) in DCE (2.25 mL). The mixture was stirred at room temperature for 40 min, before Sodium triacetoxyborohydride (0.30 g, 1.41 mmol) was added in 3 portions over 45 min. The reaction was continued overnight and diluted with DCM (50 mL). The solution was washed successively with 1M Na2CO3 (25 mL) and water (30 mL). The organic layer was dried over MgSO4, filtered and concentrated to give the crude product. Preparative LC (gradient of ACN in 25 mM aqueous NH4HCO3 from 41 to 83%) gave, upon removal of solvent, the pure product as a sticky solid. Triturating in diethyl ether (2 mL) afford a white powder (0.056 g, 18%). 1H NMR (300 MHz, Chloroform-d) δ 1.48-1.77 (m, 1H), 2.03-2.31 (m, 7H), 2.41 (s, 3H), 2.68-2.75 (m, 4H), 3.09 (m, 2H), 4.16-4.38 (m, 1H), 6.64 (d, J=3.2 Hz, 1H), 7.08 (dd, J=8.7, 1.9 Hz, 1H), 7.37 (d, J=3.3 Hz, 1H), 7.56 (m, 2H), 8.40 (d, J=2.2 Hz, 1H), 9.14 (d, J=2.2 Hz, 1H). C27H25F3N6OS MS m/z 539 (M+H)+.
Following the procedure described in Example 16, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of formula (I) of the invention were prepared.
4-(1-Cyano-cyclobutylamino)-2-fluoro-benzoic acid methyl ester (2.0 g, 8.05 mmol) and 5-Amino-3-trifluoromethyl-pyridine-2-carbonitrile (1.96 g, 10.5 mmol) were mixed in DMA (35 mL). Thiophosgene (0.92 mL, 12.1 mmol) was added via syringe. The resulting solution was stirred at 70° C. overnight and then allowed to cool to room temperature. The mixture was diluted with MeOH (8.0 mL) and 1M HCl (8.0 mL) was added. The stirring was maintained for 2 h. EA (100 mL) was added and the solution was washed with 1M Na2CO3 (100 mL) and brine (50 mL). The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 35%) to yield the desired product. Triturating in diethyl ether (20 mL) afforded 4-[7-(6-Cyano-5-trifluoromethyl-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluoro-benzoic acid methyl ester as a white solid (1.49 g, 39%). 1H NMR (300 MHz, Chloroform-d) δ 1.64-1.85 (m, 1H), 2.16-2.38 (m, 1H), 2.48-2.66 (m, 2H), 2.66-2.83 (m, 2H), 3.99 (s, 3H), 7.14-7.25 (m, 2H), 8.18 (t, J=8.0 Hz, 1H), 8.35 (d, J=2.2 Hz, 1H), 9.09 (d, J=2.2 Hz, 1H). C21H14F4N4O3S MS m/z 479 (M+H)+.
4-[7-(6-Cyano-5-trifluoromethyl-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluoro-benzoic acid methyl ester (1.49 g, 3.11 mmol) was taken in MeOH (15 mL). 1M NaOH (15 mL, 15.0 mmol) was added at room temperature. The pH of the reaction mixture was brought to 2-3 by addition of 1M HCl (ca. 48 mL). A resulting precipitate was collected by filtration and washed with water (50 mL). 4-[7-(6-Cyano-5-trifluoromethyl-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluoro-benzoic acid was collected as a solid and dried under high vacuum (2.93 g, 94%). 1H NMR (300 MHz, DMSO-d6) δ 1.48-1.74 (m, 1H), 1.85-2.11 (m, 1H), 2.38-2.58 (m, 2H), 2.59-2.74 (m, 2H), 7.41 (dd, J=8.3, 1.9 Hz, 1H), 7.49 (dd, J=10.9, 1.9 Hz, 1H), 8.10 (t, J=8.2 Hz, 1H), 8.75 (d, J=2.1 Hz, 1H), 9.21 (d, J=2.0 Hz, 1H), 13.56 (br s, 1H). C20H12F4N4O3S MS m/z 465 (M+H)+.
O-(benzotriazol-1-yl)-N,N,N′—N′-tetramethyluronium hexafluorophosphate (HBTU), (0.306 g, 0.808 mmol) was added at room temperature to a stirred solution of 4-[7-(6-Cyano-5-trifluoromethyl-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-5-yl]-2-fluoro-benzoic acid (0.25 g, 0.538 mmol), 4-Aminomethyl-piperidine-1-carboxylic acid tert-butyl ester (0.138 g, 0.646 mmol) and Diisopropylethylamine (0.275 mL, 1.615 mmol) in DCM (12 mL). The reaction was continued for 1 h and then concentrated under reduced pressure. The residue was taken in EA (40 mL) and washed with 1M Na2CO3 (25 mL). The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by flash column chromatography over silica gel (gradient of EA in heptanes from 0 to 60%) to give a white solid (0.251 g, 70%). 1H NMR (300 MHz, Chloroform-d) δ 1.17-1.31 (m, 3H), 1.46 (s, 9H), 1.65-1.91 (m, 5H), 2.18-2.40 (m, 1H), 2.46-2.64 (m, 2H), 2.64-2.78 (m, 4H), 3.35-3.54 (m, 2H), 6.79 (dt, J=11.9, 5.9 Hz, 1H), 7.18 (dd, J=11.5, 1.9 Hz, 1H), 7.28 (d, J=9.3 Hz, 1H), 8.31 (d, J=8.4 Hz, 1H), 8.35 (d, J=2.0 Hz, 1H), 9.09 (d, J=2.2 Hz, 1H). C31H32F4N6O4S MS m/z 561 (M+H-Boc)+.
The previous intermediate (0.211 g, 0.291 mmol) was taken in dioxane (5 mL) and treated with 4N HCl in dioxane (1.45 mL, 5.81 mmol) with stirring. After 16 h, the reaction mixture was concentrated to a gummy residue. Preparative LC (gradient of ACN in 25 mM aqueous NH4HCO3 from 19 to 55%) gave, upon removal of solvent, the pure product as a white solid (0.112 g, 65%). 1H NMR (300 MHz, DMSO-d6) δ 1.13-1.30 (m, 2H), 1.49-1.65 (m, 1H), 1.65-1.81 (m, 3H), 1.91-2.07 (m, 1H), 2.39-2.56 (m, 2H), 2.55-2.75 (m, 4H), 3.10 (d, J=12.3 Hz, 2H), 3.17 (t, J=6.1 Hz, 2H), 7.38 (dd, J=8.1, 2.0 Hz, 1H), 7.46 (dd, J=10.3, 1.9 Hz, 1H), 7.80 (t, J=8.0 Hz, 1H), 8.60 (t, J=6.1 Hz, 1H), 8.75 (d, J=2.1 Hz, 1H), 9.21 (d, J=2.1 Hz, 1H). C26H24F4N6O2S MS m/z 561 (M+H)+.
Following the procedure described in Example 17, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of formula (I) of the invention were prepared.
1H NMR (400 MHz, Chloroform- d) δ ppm ppm 1.67-1.94 (m, 6H) 2.07-2.24 (m, 3H) 2.34 (br. s., 2H) 2.43-2.56 (m, 2H) 2.61 (d, J = 9.04 Hz, 2H) 2.75 (d, J = 13.89 Hz, 1H) 3.01 (br. s., 3H) 3.19 (br. s., 2H) 3.44 (d, J = 6.17 Hz, 2H) 7.30 (d, J = 7.28 Hz, 2H) 7.47-7.62 (m, 2H) 8.31 (s, 1H) 9.05 (s, 1H). C28H29F3N6O2S HCl MS m/z 571.2 (M + H)+
A mixture of 4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide (0.4 g, 0.83 mmol) and Lawesson's reagent (0.34 g, 0.83 mmol) in toluene (4 mL) was stirred at 90° C. overnight. The mixture was cooled to RT and concentrated under reduced pressure. The residue was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 3%) to yield 4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzothioamide as a yellow solid (0.094 g, 23%). 1H NMR (500 MHz, DMSO-d6) δ ppm 1.59-1.62 (m, 1H), 1.95-2.01 (m, 1H), 2.48-2.5 (m, 2H), 2.64-2.67 (m, 2H), 3.92 (s, 3H), 7.46 (d, 1H, J=1.6 Hz), 7.48 (d, 1H, J=1.6 Hz), 7.55 (d, 1H, J=1.6 Hz), 8.13-8.15 (m, 1H), 8.75 (d, 1H, J=1.6 Hz), 9.22 (d, 1H, J=1.6 Hz). C21H15F4N5OS2 MS m/z 478.2 (M+H)+.
Tert-butyl 6-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate (2.5 g, 10.1 mmol) and cyclobutanone (1.51 mL, 20.1 mmol) were mixed in acetic acid (8 mL) and ethanol (8 mL). Sodium cyanide (1.97 g, 40.3 mmol) was added and the mixture was stirred at 80° C. overnight and then allowed to cool to room temperature. The mixture was then poured into water and extracted with DCM. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 50%). The fractions with product were collected and concentrated under reduced pressure to yield tert-butyl 6-((1-cyanocyclobutyl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate as an amorphous solid (2.66 g, 81%). C19H25N3O2 MS m/z 328.2 (M+H)+.
Tert-butyl 6-((1-cyanocyclobutyl)amino)-3,4-dihydroisoquinoline-2(1H)-carboxylate (1 g, 3.054 mmol), and freshly prepared 5-Isothiocyanato-3-trifluoromethyl-pyridine-2-carbonitrile (0.84 g, 3.66 mmol) were heated at 60° C. in DMA (12 mL) overnight and then allowed to cool to room temperature. The mixture was diluted with MeOH (88 mL) and 1M HCl (88 mL) and stirred at RT for 30 minutes. EA (100 mL) was added and the solution was washed with water, saturated NaHCO3, and brine. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 50%). The fractions with product were collected and concentrated under reduced pressure to yield tert-butyl 6-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate as a white solid (0.92 g, 54%). 1H NMR (300 MHz, Chloroform-d) δ 1.51 (s, 9H), 1.63-1.78 (m, 1H), 2.25 (d, J=9.9 Hz, 1H), 2.48-2.78 (m, 4H), 2.93 (t, J=6.0 Hz, 2H), 3.72 (t, J=5.9 Hz, 2H), 4.68 (s, 2H), 7.04-7.17 (m, 2H), 7.34 (d, J=8.1 Hz, 1H), 8.36 (d, J=2.2 Hz, 1H), 9.10 (d, J=2.2 Hz, 1H). C27H26F3N5O3S MS m/z 501.9 (M-55)+.
To a solution of tert-butyl 6-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-3,4-dihydroisoquinoline-2(1H)-carboxylate (0.77 g, 1.38 mmol) in DCM (8.5 mL) was added TFA (5.5 mL). After stirring at RT for 3 h the reaction mixture was concentrated and diluted with EtOAc. The solution was washed with aqueous saturated NaHCO3 (3×) and brine (1×). The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 35%) to yield 5-[5-oxo-8-(1,2,3,4-tetrahydroisoquinolin-6-yl)-7-thioxo-6, 8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl) pyridine-2-carbonitrile as a yellow pale solid (0.36 g, 57%). 1H NMR (300 MHz, Chloroform-d) δ 1.60-1.78 (m, 1H), 2.11-2.35 (m, 1H), 2.43-2.75 (m, 4H), 2.96 (t, J=5.9 Hz, 2H), 3.26 (t, J=6.0 Hz, 2H), 4.17 (s, 2H), 7.02-7.13 (m, 2H), 7.26 (s, 1H), 8.36 (d, J=2.2 Hz, 1H), 9.10 (d, J=2.2 Hz, 1H). C22H18F3N5OS MS m/z 458.0 (M+H)+.
