Bicyclic inhibitors of nicotinamide N-methyltransferase, compositions and uses thereof - Patents.com

Abstract

Disclosed are compounds and their pharma- ceutically acceptable salts that are useful as nicotinamide N-methyltransferase (NNMT) inhibitors.Disclosed are pharmaceutical compositions that include the compounds described herein.Also disclosed are related methods of treating cancer in a subject and methods of inhibiting tumor growth in a subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 295,657, filed December 31, 2021. [Background technology]

[0002] Tumors are heterogeneous and contain cancer cells and an elaborate microenvironment. Cancer-associated fibroblasts (CAFs) are myofibroblasts (fibroblasts with characteristics of smooth muscle cells) present in the tumor microenvironment that support tumors through paracrine signaling and production of extracellular matrix. CAFs play a key role in almost all aspects of tumor biology, including survival, resistance, metastasis and immune cell evasion. CAFs have now been identified in almost all histological types of solid tumors, often outnumbering any other cell type in the tumor and are associated with poor prognosis in patients. CAF-driven construction of extracellular matrix has been shown to prevent the infiltration of effector immune cells and activated T cells. Thus, reducing the presence of CAFs in tumors may improve response and resistance to immunotherapy. Given their intimate role in cancer maintenance, progression and resistance to targeted and immunotherapies, therapeutic agents that specifically target CAFs are very promising as a new approach in cancer treatment. However, few targets specific for CAFs (versus normal fibroblasts) have been identified. The most advanced efforts in targeting CAFs have relied on fibroblast activation protein (FAP)-recognizing CART cells. Although this therapy shows promise, FAPs are expressed by other cells in the body, including those that regulate bone marrow and muscle tissue, resulting in sometimes fatal toxicity.

[0003] To identify targets specific to CAFs, proteins differentially expressed in human CAFs but not in tumor cells or normal stroma have been identified. Specifically, biopsy samples from patients with high-grade serous carcinoma metastases (HGSC - the most common form of ovarian cancer) were laser microdissected to separate tumor cells from the stroma, followed by mass spectrometry. Expression of nicotinamide N-methyltransferase (NNMT) was found to be increased in the stroma of HGSC metastases compared to tumor cells or normal stroma. NNMT was also highly expressed in the stroma of breast and colon cancers. Importantly, NNMT was required to maintain the CAF phenotype. Furthermore, tumor burden in animal models was reduced when NNMT was knocked down or inhibited with a small molecule inhibitor. These studies indicate that NNMT is a CAF-selective therapeutic target and its inhibition with small molecules reverses the CAF phenotype and reduces tumor burden.

[0004] Thus, there is a continuing need to discover and develop novel compounds that target nicotinamide N-methyltransferase (NNMT). Summary of the Invention [Means for solving the problem]

[0005] In certain embodiments, the present application discloses a compound of formula I, or a pharma- ceutically acceptable salt thereof: [ka] In the formula, R 1 , Z 1 , Z 2 , Z 3 and x is as defined herein.

[0006] Additionally, pharmaceutical compositions comprising the compounds disclosed herein are provided.The present disclosure also relates to methods of treating or preventing cancer in a subject, as well as methods of inhibiting tumor growth in a subject. [Brief description of the drawings]

[0007] [Figure 1] 1 is a plot showing data from an in vitro NNMT enzyme inhibition assay for Compound 1 (IC50=105 nM). [Diagram 2] 1 is a plot showing data from an in vitro NNMT enzyme inhibition assay for compound 11 (IC50=35 nM). [Diagram 3] 1 is a plot showing data from a cellular potency assay for compound 11 (IC50=38 nM). [Figure 4A] 1 is a plot showing data from an in vitro NNMT enzyme inhibition assay for compound 18 (IC50=848 nM). [Figure 4B] 1 is a plot showing data from an in vitro NNMT enzyme inhibition assay for each enantiomer of compound 18, namely compound 19A (IC50=28 nM) and compound 19B (IC50=5.1 μM). [Figure 4C] 1 is a plot showing data from a cellular potency assay for compound 19A (IC50=12 nM). [Diagram 5] 1 is a plot showing data from an in vitro NNMT enzyme inhibition assay for compound (R)-34 (IC50=48 nM). [Figure 6] 1 is a plot showing the pH dependent solubility of compound 11. [Figure 7] 1 is a plot showing human and mouse liver microsomal stability of compound 11. [Figure 8] 1 is a plot showing hERG safety assessment by manual patch clamp system (hERG IC50=>30 μM; 14.96% inhibition at 30 μM). [Figure 9] 1 is a pair of plots showing CYP3A4 inhibition by compound 11 and a table showing inhibition of other CYP isoforms by compound 11. [Figure 10]Off-target activity panel of compound 11. [Figure 11] 1 is a plot showing the results of a Mini Ames assay with compound 11. The F values ​​are less than 2 at all concentrations tested in all strains with and without the S9 fraction. [Figure 12] 1 is a plot showing the plasma concentration of Compound 11 following PO dosing at 50 mg / kg and 100 mg / kg in female C57B16 / N mice. [Figure 13A] FIG. 1 is a plot showing plasma concentrations of 1-MNA from 0 to 8 hours following PO administration of Compound 11 at 50 mg / kg and 100 mg / kg in female C57B16 / N mice. [Figure 13B] FIG. 1 is a plot showing plasma concentrations of 1-MNA from 0 to 24 hours following PO administration of Compound 11 at 50 mg / kg and 100 mg / kg in female C57B16 / N mice. [Figure 14] Dose and data collection schedule for the PK / PD study of Compound 11 in female C57B16 / N mice. h = hour [Figure 15A] 1 is a plot showing the plasma concentration of Compound 11 14 days after QD administration of Compound 11 in female C57B16 / N mice. [Figure 15B] FIG. 1 is a plot showing plasma concentrations of 1-MNA 14 days after QD administration of compound 11 in female C57B16 / N mice. [Figure 16A] 1 is a plot showing the plasma concentration of Compound 11 following administration (by different methods) of Compound 11 at 50 mg / kg in female C57B16 / N mice. [Figure 16B] FIG. 1 is a plot showing plasma concentrations of 1-MNA following administration of compound 11 (by different methods) at 50 mg / kg in female C57B16 / N mice. [Figure 17A] 1 is a plot showing the plasma concentration of Compound 11 following administration of Compound 11 (by different methods) at 100 mg / kg in female C57B16 / N mice. [Figure 17B]FIG. 1 is a plot showing the plasma concentration of 1-MNA following administration of compound 11 (by different methods) at 100 mg / kg in female C57B16 / N mice. [Figure 18] 1 is a table showing the activity profile of compound 11. [Figure 19] 1 is a table summarizing the PK / PD data of compounds 1 to 3 and 11. [Figure 20] 1 is a table summarizing the mean PK parameters of various preparations of Compound 11 after different PO doses in female C57B16 / N mice. [Figure 21] FIG. 1 is a plot showing plasma concentration of 1-MNA versus time following PO administration of Compound 11 or 19A in female C57B16 / N mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In certain aspects, the present application discloses substituted polycyclic compounds and pharmaceutical compositions thereof. In particular, such compounds disclosed herein are useful as inhibitors of nicotinamide N-methyltransferase (NNMT).

[0009] NNMT catalyzes the methylation of nicotinamide using S-adenosylmethionine (SAM) as a cofactor, thereby generating 1-methylnicotinamide (1-MNA). Without wishing to be bound by theory, high expression of NNMT can maintain the CAF phenotype by reducing SAM levels, which results in DNA and histone hypomethylation as well as epigenetic / transcriptional changes that maintain the CAF cell state. This theory is supported by three observations: 1) NNMT is a "methyl sink" that reduces SAM levels and histone methylation; 2) NNMT knockdown in CAFs increases trimethylation of lysines 4 and 27 of histone 3; 3) inhibition of the histone methyltransferase EZH2 rescues NNMT knockdown and restores the CAF phenotype (α-SMA and collagen contractility).

[0010] Thus, the compounds disclosed herein can be used as inhibitors of NNMT and are particularly useful for the treatment of cancers, such as cancerous tumors associated with CAFs that have increased expression of NNMT.

[0011] I. Compound In certain embodiments, the present application discloses a compound of formula I, or a pharma- ceutically acceptable salt thereof: [ka] During the ceremony, Z 1 , O, NR 4 or CHR 2 and; Z 2 is O or CHR 3 and; Z 3 is O or NH; R 1 is H, halo, cyano, unsubstituted or substituted alkyl, unsubstituted or substituted cycloalkyl, or unsubstituted or substituted alkynyl; R 2 and R 3 each independently represents H, cyano, unsubstituted or substituted alkyl, unsubstituted or substituted alkynyl, alkyl-OR 5 , or alkyl-SR 6 or if x is 0, then R 2 and R 3 may, together with the carbon atom to which they are attached, form a fused 3-, 4-, 5- or 6-membered carbocyclic ring; R 4 is H, alkyl, or acyl; R 5 and R 6 are each independently H, CHF2, CF3, unsubstituted or substituted alkyl, unsubstituted or substituted alkynyl, or unsubstituted or substituted cycloalkyl; x is an integer selected from 0 and 1; However, Z 2 and Z3 and Z cannot both be O; and if x is 0, then Z 1 and Z 2 are not both O and Z 1 and Z 3 Both of them cannot be O.

[0012] In certain embodiments, the present application discloses a compound of formula I, or a pharma- ceutically acceptable salt thereof: [ka] During the ceremony, Z 1 , O, NR 4 or CHR 2 and; Z 2 is O or CHR 3 and; Z 3 is O or NH; R 1 is H, halo, cyano, unsubstituted or substituted alkyl (e.g., fluoroalkyl), unsubstituted or substituted cycloalkyl, or unsubstituted or substituted alkynyl; R 2 and R 3 are each independently H, cyano, alkyl, or alkynyl; or, when x is 0, R 2 and R 3 may, together with the carbon atom to which they are attached, form a fused 3-, 4-, 5- or 6-membered carbocyclic ring; R 4 is H, alkyl, or acyl; x is an integer selected from 0 and 1; However, Z 2 and Z 3 and Z cannot both be O; and if x is 0, then Z 1 and Z 2 Both of these cannot be O, and Z 1 and Z 3 Both of them cannot be O.

[0013] In some embodiments, Z 1 and Z 2 Both of them cannot be O.

[0014] In some embodiments, x is 0. In some embodiments, x is 1.

