Bicyclic inhibitors of nicotinamide n-methyltransferase, compositions and uses thereof

Abstract

Disclosed are compounds and pharmaceutically acceptable salts thereof, which are useful as inhibitors of nicotinamide N-methyltransferase (NNMT). Also disclosed are pharmaceutical compositions comprising the compounds disclosed herein. Related methods of treating cancer in a subject and methods of inhibiting tumor growth in a subject are also disclosed.

Description

[0001] Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 63 / 295,657, filed on December 31, 2021. Background Art

[0003] Tumors are heterogeneous, containing cancer cells and delicate microenvironments. Cancer-associated fibroblasts (CAFs) are myofibroblasts (fibroblasts with smooth muscle cell characteristics) present in the tumor microenvironment, which support tumors through paracrine signaling and the production of extracellular matrix. CAFs play a key role in almost all aspects of tumor biology, including survival, resistance, metastasis, and immune cell escape. CAFs have now been identified in solid tumors of almost all tissue types, sometimes exceeding any other cell type in the tumor, and are associated with poor prognosis in patients. The extracellular matrix accumulation driven by CAFs has been shown to prevent the infiltration of effector immune cells and activated T cells. Therefore, reducing the presence of CAFs in tumors can improve the response and resistance to immunotherapy. In view of their close role in cancer maintenance, development, and resistance to targeted therapy and immunotherapy, the therapeutic agent of specific targeting CAF has great prospects as a new method in cancer treatment. However, only a few targets (relative to normal fibroblasts) that are specific to CAFs have been identified. The most advanced efforts to target CAFs are to use CAR T cells that recognize fibroblast activation protein (FAP). Although this therapy has shown promise, FAP is expressed by other cells in the body, including those that regulate bone marrow and muscle tissue, sometimes leading to lethal toxicity.

[0004] In order to identify targets specific to CAF, proteins differentially expressed in human CAF but not in tumor cells or normal stroma have been identified. Specifically, biopsy samples from patients with high-grade serous carcinoma metastasis (the most common form of HGSC-ovarian cancer) undergo laser microdissection to separate tumor cells from the stroma, followed by mass spectrometry. It was found that the expression of nicotinamide N-methyltransferase (NNMT) increased in the stroma of HGSC metastasis compared with tumor cells or normal stroma. NNMT is also highly expressed in breast cancer and colon cancer stroma. Importantly, NNMT is necessary to maintain the CAF phenotype. In addition, when NNMT is knocked down or inhibited by small molecule inhibitors, the tumor burden in animal models is reduced. These studies show that NNMT is a CAF selective therapeutic target, and the inhibitory effect of small molecules on it reverses the CAF phenotype and reduces tumor burden.

[0005] Therefore, there is a continuing need to discover and develop new compounds that target nicotinamide N-methyltransferase (NNMT). Summary of the invention

[0006] In certain embodiments, the present application discloses a compound of Formula I:

[0007]

[0008] or a pharmaceutically acceptable salt thereof, wherein R 1 , Z 1 , Z 2 , Z 3 and x are as defined herein.

[0009] 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 and methods of inhibiting tumor growth in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a graph showing data from an in vitro NNMT enzyme inhibition assay of compound 1 (IC 50 =105nM).

[0011] Figure 2 is a graph showing data from an in vitro NNMT enzyme inhibition assay of compound 11 (IC 50 =35nM).

[0012] Figure 3 is a graph showing data from a cellular potency assay of compound 11 (IC 50 =38nM).

[0013] Figure 4A is a graph showing data from an in vitro NNMT enzyme inhibition assay of compound 18 (IC 50 =848nM).

[0014] Figure 4B Each enantiomer of compound 18, i.e., compound 19A (IC 50 =28 nM) and compound 19B (IC 50 =5.1 μM).

[0015] Figure 4C is a graph showing data from a cellular potency assay of compound 19A (IC 50 =12nM).

[0016] Figure 5 is a graph showing data from an in vitro NNMT enzyme inhibition assay of compound (R)-34 (IC 50 =48nM).

[0017] Figure 6is a graph showing the pH-dependent solubility of 11.