Formaldehyde (37% wt in water, 0.06 mL, 0.81 mmol) was added to a solution of 5-[5-oxo-8-(1,2,3,4-tetrahydroisoquinolin-6-yl)-7-thioxo-6, 8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl) pyridine-2-carbonitrile (0.124 g, 0.27 mmol) in DCE (5 mL). The mixture was stirred at room temperature for 5 min, before Sodium triacetoxyborohydride (0.172 g, 0.81 mmol) was added. The reaction was stirred at RT overnight and diluted with EtOAc (125 mL). The solution was washed successively with aqueous saturated NaHCO3. The organic layer was dried over MgSO4, filtered and concentrated to give the crude product. Chromatography over silica gel (gradient of MeOH in DCM from 0 to 10%) gave, upon removal of solvent, 5-[8-(2-methyl-3,4-dihydro-1H-isoquinolin-6-yl)-5-oxo-7-thioxo-6,8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile as a pale yellow solid (0.082 g, 83%). 1H NMR (300 MHz, Chloroform-d) δ 1.62-1.79 (m, 3H), 2.05-2.34 (m, 1H), 2.42-2.71 (m, 6H), 2.77 (s, 2H), 3.03 (d, J=6.1 Hz, 2H), 3.69 (s, 2H), 7.07 (s, 2H), 7.24 (s, 1H), 8.36 (d, J=2.2 Hz, 1H), 9.10 (d, J=2.2 Hz, 1H). C23H20F3N5OS MS m/z 472.0 (M+H)+.
5-[5-oxo-8-(1,2,3,4-tetrahydroisoquinolin-6-yl)-7-thioxo-6, 8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl) pyridine-2-carbonitrile (0.121 g, 0.26 mmol) was combined with acetic anhydride (2 mL). After stirring at RT overnight, the reaction mixture was concentrated and diluted with DCM. The solution was washed with aqueous saturated NaHCO3 (3×) and brine (1×). The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 5 to 40%) to yield impure desired product which was further purified by reverse phase preparative HPLC (0.072 g, 55%).
The raw material was purified by using a GILSON Semi-Preparative System, operated by Trilution software, equipped with a Phenomenex Gemini C18 100A column (100 mm long×30 mm I.D.; 5 μm particles) at 25° C., with a flow rate of 40 mL/min. A gradient elution was performed from 70% of a 0.1% HCOOH aqueous solution (pH 3)/30% Acetonitrile to 73% of a 0.1% HCOOH aqueous solution (pH3)/27% Acetonitrile in 20 min. The injection volume was 8000 μL. Acquisition frequency was set to 254 nm for the UV-Dual detector. 1H NMR (300 MHz, Chloroform-d) δ 1.36-1.71 (m, 4H), 2.07-2.26 (m, 2H), 2.40-2.70 (m, 3H), 2.79-3.01 (m, 2H), 3.65-3.87 (m, 2H), 4.57-4.84 (m, 2H), 7.00-7.13 (m, 2H), 7.24-7.35 (m, 1H), 8.29 (d, J=2.2 Hz, 1H), 9.03 (d, J=2.2 Hz, 1H). C24H20F3N5O2S MS m/z 500 (M+H)+.
3-Methylbutyraldehyde (0.103 mL, 0.96 mmol) was added to a solution of 3-chloro-5-(8-oxo-5-(6-(piperidin-4-yloxy)pyridin-3-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)picolinonitrile (0.15 g, 0.32 mmol) in DCE (4.8 mL). The mixture was stirred at RT for 10 min, before Sodium triacetoxyborohydride (0.203 g, 0.96 mmol) was added. The reaction was stirred at RT overnight, washed with aqueous saturated NaHCO3 and extracted with EtOAc. The organic layer was washed successively with water, brine, dried over MgSO4, filtered and concentrated to give the crude product. Chromatography was performed over silica gel (gradient of MeOH in DCM from 0 to 10%). The pure fractions were concentrated to give a residue which was further purified by reverse phase preparative HPLC to afford 3-chloro-5-(5-(6-((1-isopentylpiperidin-4-yl)oxy)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)picolinonitrile as a solid (0.014 g, 8%). 1H NMR (300 MHz, Chloroform-d) δ 0.91 (d, J=6.5 Hz, 6H), 1.37-1.52 (m, 2H), 1.54-1.78 (m, 1H), 1.84-1.99 (m, 2H), 2.05-2.31 (m, 4H), 2.34-2.58 (m, 6H), 2.63-2.74 (m, 2H), 2.78-2.94 (m, 2H), 5.01-5.25 (m, 1H), 6.90 (d, J=8.8 Hz, 1H), 7.50 (dd, J=8.7, 2.8 Hz, 1H), 8.08 (d, J=2.7 Hz, 1H), 8.11 (d, J=2.2 Hz, 1H), 8.79 (d, J=2.1 Hz, 1H). C27H31ClN6O2S MS m/z 539.1 (M+H)+.
5-(5-(6-hydroxypyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.84 g, 2 mmol), methanol (0.089 mL, 2.2 mmol) and triphenylphosphine (1.154 g, 4.4 mmol) were dissolved in dry DMF (2 mL) and dry THF (14 mL) under a nitrogen atmosphere and heated at 50° C. A solution of Diisopropyl azodicarboxylate (DIAD, 0.788 mL, 4 mmol) in THF (6 mL) was added dropwise. Once the addition was complete, the reaction was continued for 3 h at the same temperature. The mixture was allowed to cool and then diluted with EtOAc. The organic layer was washed with aqueous saturated NaHCO3, brine, dried over MgSO4, filtered and concentrated to dryness. The crude residue was chromatographed over silica gel (gradient of EtOAc in heptane from 5 to 30%). The pure fractions were concentrated to give a residue which was further purified by reverse phase preparative HPLC to afford 5-(5-(1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a solid (0.23 g, 27%).
The raw material was purified by using a GILSON Semi-Preparative System, operated by Trilution software, equipped with a Phenomenex Gemini C18 100A column (100 mm long×30 mm I.D.; 5 μm particles) at 25° C., with a flow rate of 40 mL/min. A gradient elution was performed from 59% of a 0.1% HCOOH aqueous solution (pH 3)/41% Acetonitrile to 17% of a 0.1% HCOOH aqueous solution (pH 3)/83% Acetonitrile in 20 min. The injection volume was 8000 μL. Acquisition frequency was set to 254 nm for the UV-Dual detector. 1H NMR (300 MHz, Chloroform-d) δ 1.75-1.98 (m, 1H), 2.20-2.42 (m, 1H), 2.44-2.63 (m, 2H), 2.63-2.77 (m, 2H), 3.63 (s, 3H), 6.72 (d, J=9.7 Hz, 1H), 7.19-7.28 (m, 1H), 7.40 (d, J=2.8 Hz, 1H), 8.32 (d, J=2.2 Hz, 1H), 9.06 (d, J=2.2 Hz, 1H). C19H14F3N5O2S MS m/z 433.9 (M+H)+.
A mixture of 5-[5-(6-Hydroxy-pyridin-3-yl)-8-oxo-6-thioxo-5,7-diaza-spiro[3.4]oct-7-yl]-3-trifluoromethyl-pyridine-2-carbonitrile and 1-Boc-3-hydroxymethylazetidine in a freshly prepared 0.36M solution of 2-(tributylphosphoranylidene)-acetonitrile in heptane under a nitrogen atmosphere was heated to 110° C. for 16 h. The crude material was poured onto water/NaHCO3 and extracted with Ethyl acetate. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The residue was purified by flash chromatography over silica gel (Ethyl acetate-heptane gradient from 5% to 30%). Two products were isolated. Pure fractions were combined and concentrated to dryness under high vacuum to give the mixture as Cpd 130.
(405 mg, 34%). 1H NMR (300 MHz, Chloroform-d) δ 1.44 (s, 9H), 1.65-1.83 (m, 1H), 2.15-2.35 (m, 1H), 2.44-2.61 (m, 2H), 2.63-2.78 (m, 2H), 2.92-3.12 (m, 1H), 3.73-3.89 (m, 2H), 4.02-4.16 (m, 2H), 4.51 (d, J=6.7 Hz, 2H), 6.95 (d, J=8.7 Hz, 1H), 7.54 (dd, J=8.8, 2.6 Hz, 1H), 8.11 (d, J=2.6 Hz, 1H), 8.36 (d, J=2.3 Hz, 1H), 9.09 (d, J=2.2 Hz, 1H). C27H27F3N6O4S MS m/z 588.9 (M+H)+.
(215 mg, 18%). 1H NMR (300 MHz, Chloroform-d) δ 1.35 (s, 9H), 1.67-1.85 (m, 1H), 2.12-2.34 (m, 1H), 2.35-2.53 (m, 2H), 2.56-2.77 (m, 2H), 2.95-3.15 (m, 1H), 3.56-3.71 (m, 2H), 3.94-4.01 (m, 2H), 4.09-4.19 (m, 2H), 6.64 (d, J=9.7 Hz, 1H), 7.10-7.29 (m, 1H), 7.38 (d, J=2.7 Hz, 1H), 8.26 (d, J=2.2 Hz, 1H), 8.98 (d, J=2.2 Hz, 1H). C27H27F3N6O4S MS m/z 588.8 (M+H)+.
To a solution of 5-(5-(4-(azetidin-3-yloxy)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.11 g, 0.232 mmol) and DIPEA (0.0121 mL, 0.696 mmol) in MeCN (3 mL) was added propargyl bromide (0.024 mL, 0.186 mmol). After stirring at RT for 15 h, dimethyl amine (0.024 mL, 0.232 mmol) was added and the solution stirred for another 30 min. The mixture was poured onto ice and aqueous saturated NaHCO3 and diluted with EtOAc. The organic layer was washed with aqueous saturated NaHCO3, brine, dried over MgSO4, filtered and concentrated to give 5-(8-oxo-5-(4-((1-(prop-2-yn-1-yl)azetidin-3-yl)oxy)phenyl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.004 g, 3%). 1H NMR (300 MHz, Chloroform-d) δ 1.59-1.82 (m, 1H), 2.20-2.29 (m, 1H), 2.30-2.35 (m, 1H), 2.50-2.60 (m, 2H), 2.61-2.75 (m, 2H), 3.40 (d, J=2.4 Hz, 2H), 3.43-3.50 (m, 2H), 3.75-3.93 (m, 2H), 4.70-4.95 (m, 1H), 6.96 (d, J=8.9 Hz, 2H), 7.21 (d, J=8.8 Hz, 1H), 8.36 (d, J=2.3 Hz, 1H), 9.10 (d, J=2.3 Hz, 1H). C25H20F3N5O2S MS m/z 512.0 (M+H)+.
Following the procedure described in Example 23, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of formula (I) of the invention were prepared.
A solution of 5-nitro-1H-indazole (1.19 g, 7.3 mmol) in DMF (12 mL) was added to a suspension of sodium hydride (0.321 g, 13.37 mmol) in DMF (1 mL). After stirring the mixture at RT for 30 min and at 100° C. for 15 min, a solution of tert-butyl 4-(((methylsulfonyl)oxy)methyl)piperidine-1-carboxylate (2.14 g, 7.3 mmol) (J. Med. Chem. 2012, 55, 2416-2426) in DMF (13 mL) was added dropwise in two portions at the same temperature. The reaction mixture was stirred at 100° C. overnight, allowed to cool to RT and diluted with aqueous saturated NaHCO3 and extracted with EtOAc. The organic layer was washed with aqueous saturated NaHCO3, brine, was dried over MgSO4, filtered and concentrated to dryness. The crude residue was chromatographed over silica gel (gradient of EtOAc in heptane from 30 to 100%) to give, upon removal of solvent, tert-butyl 4-((5-nitro-1H-indazol-1-yl)methyl)piperidine-1-carboxylate (0.855 g, 33%) and tert-butyl 4-((5-nitro-2H-indazol-2-yl)methyl)piperidine-1-carboxylate (0.495 g, 19%) as a mixed fraction (1.09 g).
1H NMR (300 MHz, DMSO-d6) δ 1.00-1.25 (m, 2H), 1.29-1.43 (m, 11H), 1.96-2.17 (m, 1H), 2.54-2.71 (m, 2H), 3.74-4.07 (m, 2H), 4.39 (d, J=7.0 Hz, 2H), 7.93 (d, J=9.3 Hz, 1H), 8.20 (dd, J=9.3, 2.1 Hz, 1H), 8.40 (s, 1H), 8.80 (d, J=2.1 Hz, 1H). C18H24N4O4 MS m/z 305.1 (M-55).