[0015] In some embodiments, Z 1 is CHR 2 In some embodiments, R 2 is H. In some embodiments, Z 1 is O. In some embodiments, Z 1 is NR 4 It is.

[0016] In some embodiments, Z 2 is CHR 3 In some embodiments, R 3 is H. In some embodiments, R 3 is cyano. In some embodiments, R 3 is alkyl, preferably lower alkyl, more preferably methyl. In some embodiments, R 3 is alkynyl, preferably lower alkynyl, more preferably ethynyl. In some embodiments, Z 2 is O.

[0017] In some embodiments, R 2 and R 3 taken together with the carbon atoms to which they are attached form a fused three-membered carbocyclic ring.

[0018] In some embodiments, Z 3 is NH. In some embodiments, Z 3 is O.

[0019] In some embodiments, R 1 is alkyl, preferably lower alkyl, more preferably methyl. In some embodiments, R 1is halo, preferably chloro. In some embodiments, R 1 is substituted alkyl, preferably substituted lower alkyl, more preferably fluorine-substituted lower alkyl.

[0020] In some embodiments, R 4 is alkyl, preferably lower alkyl, more preferably methyl.

[0021] In some embodiments, the compound of formula I has the following structure: [ka] has.

[0022] In some embodiments, R 1 is H or CH3.

[0023] In some embodiments, R 1 is a halo.

[0024] In some embodiments, R 1 is Cl.

[0025] In some embodiments, R 3 is unsubstituted C1-C6 alkyl.

[0026] In some embodiments, R 3 is CH3.

[0027] In some embodiments, R 3 is unsubstituted C2-C6 alkynyl. In another embodiment, R 3 is a substituted C2-C6 alkynyl. In some embodiments, when substituted, the C2-C6 alkynyl is substituted with CF3.

[0028] In some embodiments, R 3 is alkyl-OR 5 or alkyl-SR 6In some embodiments, R 5 and R 6 are each independently CHF2, CF3, CH3, CH(CH3)2, or CH(CH2)2.

[0029] In some embodiments, the compound of formula I has the following structure: [ka] has.

[0030] In some embodiments, the compound of formula I has the following structure: [ka] has.

[0031] In some embodiments, the compound of formula I has the following structure: [ka] has.

[0032] In some embodiments, the compound of formula I has the following structure: [ka] has.

[0033] In some embodiments, R 1 is H or CH3.

[0034] In some embodiments, R 1 is a halo.

[0035] In some embodiments, R 1 is Cl.

[0036] In some embodiments, R 2 is unsubstituted C1-C6 alkyl.

[0037] In some embodiments, R 3 is unsubstituted C1-C6 alkyl.

[0038] In some embodiments, R 1 , R 2 , and R 3 are CH3, respectively.

[0039] Exemplary compounds of Formula I are shown in Table 1A. [Table 1A-1] [Table 1A-2] [Table 1A-3]

[0040] Further exemplary compounds of Formula I are shown in Table IB. [Table 1B-1] [Table 1B-2] [Table 1B-3]

[0041] II. Method In certain embodiments, the present application discloses a method of treating or preventing cancer, comprising administering to a subject in need thereof an effective amount of a compound disclosed herein (e.g., a compound of Formula I) or a pharma- ceutically acceptable salt thereof, or a composition disclosed herein, thereby treating or preventing cancer.

[0042] In some embodiments, the cancer is brain cancer, head and neck cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, duodenal cancer, appendix cancer, colon cancer, rectal cancer, liver cancer, pancreatic cancer, gallbladder cancer, anal cancer, kidney cancer, ureter cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovarian cancer, or skin cancer.Preferably, the cancer is ovarian cancer, colon cancer, or breast cancer.In some embodiments, the ovarian cancer is high-grade serous carcinoma (HGSC).

[0043] In some embodiments, the cancer is a solid tumor.In some embodiments, the subject is generally a subject who has been diagnosed with cancerous tumor or a subject who has previously been treated for cancerous tumor (e.g., tumor has previously been surgically removed).Cancerous tumor can be a primary tumor and / or a secondary (e.g., metastatic) tumor.

[0044] In certain embodiments, the present application discloses a method of inhibiting tumor growth, comprising administering to a subject in need thereof an effective amount of a compound disclosed herein or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition.

[0045] In some embodiments, the methods disclosed herein further comprise conjointly administering to the subject an effective amount of one or more additional chemotherapeutic agents. In certain embodiments, the methods disclosed herein further comprise conjointly administering to the subject an effective amount of radiation therapy.

[0046] Chemotherapeutic agents that may be co-administered with the compounds of the invention include ABT-263, aminoglutethimide, amsacrine, anastrozole, asparaginase, AZD5363, Bacillus Calmette-Guerin vaccine (bcg), bicalutamide, bleomycin, bortezomib, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, cobimetinib, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridine, dexamethasone, dichloroacetate, dienestrose, , diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil and 5-fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, lenalidomide, retrozole leucovorin, leuprolide, levamisole, lomustine, lonidamine, LY2603618, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, miltefosine, mitomycin, mitotane, mitoxantrone, MK2206, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, pazopanib, perifosine, PF-046 91502, PF477736, plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, romidepsin, selumetinib, sorafenib, streptozocin, sunitinib, suramin, tamoxifen, temozolomide, temsirolimus, teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trametinib, trastuzumab, tretinoin, vinblastine, vincristine, vindesine,For example, chemotherapeutic agents that may be co-administered with the compounds of the invention include: aminoglutethimide, amsacrine, anastrozole, asparaginase, BCG, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, Cytarabine, dacarbazine, dactinomycin, daunorubicin, demethoxyviridine, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea , idarubicin, ifosfamide, imatinib, interferon, irinotecan, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidoxetine lonate, pentostatin, perifosine, plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, sorafenib, streptozocin, sunitinib, suramin, tamoxifen, temozolomide, temsirolimus, teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine. In certain embodiments, the chemotherapeutic agent is cisplatin. In certain embodiments, the additional chemotherapeutic agent is a CHK1 inhibitor.

[0047] Many combination therapies have been developed for the treatment of cancer. In certain embodiments, the compounds of the present invention can be co-administered with combination therapy. Examples of combination therapy that can be co-administered with the compounds of the present invention are listed in Table 2.

[0048] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0049] In some embodiments, the co-administered chemotherapeutic agent is an immune-oncology therapeutic, such as an inhibitor of CTLA-4, indoleamine 2,3-dioxygenase, and / or PD-1 / PD-L1.

[0050] In certain embodiments, the compound disclosed herein or a pharma- ceutically acceptable salt thereof and one or more additional chemotherapeutic agents are administered simultaneously. In alternative embodiments, the one or more additional chemotherapeutic agents are administered within about 5 minutes to about 168 hours before or after administration of the compound.

[0051] In certain embodiments, the subject is a mammal, for example a human.

[0052] In certain embodiments, disclosed herein is a method for inhibiting NNMT, comprising contacting a cell expressing NNMT with a compound of formula I. In some embodiments, the cell is a cancer cell. Such a method can be carried out in vivo or in vitro.

[0053] III. Pharmaceutical Compositions In certain embodiments, the present invention provides a pharmaceutical composition comprising a compound of formula I, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier.

[0054] The compositions and methods of the present invention can be utilized to treat individuals who need it.In certain embodiments, the individual is a mammal, such as a human, or a non-human mammal.When administered to animals, such as humans, the compositions or compounds are preferably administered as pharmaceutical compositions, for example, comprising the compounds of the present invention and pharmaceutically acceptable carriers.

[0055] Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline, or other solvents or vehicles such as, for example, glycols, glycerol, oils (e.g., olive oil), or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes such as injection or implantation that avoid transport or diffusion through epithelial barriers), the aqueous solutions are pyrogen-free (pyrogenic) or substantially pyrogen-free. Excipients can be selected, for example, to effect delayed release of the drug or to selectively target one or more cells, tissues, or organs. Pharmaceutical compositions can be in unit dosage form, such as tablets, capsules (including sprinkle capsules and gelatin capsules), granules, lyophilisates for reconstitution, powders, solutions, syrups, suppositories, injections, and the like. The compositions can also be present in transdermal delivery systems, such as skin patches. The compositions can also be present in solutions suitable for topical administration, such as eye drops.

[0056] A pharma- ceutically acceptable carrier may contain a physiologically acceptable agent that acts, for example, to stabilize, increase the solubility, or increase the absorption of a compound, such as the compound of the present invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose, or dextran, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, or other stabilizers or excipients. The choice of a pharma- ceutical acceptable carrier that includes a physiologically acceptable agent depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition may be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) may also be, for example, a liposome or other polymer matrix into which the compound of the present invention may be incorporated. For example, liposomes containing phospholipids or other lipids are relatively simple to make and administer, non-toxic, physiologically acceptable, and metabolizable carriers.

[0057] The pharmaceutical compositions (preparations) can be administered to a subject by any of a number of routes of administration, including, for example, orally (e.g., as a drench, tablet, capsule (including sprinkle capsule and gelatin capsule), bolus, powder, granule, paste for application to the tongue, such as in an aqueous or non-aqueous solution or suspension); absorption through the oral mucosa (e.g., sublingually); anally, rectally, or vaginally (e.g., as a pessary, cream, or foam); parenterally (e.g., as a sterile solution or suspension, including intramuscularly, intravenously, subcutaneously, or intrathecally); intranasally; intraperitoneally; subcutaneously; transdermally (e.g., as a patch applied to the skin); and topically (e.g., as a cream, ointment, or spray applied to the skin, or as eye drops). The compounds may also be formulated for inhalation. In certain embodiments, the compounds may simply be dissolved or suspended in sterile water. Details of suitable routes of administration and compositions suitable therefor can be found, for example, in U.S. Pat. Nos. 6,110,973, 5,731,000, 5,541,231, 5,427,798, 5,358,970, and 4,172,896, and patents cited therein.

[0058] The formulations can be conveniently provided in unit dosage form and can be prepared by any method well known in the art of pharmacy. The amount of active ingredient (active ingredient) that can be combined with carrier materials to produce a single dosage form varies depending on the host treated, the particular mode of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of compound that produces a therapeutic effect. Generally, out of 100%, this amount ranges from about 1% to about 99%, preferably from about 5% to about 70%, most preferably from about 10% to about 30% of active ingredient.

[0059] The method of preparing these formulations or compositions includes the step of bringing into association an active compound, such as the compound of the present invention, with a carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound of the present invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0060] Formulations of the present invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), lyophilisates, powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. The composition or compound may also be administered as a bolus, electuary, or paste.