[0018] Figure 7 is a graph showing the stability of 11 in human and mouse liver microsomes.

[0019] Figure 8 is a diagram showing the hERG safety evaluation performed by the manual patch clamp system (hERG IC 50 = > 30 μM; 14.96% inhibition at 30 μM).

[0020] Fig. 9 is a pair of graphs showing the inhibitory effects of 11 on CYP3A4 and a table showing the inhibitory effects of 11 on other CYP isoforms.

[0021] Fig.10 There is a panel of 11 off-target activities.

[0022] Fig.11 is a graph showing the results of Mini Ames assay with 11. In each strain with and without S9 fraction, the F value was less than 2 at all tested concentrations.

[0023] Fig.12 is a graph showing the plasma concentrations of 11 after 50 mg / kg and 100 mg / kg PO administration in female C57B16 / N mice.

[0024] Fig.13A is a graph showing plasma concentrations of 1-MNA 0-8 hours following 50 mg / kg and 100 mg / kg PO dosing of 11 in female C57B16 / N mice.

[0025] Fig. 13B is a graph showing plasma concentrations of 1-MNA 0-24 hours following 50 mg / kg and 100 mg / kg PO dosing of 11 in female C57B16 / N mice.

[0026] Fig.14 The dose and data collection schedule for the 11PK / PD study in female c57b16 / n mice.

[0027] Fig.15A is a graph showing the plasma concentration of 11 after QD administration of 11 for 14 days in female C57B16 / N mice.

[0028] Fig. 15B is a graph showing the plasma concentration of 1-MNA after QD dosing for 11 to 14 days in female C57B16 / N mice.

[0029] Fig.16Ais a graph showing the plasma concentration of 11 after 50 mg / kg administration of 11 (by different methods) in female C57B16 / N mice.

[0030] Fig. 16B is a graph showing the plasma concentration of 1-MNA after 50 mg / kg administration of 11 (by different methods) in female C57B16 / N mice.

[0031] Fig.17A is a graph showing the plasma concentration of 11 after 100 mg / kg administration of 11 (by different methods) in female C57B16 / N mice.

[0032] Fig. 17B is a graph showing the plasma concentration of 1-MNA after 100 mg / kg administration of 11 (by different methods) in female C57B16 / N mice.

[0033] Fig.18 is a table showing the activity characteristics of 11.

[0034] Fig.19 is a table summarizing the PK / PD data for compounds 1 to 3 and 11.

[0035] Fig. 20 is a table summarizing the mean PK parameters for various Compound 11 samples following different PO doses in female C57B16 / N mice.

[0036] Fig.21 is a graph showing plasma concentration of 1-MNA versus time following PO administration of Compound 11 or 19A in female C57B16 / N mice. DETAILED DESCRIPTION

[0037] In certain aspects, the present application discloses substituted polycyclic compounds and pharmaceutical compositions thereof. In particular, such compounds disclosed herein can be used as inhibitors of nicotinamide N-methyltransferase (NNMT).

[0038] NNMT catalyzes the methylation of nicotinamide using S-adenosylmethionine (SAM) as a cofactor to generate 1-methylnicotinamide (1-MNA). Without wishing to be bound by theory, high expression of NNMT could maintain the CAF phenotype by reducing SAM levels, which would lead to DNA and histone hypomethylation and 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 histone 3 lysines 4 and 27; and 3) inhibition of the histone methyltransferase EZH2 rescues NNMT knockdown and restores the CAF phenotype (α-SMA and collagen contractility).

[0039] Therefore, the compounds disclosed herein can be used as inhibitors of NNMT, which are particularly useful for treating cancers such as CAF-associated cancerous tumors with increased NNMT expression.