1H NMR (300 MHz, DMSO-d6) δ 1.03-1.26 (m, 2H), 1.31-1.54 (m, 11H), 2.18 (td, J=7.4, 3.7 Hz, 1H), 2.67 (s, 2H), 3.91 (d, J=13.1 Hz, 2H), 4.42 (d, J=7.2 Hz, 2H), 7.77 (d, J=9.5 Hz, 1H), 8.00 (dd, J=9.4, 2.2 Hz, 1H), 8.78 (s, 1H), 8.88 (d, J=2.2 Hz, 1H). C18H24N4O4 MS m/z 305.1 (M-55)+
A solution of tert-butyl 4-((5-nitro-1H-indazol-1-yl)methyl)piperidine-1-carboxylate (0.622 g, 1.72 mmol) in EtOAc (7 mL) was purged using nitrogen and vacuum. Palladium on charcoal (10% wet) was added the mixture was hydrogenated for 2 h. The reaction mixture was filtered through diatomaceous earth and the filtrate was concentrated under reduced pressure to give tert-butyl 4-((5-amino-1H-indazol-1-yl)methyl)piperidine-1-carboxylate used directly into the next step. C18H26N4O2 MS m/z 331.0 (M+H)+.
tert-Butyl 4-((5-amino-1H-indazol-1-yl)methyl)piperidine-1-carboxylate (0.54 g, 1.63 mmol) and cyclobutanone (0.244 mL, 3.268 mmol) were mixed in acetic acid (1.3 mL) and ethanol (1.3 mL). Sodium cyanide (0.32 g, 6.53 mmol) was added and the mixture was stirred at 50° C. overnight and then allowed to cool to room temperature. The mixture was then poured into water and extracted with DCM. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of MeOH in DCM from 0 to 10%). The fractions with product were collected and concentrated under reduced pressure to yield tert-butyl 4-((5-((1-cyanocyclobutyl)amino)-1H-indazol-1-yl)methyl)piperidine-1-carboxylate as an amorphous solid (0.4 g, 60%). C23H31N5O2 MS m/z 354.2 (M-55)+.
tert-Butyl 4-((5-((1-cyanocyclobutyl)amino)-1H-indazol-1-yl)methyl)piperidine-1-carboxylate (0.4 g, 0.977 mmol), and freshly prepared 5-Isothiocyanato-3-trifluoromethyl-pyridine-2-carbonitrile (0.224 g, 0.977 mmol) were heated at 60° C. in DMA (3.9 mL) overnight and then allowed to cool to room temperature. The mixture was diluted with MeOH (1.95 mL) and 1M HCl (1.95 mL) and stirred at RT for 30 min. EA (20 mL) was added and the solution washed with water, aqueous saturated NaHCO3 and brine. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 5 to 40%). The fractions with product were collected and concentrated under reduced pressure to yield tert-butyl 4-((5-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-1H-indazol-1-yl)methyl)piperidine-1-carboxylate as a foam (0.501 g, 80%). C31H32F3N7O3S MS m/z 540.2 (M-Boc)+.
To a solution of tert-butyl 4-((5-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-1H-indazol-1-yl)methyl)piperidine-1-carboxylate (0.501 g, 0.783 mmol) in DCM (4.7 mL) was added TFA (3.13 mL). After stirring at RT for 3 h the reaction mixture was concentrated and diluted with DCM. The solution was washed with aqueous saturated NaHCO3, water and brine. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of MeOH in DCM from 0 to 10%) to yield 5-(8-oxo-5-(1-(piperidin-4-ylmethyl)-1H-indazol-5-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a solid (0.19 g, 44%). 1H NMR (300 MHz, Chloroform-d) δ 1.59-1.97 (m, 5H), 2.14-2.34 (m, 1H), 2.34-2.52 (m, 1H), 2.55-2.78 (m, 4H), 2.85-3.07 (m, 2H), 3.38-3.62 (m, 2H), 4.39 (d, J=7.0 Hz, 2H), 7.34 (dd, J=8.9, 1.9 Hz, 1H), 7.63 (d, J=8.9 Hz, 1H), 7.74 (d, J=1.9 Hz, 1H), 8.18 (s, 1H), 8.40 (d, J=2.2 Hz, 1H), 9.12 (d, J=2.2 Hz, 1H). C26H24F3N7OS MS m/z 540.0 (M+H)+.
Formaldehyde (37% wt in water, 0.095 mL, 1.28 mmol) was added to a solution of 5-(8-oxo-5-(1-(piperidin-4-ylmethyl)-1H-indazol-5-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.231 g, 0.428 mmol) in DCE (6 mL). The mixture was stirred at room temperature for 5 min, before Sodium triacetoxyborohydride (0.272 g, 1.28 mmol) was added. The reaction was stirred at RT overnight and diluted with aqueous saturated NaHCO3. The solution was extracted with EtOAc and the organic layer was washed successively with water, brine, dried over MgSO4, filtered and concentrated to give 5-(5-(1-((1-methylpiperidin-4-yl)methyl)-1H-indazol-5-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a solid (0.163 g, 67%). 1H NMR (300 MHz, Chloroform-d) δ 1.47-1.62 (m, 2H), 1.69 (d, J=11.5 Hz, 3H), 1.94-2.17 (m, 2H), 2.18-2.28 (m, 1H), 2.31 (s, 3H), 2.52-2.80 (m, 4H), 2.93 (d, J=11.5 Hz, 2H), 4.32 (d, J=6.9 Hz, 2H), 7.22-7.30 (m, 1H), 7.58 (d, J=8.9 Hz, 1H), 7.70 (d, J=1.9 Hz, 1H), 8.12 (s, 1H), 8.39 (d, J=2.2 Hz, 1H), 9.13 (d, J=2.2 Hz, 1H). C27H26F3N7OS MS m/z 554.0 (M+H)+.
Following the procedure described in Example 24, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of Formula (I) of the invention were prepared.
To a mixture of 5-(8-oxo-5-(1-(piperidin-4-ylmethyl)-1H-indazol-5-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.134 g, 0.248 mmol), Et3N (0.052 mL, 0.372 mmol) and DMAP (3 mg) in DCM (0.62 mL) was added acetic anhydride (0.028 mL, 0.298 mmol). After stirring at RT overnight the reaction mixture was concentrated and diluted with DCM. The solution was washed with aqueous saturated NaHCO3 and brine. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of MeOH in DCM from 0 to 10%) to yield 5-(5-(1-((1-acetylpiperidin-4-yl)methyl)-1H-indazol-5-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a solid (0.087 g, 60%). 1H NMR (300 MHz, Chloroform-d) δ 1.32 (ddd, J=17.7, 12.6, 4.6 Hz, 2H), 1.63-1.85 (m, 3H), 2.10 (s, 3H), 2.21-2.41 (m, 2H), 2.46-2.81 (m, 5H), 2.97-3.14 (m, 1H), 3.86 (d, J=13.6 Hz, 1H), 4.34 (d, J=6.9 Hz, 2H), 4.67 (d, J=13.4 Hz, 1H), 7.21-7.34 (m, 1H), 7.59 (d, J=8.9 Hz, 1H), 7.73 (d, J=1.9 Hz, 1H), 8.14 (s, 1H), 8.41 (d, J=2.2 Hz, 1H), 9.14 (d, J=2.2 Hz, 1H). C28H26F3N7O2S MS m/z 581.9 (M+H)+.
To a solution of 2-methylpropane-2-sulfinamide (2.424 g, 20 mmol) and oxetan-3-one (1.41 mL, 24 mmol) in THF (40 mL) was added dropwise over 15 min Titanium (IV) ethoxide (8.38 mL, 40 mmol) under a nitrogen atmosphere at RT and the mixture was stirred at 60° C. overnight. The reaction mixture was allowed to cool to RT. The mixture was quenched with chilled aqueous saturated NaHCO3 and diluted with EtOAc. The suspension was filtered through diatomaceous earth and the organic layer was washed with aqueous saturated NaHCO3, brine, dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 10 to 100%). The fractions with product were collected and concentrated under reduced pressure to yield 2-methyl-N-(oxetan-3-ylidene)propane-2-sulfinamide (1 g, 29%). 1H NMR (300 MHz, Chloroform-d) δ 1.28 (s, 9H), 5.37-5.58 (m, 2H), 5.61-5.87 (m, 2H). C7H13NO2S MS m/z 176.1 (M+H)+.
n-Butyl lithium (1.65M in hexanes, 3.29 mL, 5.27 mmol) was added to a solution of 4-Bromo-N,N-bis(trimethylsilyl)aniline (1.488 mL, 5.27 mmol) in THF (18 mL) at −78° C. under nitrogen. After stirring at −78° C. for 1 h, a solution of 2-methyl-N-(oxetan-3-ylidene)propane-2-sulfinamide (0.77 g, 4.39 mmol) was added over 15 min at −78° C. under nitrogen. After stirring at −78° C. for 1 h, the mixture was allowed to warm to RT and stirred at RT overnight. The mixture was poured into chilled aqueous saturated NaHCO3 and extracted with EtOAc. The organic layer was acidified with acetic acid and oxalic acid then the mixture was basified with NaHCO3 and Na2CO3. The organic layer was dried over MgSO4, filtered and concentrated under high vacuum to yield N-(3-(4-aminophenyl)oxetan-3-yl)-2-methylpropane-2-sulfinamide (0.76 g, 64%) used directly into the next step. 1H NMR (300 MHz, Chloroform-d) δ 1.20 (s, 9H), 4.95-5.05 (m, 4H), 5.11-5.19 (m, 1H), 6.70 (d, J=8.5 Hz, 2H), 7.13 (d, J=8.5 Hz, 2H). C13H20N2O2S MS m/z 269.2 (M+H)+.
N-(3-(4-aminophenyl)oxetan-3-yl)-2-methylpropane-2-sulfinamide (0.76 g, 2.83 mmol) and cyclobutanone (0.423 mL, 5.664 mmol) were mixed in acetic acid (2.3 mL) and ethanol (2.3 mL). Sodium cyanide (0.555 g, 11.328 mmol) was added and the mixture was stirred at 50° C. overnight and then allowed to cool to room temperature. The mixture was then poured into water and extracted with DCM. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of MeOH in DCM from 0 to 10%). The fractions with product were collected and concentrated under reduced pressure to yield N-(3-(4-((1-cyanocyclobutyl)amino)phenyl)oxetan-3-yl)-2-methylpropane-2-sulfinamide as a solid (0.785 g, 80%). C18H25N3O2S MS m/z 348.2 (M+H)+.
N-(3-(4-((1-cyanocyclobutyl)amino)phenyl)oxetan-3-yl)-2-methylpropane-2-sulfinamide (0.353 g, 1.016 mmol), and freshly prepared 5-Isothiocyanato-3-trifluoromethyl-pyridine-2-carbonitrile (0.379 g, 1.125 mmol) were heated at 60° C. in DMA (4.1 mL) overnight and then allowed to cool to room temperature. The mixture was diluted with MeOH (5.12 mL) and 1M HCl (2.03 mL) and stirred at RT for 30 min. EA was added and the solution washed with water, aqueous saturated NaHCO3 and brine. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 100%). The fractions with products were collected and concentrated under reduced pressure. The raw material was purified by using a GILSON Semi-Preparative System, operated by Trilution software, equipped with a Phenomenex Gemini C18 100A column (100 mm long×30 mm I.D.; 5 μm particles) at 25° C., with a flow rate of 40 mL/min. A gradient elution was performed from 70% of a 65 mM aqueous solution of Ammonium acetate (pH 7)/30% Acetonitrile to 73% of a 65 mM aqueous solution of Ammonium acetate (pH 7)/27% Acetonitrile in 20 min. Pure fractions were combined, neutralized with NaHCO3 solution and extracted with dichloromethane. The organic layer was washed with brine, dried over MgSO4 and concentrated to afford pure N-(3-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)oxetan-3-yl)-2-methylpropane-2-sulfinamide (0.192 g, 33%) and pure 5-(5-(4-(3-aminooxetan-3-yl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.041 g, 9%)
1H NMR (300 MHz, Chloroform-d) δ 1.63-1.80 (m, 1H), 2.15-2.37 (m, 1H), 2.51-2.84 (m, 4H), 4.80 (d, J=6.5 Hz, 2H), 5.02 (d, J=6.5 Hz, 2H), 7.37 (d, J=8.5 Hz, 1H), 7.85 (d, J=8.5 Hz, 2H), 8.37 (d, J=2.3 Hz, 1H), 9.11 (d, J=2.3 Hz, 1H). C22H18F3N5O2S MS m/z 473.9 (M+H)+.