[0061] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharma- ceutically acceptable carriers, e.g., sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, e.g., carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) cold storage agents, such as glycerol; (5) refrigerants, such as glycerol; (6) glycerol; (7) glycerol; (8) glycerol; (9) glycerol; (10) glycerol; (11) glycerol; (12) glycerol; (13) glycerol; (14) glycerol; (15) glycerol; (16) glycerol; (17) glycerol; (18) glycerol; (19) glycerol; (20) glycerol; (21) glycerol; (22) glycerol; (23) glycerol; (24) glycerol; (25) glycerol; (26) glycerol; (27) glycerol; (28) glycerol; (29) glycerol; (30) glycerol; (31) glycerol; (32) glycerol; (33) glycerol; (34) glycerol; (35) glycerol; (36) glycerol; (37) glycerol; (38) glyc Disintegrating agents such as starch, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution retarders such as paraffin; (6) absorption enhancers such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glycerol monostearate; (8) absorbents such as kaolin and bentonite clay; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents such as modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets, and pills, the pharmaceutical composition may also include buffering agents. Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0062] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants or dispersants. Molded tablets can be made by molding a mixture of powdered compound moistened with an inert liquid diluent in a suitable machine.

[0063] Tablets, and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation field, optionally scored or. They can also be formulated to provide slow or controlled release of the active ingredient contained therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres to provide a desired release profile. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can also optionally contain opacifying agents, and can be compositions that release the active ingredient only or preferentially in a certain part of the digestive tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0064] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsion, lyophilized matter for reconstitution, microemulsion, solution, suspension, syrup and elixir.In addition to active ingredient, liquid dosage forms can contain inert diluents commonly used in the art, such as water or other solvents, cyclodextrin and its derivatives, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oil (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid ester of sorbitan, and mixtures thereof.

[0065] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0066] Suspensions may contain, in addition to the active compounds, suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, hydroxyaluminum oxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0067] Formulations of pharmaceutical compositions for rectal, vaginal or urethral administration may be provided as suppositories, which can be prepared by mixing one or more active compounds with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, which are solid at room temperature but liquid at body temperature and will therefore melt in the rectum or vaginal cavity and release the active compound.

[0068] Formulations of the pharmaceutical composition for administration by mouth may be provided as a mouthwash, or an oral spray, or an oral ointment.

[0069] Alternatively, or in addition, the compositions may be formulated for delivery via a catheter, stent, wire, or other intraluminal device, which may be particularly useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

[0070] Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.

[0071] Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.The active compound may be mixed under sterile conditions with a pharma- ceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0072] The ointments, pastes, creams and gels may contain, in addition to the active compound, excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0073] Powders and sprays can contain, in addition to the active compounds, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0074] Transdermal patch has the additional advantage of providing controlled delivery of the compound of the present invention to the body.Such dosage forms can be made by dissolving or dispersing active compound in suitable medium.Absorption enhancers can also be used to increase the flux of compound across skin.The rate of such flux can be controlled by providing a rate-controlling membrane or dispersing compound in a polymer matrix or gel.

[0075] Ophthalmic formulations, eye ointments, powders, solutions, etc. are also considered to be within the scope of the present invention.Exemplary ophthalmic formulations are described in U.S. Patent Application Publication Nos. 2005 / 0080056, 2005 / 0059744, 2005 / 0031697, and 2005 / 004074, and U.S. Patent No. 6,583,124, the contents of which are incorporated herein by reference.If desired, liquid ophthalmic formulations have properties similar to those of tears, aqueous humor, or vitreous humor, or are compatible with such fluids.The preferred route of administration is topical administration (e.g., topical administration such as eye drops, or administration via implant).

[0076] The phrases "parenteral administration" and "administered parenterally" as used herein refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, spinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration include one or more active compounds in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0077] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0078] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers and dispersing agents. Prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as paraben, chlorobutanol, phenol sorbic acid, etc. It may be desirable to include isotonic agents such as sugars, sodium chloride, etc. in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0079] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of drugs.This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility.The absorption rate of the drug then depends on its dissolution rate, which may depend on the crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0080] Injectable depot forms are made by forming microencapsulated matrices of the subject compound in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot preparations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0081] For use in the methods of the invention, the active compound may be provided per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0082] The introduction method may also be provided by rechargeable or biodegradable devices.In recent years, various slow-release polymeric devices for controlled delivery of drugs, including proteinaceous biologics, have been developed and tested in vivo.Various biocompatible polymers, including both biodegradable and non-degradable polymers (including hydrogels), can be used to form implants for sustained release of compounds at specific target sites.

[0083] Actual dosage levels of the active ingredients in pharmaceutical compositions may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient.

[0084] The selected dosage level will depend on a variety of factors, including the activity of the particular compound or combination of compounds, or esters, salts or amides thereof, used, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and / or substances used in combination with the particular compound used, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical arts.

[0085] A physician or veterinarian of ordinary skill in the art can easily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start the dose of the pharmaceutical composition or compound at a level lower than required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. By "therapeutically effective amount" is meant the concentration of the compound sufficient to induce the desired therapeutic effect. It is generally understood that the effective amount of the compound varies according to the subject's weight, sex, age, and medical history. Other factors that affect the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent administered together with the compound of the present invention. A larger total dose can be delivered by multiple administrations of the drug. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, incorporated herein by reference).

[0086] In general, a suitable daily dose of an active compound used in the compositions and methods of the present invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0087] If desired, the effective daily dose of active compound may be administered as 1, 2, 3, 4, 5, 6 or more subdoses administered separately at appropriate intervals throughout the day, and may be administered in unit dosage form as needed.In certain embodiments of the present invention, active compound may be administered two or three times a day.In a preferred embodiment, active compound is administered once a day.

[0088] The patients receiving this treatment may be any animal in need, including primates, particularly humans, and other mammals such as horses, cows, pigs and sheep; as well as poultry and pets in general.

[0089] In certain embodiments, the compounds of the present invention can be used alone or can be administered in combination with another type of therapeutic agent. As used herein, the phrase "conjoint administration" refers to any form of administration of two or more different therapeutic compounds, such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are effective in the patient at the same time, which may include the synergistic effect of the two compounds). For example, the different therapeutic compounds can be administered simultaneously or sequentially, either in the same formulation or in separate formulations. In certain embodiments, the different therapeutic compounds can be administered within 1 hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 1 week of each other's administration. Thus, individuals who undergo such treatment can benefit from the combined effect of the different therapeutic compounds.

[0090] In certain embodiments, co-administration of a compound of the invention with one or more additional therapeutic agents (e.g., one or more additional chemotherapeutic agents) provides improved efficacy compared to individual administration of a compound of the invention (e.g., a compound of Table 1A or 1B) or each of the one or more additional therapeutic agents. In certain such embodiments, the co-administration provides an additive effect, where additive effect refers to the sum of the effects of each of the individual administration of a compound of the invention and one or more additional therapeutic agents.

[0091] The present invention includes the use of pharma- ceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. The term "pharma- ceutically acceptable salts" as used herein includes salts derived from inorganic or organic acids, including, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, phosphoric acid, formic acid, acetic acid, lactic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, glycolic acid, salicylic acid, citric acid, methanesulfonic acid, benzenesulfonic acid, benzoic acid, malonic acid, trifluoroacetic acid, trichloroacetic acid, naphthalene-2-sulfonic acid, and other acids. Pharmaceutically acceptable salt forms may include forms in which the ratio of the molecules comprising the salt is not 1:1. For example, the salt may contain more than one inorganic or organic acid molecule per molecule of base, such as two hydrochloric acid molecules per molecule of the compounds of Tables 1A, 1B, and 2. As another example, a salt may contain less than one inorganic or organic acid molecule per molecule of base, such as two molecules of a compound of Tables 1A, 1B and 2 per molecule of tartaric acid.

[0092] In further embodiments, the contemplated salt of the present invention includes, but is not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salt.In certain embodiments, the contemplated salt of the present invention includes, but is not limited to, L-arginine, benethamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, l-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine and zinc salt.In certain embodiments, the contemplated salt of the present invention includes, but is not limited to, Na, Ca, K, Mg, Zn or other metal salt.

[0093] Pharmaceutically acceptable acid addition salts may also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, etc. Mixtures of such solvates may also be prepared. The source of such solvates may be from the solvent of crystallization, may be inherent in the solvent of preparation or crystallization, or may be associated with such solvent.

[0094] Wetting agents, emulsifying agents and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0095] Examples of pharma- ceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0096] IV. Definition For purposes of the present invention, the following definitions will be used (unless otherwise stated).

[0097] As used herein, the term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0098] As used herein, the term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, -OCF3, ethoxy, propoxy, tert-butoxy, and the like.

[0099] As used herein, the term "alkenyl" refers to an aliphatic group containing at least one double bond, and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl", the latter of which refers to an alkenyl moiety having a substituent replacing hydrogen on one or more carbons of the alkenyl group. Such substituents may be present on one or more carbons that are included or not included in one or more double bonds. Furthermore, such substituents include all of the substituents contemplated for the alkyl groups discussed below, except where stability is prevented. For example, substitution of alkenyl groups with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0100] As used herein, an "alkyl" group or "alkane" is a straight-chain or branched non-aromatic hydrocarbon that is fully saturated. Typically, a straight-chain or branched alkyl group has from 1 to about 20 carbon atoms, preferably from 1 to about 10 carbon atoms, unless otherwise defined. Examples of straight-chain and branched alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl. C1-C6 straight-chain or branched alkyl groups are also referred to as "lower alkyl" groups.

[0101] Furthermore, the term "alkyl" (or "lower alkyl") as used throughout the specification, examples, and claims is intended to include both "unsubstituted alkyl" and "substituted alkyl," the latter of which refers to an alkyl moiety having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents include, unless otherwise specified, for example, halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, where appropriate. For example, substituents of substituted alkyls can include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonates and phosphinates), sulfonyl (including sulfates, sulfonamides, sulfamoyl and sulfonates), and silyl groups, as well as ethers, alkylthio, carbonyl (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN, and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.

[0102] As used herein, "C x-y The term "C" when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, is meant to include groups containing x to y carbons in the chain. For example, "C x-yThe term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain and branched-chain alkyl groups containing x to y carbons in the chain, and includes haloalkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl. CO alkyl indicates hydrogen if the group is in a terminal position, or a bond if internal. 2-y alkenyl" and "C 2-y The term "alkynyl" refers to substituted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.