[0040] I. Compounds

[0041] In certain embodiments, the present application discloses a compound of Formula I or a pharmaceutically acceptable salt thereof:

[0042]

[0043] in

[0044] Z 1 Yes O, NR 4 or CHR 2 ;

[0045] Z 2 O or CHR 3 ;

[0046] Z 3 is O or NH;

[0047] R 1 is H, halo, cyano, unsubstituted or substituted alkyl, unsubstituted or substituted cycloalkyl, or unsubstituted or substituted alkynyl;

[0048] 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

[0049] If x is 0, then R 2 and R 3 Together with the carbon atoms to which they are attached, they can form a 3-, 4-, 5-, or 6-membered fused carbocyclic ring;

[0050] R 4 is H, alkyl or acyl;

[0051] R 5 and R 6 are each independently H, CHF2, CF3, unsubstituted or substituted alkyl, unsubstituted or substituted alkynyl, or unsubstituted or substituted cycloalkyl; and

[0052] x is an integer selected from 0 and 1;

[0053] The condition is Z 2 and Z 3 are not all O; and if x is 0, then Z 1 and Z2 Not all O and Z 1 and Z 3 Not all O.

[0054] In certain embodiments, the present application discloses a compound of Formula I or a pharmaceutically acceptable salt thereof:

[0055]

[0056] in

[0057] Z 1 Yes O, NR 4 or CHR 2 ;

[0058] Z 2 O or CHR 3 ;

[0059] Z 3 is O or NH;

[0060] R 1 is H, halo, cyano, unsubstituted or substituted alkyl (e.g., fluoroalkyl), unsubstituted or substituted cycloalkyl, or unsubstituted or substituted alkynyl;

[0061] R 2 and R 3 are each independently H, cyano, alkyl or alkynyl; or

[0062] If x is 0, then R 2 and R 3 Together with the carbon atoms to which they are attached, they can form a 3-, 4-, 5-, or 6-membered fused carbocyclic ring;

[0063] R 4 is H, alkyl or acyl; and

[0064] x is an integer selected from 0 and 1;

[0065] The condition is Z 2 and Z 3 are not all O; and if x is 0, then Z 1 and Z 2 Not all O and Z 1 and Z 3 Not all O.

[0066] In some embodiments, Z 1 and Z 2 Not all O

[0067] In some embodiments, x is 0. In some embodiments, x is 1.

[0068] In some embodiments, Z1 It is CHR 2 In some embodiments, R 2 is H. In some embodiments, Z 1 is O. In some embodiments, Z 1 YesNR 4 .

[0069] In some embodiments, Z 2 It is CHR 3 In some embodiments, R 3 is H. In some embodiments, R 3 In some embodiments, R 3 is alkyl, preferably lower alkyl, more preferably methyl. 3 is alkynyl, preferably lower alkynyl, more preferably ethynyl. 2 It's O.

[0070] In some embodiments, R 2 and R 3 Together with the carbon atoms to which they are attached they form a 3-membered fused carbocyclic ring.

[0071] In some embodiments, Z 3 is NH. In some embodiments, Z 3 It's O.

[0072] In some embodiments, R 1 is alkyl, preferably lower alkyl, more preferably methyl. 1 is halo, preferably chloro. 1 It is a substituted alkyl group, preferably a substituted lower alkyl group, more preferably a fluorine-substituted lower alkyl group.

[0073] In some embodiments, R 4 It is an alkyl group, preferably a lower alkyl group, more preferably a methyl group.

[0074] In some embodiments, the compound of Formula I has the following structure:

[0075]

[0076] In some embodiments, R 1 It is H or CH3.

[0077] In some embodiments, R 1 It is halogenated.

[0078] In some embodiments, R 1 It is Cl.

[0079] In some embodiments, R 3 is unsubstituted C 1- C6 alkyl.

[0080] In some embodiments, R 3 It is CH3.

[0081] In some embodiments, R 3 is unsubstituted C 2- C6 alkenyl. In other embodiments, R 3 is replaced by C 2- C6 alkynyl. In some embodiments, when substituted, C 2- The C6 alkynyl group is substituted by CF3.

[0082] In some embodiments, R 3 is alkyl-OR 5 or alkyl-SR 6 In some embodiments, R 5 and R 6 Each is independently CHF2, CF3, CH3, CH(CH3)2 or CH(CH2)2.

[0083] In some embodiments, the compound of Formula I has the following structure:

[0084]

[0085] In some embodiments, the compound of Formula I has the following structure:

[0086]

[0087] In some embodiments, the compound of Formula I has the following structure:

[0088]

[0089] In some embodiments, the compound of Formula I has the following structure:

[0090]

[0091] In some embodiments, R 1 It is H or CH3.