To a solution of N-(3-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)oxetan-3-yl)-2-methylpropane-2-sulfinamide (0.168 g, 0.157 mmol) in DMA (3.5 mL) and MeOH (3.5 mL) was added aqueous 2.0M HCl (0.7 mL, 1.4 mmol). The mixture was stirred for 30 min at RT; additional aqueous 2.0M HCl (0.2 mL, 0.4 mmol) was added. After stirring at RT for 1 day the mixture was poured onto ice and diluted with EtOAc. The organic layer was washed with water, aqueous saturated NaHCO3, brine, dried over MgSO4, filtered and concentrated to dryness to yield 5-(5-(4-(3-aminooxetan-3-yl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a solid (0.074 g, 100%). 1H NMR (300 MHz, Chloroform-d) δ 1.63-1.80 (m, 1H), 2.15-2.37 (m, 1H), 2.51-2.84 (m, 4H), 4.80 (d, J=6.5 Hz, 2H), 5.02 (d, J=6.5 Hz, 2H), 7.37 (d, J=8.5 Hz, 1H), 7.85 (d, J=8.5 Hz, 2H), 8.37 (d, J=2.3 Hz, 1H), 9.11 (d, J=2.3 Hz, 1H). C22H18F3N5O2S MS m/z 473.9 (M+H)+.
Formaldehyde (37% wt in water, 0.013 mL, 0.471 mmol) was added to a solution of yield 5-(5-(4-(3-aminooxetan-3-yl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.074 g, 0.157 mmol) in DCE (3 mL). The mixture was stirred at room temperature for 5 min, before Sodium triacetoxyborohydride (0.099 g, 0.471 mmol) was added. The reaction was stirred at RT overnight and diluted with aqueous saturated NaHCO3. The solution was extracted with DCM and the organic layer was dried over MgSO4, filtered and concentrated to give a residue The raw material was purified by using a GILSON Semi-Preparative System, operated by Trilution software, equipped with a Phenomenex Gemini C18 100A column (100 mm long×30 mm I.D.; 5 m particles) at 25° C., with a flow rate of 40 mL/min. A gradient elution was performed from 70% of a 25 mM aqueous solution of Ammonium bicarbonate (pH 8)/30% Acetonitrile-Methanol (1:1 mixture) to 73% of a 25 mM aqueous solution of Ammonium bicarbonate (pH 8)/27% Acetonitrile-Methanol (1:1 mixture) in 20 min. Pure fractions were combined and extracted with dichloromethane. The organic layer was washed with brine, dried over MgSO4 and concentrated to afford pure 5-(5-(4-(3-(dimethylamino)oxetan-3-yl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a pale yellow solid (0.048 g, 61%). 1H NMR (300 MHz, Chloroform-d) δ 1.62-1.80 (m, 1H), 2.15 (s, 6H), 2.18-2.37 (m, 1H), 2.50-2.78 (m, 4H), 4.79-5.11 (m, 4H), 7.29-7.41 (m, 4H), 8.38 (d, J=2.2 Hz, 1H), 9.11 (d, J=2.2 Hz, 1H). C24H22F3N5O2S MS m/z 502.1 (M+H)+.
Following the procedure described in Example 26, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compound of Formula (I) of the invention was prepared.
1-((4-bromophenyl)amino)cyclobutanecarbonitrile (9.79 g, 39 mmol), and freshly prepared 5-Isothiocyanato-3-trifluoromethyl-pyridine-2-carbonitrile (9.3 g, 32.46 mmol) were heated at 60° C. in DMA (50 mL) for 2 h and then allowed to cool to room temperature. The mixture was diluted with MeOH (50 mL) and 2M HCl (50 mL) and stirred at 60° C. for another 2 h. EA was added and the organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The crude material was purified by reverse phase preparative HPLC (Column: SYNERGI, Flow rate: 80 mL/min, Mobile Phase A: Purified water (containing 0.1% TFA), Mobile Phase B: acetonitrile, Gradient: 55-98(% B) from 0-35 min). The desired fractions were collected and the pH adjusted to 8 using 10% aqueous NaHCO3. The solution was concentrated under reduced pressure and extracted with EtOAc. The organic layers were washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure to yield 5-(5-(4-bromophenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a solid (5 g, 32%). 1H NMR (400 MHz, Chloroform-d) δ ppm 1.68-1.78 (m, 1H) 2.18-2.34 (m, 1H) 2.49-2.62 (m, 2H) 2.66-2.77 (m, 2H) 7.18-7.24 (m, 2H) 7.72-7.79 (m, 2H) 8.36 (d, J=2.26 Hz, 1H) 9.10 (d, J=2.26 Hz, 1H). C19H12BrF3N4OS MS m/z 483 (M+H)+.
A mixture of 1-bromo-4-nitrobenzene (0.3 g, 1.485 mmol), tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate (0.647 g, 1.931 mmol), potassium carbonate (0.616 g, 4.455 mmol), Pd(dppf)Cl2 (0.121 g, 0.148 mmol) in 1,4-Dioxane (6 mL0 and water (3 mL) was stirred at 90° C. overnight then allowed to cool to RT. The mixture was filtered through diatomaceous earth; the organic layer was washed with water, brine, dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of MeOH in DCM from 0 to 10%). The fractions with products were collected and concentrated under reduced pressure to yield tert-butyl 3-(4-nitrophenyl)-8-azabicyclo[3.2.1]oct-3-ene-8-carboxylate as a solid (0.490 g, 100%). C18H22N2O4 MS m/z 275.1 (M-55)+
A solution of tert-butyl 3-(4-nitrophenyl)-8-azabicyclo[3.2.1]oct-3-ene-8-carboxylate (0.491 g, 1.485 mmol) in EtOAc 4.5 mL) was purged using nitrogen and reduced pressure. Palladium on charcoal (10% wet) was added and the mixture was hydrogenated overnight. The reaction mixture was filtered through diatomaceous earth and concentrated under reduced pressure to give tert-butyl 4-((5-amino-1H-indazol-1-yl)methyl)piperidine-1-carboxylate used directly into the next step. C18H26N2O2 MS m/z 303.2 (M+H)+.
tert-Butyl 4-((5-amino-1H-indazol-1-yl)methyl)piperidine-1-carboxylate (0.449 g, 1.485 mmol) and cyclobutanone (0.222 mL, 2.97 mmol) were mixed in acetic acid (1.2 mL) and ethanol (1.2 mL). Sodium cyanide (0.291 g, 5.94 mmol) was added and the mixture was stirred at 50° C. overnight and then allowed to cool to room temperature. The mixture was then poured into water and extracted with DCM. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EtOAc in Heptane from 5 to 80%). The fractions with product were collected and concentrated under reduced pressure to yield tert-butyl 3-(4-((1-cyanocyclobutyl)amino)phenyl)-8-azabicyclo[3.2.1]octane-8-carboxylate as a solid (0.486 g, 86%). C23H31N3O2 MS m/z 382.3 (M+H)+.
tert-Butyl 3-(4-((1-cyanocyclobutyl)amino)phenyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (0.53 g, 1.39 mmol), and freshly prepared 5-Isothiocyanato-3-trifluoromethyl-pyridine-2-carbonitrile (0.516 g, 1.53 mmol) were heated at 60° C. in DMA (4.06 mL) overnight and then allowed to cool to room temperature. The mixture was diluted with MeOH (3 mL) and 1M HCl (3 mL) and stirred at RT for 30 min. EA was added and the solution was washed with water, aqueous saturated NaHCO3 and brine. The organic layer was dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 5 to 40%). The fractions with product were collected and concentrated under reduced pressure to yield tert-butyl 3-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)-8-azabicyclo[3.2.1]octane-8-carboxylate as a yellow solid (0.445 g, 52%). Another aliquot (0.073 g) was purified by reverse phase preparative HPLC to yield titled product as a yellow solid (0.038 g).
HPLC Conditions: GILSON Semi-Preparative System, operated by Trilution software, equipped with a Phenomenex Gemini C18 100A column (100 mm long×30 mm I.D.; 5 μm particles) at 25° C., with a flow rate of 40 mL/min. A gradient elution was performed from 49% of a 0.1% HCOOH aqueous solution (pH 3)/51% Acetonitrile to 6% of a 0.1% HCOOH aqueous solution (pH 3)/94% Acetonitrile in 20 min. The injection volume was 8000 μL. Acquisition frequency was set to 254 nm for the UV-Dual detector. 1H NMR (300 MHz, Chloroform-d) δ 1.51 (s, 9H), 1.53-1.56 (m, 4H), 1.59-1.74 (m, 1H), 1.75-1.89 (m, 1H), 1.96-2.15 (m, 2H), 2.15-2.34 (m, 1H), 2.62 (dq, J=20.8, 10.6, 10.1 Hz, 6H), 4.21-4.51 (m, 2H), 7.20-7.28 (m, 2H), 7.43 (t, J=8.3 Hz, 2H), 8.37 (d, J=2.2 Hz, 1H), 9.11 (d, J=2.2 Hz, 1H). C31H32F3N5O3S MS m/z 512.1 (M-100+H)+.
To a solution of tert-butyl 3-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)-8-azabicyclo[3.2.1]octane-8-carboxylate (0.445 g, 0.728 mmol) in DCM (6 mL) was added TFA (4 mL). After stirring at RT for 3 h the reaction mixture was concentrated and evaporated with toluene. The mixture was poured into aqueous saturated NaHCO3 and extracted with EtOAc. The organic layer was washed with water, brine, dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of 2 M NH3/MeOH in DCM from 0 to 10%) to yield 5-(5-(4-(8-azabicyclo[3.2.1]octan-3-yl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a solid (0.195 g, 52%). Another aliquot (0.087 g) was purified by reverse phase preparative HPLC to yield the title product as a yellow solid (0.068 g).
HPLC Conditions: GILSON Semi-Preparative System, operated by Trilution software, equipped with a Phenomenex Gemini C18 100A column (100 mm long×30 mm I.D.; 5 μm particles) at 25° C., with a flow rate of 40 mL/min. A gradient elution was performed from 70% of a 0.1% HCOOH aqueous solution (pH 3)/30% Acetonitrile to 73% of a 0.1% HCOOH aqueous solution (pH 3)/27% Acetonitrile in 20 min. The injection volume was 8000 μL. Acquisition frequency was set to 254 nm for the UV-Dual detector. 1H NMR (300 MHz, Chloroform-d) δ 1.58-1.77 (m, 4H), 1.83-2.00 (m, 1H), 2.07-2.30 (m, 1H), 2.34-2.80 (m, 10H), 3.71-3.97 (m, 2H), 7.21-7.29 (m, 2H), 7.46-7.54 (m, 2H), 8.37 (d, J=2.2 Hz, 1H), 9.11 (d, J=2.2 Hz, 1H). C26H24F3N5OS MS m/z 512.0 (M+H)+.
Formaldehyde (37% wt in water, 0.047 mL, 0.633 mmol) was added to a solution of 5-(5-(4-(8-azabicyclo[3.2.1]octan-3-yl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.108 g, 0.211 mmol) in DCE (5 mL). The mixture was stirred at room temperature for 5 min, before Sodium triacetoxyborohydride (0.134 g, 0.633 mmol) was added. The reaction was stirred at RT overnight and diluted with aqueous saturated NaHCO3. The solution was extracted with DCM and the organic layer was washed, dried over MgSO4, filtered and concentrated to give a residue that was then purified by chromatography over silica gel (gradient of MeOH in DCM from 0 to 10%) to yield 5-(5-(4-(8-methyl-8-azabicyclo[3.2.1]octan-3-yl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a beige solid (0.05 g, 45%). 1H NMR (300 MHz, Chloroform-d) δ 1.60-1.76 (m, 1H), 1.78-1.97 (m, 1H), 2.02-2.18 (m, 4H), 2.21-2.40 (m, 2H), 2.49-2.81 (m, 8H), 2.87-3.17 (m, 1H), 3.38-3.51 (m, 1H), 3.72-3.91 (m, 2H), 7.20-7.34 (m, 2H), 7.51-7.66 (m, 2H), 8.36 (d, J=2.3 Hz, 1H), 9.11 (d, J=2.3 Hz, 1H). C27H26F3N5OS MS m/z 526.0 (M+H)+.
Following the procedure described in Example 28, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of Formula (I) of the invention were prepared.