[0103] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond, and is intended to include both "unsubstituted alkynyl" and "substituted alkynyl", the latter of which refers to an alkynyl moiety having a substituent replacing hydrogen on one or more carbons of the alkynyl group. Such substituents may be present on one or more carbons that are included or not included in one or more triple bonds. Furthermore, such substituents include all of the substituents contemplated for the alkyl group discussed above, except where stability is prevented. For example, substitution of alkynyl groups with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0104] As used herein, the term "amide" refers to the following group: [ka] Refers to, In the formula, each R 10 independently represent hydrogen or a hydrocarbyl group, or two R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0105] As used herein, the terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines, and salts thereof, e.g., [ka] "The term '" refers to a portion that can be represented by In the formula, each R 10 independently represent hydrogen or a hydrocarbyl group, or two R 10 together with the N atom to which they are attached complete a heterocycle having 4 to 8 atoms in the ring structure.

[0106] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups in which each atom of the ring is carbon. Preferably, the ring is a 5- to 7-membered ring, more preferably a 6-membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings, in which two or more carbons are common to two adjacent rings, and at least one of the rings is aromatic, e.g., the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Aryl groups include benzene, naphthalene, phenanthrene, phenol, aniline, and the like.

[0107] As used herein, the terms "carbocycle" and "carbocyclic" refer to a saturated or unsaturated ring in which each atom of the ring is carbon. The term carbocycle includes both aromatic and non-aromatic carbocycles. Non-aromatic carbocycles include both cycloalkane rings in which all carbon atoms are saturated and cycloalkene rings that contain at least one double bond. "Carbocycle" includes 3-7 membered monocyclic rings and 8-12 membered bicyclic rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated and aromatic rings. Carbocycles include bicyclic molecules in which one, two or three or more atoms are shared between the two rings. The term "fused carbocycle" refers to a bicyclic carbocycle in which each of the rings shares two adjacent atoms with the other ring. Each ring of a fused carbocycle may be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings is included in the definition of carbocycle, as long as valence allows. Exemplary "carbocycles" include cyclopentane, cyclohexane, bicyclo[2.2.1]heptane, 1,5-cyclooctadiene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]oct-3-ene, naphthalene and adamantane. Exemplary fused carbocycles include decalin, naphthalene, 1,2,3,4-tetrahydronaphthalene, bicyclo[4.2.0]octane, 4,5,6,7-tetrahydro-1H-indene and bicyclo[4.1.0]hept-3-ene. A "carbocycle" can be substituted at any one or more positions that can have a hydrogen atom.

[0108] As used herein, a "cycloalkyl" group is a cyclic hydrocarbon that is fully saturated. "Cycloalkyl" includes monocyclic and bicyclic rings. Typically, a monocyclic cycloalkyl group has 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms, unless otherwise defined. The second ring of a bicyclic cycloalkyl can be selected from saturated, unsaturated, and aromatic rings. Cycloalkyl includes bicyclic molecules in which one, two, or three or more atoms are shared between the two rings. The term "fused cycloalkyl" refers to a bicyclic cycloalkyl in which each of the rings shares two adjacent atoms with the other ring. The second ring of a fused bicyclic cycloalkyl can be selected from saturated, unsaturated, and aromatic rings. "Cycloalkenyl" groups are cyclic hydrocarbons that contain one or more double bonds.

[0109] The terms "halo" and "halogen" as used herein mean halogen and include chloro, fluoro, bromo and iodo.

[0110] The terms "hetaralkyl" and "heteroaralkyl," as used herein, refer to an alkyl group substituted with a heteroaryl group.

[0111] The term "heteroalkyl," as used herein, refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, wherein the two heteroatoms are not adjacent.

[0112] As used herein, the terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5-7 membered rings, more preferably 5-6 membered rings, in which the ring structure contains at least one heteroatom, preferably 1-4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjacent rings, in which at least one of the rings is heteroaromatic, e.g., the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine.

[0113] The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur.

[0114] As used herein, the terms "heterocyclyl", "heterocycle" and "heterocyclic" refer to a substituted or unsubstituted non-aromatic ring structure, preferably a 3- to 10-membered ring, more preferably a 3- to 7-membered ring, whose ring structure contains at least one heteroatom, preferably 1-4 heteroatoms, more preferably 1 or 2 heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more cyclic rings, where two or more carbons are common to two adjacent rings, and at least one of the rings is heterocyclic, e.g., the other cyclic rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, and / or heterocyclyl. Heterocyclyl groups include, for example, piperidine, piperazine, pyrrolidine, morpholine, lactones, lactams, and the like. Heterocyclyl groups may also be substituted by an oxo group. For example, "heterocyclyl" encompasses both pyrrolidine and pyrrolidinone.

[0115] As used herein, the term "hydrocarbyl" refers to a group that does not have =O or =S substituents and typically has at least one carbon-hydrogen bond and a primarily carbon backbone, but is bonded through a carbon atom that may optionally contain heteroatoms. Thus, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered hydrocarbyl for purposes of this application, while substituents such as acetyl (which has =O substituent on the linking carbon) and ethoxy (which is linked through an oxygen rather than a carbon) are not. Hydrocarbyl groups include, but are not limited to, aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

[0116] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0117] As used herein, the term "lower", when used in conjunction with chemical moieties such as, for example, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy, means to include groups in which there are 10 or fewer non-hydrogen atoms in the substituent, preferably 6 or fewer. "Lower alkyl" refers, for example, to alkyl groups containing 10 or fewer carbon atoms, preferably 6 or fewer carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy substituents defined herein are lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy, respectively, whether they appear alone or in combination with other substituents, such as in the descriptions of hydroxyalkyl and aralkyl (where, for example, atoms in the aryl group are not counted when counting the carbon atoms in the alkyl substituent).

[0118] As used herein, the term "oxo" refers to a carbonyl group. When an oxo substituent occurs on an otherwise saturated group, such as an oxo-substituted cycloalkyl group (e.g., 3-oxo-cyclobutyl), the substituted group is still intended to be a saturated group. When a group is referred to as being substituted by an "oxo" group, this may mean that a carbonyl moiety (i.e., -C(=O)-) replaces a methylene unit (i.e., -CH2-).

[0119] As used herein, the terms "polycyclyl", "polycyclic", and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl) in which two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings". Each of the rings of a polycycle may be substituted or unsubstituted. In certain embodiments, each ring of a polycycle contains 3 to 10, preferably 5 to 7, atoms in the ring.

[0120] As used herein, the term "substituted" refers to a moiety having a substituent replacing a hydrogen on one or more carbons of the backbone. It will be understood that "substituted" or "substituted with" includes the implicit proviso that such substitution is in accordance with the allowed valences of the substituted atom and substituent, and that the substitution results in a stable compound that does not spontaneously undergo transformation, for example, by rearrangement, cyclization, elimination, and the like. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituent of organic compounds described herein that satisfies the valence of the heteroatom. The substituents may include any of the substituents described herein, such as halogen, hydroxyl, carbonyl (e.g., carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (e.g., thioester, thioacetate, or thioformate), alkoxyl, phosphoryl, phosphate, phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. Those skilled in the art will understand that the substituents themselves may be substituted, where appropriate. Unless specifically stated as "unsubstituted," references to chemical moieties herein are understood to include substituted variants. For example, references to "aryl" groups or moieties implicitly include both substituted and unsubstituted variants.

[0121] As used herein, the term "administer" refers to the actual physical introduction of a composition into or onto a subject (as appropriate). Any method of introducing a composition into a subject is contemplated according to the present invention; the method is not dependent on any particular means of introduction, and should not be construed as such. Means of introduction are well known to those of skill in the art, and are exemplified herein.

[0122] As used herein, the terms "effective amount," "effective dose," "sufficient amount," "amount effective for," "therapeutically effective amount," or grammatical equivalents thereof, refer to a dosage sufficient to produce a desired result, and refer to a dosage sufficient to ameliorate or in some manner alleviate symptoms, or to halt or reverse the progression of symptoms, and provide either a subjective alleviation of symptoms or an objectively identifiable improvement as noted by a clinician or other qualified observer. Amelioration of symptoms of a particular condition by administration of a pharmaceutical composition described herein refers to any relief that may be associated with administration of the pharmaceutical composition, whether permanent or temporary, persistent or transient.

[0123] As used herein, the term "prodrug" is intended to include compounds that are converted to the therapeutically active agents of the present invention under physiological conditions. The general method for making prodrugs is to include one or more selected moieties that are hydrolyzed under physiological conditions to produce the desired molecule. In other embodiments, prodrugs are converted by the enzyme activity of the host animal. For example, esters or carbonates (e.g., esters or carbonates of alcohols or carboxylic acids) are preferred prodrugs of the present invention. In certain embodiments, some or all of the compounds in the formulations represented above can be replaced with the corresponding suitable prodrugs, for example, hydroxyls in the parent compound are presented as esters, or carbonates or carboxylic acids present in the parent compound are presented as esters.

[0124] As used herein, the term "pharmaceutical acceptable" refers to a composition that is physiologically tolerable and typically does not produce an allergic or similar adverse reaction when administered to a subject, preferably a human subject.Preferably, as used herein, the term "pharmaceutical acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, particularly humans.

[0125] As used herein, the phrase "pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of substances that can serve as pharma-ceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and the like. (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffers; and (21) other non-toxic compatible substances used in pharmaceutical preparations.

[0126] As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the onset of the disorder or condition in a treated sample compared to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition compared to an untreated control sample.

[0127] As used herein, "subject" means a human or an animal (and, in the case of an animal, more typically a mammal). In one embodiment, the subject is a human.

[0128] As used herein, the term "treating" is art-recognized and includes, for example, administering to a host one or more compositions of the invention to reduce, ameliorate, or stabilize an existing undesirable condition or its side effects. All definitions defined and used herein should be understood to govern dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms. EXAMPLES

[0129] Example 1: Synthesis of Compound 1 [ka]

[0130] As shown in Figure 1, compound 1 showed an IC 50 = 105nM.