[0092] In some embodiments, R 1 It is halogenated.

[0093] In some embodiments, R 1 It is Cl.

[0094] In some embodiments, R 2 is unsubstituted C 1-C6 alkyl.

[0095] In some embodiments, R 3 is unsubstituted C 1- C6 alkyl.

[0096] In some embodiments, R 1 , R 2 and R 3 Each is CH3. Exemplary compounds of Formula I are depicted in Table 1A.

[0097] Table 1A. Exemplary compounds of Formula I

[0098]

[0099]

[0100]

[0101] Additional exemplary compounds of Formula I are depicted in Table IB.

[0102] Table 1B. Exemplary compounds of Formula I

[0103]

[0104]

[0105]

[0106]

[0107] II. Methods

[0108] In certain embodiments, the present application discloses a method for treating or preventing cancer, comprising administering an effective amount of a compound disclosed herein (e.g., a compound of Formula I) or a pharmaceutically acceptable salt thereof, or a composition disclosed herein to a subject in need thereof, thereby treating or preventing the cancer.

[0109] In some embodiments, the cancer is brain cancer, head and neck cancer, breast cancer, lung cancer, esophageal cancer, stomach cancer, duodenum 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).

[0110] In certain embodiments, the cancer is a solid tumor. In some embodiments, the subject is typically a subject who has been diagnosed with a cancerous tumor or a subject who has previously been treated for a cancerous tumor (e.g., where the tumor has previously been surgically removed). The cancerous tumor can be a primary tumor and / or a secondary (e.g., metastatic) tumor.

[0111] In certain embodiments, the present application discloses a method of inhibiting tumor growth, which comprises administering to a subject in need thereof an effective amount of a compound disclosed herein or a pharmaceutically acceptable salt or pharmaceutical composition thereof.

[0112] In some embodiments, the methods disclosed herein further comprise co-administering to the subject an effective amount of one or more additional chemotherapeutic agents. In certain embodiments, the methods disclosed herein further comprise co-administering to the subject an effective amount of radiation therapy.

[0113] Chemotherapeutic agents that can be administered in combination with the compounds of the present invention include: ABT-263, aminoglutethimide, amsacrine, anastrozole, asparaginase, AZD5363, bacillus Calmette-Guérin (bcg), bicalutamide, bleomycin, bortezomib, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, cobimetinib, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, azine, dactinomycin, daunorubicin, demethylglucosidase, dexamethasone, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, 5-fluorouracil and 5-fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, lenalidomide adominis, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, LY2603618, nitrogen mustard, medroxyprogesterone, megestrol acetate, melphalan, mercaptopurine, mesna, metformin, methotrexate, miltefosine, mitomycin, mitotane, mitoxantrone, MK2206, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, pazopanib, perifosine, PF-04691502, P F477736, plicamycin, polylidomide, porfimer sodium, procarbazine, raltitrexed, rituximab, romidepsin, selecitinib, sorafenib, streptozotocin, sunitinib, suramin, tamoxifen, temozolomide, temsirolimus, teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan, trametinib, trastuzumab, retinoic acid, vinblastine, vincristine, vindesine, vinorelbine, and vorinostat (SAHA).For example, chemotherapeutic agents that can be administered in combination with the compounds of the present invention include aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg, bicalutamide, bleomycin, bortezomib, buserelin, busulfan, camptothecin, capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil, chloroquine, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, glycosides, dacarbazine, dactinomycin, daunomycin, demethylglucosidase, dichloroacetate, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, isocyclic Phosphamide, imatinib, interferon, irinotecan, lenalidomide, letrozole, leucovorin, leuprolide, levamisole, lomustine, lonidamine, nitrogen mustard, medroxyprogesterone, megestrol acetate, melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostat In some embodiments, the chemotherapeutic agent is cisplatin. In some embodiments, the additional chemotherapeutic agent is a CHK1 inhibitor.

[0114] Many combination therapies have been developed for the treatment of cancer. In certain embodiments, the compounds of the present invention can be administered in combination with combination therapies. Examples of combination therapies that can be administered in combination with the compounds of the present invention are included in Table 2.