To a solution of 1-fluoro-4-nitrobenzene (5 g, 35.43 mmol) and tert-butyl piperazine-1-carboxylate (6.6 g, 35.43 mmol) in DMF (100 mL) was added potassium carbonate (14.7 g, 106.36 mmol) and the mixture was stirred at 50° C. for 18 h, then allowed to cool to room temperature and concentrated under reduced pressure. The oily residue was washed with diethyl ether (3×) to give tert-butyl 4-(4-nitrophenyl)piperazine-1-carboxylate as a yellow solid (8.2 g, 95%). 1H NMR (400 MHz, Chloroform-d) δ ppm 1.48 (s, 9H) 3.38-3.45 (m, 4H) 3.56-3.63 (m, 4H) 6.75-6.86 (m, 2H) 8.07-8.20 (m, 2H). C15H21N3O4 MS m/z 308.1 (M+H)+
A solution of tert-butyl 4-(4-nitrophenyl)piperazine-1-carboxylate (8.2 g, 26.68 mmol) in MeOH (100 mL) was purged using nitrogen and reduced pressure. Palladium on charcoal (10% wet) was added and the mixture was hydrogenated (50 psi) for 16 h. The reaction mixture was filtered through diatomaceous earth and concentrated under reduced pressure to give tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (7.4 g, 97%) as a dark blue oil used directly into the next step. C15H23N3O2 MS m/z 277.9 (M+H)+.
To a solution of tert-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (7.6 g, 27.4 mmol) and cyclobutanone (3.06 mL, 40.94 mmol) in MeOH (50 mL) was added zinc iodide (0.44 g, 1.378 mmol) at RT. Trimethylsilyl cyanide (4.1 g, 41.328 mmol) was then added and the mixture was stirred at 90° C. for 16 h and then allowed to cool down to RT and concentrated under reduced pressure. The residue was partitioned between water and EtOAc. The organic layer was concentrated under reduced pressure to give a dark oil, then the oil was further washed with diethyl ether to afford tert-butyl 4-(4-((1-cyanocyclobutyl)amino)phenyl)piperazine-1-carboxylate as a purple solid (7.4 g, 56%). C20H28N4O2 MS m/z 356.9 (M+H)+.
A solution of 5-isothiocyanato-3-(trifluoromethyl)picolinonitrile (0.4 g, 1.75 mmol) in DMA (5 mL) was added to a solution of tert-butyl 4-(4-((1-cyanocyclobutyl) amino)phenyl)piperazine-1-carboxylate (0.518 g, 1.45 mmol) in DMA (5 mL). The mixture was heated at 60° C. for 2 h and then allowed to cool to room temperature. The mixture was treated with MeOH (5 mL) and 2M HCl (5 mL). The resulting suspension stirred at 60° C. for 2 h and poured into water. The precipitate was collected by filtration, washed with water and dried to give tert-butyl 4-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)piperazine-1-carboxylate (0.8 g, 94%) as a grey solid. The compound was directly used into the next step without purification.
To a solution of tert-butyl 4-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenyl)piperazine-1-carboxylate (0.8 g, 1.36 mmol) in DCM (30 mL) was added TFA (1.55 g, 13.64 mmol). After stirring at RT for 18 h the reaction mixture was concentrated to give the title compound as an oil (0.74 g, 90%). A portion of the crude material was purified by preparative reverse phase HPLC (column: Synergi Max-RP 150×30 mm×4u, mobile phase: 32-52% CH3CN/H2O(HCl)) to yield 5-(8-oxo-5-(4-(piperazin-1-yl)phenyl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl) picolinonitrile as a solid. 1H NMR (400 MHz, Chloroform-d) δ ppm 1.67-1.76 (m, 1H) 2.17-2.33 (m, 1H) 2.51-2.63 (m, 2H) 2.64-2.74 (m, 2H) 3.42-3.54 (m, 4H) 3.60-3.76 (m, 4H) 7.11-7.18 (m, 2H) 7.24-7.26 (m, 2H) 8.34-8.38 (m, 1H) 9.07-9.14 (m, 1H). C23H21F3N6OS, HCl MS m/z 487.1 (M+H)+.
To a mixture of 5-(8-oxo-5-(4-(piperazin-1-yl)phenyl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl) picolinonitrile (0.22 g, 0.452 mmol), DIEA (0.233 g, 1.808 mmol) in DCM (8 mL) was added 1-Pyrrolidinecarbonyl chloride (0.120 g, 0.904 mmol). After stirring at RT overnight the reaction mixture was concentrated to afford the title compound as an oil. The crude oil was purified by reverse phase preparative HPLC (column: Synergi Max-RP 150*30 mm*4u, mobile phase: 54-84% CH3CN/H2O (HCl)) to give 5-(8-oxo-5-(4-(4-(pyrrolidine-1-carbonyl)piperazin-1-yl)phenyl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a light yellow solid (0.089 g, 32%). 1H NMR (400 MHz, Chloroform-d) δ ppm 1.88 (br. s., 4H) 2.26 (br. s., 2H) 2.54 (br. s., 2H) 2.70 (br. s., 2H) 3.42 (br. s., 4H) 3.51 (br. s., 4H) 3.89 (br. s., 4H) 7.40 (br. s., 2H) 7.80 (br. s., 2H) 8.35 (s, 1H) 9.08 (s, 1H). C28H28F3N7O2S. HCl MS m/z 584.2 (M+H)+.
Following the procedure described in Example 29, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of formula (I) of the invention were prepared.
To a solution of (3,4-dihydro-2H-pyran-2-yl)methanol (5.00 mL, 46.9 mmol) in dry DMF (18 mL) was added imidazole (7.98 g, 117 mmol). The solution was cooled to 0° C., TBDMSCl (8.48 g, 56.2 mmol) was added and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with Et2O and washed with brine (3×). The combined organic layers were dried over MgSO4, filtered and concentrated in vacuo to give tert-butyl((3,4-dihydro-2H-pyran-2-yl)methoxy)dimethylsilane as an oil (9.6 g, 90%).
In a Schlenk tube, 2-methyltetrahydrofuran (5 mL) was carefully degassed in vacuo and back-filled with N2, then, bis(pinacolato)diboron (0.695 g, 2.74 mmol), bis(1,5-cyclooctadiene)di-μ-methoxydiiridium (I) (22 mg, 32.8 μmol) and 4,4′-di-tert-butyl-2,2′-bipyridine (18 mg, 0.066 mmol) were added. The brown mixture was stirred for 10 min. In another flask, a solution of tert-butyl ((3,4-dihydro-2H-pyran-2-yl)methoxy) dimethylsilane (0.5 g, 2.19 mmol) in 2-Me-THF (5 mL) was degassed and transferred via cannula to the Schlenk tube. The reaction was heated at 80° C. for 2 h then bis(1,5-cyclooctadiene)di-t-methoxydiiridium (I) (22 mg, 32.8 μmol) was added, and the reaction mixture was degassed and back-filled with N2 and stirred at 80° C. for 24 h to give tert-butyldimethyl((6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-pyran-2-yl)methoxy)silane in solution in 2-Me-THF used as such into the next step.
A mixture of tert-butyldimethyl((6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-pyran-2-yl)methoxy)silane (155 mg, 0.437 mmol), water (200 μL), acetone (3 mL, 40.6 mmol) and K3PO4 (247 mg, 1.17 mmol) was purged with N2 and stirred at room temperature for 2 min. Then 5-(5-(4-bromophenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (140 mg, 0.292 mmol) and Pd(dppf)2Cl2 (19 mg, 29.2 μmol) were added. The mixture was purged again with N2 and stirred at room temperature for 90 min. The mixture was diluted with EtOAc and water, the layers were separated and the organic layer was dried over MgSO4, filtered and the solvent was removed under reduced pressure to give 5-(5-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3,4-dihydro-2H-pyran-6-yl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl) picolinonitrile as a brown oil, used as such in the next step.
To a solution of 5-(5-(4-(2-(((tert-butyldimethylsilyl)oxy)methyl)-3,4-dihydro-2H-pyran-6-yl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl) picolinonitrile (267 mg, 0.425 mmol) in THF (2 mL) was added TBAF (0.51 mL, 0.510 mmol). The mixture was stirred at room temperature for 24 h. Silica was added and the mixture was concentrated in vacuo to give a dry load which was purified by preparative LC (irregular SiOH 15-40 μm, 12 g Grace, dry loading, mobile phase (gradient of MeOH in DCM from 0 to 20%) to give a crude residue further purified by achiral SFC (Stationary phase: Cyano 6 μm 150×21.2 mm, mobile phase: 80% C02, 20% MeOH) to yield 5-(5-(4-(2-(hydroxymethyl)-3,4-dihydro-2H-pyran-6-yl)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile as a white solid (0.018 g, 8%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.50-1.65 (m, 2H) 1.89-2.00 (m, 2H) 2.16-2.34 (m, 2H) 2.41-2.50 (m, 2H) 2.60-2.68 (m, 2H) 3.55-3.68 (m, 2H) 3.93-4.06 (m, 1H) 4.86 (t, J=5.8 Hz, 1H) 5.59 (t, J=4.0 Hz, 1H) 7.38 (d, J=8.6 Hz, 2H) 7.80 (d, J=8.1 Hz, 2H) 8.77 (d, J=2.0 Hz, 1H) 9.22 (d, J=2.0 Hz, 1H). C25H21F3N4O3S MS m/z 515.2 (M+H)+
To a solution of (3R,4R)-3,4-dihydro-2H-pyran-3,4-diol (2.30 g, 19.8 mmol) in THF (140 mL) at 0° C. were added TEA (12.4 mL, 89.1 mmol), then dropwise TMSCl (8.80 mL, 69.3 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The organic solvent was concentrated in vacuo, and the crude product was diluted with Et2O and water. The organic layer was washed with brine, dried over MgSO4, filtered and evaporated in vacuo to yield (((3R,4R)-3,4-dihydro-2H-pyran-3,4-diyl)bis(oxy))bis(trimethylsilane) as a yellow oil (4.79 g, 93%).
In a Schlenck flask, a stirred mixture of bis(1,5-cyclooctadiene)di-t-methoxydiiridium (I) (25 mg, 0.038 mmol) and 4,4′-di-tert-butyl-2,2′-bipyridine (21 mg, 0.077 mmol) in 2-Me-THF (6.25 mL) was degassed under reduced pressure and purged with N2. The black mixture was stirred at rt for 15 min. In another flask, a solution of (((3R,4R)-3,4-dihydro-2H-pyran-3,4-diyl)bis(oxy))bis(trimethylsilane) (500 mg, 1.92 mmol) and bis(pinacolato)diboron (0.731 g, 2.88 mmol) in 2-Me-THF (6.25 mL) was degassed and transferred to the first flask. The reaction mixture was degassed in vacuo and purged again with N2, then stirred at 80° C. for 2 h to give (((3R,4R)-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-pyran-3,4-diyl)bis(oxy))bis(trimethylsilane), used as such into the next step.
To a stirred solution of (2S,3R,4S)-2-methyl-3,4-dihydro-2H-pyran-3,4-diol (3.18 g, 24.4 mmol), TEA (17.0 mL, 122 mmol) and DMAP (149 mg, 1.22 mmol) in DMF (100 mL) at 0° C. was added TMSCl (9.30 mL, 73.3 mmol), and the reaction mixture was stirred at room temperature for 17 h. The crude mixture was diluted with Et2O and water. The organic layer was washed with saturated NH4Cl (2×), brine (3×), dried over MgSO4, filtered and evaporated in vacuo to yield (((2S,3R,4S)-2-methyl-3,4-dihydro-2H-pyran-3,4-diyl)bis(oxy))bis(trimethylsilane) as an orange oil (6.76 g, 100%).
In a Schlenck flask, a stirred mixture of bis(1,5-cyclooctadiene)di-t-methoxydiiridium (I) (19 mg, 0.029 mmol) and 4,4′-di-tert-butyl-2,2′-bipyridine (16 mg, 0.058 mmol) in 2-Me-THF (5 mL) was degassed under reduced pressure and purged with N2. The black mixture was stirred at room temperature for 15 min. In another flask, a solution of (((2S,3R,4S)-2-methyl-3,4-dihydro-2H-pyran-3,4-diyl)bis(oxy))bis(trimethylsilane) (400 mg, 1.46 mmol) and bis(pinacolato)diboron (555 mg, 2.19 mmol) in 2-Me-THF (5 mL) was degassed and transferred to the first flask. The reaction mixture was degassed in vacuo and purged again with N2, then stirred at 80° C. for 2 h to give (((2S,3R,4S)-2-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,4-dihydro-2H-pyran-3,4-diyl)bis(oxy)) bis(trimethylsilane), used as such into the next step.