[0131] Example 2: Synthesis of Compound 11 [ka]

[0132] compound 2 To a stirred mixture of 4-chloro-2-fluoro-pyridine (5.0 g, 38.17 mmol, 1.00 equiv) in THF (20.0 mL) at -78°C was added dropwise a solution of n-butyllithium (2.5 M in hexanes, 18.3 mL, 45.80 mmol, 1.20 equiv) under N2 atmosphere. The resulting mixture was stirred at this temperature for 0.5 h. To this was added a solution of CuI (290.0 mg, 1.53 mmol, 0.04 equiv) and tert-butyl 2,2-dioxooxathiazolidine-3-carboxylate (8.1 g, 34.4 mmol, 0.90 equiv) in THF (20.0 mL) at -78°C and the mixture was stirred at this temperature for 0.5 h. The resulting mixture was quenched with citric acid solution and extracted with EtOAc. The organic layer was dried over Na2SO4 and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (0-60% EtOAc in petroleum ether) to give tert-butyl N-[rac-(1R)-2-(4-chloro-6-fluoro-3,4-dihydropyridin-5-yl)-l-methyl-ethyl]carbamate (4.8 g, 17.5%) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ8.05(d,J=5.2Hz,1H),7.45(d,J=5.2Hz,1H),6.86(d,J=8.8Hz,1H),3.97-3.7 1(m,1H),2.79(d,J=7.2Hz,2H),1.34-1.13(m,9H),1.09(d,J=6.8Hz,3H).MS(ESI,m / z):289,291(M+H) + .

[0133] Compound 2 showed an IC 50 = 53 to 150 nM.

[0134] compound 3 A mixture of tert-butyl N-[rac-(1R)-2-(4-chloro-6-fluoro-3,4-dihydropyridin-5-yl)-l-methyl-ethyl]carbamate (4.8 g, 16.70 mmol, 1.00 equiv.) and TFA (10.0 mL, 134.77 mmol, 8.07 equiv.) in DCM (10.0 mL) was stirred at ambient temperature for 2 h and concentrated in vacuo to give (R)-l-(4-chloro-2-fluoropyridin-3-yl)propan-2-amine 2,2,2-trifluoroacetate (crude, 4.2 g) as a pale yellow oil, which was used in the next step without further purification. MS (ESI, m / z). 189, 191 (M+H). + .

[0135] compound 4 A mixture of (R)-l-(4-chloro-2-fluoropyridin-3-yl)propan-2-amine 2,2,2-trifluoroacetate (4.2 g, 13.91 mmol, 1.00 equiv.) and TEA (7.0 g, 13.91 mmol, 5.00 equiv.) in DMF (50.0 mL) was stirred at 80° C. for 16 h and then concentrated under vacuum. The residue was purified by reverse-phase flash chromatography on C18 gel (0-60% acetonitrile in water (containing 0.05% NH4HCO3)) to give (R)-4-chloro-2-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine (1.3 g, 55.8%) as a pale yellow solid. 1 H NMR(400MHz,DMSO-d6)δ7.63(d,J=5.6Hz,1H),7.01(brs,1H),6.44(d,J=5.6Hz,1H),4.03-3.89( m,1H),3.23-3.09(m,1H),2.58-2.51(m,1H),1.19(d,J=6.4Hz,3H).MS(ESI,m / z):169,171(M+H) + .

[0136] compound 5 A mixture of (R)-4-chloro-2-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine (1.0 g, 5.95 mmol, 1.00 equiv) and NIS (1.61 g, 7.14 mmol, 1.20 equiv) in acetonitrile (10.0 mL) was stirred at ambient temperature for 4 h. The resulting mixture was diluted with water and extracted three times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel (0-30% EtOAc in petroleum ether) to give (R)-4-chloro-5-iodo-2-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine (860.0 mg, 49.1%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6)δ7.99(s,1H),7.15(brs,1H),4.06-3.89(m,1H),3.30-3. 12(m,1H),2.70-2.54(m,1H),1.19(d,J=6.4Hz,3H).MS(ESI,m / z):295,297(M+H) + .

[0137] compound 6 A mixture of (R)-4-chloro-5-iodo-2-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine (700.0 mg, 2.38 mmol, 1.00 equiv), Zn(CN)2 (473.6 mg, 4.05 mmol, 1.70 equiv), and Pd(PPh3)4 (277.2 mg, 0.24 mmol, 0.10 equiv) in NMP (7.0 mL) was stirred at 120° C. for 8 h under a N2 atmosphere. The resulting mixture was diluted with water and extracted three times with EtOAc. The organic layers were combined, washed with brine, and concentrated under reduced pressure. The residue was purified by reverse-phase flash chromatography on C18 gel (0-80% acetonitrile in water (containing 0.05% NH4HCO3)) to give (R)-4-chloro-2-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile (350.0 mg, 76.1%) as a white solid. 1H NMR(400MHz,DMSO-d6)δ8.24(brs,1H),8.19(s,1H),4.17-4.04(m,1H),3.31-3. 16(m,1H),2.71-2.56(m,1H),1.22(d,J=6.2Hz,3H).MS(ESI,m / z):194,196(M+H) + .

[0138] compound 7 A mixture of (2R)-4-chloro-2-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-5-carbonitrile (100.0 mg, 0.52 mmol, 1.00 equiv) and concentrated sulfuric acid (1.0 ml) was stirred at 60° C. for 3 h. The resulting mixture was diluted with water, basified to pH 10 with NaOH (1M) at 0° C., and extracted with DCM. The organic layer was dried over Na2SO4 and concentrated in vacuo to give (2R)-4-chloro-2-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (crude, 100.0 mg) as a white solid, which was used in the next step without further purification. 1 H NMR(400MHz,DMSO-d6)δ7.88(s,1H),7.58(brs,1H),7.31(brs,1H),7.25(brs,1H),4.08-3.90( m,1H),3.28-3.10(m,1H),2.63-2.50(m,1H),1.19(d,J=6.4Hz,3H).MS(ESI,m / z):212,214(M+H) + .

[0139] compound 11 To a stirred solution of (2R)-4-chloro-2-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (80.0 mg, 0.38 mmol, 1.00 equiv) and Pd / C (wet, 10%, 40.0 mg) in EtOH (6.0 mL) at ambient temperature was added ammonium formate (71.8 mg, 1.14 mmol, 3.00 equiv) in portions. The resulting mixture was stirred at 70° C. for 3 h and then filtered through a pad of Celite. The filtrate was concentrated under vacuum. The residue was purified by preparative HPLC to give (2R)-2-methyl-2,3-dihydro-1H-pyrrolo[2,3-b]pyridine-5-carboxamide (18.4 mg) as a white solid. 1 H NMR(400MHz,DMSO-d6)δ8.27(d,J=2.0Hz,1H),7.67-7.49(m,2H),7.12(s,1H),6.97(brs,1H),4.04 -3.89(m,1H),3.20-3.07(m,1H),2.58-2.51(m,1H),1.18(d,J=6.4Hz,3H).MS(ESI,m / z):178(M+H) + .

[0140] Example 3: Synthesis of additional compounds Compound 2 is prepared by the synthetic route outlined in Scheme 3: [ka]

[0141] Compound 3 is prepared by the synthetic route outlined in Scheme 4: [ka]

[0142] Compound 5 is prepared by the synthetic route outlined in Scheme 5: [ka]

[0143] Compound 6 is prepared by the synthetic route outlined in Scheme 6: [ka]

[0144] Compound 7 is prepared by the synthetic route outlined in Scheme 7: [ka]

[0145] Compound 8 is prepared by the synthetic route outlined in Scheme 8: [ka]

[0146] Compound 15 is prepared by the synthetic route outlined in Scheme 9: [ka]

[0147] Compound 16 is prepared by the synthetic route outlined in Scheme 10: [ka]

[0148] Compound 18 is prepared by the synthetic route outlined in Scheme 11: [ka]

[0149] Chiral chromatography was used to separate the enantiomers of compound 18, namely compounds 19A and 19B. Chiral separation of 24 mg of compound 18 by HPLC under basic conditions identified 5.9 mg of the first enantiomer by the first peak (peak-1: rt=2.134 min, ee=100%, chemical purity=99.62%) and 4.8 mg of the second enantiomer by the second peak (peak-2: 2.933 min, ee=99.65%, chemical purity=99.60%). Preparative HPLC conditions are provided below. TIFF2025503521000035.tif67158

[0150] Compound 21 is prepared by the synthetic route outlined in Scheme 12: [ka]

[0151] Compound 24 is prepared by the synthetic route outlined in Scheme 13: [ka]

[0152] Compound 28 is prepared by the synthetic route outlined in Scheme 14: [ka]

[0153] Compound 26B was prepared by the synthetic route outlined in Scheme 15: [ka]

[0154] Example 4: Biological Data NNMT biochemical activity was evaluated using the Promega Mtase-Glo Methyltransferase Assay kit. The assay was performed in a white flat-bottom 96-well plate. Each reaction well had a final volume of 20 μL and contained 40 nM recombinant human NNMT, 8 μM nicotinamide, and 6.7 μM SAM in 1× reaction buffer (20 mM Tris buffer, pH 8.0, 0.50 mM NaCl, 1 mM EDTA, 3 mM MgCl2, 0.1 mg / mL BSA, 1 mM DTT). The reaction was carried out for 20 min at room temperature (rt). MTase-Glo reagent and MTase-Glo detection solution were added according to the manufacturer's instructions, the luminescence signal was measured on a SpectraMax i3 plate reader, and the data was analyzed with GraphPad Prism version 9. Scheme 16 shows a visual overview of the Promega MTase-Glo Methyltransferase assay used to evaluate the biochemical activity of novel inhibitors.

[0155] Compound 11 (IC 50 =35~42nM), Compound 18 (IC 50 =848nM), Compound 19A (IC 50 = 28–43 nM) and compound 19B (IC 50 Compound 26A (IC = 5.1 μM) was also evaluated for NNMT biochemical activity. 50 = 20 nM) was also assessed for NNMT biochemical activity.

[0156] [ka]

[0157] Cell titers were determined in K562 (ATCC) cells by measuring (1-methylnicotinamide) 1-MNA levels after overnight incubation of compounds at various concentrations. Cells were plated at 1 million per well and incubated with compounds at the indicated concentrations for 24 hours. Cells were harvested by pipetting and extracted with 200 μl of acetonitrile containing 10 ng / ml of d4-1-MNA as an internal deuterated standard. Samples were analyzed on an Agilent 64-60 Triple Quad LC / MS equipped with an Agilent 1290 Infinity HPLC using a Restek Allure 5 μm PPFP column 150 mm x 2.1. Mobile phase A was 2.5 mM ammonium formate in water and mobile phase B was methanol with 0.1% formic acid delivered at 0.25 mL / min. The capillary voltage was set at 2400 V and the nozzle was set at 300 V. The drying gas temperature was 240° C., the drying gas flow rate was 4 L / min, and the nebulizer pressure was 40 psi. The mass spectrometer was operated in multiple reaction monitoring mode. Mass transitions were m / z 137.1-94.1 for 1-MNA and m / z 141.1-98.1 for d4-MNA.