[0115] Table 2: Exemplary combination therapies for treating cancer.

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124] In some embodiments, the co-administered chemotherapeutic agent is an immuno-oncology therapeutic, such as an inhibitor of CTLA-4, indoleamine 2,3-dioxygenase, and / or PD-1 / PD-L1.

[0125] In certain embodiments, a compound disclosed herein or a pharmaceutically acceptable salt thereof and the 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.

[0126] In certain embodiments, the subject is a mammal, such as a human.

[0127] In certain embodiments, disclosed herein are methods of inhibiting NNMT comprising contacting a cell expressing NNMT with a compound of Formula I. In certain embodiments, the cell is a cancer cell. Such methods can be performed in vivo or in vitro.

[0128] III. Pharmaceutical Compositions

[0129] In certain embodiments, the present invention provides a pharmaceutical composition comprising a compound of Formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0130] The compositions and methods of the present invention can be used to treat individuals in need thereof. In certain embodiments, the individual is a mammal, such as a human or a non-human mammal. When administered to an animal such as a human, the composition or compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the present invention and a pharmaceutically acceptable carrier.

[0131] 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 ethylene glycol, glycerol, oils such as olive oil or injectable organic esters. In a preferred embodiment, when such pharmaceutical compositions are used for human administration, particularly for invasive administration routes (i.e., routes such as injection or implantation, which avoid transport or diffusion through epithelial barriers), the aqueous solution is pyrogen-free or substantially pyrogen-free. Excipients can be selected, for example, to achieve delayed release of medicaments or selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage forms such as tablets, capsules (including spray capsules and gelatin capsules), granules, lyophilized agents for reconstruction, powders, solutions, syrups, suppositories, injections, etc. The composition can also be present in transdermal delivery systems, such as skin patches. The composition can also be present in solutions suitable for topical administration such as eye drops.

[0132] Pharmaceutically acceptable carriers can contain physiologically acceptable agents, and their effect is, for example, to stabilize, increase solubility or increase the absorption of compounds such as the compounds 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 selection of pharmaceutically acceptable carriers (including physiologically acceptable agents) depends on, for example, the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-microemulsifying drug delivery system. The pharmaceutical composition (preparation) can also be a liposome or other polymer matrix, and for example, the compounds of the present invention can be incorporated therein. For example, liposomes comprising phospholipids or other lipids are nontoxic, physiologically acceptable and metabolizable carriers, and their preparation and application are relatively simple.

[0133] The pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration, including, for example, oral administration (e.g., as an infusion in an aqueous or non-aqueous solution or suspension, tablet, capsule (including spray capsules and gelatin capsules), bolus, powder, granules, paste applied to the tongue); absorption through the oral mucosa (e.g., sublingually); anus, rectum or vagina (e.g., as a vaginal suppository, cream or foam); parenteral administration (including intramuscular, intravenous, subcutaneous or intrathecal, e.g., as a sterile solution or suspension); nasal; intraperitoneal; subcutaneous; transdermal (e.g., applied to the skin as a patch); and topical (e.g., applied to the skin as a cream, ointment or spray, or as eye drops). The compound can also be formulated for inhalation. In certain embodiments, the compound can simply be dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for such routes of administration 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.

[0134] The preparation can be conveniently present in unit dosage form and can be prepared by any method well known in the pharmaceutical field. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the subject being treated, the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces the therapeutic effect. Typically, among the hundred percent, this amount will be in the range of about 1% to about 99%, preferably about 5% to about 70%, and most preferably about 10% to about 30% of the active ingredient.

[0135] The method for preparing these preparations or compositions comprises the step of combining active compound such as compound of the present invention with carrier and optional one or more auxiliary components.Usually, preparation is prepared by the following mode: compound of the present invention is combined with liquid carrier or finely divided solid carrier or both uniformly and intimately, and then (if necessary) the product is shaped.

[0136] Formulations of the present invention suitable for oral administration may be in the form of capsules (including spray capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth), lyophilizates, 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 gum arabic) and / or as a mouthwash, etc., each containing a predetermined amount of the compound of the present invention as the active ingredient. The composition or compound may also be administered as a bolus, electuary or paste.