Following the procedure described in Example 30, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of formula (I) of the invention were prepared.
To a solution of 5-(5-(4-bromophenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-methylpicolinonitrile (0.306 g, 0.72 mmol) and (1H-pyrazol-3-yl)boronic acid (0.12 g, 1.08 mmol) in DME (3 mL) was added, under an Argon atmosphere, aqueous 2M Na2CO3 (0.75 mL, 1.51 mmol) and Pd(PPh3)4 (0.032 g, 0.028 mmol). The reaction mixture was refluxed for 16 h, filtered and concentrated under reduced pressure and the resulting residue was purified by preparative reverse-phase chromatography to yield 3-methyl-5-[5-oxo-8-(4-pyrazol-4-ylphenyl)-7-thioxo-6, 8-diazaspiro[3.4]oct-6-yl]pyridine-2-carbonitrile as a solid (0.179 g, 60%). 1H NMR (500 MHz, DMSO-d6) d ppm 1.53-1.61 (m, 1H), 1.92-2.02 (m, 1H), 2.37-2.49 (m, 2H), 2.55-2.66 (m, 2H), 2.59 (s, 3H), 7.39-7.42 (m, 2H, J=8.2 Hz), 7.81-7.85 (m, 2H, J=8.2 Hz), 7.97-8.11 (m, 1H), 8.16 (d, 1H, J=1.9 Hz), 8.22-8.37 (m, 1H), 8.73 (d, 1H, J=1.9 Hz), 13.00-13.21 (m, 1H). C22H18N60S MS m/z 415.2 (M+H)+.
Following the procedure described in Example 31, above, selecting and substituting the appropriate reagents, starting materials, and purification methods, and adjusting reaction temperatures, times and other variables or parameters, as needed or desirable, as would be readily recognized by those skilled in the art, the following compounds of Formula (I) of the invention were prepared.
To a solution of 2,6-dibromopyridine (2.37 g, 10 mmol) in THF (7.5 mL) was added a solution of 2.0M isopropylmagnesium bromide in THF (7.5 mL, 15 mmol) and the mixture was stirred at RT for 90 min to give a dark solution. This solution was added dropwise to a solution of sulfuryl chloride (1.62 mL, 20 mmol) in hexanes (75 mL) and cooled at 0° C. The resulting yellow solution was stirred at 0° C. for 15 min and concentrated under reduced pressure. The residue was diluted with heptane and the solution concentrated under reduced pressure to give a yellow residue. The residue was dissolved in DCM (35 mL) and cooled to 0° C. Et3N (2.5 mL, 18 mmol) was added followed by dibenzylamine (1.93 mL, 10 mmol). The mixture was stirred for 1 h at RT, diluted with water and extracted with DCM. The organic layer was washed with brine, dried over MgSO4, filtered and concentrated under reduced pressure. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 10 to 50%). The fractions with product were collected and concentrated under reduced pressure to yield N,N-dibenzyl-6-bromopyridine-2-sulfonamide as an orange oil (1.45 g, 34%).
A mixture of N,N-dibenzyl-6-bromopyridine-2-sulfonamide (0.65 g, 1.56 mmol), 1-aminocyclobutanecarboxylic acid (0.197 g, 1.71 mmol), DBU (0.6 mL, 4 mmol), copper (I) iodide (0.029 g, 0.152 mmol) in DMA (3 mL) was stirred at 110° C. for 3 h. More copper (I) iodide (0.089 g, 0.476 mmol) was added and the mixture stirred at 110° C. overnight. The mixture was allowed to cool to RT, then diluted with water and EtOAc. The aqueous layer was then acidified with aqueous 1M HCl to pH 5 and extracted with EtOAc. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. The crude material was purified by chromatography over silica gel (gradient of MeOH in DCM from 0 to 20%). The fractions with product were collected and concentrated under reduced pressure to yield 1-((6-(N,N-dibenzylsulfamoyl)pyridin-2-yl)amino) cyclobutanecarboxylic acid (0.3 g, 39%).
A mixture of 1-((6-(N,N-dibenzylsulfamoyl)pyridin-2-yl)amino) cyclobutanecarboxylic acid (0.3 g, 0.66 mmol), 2.0M solution of (trimethylsilyl) diazomethane (0.66 mL, 1.33 mmol) in toluene (2 mL) and methanol (2 mL) was stirred at RT for 90 min. The mixture was then absorbed on silica gel and purified by chromatography over silica gel (gradient of EtOAc in hexanes from 10 to 100%) to give methyl 1-((6-(N,N-dibenzylsulfamoyl) pyridin-2-yl)amino)cyclobutanecarboxylate as a white solid (0.209 g, 68%).
Methyl 1-((6-(N,N-dibenzyl sulfamoyl)pyridin-2-yl)amino)cyclobutanecarboxylate (0.209 g, 0.45 mmol), DMAP (0.137 g, 1.12 mmol) and freshly prepared 5-Isothiocyanato-3-trifluoromethyl-pyridine-2-carbonitrile (0.308 g, 1.35 mmol) were heated at 105° C. in toluene (4 mL) overnight and then allowed to cool to room temperature. The mixture was concentrated and the residue purified by chromatography over silica gel (gradient of EA in heptane from 0 to 50%). The fractions with product were collected and concentrated under reduced pressure and purified by reverse phase preparative HPLC to yield N,N-dibenzyl-6-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)pyridine-2-sulfonamide as a yellow solid (0.046 g, 15%).
A solution of N,N-dibenzyl-6-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)pyridine-2-sulfonamide (0.046 g, 0.069 mmol) in sulfuric acid (1 mL) was vortexed for 1 h and diluted with ice-water and EtOAc. The organic layer was separated, washed with aqueous saturated NaHCO3, brine, dried over MgSO4, filtered and concentrated to dryness. The crude material was purified by chromatography over silica gel (gradient of EA in heptane from 0 to 50%) to yield 6-{7-[6-cyano-5-(trifluoromethyl)(3-pyridyl)]-8-oxo-6-thioxo-5,7-diazaspiro[3.4]oct-5-yl}pyridine-2-sulfonamide as a white solid (0.025 g, 75%). 1H NMR (500 MHz, DMSO-d6) d ppm 1.80 (ddt, 1H, J=15.8, 10.6, 5.2 Hz), 1.92-2.04 (m, 1H), 2.55-2.73 (m, 2H), 2.87-2.98 (m, 2H), 7.70 (s, 2H), 8.03-8.08 (m, 2H), 8.33 (t, 1H, J=7.9 Hz), 8.81 (d, 1H, J=1.9 Hz), 9.25 (d, 1H, J=1.9 Hz). C18H13F3N6O3S2 MS m/z 483.1 (M+H)+.
Cyclobutanone (1.074 mL, 14.37 mmol) was added to a solution of methyl 4-aminocyclohexanecarboxylate (0.753 g, 4.79 mmol) in Acetic acid (0.5 mL) and MeOH (4.5 mL). The solution was stirred 15 min at room temperature before trimethylsilyl cyanide (1.798 mL, 14.37 mmol) was added dropwise. After stirring at RT overnight, aqueous 1.0M Na2CO3 (50 mL) was added carefully and the solution was extracted with EtOAc. The combined organic layers were dried over MgSO4, filtered and concentrated to give methyl 4-((1-cyanocyclobutyl)amino)cyclohexanecarboxylate (1.13 g, 100%), directly used into the next step. C13H20N2O2 MS m/z 237 (M+H)+.
A solution of 5-isothiocyanato-3-(trifluoromethyl)picolinonitrile (1.215 g, 5.3 mmol) in DMA (6 mL) was added to a solution of methyl 4-((1-cyanocyclobutyl)amino) cyclohexanecarboxylate (1.132 g, 4.79 mmol) in DMA (5 mL). The mixture was heated at 60° C. for 3 h and then allowed to cool to room temperature. The mixture was treated with MeOH (9 mL) and 2M HCl (9 mL). The resulting mixture was stirred at 40° C. for 2 h and extracted with EtOAc (100 mL). The organic layer was washed with aqueous saturated NaHCO3, water, brine and dried over MgSO4, filtered and concentrated under reduced pressure. Chromatography was performed on silica gel (gradient of EA in heptane from 0 to 100%), followed by purification by preparative reverse phase HPLC [C18 column using gradient of a mixture 50% (65 mM aqueous NH4OAc+ACN (90:10))−50% (MeOH) to 25% (65 mM NH4OAc+ACN (90:10))−75% (MeOH)). The pure fractions were collected and concentrated to dryness to give methyl 4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)cyclohexanecarboxylate as a solid (0.354 g, 16%). 1H NMR (300 MHz, Chloroform-d) δ 1.65 (tt, J=13.7, 4.4 Hz, 2H), 1.75-1.90 (m, 2H), 1.94-2.13 (m, 1H), 2.35-2.45 (m, 3H), 2.58-2.69 (m, 2H), 2.69-2.95 (m, 5H), 3.75 (s, 3H), 4.15-4.35 (m, 1H), 8.26 (d, J=2.2 Hz, 1H), 8.99 (d, J=2.2 Hz, 1H). C21H21F3N4O3S MS m/z 467 (M+H)+.
Trans-tert-butyl 4-hydroxycyclohexyl)carbamate (1.705 g, 7.92 mmol), phenol (0.745 g, 7.92 mmol) and triphenylphosphine (3.74 g, 14.25 mmol) were dissolved in dry THF (15 mL) under a nitrogen atmosphere. A solution of Diisopropyl azodicarboxylate (DIAD, 2.81 mL, 14.25 mmol) in THF (30 mL) was added dropwise over 15-20 min. Upon completion of the addition, the reaction was continued for 3 h at RT. The mixture was then concentrated and the crude residue was chromatographed over silica gel (gradient of EtOAc in Heptane from 0 to 50%). The pure fractions were concentrated to give Trans-tert-butyl 4-phenoxycyclohexyl)carbamate (0.853 g, 30%), directly used into the next step. C17H25NO3 MS m/z 192 (M+H-Boc)+.
(Trans)-tert-butyl 4-phenoxycyclohexyl)carbamate (0.853 g, 2.92 mmol) was taken in dioxane (15 mL). Dry 4N HCl in dioxane (7.32 mL, 29.27 mmol) was added with stirring. The mixture was stirred overnight at room temperature and concentrated under reduced pressure. The residue was diluted with DCM and washed with aqueous 1M Na2CO3. The organic layer was dried over MgSO4, filtered and concentrated under reduced pressure. Chromatography over silica gel (gradient of DCM/MeOH/NH3 (9/1/0.1)/DCM, from 0 to 100% and then 100%) yielded (trans)-4-phenoxycyclohexanamine as a pale foam (0.28 g, 50%), which was directly used into the next step. C12H17NO MS m/z 192 (M+H)+.
Cyclobutanone (0.328 mL, 4.39 mmol) was added to a solution of (trans)-4-phenoxycyclohexanamine (0.28 g, 1.46 mmol) in Acetic acid (0.5 mL) and MeOH (4.5 mL). The solution was stirred 15 min at room temperature before trimethylsilyl cyanide (0.549 mL, 4.39 mmol) was added dropwise. After stirring at RT overnight, additional cyclobutanone (0.164 mL, 2.19 mmol) and trimethylsilyl cyanide (0.275 mL, 2.19 mmol) were added. After stirring at RT overnight, water was added carefully and the solution was extracted with EtOAc. The organic layers were dried over MgSO4, filtered and concentrated. Chromatography over silica gel (gradient of AcOEt in Heptane, from 0 to 50%) yielded 1-((trans)-4-phenoxycyclohexyl)amino)cyclobutanecarbonitrile (0.109 g, 28%) directly used into the next step. C17H22N2O MS m/z 271 (M+H)+.