[0158] Compound 11 (IC 50 =38nM), Compound 19A (IC 50 A cell titration assay was also performed on compound 26A (IC 50 =114nM).

[0159] Example 5: Microsomal stability Table 3 summarizes the results of metabolic stability assays performed with (R)-11 in pooled human and male mouse liver microsomes. [Table 3]

[0160] A master solution was prepared according to the table below. TIFF2025503521000042.tif50149

[0161] 40 μL of 10 mM NADPH solution was added to each well. The final concentration of NADPH was 1 mM. The mixture was prewarmed at 37°C for 5 min. A negative control sample was prepared by replacing the NADPH solution with 40 μL of ultra-pure H2O. The negative control was used to exclude misleading factors arising from the instability of the chemical itself. Samples containing NADPH were prepared in duplicate. The negative control was prepared in singlet.

[0162] The reaction was started by adding 4 μL of a solution of 200 μM of the control or test compound. In this study, verapamil was used as a positive control. The final concentration of the test or control compound was 2 μM. Aliquots of 50 μL were taken from the reaction solution at 0, 15, 30, 45 and 60 min. The reaction was stopped by adding 4 volumes of cold acetonitrile containing IS (100 nM alprazolam, 200 nM imipramine, 200 nM labetalol and 2 μM ketoprofen). The samples were centrifuged at 3,220 g for 40 min. An aliquot of 90 μL of the supernatant was mixed with 90 μL of ultrapure water and then used for LC-MS / MS analysis.

[0163] For metabolite standard samples: 1 μL of the 400 μM working solution was added to 89 μL of the master solution, followed by 400 μL of cold acetonitrile containing internal standards (IS) (100 nM alprazolam, 200 nM imipramine, 200 nM labetalol and 2 μM ketoprofen). 10 μL of 10 mM NADPH solution or ultrapure water was then added to the mixture. The samples were centrifuged at 3,220 g for 40 min to precipitate proteins. An aliquot of 90 μL of the supernatant diluted with 90 μL of water was used for LC-MS / MS analysis.

[0164] Example 6: Permeability Table 4 summarizes the results of the permeability assay performed with compound 11 in Caco-2 cells. Table 5 summarizes the results of the bidirectional assay performed with compound 11 in Caco-2 cells.

[0165] [Table 4]

[0166] [Table 5]

[0167] Preparation of Caco-2 cells: 50 μL and 25 mL of cell culture medium were added to each well of the Transwell insert and reservoir, respectively. The HTS Transwell plate was then incubated at 37 °C and 5% CO2 for 1 h before seeding the cells. Caco-2 cells were cultured at 6.86 × 10 5 The cells were diluted to 1000 cells / mL and 50 μL of the cell suspension was dispensed into the filter wells of a 96-well HTS Transwell plate. The cells were cultured for 14–18 days in a cell culture incubator at 37°C, 5% CO2, and 95% relative humidity. Cell medium was changed every other day, starting no later than 24 h after initial plating.

[0168] Preparation of stock solutions: Stock solutions of control and test compounds were prepared in DMSO at a concentration of 10 mM. Digoxin, prazosin and propranolol were used as control compounds in this assay.

[0169] Assessment of cell monolayer integrity: The medium was removed from the reservoir and each Transwell insert and replaced with pre-warmed fresh medium. The transepithelial electrical resistance (TEER) across the monolayer was measured using a Millicell Epithelial Volt-Ohm measurement system (Millipore, USA). After measurements, the plates were returned to the incubator.

[0170] Assay procedure: Caco-2 plates were removed from the incubator and washed twice with pre-warmed HBSS (10 mM HEPES, pH 7.4) and then incubated at 37°C for 30 min. Stock solutions of control and test compounds were diluted in DMSO to 1 mM solutions and then diluted in HBSS (10 mM HEPES, pH 7.4) to 5 μM working solutions. The final concentration of DMSO in the incubation system was 0.5%. To determine the rate of drug transport in the apical to basolateral direction. Add 125 μL of test compound to the Transwell insert (apical compartment) and immediately transfer 50 μL of sample (D0 sample) from the apical compartment to a new 96-well plate. Fill the wells of the receiver plate (basolateral compartment) with 235 μL of HBSS (10 mM HEPES, pH 7.4). To determine the rate of drug transport in the basolateral to apical direction. Add 285 μL of test compound to the receiver plate wells (basolateral compartment) and immediately transfer 50 μL of sample (D0 sample) from the basolateral compartment to a new 96-well plate. Fill the Transwell insert (apical compartment) with 75 μL of HBSS (10 mM HEPES, pH 7.4). Time 0 samples were prepared by transferring 50 μL of 5 μM working solution to a well of a 96-deep-well plate, followed by the addition of 200 μL of cold acetonitrile containing the appropriate internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine and 200 nM diclofenac). The plate was incubated at 37 °C for 2 h. At the end of the incubation, 50 μL of sample from the donor side (apical compartment for Ap→Bl flux, basolateral compartment for Bl→Ap) and receiver side (basolateral compartment for Ap→Bl flux, apical compartment for Bl→Ap) were transferred to wells of a new 96-well plate, followed by the addition of 4 volumes of cold methanol containing the appropriate internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine and 200 nM diclofenac).The samples were vortexed for 5 min and then centrifuged at 3,220 g for 40 min. 100 μL of the supernatant was mixed with 100 μL of ultrapure water and used for LC-MS / MS analysis. To determine the leakage of Lucifer Yellow after a 2-h transport period, a stock solution of Lucifer Yellow was prepared in water and diluted with HBSS (10 mM HEPES, pH 7.4) to a final concentration of 100 μM. 100 μL of Lucifer Yellow solution was added to each Transwell insert (apical compartment), followed by filling the wells of the receiver plate (basolateral compartment) with 300 μL of HBSS (10 mM HEPES, pH 7.4). The plate was incubated at 37 °C for 30 min. 80 μL of sample was directly removed from the apical and basolateral wells (using the basolateral access holes) and transferred to the wells of a new 96-well plate. Lucifer yellow fluorescent signal (to monitor monolayer integrity) was measured in a fluorescent plate reader with excitation at 485 nM and emission at 530 nM.

[0171] Example 7: Pharmacokinetic and Pharmacodynamic Studies Table 6A summarizes the results of mouse PK studies performed with compound 11. [Table 6A]

[0172] Table 6B summarizes the results of additional mouse PK studies performed with compound 11. [Table 6B]

[0173] Table 7 summarizes the results of additional mouse PK studies performed with compound 11. [Table 7]

[0174] Table 8 summarizes the PK parameters of 1-MNA in mice following administration of compound 11. [Table 8]

[0175] Doses were freshly prepared on the day of administration. The vehicle composition for each dose is listed below. For IV, an appropriate amount of test article was dissolved in the vehicle using vortexing and / or sonication to obtain a solution with the intended concentration level. For PO, an appropriate amount of test article was dissolved in the appropriate vehicle; vortexing and / or sonication can be used to achieve a solution formulation with the intended concentration level. IV (compound): 0.5% Tween 80 in saline PO: (a) 0.2% Tween 80 / 10% PEG-300 in water. Adjust pH with 1N HCl to obtain a clear solution, or (b) 0.2% Tween 80 / 20% PEG-300 in water. Adjust pH with 1N HCl to obtain a clear solution.

[0176] Study arms and dosing information are shown in the table below. TIFF2025503521000049.tif56161

[0177] After dosing, 0.03 mL blood samples (0.2 mL at the final time point by cardiac puncture) were collected from the dorsal metatarsal vein at the following time points: IV: Pre-dose, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 24 hours post-dose PO: Pre-dose, 0.25, 0.5, 1, 2, 4, 6, 8, 24 hours post-dose

[0178] Blood from each sample was transferred to a plastic microcentrifuge tube containing EDTAK2 as an anticoagulant. Blood samples were centrifuged at 4,000 g for 5 min at 4° C. to obtain plasma. Samples were immediately frozen in an upright position and stored at −75±15° C. prior to analysis.

[0179] Samples were analyzed on an AB Sciex Triple Quad 5500 LC / MS / MS instrument using a Gemini 5 μm C18 110A 150x3mm column (for JBSNF-000265, an XBridge BEH C18 2.5 μm 4.6×75mm) equipped with the following equipment:

[0180] Prominence Degasser DGU-20A5R(C), serial number: L20705414138 IX; Liquid chromatograph LC-30AD, serial numbers: L20555408197 AE and L20555408195 AE; Communication bus module CBM-20A, serial number: L20235429486 CD; Autosampler SIL-30AC, serial number: L20565403814 AE; Rack changer II: L20585400900 SS

[0181] Mobile phase A was 0.1% formic acid in water and mobile phase B was 95% acetonitrile in water (0.1% formic acid) delivered at 0.4 mL / min.

[0182] Sample preparation: Working solutions of the desired serial concentrations were obtained by diluting the stock solutions of the analytes with 50% aqueous acetonitrile. 5 µL of working solution (1, 2, 3, 5, 10, 50, 100, 500, 1000 ng / mL) were added to 5 µL of blank C57B16 / N mouse plasma to obtain calibration standards from 1 to 1000 ng / mL (1, 2, 3, 5, 10, 50, 100, 500, 1000 ng / mL) in a total volume of 10 µL. Four quality control samples of 3 ng / mL, 5 ng / mL, 100 ng / mL, and 800 ng / mL for plasma were prepared separately from those used for the calibration curve. These QC samples were prepared on the day of analysis in the same manner as the calibration standards.

[0183] 10 μL of standard, 10 μL of QC sample and 10 μL of unknown sample (5 μL of plasma and 5 μL of blank solution) were added to 100 μL of acetonitrile (containing 50 ng / mL of D4-1-MNA) respectively to precipitate proteins. The samples were then vortexed for 30 seconds. After centrifugation at 4,000 rpm for 15 min at 4°C, the supernatant was diluted 3-fold with water. 10 μL of the diluted supernatant (20 μL for JBSNF-000265) was injected into the LC / MS / MS system for quantitative analysis.