[0137] To prepare solid dosage forms for oral administration (capsules (including spray capsules and gelatin capsules), tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as 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, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) wetting agents, Such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) solution retardants, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (8) absorbents, such as kaolin and bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, 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 spray capsules and gelatin capsules), tablets and pills, the pharmaceutical composition may also contain a buffer. 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 and the like.

[0138] Tablets can be prepared by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant or a dispersant. Molded tablets can be prepared by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine.

[0139] Tablets and other solid dosage forms of pharmaceutical compositions, such as dragees, capsules (including spray capsules and gelatin capsules), pills and granules, can be optionally scored or prepared with coatings and shells such as enteric coatings and other coatings well known in the field of pharmaceutical formulation. They can also be formulated to provide slow or controlled release of active ingredients therein using, for example, hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres in different proportions that provide the desired release profile. They can be sterilized by, for example, filtering through a bacteria-retaining filter or by incorporating a sterilizing agent in the form of a sterile solid composition, which can be dissolved in sterile water or some other sterile injectable medium just before use. These compositions can also optionally contain an opacifier, and can be compositions that release the active ingredient only or preferably in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. If appropriate, the active ingredient can also be in a microencapsulated form with one or more of the above-mentioned excipients.

[0140] Liquid dosage forms that can be used for oral administration include pharmaceutically acceptable emulsions, lyophilized agents for reconstitution, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage form may also contain an inert diluent commonly used in the art, such as, for example, water or other solvents, cyclodextrins and their derivatives, solubilizers and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (particularly cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuran alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0141] 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.

[0142] Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0143] Formulations of pharmaceutical compositions for rectal, vaginal or urethral administration may be presented as suppositories, which may 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, suppository wax or salicylates, 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.

[0144] The formulation of the pharmaceutical composition for administration to the oral cavity may be presented as a mouthwash, or an oral spray, or an oral ointment.

[0145] Alternatively or additionally, the composition can be formulated for delivery via a catheter, stent, wire or other intraluminal device. Delivery via such a device may be particularly useful for delivery to the bladder, urethra, ureter, rectum or intestine.

[0146] Formulations 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.

[0147] 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 pharmaceutically acceptable carrier and any preservatives, buffers or propellants that may be required.

[0148] Ointments, pastes, creams and gels may contain, in addition to the active compounds, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0149] In addition to the active compound, powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0150] Transdermal patches have the additional advantage of providing controlled delivery of the compounds of this invention to health. Such dosage forms can be prepared by dissolving or dispersing the active compound in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound through the skin. The rate of such flux can be controlled by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

[0151] Ophthalmic preparations, ophthalmic ointments, powders, solutions, etc. are also considered within the scope of the present invention. Exemplary ophthalmic preparations are described in U.S. 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 preparations have the characteristics of or are compatible with tears, aqueous humor, or vitreous humor. Preferred routes of administration are topical administration (e.g., topical administration, such as eye drops, or administration via implants).

[0152] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds and one or more pharmaceutically 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 prior to 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.

[0153] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. Suitable fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0154] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. The prevention of microbial action can be ensured by including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It can also be expected that isotonic agents such as sugar, sodium chloride, etc. are included in the composition. In addition, the extended absorption of injectable drug forms can be achieved by including agents that delay absorption such as aluminum monostearate and gelatin.

[0155] In some cases, in order to prolong the effect of the drug, it is desirable to slow down the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous substance with poor water solubility. The absorption rate of the drug depends on its dissolution rate, which in turn depends on crystal size and crystalline form. Alternatively, delayed absorption of a parenteral drug form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0156] Injectable reservoir forms are prepared by forming a microencapsulation matrix of the subject compound in a biodegradable polymer such as polylactide-polyglycolide. According to the ratio of drug to polymer and the properties of the specific polymer used, the drug release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Reservoir-type injectable formulations are also prepared by capturing the drug in a liposome or microemulsion compatible with body tissues.

[0157] For use in the methods of the invention, the active compound may be administered per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably, 0.5% to 90%) active ingredient and a pharmaceutically acceptable carrier.