A solution of 5-isothiocyanato-3-(trifluoromethyl)picolinonitrile (0.092 g, 0.403 mmol) and 1-((trans)-4-phenoxycyclohexyl)amino)cyclobutanecarbonitrile (0.109 g, 0.403 mmol) in DMA (5 mL) was heated at 60° C. for 6 h and then allowed to cool to room temperature. The mixture was treated with MeOH (5 mL) and 2M HCl (5 mL). The resulting mixture was stirred at RT overnight and extracted with EtOAc (50 mL). The organic layer was washed with aqueous saturated NaHCO3, water, brine, and dried over MgSO4, then filtered and concentrated under reduced pressure. Chromatography over silica gel (gradient of EA in heptane from 0 to 50%) yielded a residue which was further recrystallized from diethyl ether to yield 5-[5-oxo-8-(4-phenoxycyclohexyl)-7-thioxo-6,8-diazaspiro[3.4]octan-6-yl]-3-(trifluoromethyl)pyridine-2-carbonitrile as a solid (0.07 g, 34%). 1H NMR (300 MHz, Chloroform-d) δ 1.58-1.87 (m, 4H), 1.96-2.17 (m, 1H), 2.10-2.45 (m, 3H), 2.60-2.75 (m, 2H), 2.82-3.02 (m, 4H), 4.30-4.54 (m, 1H), 4.64 (br s, 1H), 6.95-6.99 (m, 3H), 7.28-7.34 (m, 2H), 8.28 (d, J=2.1 Hz, 1H), 9.02 (d, J=2.1 Hz, 1H). C25H23F3N4O2S MS m/z 501 (M+H)+.
Cyclobutanone (0.314 mL, 4.2 mmol) was added to a solution of tert-butyl (4-aminocyclohexyl)carbamate (0.3 g, 1.4 mmol) in acetic acid (0.5 mL) and MeOH (4.5 mL). The solution was stirred 15 min at room temperature before trimethylsilyl cyanide (0.525 mL, 4.2 mmol) was added dropwise. After stirring at RT overnight, water was added carefully and the solution was extracted with EtOAc. The organic layers were dried over MgSO4, filtered and concentrated to yield tert-butyl (4-((1-cyanocyclobutyl)amino) cyclohexyl)carbamate, used directly into the next step.
A solution of 5-isothiocyanato-3-(trifluoromethyl)picolinonitrile (0.367 g, 1.6 mmol) and tert-butyl (4-((1-cyanocyclobutyl)amino) cyclohexyl)carbamate (0.411 g, 1.4 mmol) in DMA (6 mL) was heated at 60° C. for 3 h and then allowed to cool to room temperature. The mixture was treated with MeOH (9 mL) and 1N HCl (9 mL). The resulting mixture was stirred at RT overnight and extracted with EtOAc (100 mL). The organic layer was washed with aqueous saturated NaHCO3, water, brine and dried over MgSO4, filtered and concentrated under reduced pressure to give tert-butyl (4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)cyclohexyl)carbamate (0.64 g, 87%), used directly into the next step. C24H28F3N5O3S MS m/z 424 (M+H-Boc)+.
Trifluoroacetic acid (2 mL) was added to a solution of tert-butyl (4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)cyclohexyl) carbamate (0.64 g, 1.222 mmol). After stirring for 2 h at RT, the solvent was concentrated under reduced pressure and the residue co-evaporated twice with toluene to yield 5-(5-(4-aminocyclohexyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.97 g, 100%), used directly into the next step. C19H20F3N5OS MS m/z 424 (M+H)+.
To a solution of 5-(5-(4-aminocyclohexyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (0.517 g, 1.222 mmol) in DCM (10 mL) was added at 0° C. Et3N (0.186 mL, 1.344 mmol) and benzoyl chloride (0.156 mL, 1.344 mmol). After stirring at RT for 2 h, the mixture was washed with aqueous saturated NH4Cl, brine, dried over MgSO4, filtered and concentrated under reduced pressure. Chromatography over silica gel (gradient of MeOH in DCM from 0 to 5%) gave a residue, which was then purified by preparative reverse phase HPLC (isocratic method, eluting 50% acetonitrile and 50% 25 mM NH4HCO3). The pure fractions were collected and concentrated to dryness to give N-[4-[6-[6-cyano-5-(trifluoromethyl)-3-pyridyl]-5-oxo-7-thioxo-6,8-diazaspiro[3.4]octan-8-yl]cyclohexyl]benzamide (0.045 g, 7%) as a solid. 1H NMR (300 MHz, Chloroform-d) δ 1.65-1.85 (m, 4H), 1.96-2.19 (m, 3H), 2.27-2.50 (m, 1H), 2.62-2.78 (m, 4H), 3.26-3.51 (m, 2H), 3.77-3.95 (m, 1H), 4.49-4.64 (m, 1H), 6.74 (d, J=8.5 Hz, 1H), 7.37-7.53 (m, 3H), 7.86 (d, J=6.7 Hz, 2H), 8.25 (d, J=2.2 Hz, 1H), 8.99 (d, J=2.1 Hz, 1H). C26H24F3N5O2S MS m/z 528 (M+H)+.
Preparation of Blend in Capsule of 10 mg, 40 mg and 100 mg of Cpd 43, HCl Salt
Formulations were assigned the following formulation numbers:
The stability of 10 mg Screen Batch was performed as follows using an UPLC method. Samples were pulled at the time points predefined and analyzed by UPLC. The detailed chromatographic conditions are listed below. The parent drug and related substance were integrated for calculation.
Conclusion: The prototype formula is chemically stable stressed under 40° C./75% RH open up to 3 months.
The term “biological sample”, as used herein, includes, without limitation, cell cultures or extracts thereof; biopsied material obtained from a mammal or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof.
Antagonism of receptors in a biological sample is useful for a variety of purposes that are known to one of skill in the art. Examples of such purposes include, but are not limited to, biological assays, gene expression studies, and biological target identification.
Certain embodiments of the present invention are directed to a method of treatment by antagonizing AR in a patient or a subject in need of such treatment comprising the step of administering to said patient a compound of Formula (I) of the present invention, or a composition comprising said compound.
The activity of a compound of Formula (I) as an antagonist of AR or for the treatment of an AR-mediated disease, disorder or condition, may be assayed in vitro or in vivo. An in vivo assessment of the efficacy of the compounds of the invention may be made using an animal model of an AR-mediated disease, disorder or condition, e.g., a rodent or primate model. The in vivo assessment may be further defined as an androgen dependent organ development (Hershberger) assay or as a tumor xenograft model. Cell-based assays may be performed using, e.g., a cell line isolated from a tissue that expresses either wild type or mutant AR. Additionally, biochemical or mechanism based assays, e.g., transcription assays using a purified protein, Northern blot, RT-PCR, etc., may be performed.
In vitro assays include assays that determine cell morphology, protein expression, and/or the cytotoxicity, enzyme inhibitory activity, and/or the subsequent functional consequences of treatment of cells with compounds of the invention. Alternate or additional in vitro assays may be used to quantitate the ability of the inhibitor to bind to protein or nucleic acid molecules within the cell.
Inhibitor binding may be measured by radiolabelling the inhibitor prior to binding, isolating the inhibitor/target molecule complex and determining the amount of radiolabel bound. Alternatively or additionally, inhibitor binding may be determined by running a competition experiment where new inhibitors are incubated with purified proteins or nucleic acids bound to known radioligands. Detailed conditions of exemplary systems for assaying a compound of Formula (I) of the present invention as an antagonist of AR are set forth in the Biological Examples below.
Such assays are exemplary and not intended to limit the scope of the invention. The skilled practitioner can appreciate that modifications can be made to conventional assays to develop equivalent or other assays that can be employed to comparably assess activity or otherwise characterize compounds and/or compositions as described herein.
Radioligand binding assays were performed with the cell extracts and ligands as detailed below. Complete methodology is contained within the cited publications. Kd values were determined by Non-Specific Incubation Detection Method.
Receptors
The results are expressed as a percent of control specific binding measured specific binding*100 control specific binding and as a percent inhibition of control specific binding 100−(measured specific binding*100) control specific binding obtained in the presence of compound.
The IC50 values (concentration causing a half-maximal inhibition of control specific binding) and Hill coefficients (nH) were determined by non-linear regression analysis of the competition curves generated with mean replicate values using Hill equation curve fitting.
Y=D+[A−D]
1+(C/C50)nH
wherein Y=specific binding, A=left asymptote of the curve, D=right asymptote of the curve, C=compound concentration, C50=IC50, and nH=slope factor. This analysis was performed using software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot® 4.0 for Windows® (© 1997 by SPSS Inc.).
The inhibition constants (Ki) were calculated using the Cheng Prusoff equation:
Ki=IC50(1+L/KD)
wherein L=concentration of radioligand in the assay, and KD=affinity of the radioligand for the receptor. A scatchard plot is used to determine the KD. Resultant data are shown in Table 2.
Radioligand binding inhibition and affinity calculations were determined using [3H]-methyltrienolone, [3H]-dexamethasone and [3H]-estradiol for AR, GR and ER, respectively. For ER, it was not possible to determine inhibition or affinity and data are not shown.
AR=androgen receptor, ER=estrogen receptor, GR=glucocorticoid receptor
LNCaP AR (cs) and LNCaP F876L luciferase cell lines were generated by transduction of each cell line (description of cell line generation Joseph J D, Lu N, Qian J, Sensintaffar J, Shao G, Brigham D, Moon M, Maneval E C, Chen I, Darimont B, Hager J H. A clinically relevant androgen receptor mutation confers resistance to second-generation antiandrogens enzalutamide and ARN-509. Cancer Discov 2013; 3:1020-1029) with an Androgen Response Element Firefly Luciferase lentiviral construct at an MOI (multiplicity of infection) of 50 following the manufacturer's instructions (Qiagen). A stable pooled-population cell line was generated using puromycin (Life Technologies) selection at 1:10,000 v/v. The protocol below was used for both cell lines and for testing of the compounds of Formula (I) of the present invention.
LNCaP cells were grown to about 80% confluence, media removed and cells rinsed in Hank's balanced salt solution prior to separation from the plate with 0.05% Trypsin EDTA. Cells were lifted and trypsin negated in complete CSS (charcoal stripped serum) culture media. CSS was maintained on cells for 24 h prior to assay, at which time 5,000 cells/20 μL were seeded in Greiner 384 well White/White Tissue Culture Treated Plates and incubated for a further 1-2 hours at 37° C., 5% CO2, prior to addition of 10 μL of 4× Test Compounds (compounds described herein) or Assay Controls (all diluted in complete media containing 10% css). A further 10 μL of 4×R-1881 Agonist Challenge (antagonist assay) or Buffer (agonist assay) was then added (all diluted in complete media containing 10% css). Agonist challenge was at 400 pM for WT assay and 600 pM for F876L assay. Plates containing cells and compounds herein were incubated for a further 20-24 hours at 37° C., 5% CO2 before addition of 40 μL/well of Steady-Glo Luciferase Assay System Reagent (Promega# E2520). After 1 h, plates were read for luminescence on a BMG Pherastar.
RLU results were collected from the Pherastar and used directly for data calculation.
Percent Max & Inhibition Calculated for Assays:
% Inhibition:
(1−(Sample RLU−Ave Low Control RLU[10 μM Antagonist Control])/(Ave High Control RLU [400 pM R−1881]−Ave Low Control RLU[10 μM Antagonist Control]))*100.
% of 1 μM R-1881 Agonist Max:
((Sample RLU−Ave Low Control RLU[DMSO/Buffer])/(Ave High Control RLU [1 μM R-1881]−Ave Low Control RLU[DMSO/Buffer]))*100.
EC/IC50 calculations were achieved utilizing calculated RLU data and data fitting macros. Data were fit using least-squares methods to the following formula:
wherein
Resultant data are shown in Table 3.
As used herein:
LNCaP cells (8,000/well) are plated in RPMI media containing 10% Charcoal Dextran Stripped Serum into plates coated with poly-d-lysine. After 24 h cells are treated with compound from 30 μM to 0.0003 μM. At 20 h post compound addition the cells were fixed (30% formaldehyde in PBS) for 20′. Cells are permeabilized in PBS 0.1% Triton (50 μL/well, three times for 5′ each) and blocked with LiCor blocking buffer (50 μL/well, 90′). The wells are then incubated overnight at 4° C. with the rabbit IgG androgen receptor antibody (AR-N20, Santa Cruz antibody) diluted 1:1000 in LiCor blocking buffer/0.1% Tween-20. Wells are washed with 0.1% Tween-20/PBS (50 μL/well, 5′ each) and then incubated in goat anti-rabbit IRDye™ 800CW (1:1000) and DRAQ5 DNA dye (1:10,0000 for 5 mM stock) diluted in 0.2% Tween-20/0.01% SDS/LiCor blocking buffer in the dark (90′). Cells are washed (50 μL/well, 5′ each) in 0.1% Tween-20/PBS. Wash buffer is removed and plates were read using the LiCor Odyssey.