[0184] Sample preparation: 1-Methylnicotinamide (1-MNA): Working solutions of the desired serial concentrations were obtained by diluting the stock solution of the analyte with 50% acetonitrile in water. 5 µL of working solution (1, 2, 3, 5, 10, 50, 100, 500, 1000 ng / mL) was added to 5 µL of water to obtain calibration standards from 1 to 1000 ng / mL (1, 2, 3, 5, 10, 50, 100, 500, 1000 ng / mL) in a total volume of 10 µL. Four quality control samples of 3 ng / mL, 5 ng / mL, 100 ng / mL, and 800 ng / mL for water were prepared separately from those used for the calibration curve. These QC samples were prepared on the day of analysis in the same manner as the calibration standards.

[0185] 10 μL of standard, 10 μL of QC sample and 10 μL of unknown sample (5 μL of plasma and 5 μL of blank solution) were added to 100 μL of acetonitrile (containing 100 ng / mL of D4-1-MNA) respectively to precipitate proteins. The samples were then vortexed for 30 seconds. After centrifugation at 4000 rpm for 15 min at 4° C., the supernatant was diluted 3-fold with water. 5 μL of the diluted supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0186] Recovery: 5 μL of working solution (10, 100, 800 ng / mL) was added to 5 μL of blank C57B16 / N mouse plasma to obtain three samples of 10 ng / mL, 100 ng / mL, and 800 ng / mL, and 30 μL of the samples were added to 100 μL of IS mixture containing acetonitrile, respectively, to precipitate the proteins. The samples were then vortexed for 30 s. After centrifugation at 4000 rpm for 15 min at 4 °C, the supernatant was diluted 3 times with water. 5 μL of the diluted supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0187] Example 8: pH-Dependent Solubility The pH-dependent solubility of compound 11 (FIG. 6) was analyzed according to the following procedure.

[0188] Preparation of stock solutions: Test compounds and control compounds stock solutions were prepared in DMSO at a concentration of 30 mM (50 mM: R-NA47). 50 mM DMSO compound stock solutions were diluted to 30 mM with DMSO. Control compounds stock solutions were prepared in DMSO at a concentration of 30 mM. Diclofenac was used as a positive control in the assay.

[0189] Solubility Determination Procedure: 10 μL of each compound's stock solution was placed in their appropriate 96-well rack in sequence, followed by the addition of 990 μL of PBS pH 1.5, pH 3.0, pH 4.5, pH 6.8, pH 7.4 and pH 9.0 to each vial of the capless solubility sample plate. This test was performed in duplicate. One stir stick was added to each vial, which was then sealed using a molded PTDE / SIL 96-well plate cover. The solubility sample plate was transferred to a Thermomixer Comfort plate shaker and incubated for 2 hours at room temperature (RT) with shaking at 1100 rpm. After the 2 hour incubation, the stir stick was removed using a large magnet and all samples from the solubility sample plate were transferred to a filter plate. All samples were filtered using a Vacuum Manifold. The filtered samples were diluted with methanol. The dilution factor may be altered according to the solubility value and LC / MS signal response.

[0190] Preparation of 0.3 μM standard solution (STD): 30 mM DMSO compound stock solution was diluted to 300 μM with DMSO and then diluted with methanol to obtain 0.3 μM STD.

[0191] Sample Analysis and Data Analysis: Samples were analyzed by LC-MS / MS. All calculations were performed using Microsoft Excel. Filtered solutions were analyzed and quantified against standards of known concentration in DMSO using LC coupled with mass spectral peak identification and quantification. Solubility values ​​of test compounds were calculated as follows:

number

[0192] Example 9: hERG Safety Evaluation A hERG safety evaluation was performed on compound 11 according to the following protocol (Figure 8).

[0193] Cell lines and cell culture: hERG stable expressing HEK293 cell line (catalog number K136) was purchased from Invitrogen. Cells are cultured in 85% DMEM, 10% dialyzed FBS, 0.1 mM NEAA, 25 mM HEPES, 100 U / mL penicillin-streptomycin, 5 μg / mL Blasticidin and 400 μg / mL Geneticin. Cells are split approximately three times a week using TrypLE™ Express and maintained at approximately 40% to approximately 80% confluence. Prior to the assay, 5 × 10 cells were cultured per 6 cm cell culture dish. 5 Cells were plated on coverslips and induced with 1 μg / mL doxycycline for 48 h.

[0194] Solution preparation: 1) Extracellular solution: 132 mM NaCl, 4 mM KCl, 3 mM CaCl2, 0.5 mM MgCl2, 11.1 mM glucose, and 10 mM HEPES (adjust pH to 7.35 with NaOH); 2) Intracellular solution: 140 mM KCl, 2 mM MgCl2, 10 mM EGTA, 10 mM HEPES and 5 mM MgATP (adjust pH to 7.35 with KOH).

[0195] Preparation of working solutions for test compounds: 1) Test compounds were initially prepared as stock solutions, at a final concentration of 10 mM in DMSO; 2) The stock solution of each compound was then serially diluted in a 1:3 ratio with DMSO to prepare three additional intermediate solutions, including 3.33, 1.11, and 0.37 mM; 3) Prior to the hERG assay, working solutions were prepared by diluting the 10, 3.33, 1.11 and 0.37 mM intermediate solutions 1000-fold with extracellular solution such that the final concentrations of the working solutions were 30, 10, 3.33, 1.11 and 0.37 µM, whereas the 30 µM working solution was prepared by a 333.333-fold dilution of the 10 mM DMSO stock. Final DMSO concentrations ranged from 0.1-0.3%; 4) hERG currents in the presence of five doses, including 30, 10, 3.33, 1.11 and 0.37 µM, were measured for IC50 determination.

[0196] Experimental procedure: 1) Remove the coverslip from the cell culture dish and place it on the microscope stage in the bath chamber; 2) Locate the desired cell using the x10 objective. Locate the tip of the electrode under the microscope using the x10 objective by focusing on the plane of the cell; Once the tip is in focus, use the coarse control of the manipulator to advance the electrode downwards towards the cell while simultaneously moving the objective to keep the tip in focus; 3) Once directly above the cell, switch to the x40 objective and use the fine control of the manipulator to gradually approach the surface of the cell; 4) Apply gentle suction through the side port of the electrode holder to form a gigaohm seal; 5) Use Cfast to remove capacitive currents that coincide with voltage steps. Obtain the whole cell configuration by repeatedly applying short periods of strong suction until the membrane patch ruptures; 6) Set the membrane potential to -60mV at this point to ensure that the hERG channels are not open. Spikes in capacitive current should be cancelled using the Cslow of the amplifier; 7) Holding potential set to -90mV for 500ms; Currents recorded at 20kHz and filtered at 10kHz. Leak currents were tested at -80mV for 500ms; 8) hERG currents were induced by depolarizing at +30mV for 4.8s, then voltage was returned to -50mV for 5.2s to remove inactivation and observe the inactivation tail current. The maximum amount of tail current size was used to determine hERG current amplitude; 9) Currents were recorded for 120s to assess current stability. Only stable cells with recording parameters above threshold were used for drug administration; 10) First, vehicle control was applied to the cells to establish a baseline. Once it was confirmed that the hERG current was stable for 5min, the working solution was applied. hERG currents in the presence of test compounds were recorded for approximately 5min until steady state was reached, after which 5 sweeps were acquired. For dose-response studies, five doses of test compound were cumulatively applied to the cells, from low to high. A positive control, dofetilide at a concentration of 150 nM, was also applied to each cell after measuring the hERG current with the highest concentration of test compound, as an internal low control for normalization of the percentage inhibition.To ensure good performance of the cultured cells and the manipulation, five doses of dofetilide were also used to test the same batch of cells.

[0197] Data Acceptability Criteria: The following criteria were used to determine data acceptability: 1) initial seal resistance >1 GΩ; 2) leak current <50% of control peak tail current; 3) peak tail amplitude >250 pA; 4) membrane resistance Rm >500 MΩ; 5) access resistance (Ra) <10 MΩ; 6) apparent rundown of peak current <2.5% / min.

[0198] Data Analysis: Data meeting the above criteria for hERG current quality were further analyzed as follows: 1) Percentage of hERG current inhibition was calculated using the following formula; 2) Dose-response curves of test compounds were plotted as percent hERG current inhibition versus test compound concentration using Graphpad Prism 8.0 and fitted to a sigmoidal dose-response curve with variable slope.

number

[0199] Example 10: CYP inhibition test Inhibition tests of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E and CYP3A4 were performed according to the following protocol (Figure 9). Compound 11 did not inhibit any of the CYP isoforms tested.

[0200] Inhibitory effects of compound 11 in human liver microsomes. 1. 1 μL of multiple concentrations of test compounds or positive control compounds (CYP1A2: furafylline, CYP2A6: tranylcypromine, CYP2B6: ketoconazole, CYP2C8: quercetin, CYP2C9: sulfaphenazole, CYP2C19: N-3-benzylnirvanol, CYP2D6: quinidine, CYP2E1: disulfiram, and CYP3A4: ketoconazole) were transferred to the "compound plate". The concentrations of test compounds were 0, 0.6, 2, 6, 20, 60, 2000, 6000, and 20000 μM. The concentrations of positive control compounds were 0, 0.2, 1, 2, 10, 50, 200, 2000, and 10000 μM.

[0201] 2. Master solutions were prepared according to Tables 9A and 9B and pre-warmed in a water bath at 37°C for 5 minutes. 179 μL of master solution was transferred to the "incubation plate". In the mixed system, the final concentrations of the test compounds were 0, 0.003, 0.01, 0.03, 0.1, 0.3, 10, 30 and 100 μM, and the final concentrations of the positive control compounds were 0, 0.001, 0.005, 0.01, 0.05, 0.25, 1, 10 and 50 μM. All experiments were performed in duplicate.

[0202] 3. The reaction was initiated by adding 20 μL of a 10 mM NADPH solution to a final concentration of 1 mM and was carried out at 37°C.

[0203] 4. At the designated time points listed in Table 9B, the reaction was stopped by adding 1.5 volumes of cold methanol containing IS (100 nM alprazolam, 200 nM imipramine, 200 nM labetalol and 2 μM ketoprofen) to the "incubation plate". The "incubation plate" was centrifuged at 3,220 g for 40 min to precipitate proteins. An aliquot of 100 μL of the supernatant was diluted with 100 μL of ultrapure water and the mixture was used for LC-MS / MS analysis.

[0204] 5. Data analysis - All calculations were performed using Microsoft Excel. Metabolite formation was analyzed using LC-MS / MS. The reduction in metabolite formation in peak area relative to vehicle control was used to calculate IC 50 The value (concentration of test compound resulting in 50% inhibition) was calculated.