LNCaP cells were expanded in RPMI 10% FBS in T150 flasks. The cells were dislodged with 0.25% Trypsin, washed in complete media, centrifuged (300 g, 3 min), and the supernatant aspirated. The cells were resuspended in RPMI phenol-red free media with 1% charcoal-stripped serum (CSS) and counted using a ViCELL (Beckman-Coulter). Three million cells in 7 mL of RPMI CSS were seeded into 100 mm dishes and incubated overnight at 37° C. 5% CO2. The following morning, compound dilutions were prepared in RPMI CSS using 50 mM stock solutions and added directly to the cells to obtain the final concentration of 10 μM. The dishes were placed into the incubator for 4 h. After 4 h, cell scrapers were used to dislodge the cells and the media/cell solution was centrifuged, supernatant aspirated, and then washed with Cell Wash Buffer (Protein Simple). The pellets were stored at −80° C. or processed immediately using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo-Fisher). The fractions obtained from this kit were stored at −80° C. Later, the samples' protein concentration was determined using the BCA Assay Kit (Thermo-Fisher) and the fraction lysates normalized to 0.6 mg/mL. The lysates were run on the Wes Simple Western platform (ProteinSimple). The data was analyzed, normalized to the total AR, and plotted in GraphPad Prism. ANOVA with Tukey's Multiple Comparisons Test was used for statistical analyses.
A compound of Formula (I) may be diluted to 2× the final desired concentration in AR Green Assay Buffer (final DMSO: 0.6%). Fluormone AL Green and the rat AR Ligand Binding Domain is diluted to 2× the final desired concentration (Fluormone: 2 nM, AR LBD: 50 nM) in AR Green Assay Buffer containing 2 mM DTT. The AR LBD/Fluormone solution is added to all the wells of a 384 well black plate (10 μL/well). A compound of Formula (I) may be added to the AR LBD/Fluormone solution (10 μL/well). The plate is incubated for 4 h in the dark. The fluorescence polarization of each well is measured using an excitation wavelength of 485 nm and emission wavelength of 530 nm.
VCaP cells were counted and seeded into black 384-well plates with clear bottoms at a concentration of 125,000 cells per mL in phenol red-free DMEM containing 10% Charcoal Stripped Serum. 16 μL of the suspension was added per well and incubated for 48 h to allow the cells to adhere. After 48 hours, a 12 point serial semilog dilution of each compound was added to the cells in 16 μL at a final concentration of 100 μM to 0.0003 μM. The compounds of Formula (I) were also run in antagonist mode using 30 μM R1881 in which 8 μL of the compound was added to the cells followed by 8 μL of R1881. After 5 days of incubation at 37° C., 16 μL Of CellTiter-Glo (Promega) was added to the cells and the relative luminescence units (RLUs) of each well determined using the Envision. The percent stimulation and % inhibition were determined for each sample and plotted using GraphPad Prism. Resultant data are shown in Table 4.
LNCaP cells were expanded in RPMI 10% FBS in T150 flasks. The cells were dislodged with 0.25% Trypsin, washed in complete media, centrifuged (300 g, 3 min), and the supernatant aspirated. The cells were resuspended in RPMI phenol-red free media with 1% charcoal-stripped serum (CSS) and counted using a ViCELL (Beckman-Coulter). 7500 cells were added to each well of a white optical bottom 384-well plate and incubated for 2 days at 37° C. 5% CO2. Compound dilutions were prepared in RPMI CSS using 50 mM stock solutions and added to the cells either alone (agonist mode) or in combination with 0.1 nM R1881 (antagonist mode). The plates were incubated for 4 days, followed by addition of CellTiter-Glo Luminescent Cell Viability kit reagent (Promega). The plates were placed on a shaker at 3000 rpm for 10 minutes and then read on an EnVision plate reader (Perkin Elmer) using Luminescence assay default settings. The data was analyzed, normalized to 0.1 nM R1881 stimulation, and plotted in GraphPad Prism. Resultant data are shown in Table 5.
HepG2 cells were maintained in EMEM supplemented with 10% FBS. One day before transfection, the media was changed to EMEM with 10% CSS. T-150 flasks were transiently transfected using 120 μL Lipofectamine 2000 (Life Technologies), 30 μg mutant cDNA (expression vector)—mutant cDNA tested were L701H, T877A, W741C and H874Y—and 40 μg 4×ARE-Luciferase (reporter vector) in OptiMEM and the flasks were incubated overnight. Cells were then trypsinized, counted and resuspended at 500,000 cells/mL. For agonist mode, the compounds of Formula (I) were serially diluted and 50 μL of the compound was added per well. 50 μL of the cells were added to each well and incubated for 48 hours. For antagonist mode, a final concentration of 90 pM R1881 was added to the diluted compounds and incubated for 48 hours. The plates were then assayed using SteadyGlo and read on the Envision. Percent Stimulation and Inhibition were determined and analyzed using GraphPad Prism. Resultant data are shown in Table 6.
F876L agonism was evident at 3 and 10 μM (5%) but zero at 30 μM.
The antagonistic (IC50) and agonistic (EC50) values for each of the AR cDNA used in the reporter assays are summarized.
All values are calculated relative to the activity of R1881 induced androgen receptor activity (n≥3). Also indicated are the maximal inhibition and extent of induction of androgen dependent signaling (%).
HepG2 cells were maintained in EMEM supplemented with 10% FBS. One day before transfection, the media was changed to EMEM with 10% CSS. T-150 flasks were transiently transfected using 120 μL Lipofectamine 2000 (Life Technologies), 24.5 μg AR-VP16 or F876L-VP16 (expression vector) and 49 μg 4×ARE-Luciferase (reporter vector) in OptiMEM and the flasks were incubated overnight. Cells were then trypsinized, counted and resuspended at 500,000 cells/mL. For agonist mode, the compounds were serially diluted and 50 μL of the compound was added per well. 50 μL of the cells were added to each well and incubated for 48 hours. For antagonist mode, a final concentration of 90 pM (VP16 AR) or 1 nM (VP16 F876L) R1881 was added to the plate and incubated for 48 hours. The plates were then assayed using SteadyGlo and read on the Envision. Percent Stimulation and Inhibition were determined and analyzed using GraphPad Prism. Resultant data are shown in Table 7.
No agonism of DNA binding observed
GABA-gated Cl Channel assays were performed at CEREP according to the following method. Membrane homogenates of cerebral cortex (120 μg protein) were incubated for 120 min at 22° C. with 3 nM [35S]-TBPS in the absence or presence of the test compound in a buffer containing 50 mM Na2HPO4/KH2PO4 (pH 7.4) and 500 mM NaCl. Nonspecific binding was determined in the presence of 20 μM picrotoxinin. Following incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters were dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radio ligand specific binding. The standard reference compound is picrotoxinin, which was tested in each experiment at several concentrations to obtain a competition curve from which its IC50 was calculated. In this assay, the following representative compounds disclosed herein had recorded activities at a 10 μM single point concentration in the GABA-gated Cl-Channel Binding Assay. Resultant data are shown in Table 8.
22RV1 were obtained from ATCC and cultured in RPMI 1640 supplemented with 10% FBS. Cells were seeded at a density of 15,000 cells/well into Greiner 384-well, white, optical-bottom plates in RPMI 1640 phenol red-free medium with 1% charcoal stripped FBS and incubated for 2 days at 37° C. The cells were treated with a compound or Formula (I) in 12-point dose-response, both in the presence and absence of 0.1 nM R1881 for 18-24 hrs. At the end of assay, Steady-Glo reagent (Promega) was added to each well according to the manufacturer's instructions, placed on a shaker at 300 rpm for 10 min, and luminescence recorded using an EnVision plate reader (Perkin Elmer). The data was exported to GraphPad Prism and analyzed using the Four Parameter equation to obtain IC50 values for each compound. Each compound was tested in at least three independent experiments.
Resultant Data
Antagonist mode: In the presence of 0.1 nM R1881, the IC50 of Cpd 43 was 347.6 nM.
Agonist mode: In the absence of 0.1 nM R1881, the IC50 of Cpd 43 was 120.8 μM.
22RV1 were obtained from ATCC and cultured in RPMI 1640 supplemented with 10% FBS. Cells were seeded at a density of 4,500 cells/well into Greiner 384-well, white, optical-bottom plates in RPMI 1640 phenol red-free medium with 1% charcoal stripped FBS and incubated for 2 days at 37° C. The cells were treated with a compound of Formula (I) in 12-point dose-response both in the presence and absence of 0.1 nM R1881 for 5 days. To determine cell viability, CellTiter-Glo reagent (Promega) was added to each well according to the manufacturer's instructions, placed on a shaker at 300 rpm for 10 min, and luminescence recorded using an EnVision plate reader (Perkin Elmer). The data was exported to GraphPad Prism and analyzed using the Four Parameter equation to obtain IC50 values for each compound. Each compound was tested in at least three independent experiments.
Resultant Data
Antagonist mode: In the presence of 0.1 nM R1881, the IC50 of Cpd 43 was 259.8 μM.
Agonist mode: In the absence of 0.1 nM R1881, the IC50 of Cpd 43 was 491.5 μM.
The effect of AR antagonists on androgen dependent signaling in vivo was assessed using the Hershberger assay. In this assay, peripubertal castrated male Sprague-Dawley rats were administered AR antagonists described herein in the presence of testosterone (0.4 mg/kg testosterone propionate) and the weights of androgen dependent organs measured. Dosing was continued for 10 days and measurements taken 24 h after the last dose. The extent of antagonism of AR and consequent inhibition of organ growth was evaluated by comparison to the castration control. Compounds of Formula (I) were dosed orally QD and an endpoint assessment made by change in weight of 5 androgen sensitive organs (ASO): Paired Cowper's Glands (CG), Seminal Vesicles with Fluids and Coagulating Glands (SVCG), Glans Penis (GP), Ventral Prostate (VP) and Levator Ani-Bulbocavernosus Complex (LABC)). According to assay guidelines, statistically significant suppression of ASO is required in 2 of 5 organs for a compound to be classified as an anti-androgen (analysis was performed by t-test/Mann-Whitney).
Unless otherwise stated, compounds defined herein were administered at 30 mg/kg and flutamide (FT), positive control, at 3 mg/kg. All compounds were co-administered with testosterone propionate (TP, 0.4 mg/kg) which was also administered alone, untreated control, (castrated only rats served as the control for complete androgen blockade). A statistically significant change in ASO achieved in at least 2 of 5 organs was indicative of an active compound. Administration of Compound 43 resulted in significant reduction in ASO versus TP control (p≤0.05) in all 5 organs. Data for the inhibition of growth of the Seminal Vesicle and Coagulating Glands (SVCG) and Ventral Prostate (VP) was reported for all studies (mean organ weight (% of TP control)+SD (n=6)).
Castrate six to seven week old male SCID Hairless Outbred mice (SHO, Charles Rivers Laboratories) were used as the host strain for xenograft studies. LNCaP SRaF876L cells were cultured as 3-D spheroids and expanded prior to subcutaneous injection on the flank of the animals (supplied post castration). Briefly, 5 mls of cells in media+5 mls of cultrex were premixed prior to plating of 500 μl=2×105 cells per well of a 24-well plate. Plates were incubated @ 37° C. for 30 min before addition of complete media on top and returned to incubator for growth of 3-D colonies. After 7 days, media was removed, plates chilled and contents of each well, 500 μl cultrex and cells, injected into flank of a recipient mouse. Tumor volume (length×width2/2) was monitored weekly. When tumors reached an average volume of ˜200 mm3, animals were randomized into treatment groups. During the treatment period tumor volume was monitored bi-weekly. At study end, tumor growth inhibition (TGI) was calculated: 100−(Treated/Control*100). At the termination of study tumors were collected and stored for further analyses.
While the foregoing specification teaches the principles of the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations and/or modifications as come within the scope of the following claims and their equivalents.
This application claims the benefit of U.S. Provisional Application Nos. 62/277,009, filed Jan. 11, 2016, and 62/363,534, filed Jul. 18, 2016, the entireties of which are incorporated by reference herein.
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| Number | Date | Country | |
|---|---|---|---|
| 20170233401 A1 | Aug 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62636534 | Jul 2016 | US | |
| 62277009 | Jan 2016 | US |