[0205] [Table 9A]

[0206] [Table 9B]

[0207] Evaluation of direct and time-dependent inhibition of CYP3A4 by compound 11 1. Master solutions were prepared according to Table 10A. Substrate solutions were prepared in a mixture of acetonitrile and ultrapure water (1:9 (v:v)) immediately before use as listed in Table 10A.

[0208] 2. The "Master solution" was pre-warmed for 5 minutes at 37° C. 169 μL of "Pre-incubation solution" and 1 μL of test compounds at multiple concentrations (0, 0.6, 2, 6, 20, 60, 2000, 6000 and 20000 μM) or 1 μL of positive control compounds (0, 0.2, 1, 2, 10, 50, 200, 2000 and 10000 μM) were transferred from the "Compound Plate" to the "Incubation Plate".

[0209] 3. For 0 minute pre-incubation, 10 μL of substrate was added to the incubation plate, then 20 μL of 10 mM NADPH solution was added to initiate the reaction at a final concentration of 1 mM, and then incubated for the indicated time listed in Table 10B.

[0210] 4. For 30 min pre-incubation without NADPH, the incubation plate was pre-incubated for 30 min in a 37°C water bath. After 30 min incubation, 10 μL of substrate was added to the incubation plate, and 20 μL of 10 mM NADPH solution was added to start the reaction at a final concentration of 1 mM. Then, incubated for the indicated time listed in Table 10B.

[0211] 6. Data analysis - All calculations were performed using Microsoft Excel. Metabolite formation was analyzed using LC-MS / MS. The reduction in metabolite formation in peak area ratio to the vehicle control was used to calculate the three IC 50 The IC values ​​for each of the three inhibitors (0 min preincubation, 30 min preincubation with NADPH and 30 min preincubation without NADPH) were calculated to evaluate the mechanism of inhibition. 50 The shift was calculated.

[0212] [Table 10A]

[0213] [Table 10B]

[0214] Example 11: Off-target studies An off-target safety panel screen was performed for compound 11. Target classes tested included ion channels, GPCRs, transporters, kinases, enzymes, and nuclear hormone receptors. Compound 11 showed no significant off-target activity at 10 μM (Figure 10).

[0215] Example 12: Ames test Mini-Ames tests of compound 11 were performed in various bacterial strains. Based on these results, compound 11 was not mutagenic (Figure 11).

[0216] 1. This study evaluated the mutagenic potential of a test article (or its metabolites) by measuring its ability to induce reverse mutations at selected loci in Salmonella typhimurium (TA98, TA1535, TA1537) and Escherichia coli WP2uvrA (pKM101) both in the presence and absence of microsomal enzymes.

[0217] 2. Standard 6-well culture plates had an approximate well diameter of 33 mm. Top agar containing 0.6% (w / v) agar and 0.5% (w / v) sodium chloride was supplemented with 0.5 mM D-biotin and 0.5 mM L-histidine when using Salmonella typhimurium strains, or 0.5 mM D-biotin and 0.5 mM L-tryptophan when using E. coli WP2uvrA(pKM101).

[0218] 3. For the Mini-Ames test, four strains were selected for testing: Salmonella typhimurium (TA98, TA1535, TA1537) and E. coli WP2uvrA (pKM101). Test strains were prepared from frozen working stocks. 10 μL of frozen working stock was added to 5 mL of nutrient broth and incubated at 37 ± 2 °C for 10 h with shaking at 220 rpm until an optical density (at 650 nm) of 0.6-0.8 was reached. Overnight cultures were used for mutagenicity testing.

[0219] 4. Test articles were supplied as powders and kept at -20°C until use. Stock solutions were prepared in DMSO at 50 mg / mL (Table 11A). Subdoses were prepared by dilution from the stock with DMSO immediately prior to use. If the test article was not soluble at 50 mg / mL, the highest concentration was reduced to the lowest insoluble concentration.

[0220] 5. Human liver S9 mix was prepared by mixing the following in reverse order and kept on ice: S9 (110 μL); 1.65 M KCl + 0.4 M MgCl2 (20 μL); sterile water (380 μL); 0.2 M sodium phosphate buffer (500 μL); NADP (4 μmol); and G-6-P (5 μmol).

[0221] 6. Control-Negative (DMSO); Positive (Table 11B)

[0222] 7. For each test article, six concentrations in triplicate for each strain are required. The highest dose level we recommend is 1000 micrograms (μg) / well (or 1 microliter (μL) / well for liquid test substances), if not limited by solubility or cytotoxicity. If the test article has solubility problems, precipitation is scored according to the following evaluation: P0: no precipitation in the solution and no crystal growth on the plate; P1: 0-20% crystal growth; P2: 20%-60% crystal growth; P3: 60%-100% crystal growth. Precipitating doses are scored, provided that precipitation does not interfere with scoring (counting the number of revertants). If precipitation interferes with scoring (counting the number of revertants), the lowest precipitating dose should be used as the highest dose scored.

[0223] 8. Mini Ames Assay- Melt top agar in microwave and keep at ≥47°C in water bath. Assay was performed using heat block set at 45°C ± 2°C. Set up 12 x 75mm tubes in duplicate. Assay was performed using heat block set at 45°C ± 2°C. Set up 12 x 75mm tubes in duplicate. The following was added to each concentration tube in order: a. 1600μL top agar; b. 80μL drug or control; c. 400μL S9 mix or PBS buffer; d. 80μL overnight culture. Vortex and dispense 540μL / well using disposable pipette. Plates were incubated at 37±2°C for approximately 48-72 hours.

[0224] 9. Scoring and Positivity Criteria--A compound is considered mutagenic if F>2 for strains TA98, WP2uvrA(pKM101), and F>3 for TA1535, TA1537, as well as a dose-related increase in mean revertants. Assessment of background lawn: 0 represents no growth or complete cytotoxicity of the background lawn; 1 represents about 25% growth; 2 represents about 50% growth; 3 represents about 75% growth; 4 represents complete "100%" growth.

number

[0225] Background lawns are measured microscopically using a stereoscope. All background lawn evaluations are performed relative to vehicle controls. In complete background lawns, lawn colonies are very small and cannot be visualized. However, in toxic plates, lawn colonies grow into microcolonies (small in diameter) or colonies (similar in diameter to revertant colonies) and can be visualized without the aid of a magnifying glass.

[0226] [Table 11A]

[0227] [Table 11B]

[0228] INCORPORATION BY REFERENCE All publications and patents mentioned herein are incorporated herein by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0229] Equivalent While specific embodiments of the invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

1. A compound having the structure of Formula I, or a pharmaceutically acceptable salt thereof: 【Chemistry 1】 [In the formula, Z １ , O, NR ４ or CHR ２ and Z ２ is O or CHR ３ and Z ３ is O or NH; R １ is H, halo, cyano, unsubstituted or substituted alkyl, unsubstituted or substituted cycloalkyl, or unsubstituted or substituted alkynyl; R ２ and R ３ are each independently H, cyano, unsubstituted or substituted alkyl, unsubstituted or substituted alkynyl, alkyl-OR ５ , or alkyl-SR ６ or, when x is 0, R ２ and R ３ may, together with the carbon atoms to which they are attached, form a fused 3-, 4-, 5-, or 6-membered carbocyclic ring; R ４ is H, alkyl, or acyl; R ５ and R ６ are each independently H, CHF ２ , C.F. ３ , unsubstituted or substituted alkyl, unsubstituted or substituted alkynyl, or unsubstituted or substituted cycloalkyl; x is an integer selected from 0 and 1; However, Z ２ and Z ３ and when x is 0, Z １ and Z ２ are not both O and Z １ and Z ３ and ∇ are never both O.

2. R ２ and R ３ are each independently H, cyano, alkyl, or alkynyl; or when x is 0, R ２ and R ３ may be taken together with the carbon atoms to which they are attached to form a fused 3-, 4-, 5-, or 6-membered carbocyclic ring.

3. Z １ and Z ２ and (b) are not both O.

4. Z １ is CHR ２ and / or 2. The compound of claim 1, wherein Z2 is CHR3.

5. R ２ The compound of claim 4, wherein R is H or alkyl and R 3 is H, alkyl, alkynyl, or cyano.

6. Z 1 is CHR 2 and Z 2 is CHR 3 , and R ２ and R ３ and the carbon atoms to which they are attached form a fused three-membered carbocyclic ring.

7. R １ The compound of claim 1 , wherein is alkyl or halo.

8. The following structure: 【Chemistry 2】 2. The compound of claim 1 having the formula:

9. R １ is H or CH ３ and / or 9. The compound of claim 8, wherein R 3 is unsubstituted C 1 -C 6 alkyl, unsubstituted C 2 -C 6 alkynyl, substituted C 2 -C 6 alkynyl, alkyl-OR 5 or alkyl-SR 6 , and R 5 and R 6 are each independently CHF 2 , CF 3 , CH 3 , CH(CH 3 ) 2 , or CH(CH 2 ) 2 .

10. The following structure: 【Transformation 3】 2. The compound of claim 1 having the formula:

11. The following structure: 【Chemistry 4】 or 【Transformation 5】 11. The compound of claim 10, having the formula:

12. R １ is H or CH ３ and / or R 2 is unsubstituted C 1 -C 6 alkyl, and / or The compound of claim 10, wherein R 3 is unsubstituted C 1 -C 6 alkyl. 【Request Item 13】 【Chemistry 6】 【Transformation 7】 【Transformation 8】 or a pharmaceutically acceptable salt thereof.

14. A pharmaceutical composition comprising a compound according to any one of claims 1 to 13 and a pharmaceutically acceptable excipient or carrier.

15. A composition for treating or preventing cancer or inhibiting tumor growth in a subject in need thereof, the composition comprising a compound described in any one of claims 1 to 13.

16. 16. The composition of claim 15, wherein the cancer is ovarian cancer, colon cancer, or breast cancer.

17. The composition of claim 15, wherein the cancer or tumor is high-grade serous carcinoma (HGSC).

18. 16. The composition of claim 15, conjointly administered with an effective amount of one or more additional chemotherapeutic agents.

19. The composition of claim 15 , wherein the subject is a mammal.

20. The composition of claim 15, wherein the mammal is a human, dog, or cat.

21. 14. An in vitro method of inhibiting NNMT, comprising contacting a cell expressing NNMT with an effective amount of a compound according to any one of claims 1 to 13.

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