[0158] The introduction method can also be provided by a rechargeable or biodegradable device. In recent years, various slow-release polymer devices have been developed and tested in vivo for the controlled delivery of drugs including protein biopharmaceuticals. A variety of biocompatible polymers (including hydrogels), including both biodegradable polymers and non-degradable polymers, can be used to form implants that continuously release compounds at specific target sites.

[0159] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0160] The selected dosage level will depend upon a variety of factors including the activity of the specific compound or combination employed, or the esters, salts or amides thereof, the route of administration, the time of administration, the rate of excretion of the specific compound employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the specific compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0161] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the therapeutically effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start the administration of a pharmaceutical composition or compound at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. "Therapeutically effective amount" means a concentration of a compound sufficient to cause the desired therapeutic effect. It is generally understood that the effective amount of a compound will vary according to the subject's weight, sex, age, and medical history. Other factors affecting 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 necessary, another type of therapeutic agent administered with the compound of the present invention. A larger total dose can be delivered by multiple administrations of the agent. Methods for determining efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13th edition, 1814-1882, incorporated herein by reference).

[0162] In general, a suitable daily dose of an active compound for use in the compositions and methods of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above.

[0163] If desired, the effective daily dose of the active compound can be administered in one, two, three, four, five, six or more sub-doses at appropriate intervals throughout the day, optionally in unit dosage form. In certain embodiments of the invention, the active compound can be administered twice or three times a day. In a preferred embodiment, the active compound will be administered once a day.

[0164] The patient receiving such treatment is any animal in need thereof, including primates, particularly humans, and other mammals such as horses, cattle, pigs, and sheep; and poultry and pets in general.

[0165] In certain embodiments, the compounds of the present invention can be used alone or in combination with another type of therapeutic agent. As used herein, the phrase "combined administration" refers to any form of administration of two or more different therapeutic compounds, so that the previously administered therapeutic compound is still effective in vivo while the second compound is administered (for example, the two compounds are effective in the patient at the same time, which can include the synergistic effect of the two compounds). For example, different therapeutic compounds can be administered simultaneously or sequentially in the same preparation or in separate preparations. In certain embodiments, different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours or a week of each other. Therefore, individuals receiving this treatment can benefit from the combined effects of different therapeutic compounds.

[0166] In certain embodiments, the compounds of the invention are administered in combination with one or more additional therapeutic agents (e.g., one or more additional chemotherapeutic agents) to provide improved efficacy relative to the administration of each of the compounds of the invention (e.g., compounds of Table 1A or 1B) or the one or more additional therapeutic agents alone. In certain such embodiments, the combined administration provides an additive effect, wherein additive effect refers to the sum of the effects of each of the compounds of the invention and the one or more additional therapeutic agents administered alone.

[0167] The present invention includes the use of pharmaceutically acceptable salts of the compounds of the present invention in the compositions and methods of the present invention. As used herein, the term "pharmaceutically acceptable salt" includes salts derived from inorganic or organic acids, and the acid includes, 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 a form in which the ratio of the molecule comprising the salt is not 1:1. For example, the salt may include more than one inorganic or organic acid molecule per molecule of base, such as two hydrochloric acid molecules per molecule of Table 1A, 1B and 2. As another example, the salt may include less than one inorganic or organic acid molecule per molecule of base, such as two molecules of Table 1A, 1B and 2 per molecule of tartaric acid.

[0168] In other embodiments, the expected salts of the present invention include but are not limited to alkyl, dialkyl, trialkyl or tetraalkyl ammonium salts. In certain embodiments, the expected salts of the present invention include but are not limited to the following salts: L-arginine, phenylethylamine, benzathine, betaine, calcium hydroxide, choline, dimethylethanolamine (deanol), diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucosamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2-hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine and zinc. In certain embodiments, the expected salts of the present invention include but are not limited to Na, Ca, K, Mg, Zn or other metal salts.

[0169] Pharmaceutically acceptable acid addition salts may also exist in the form of various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates may also be prepared. The source of such solvates may be from the solvent of crystallization, inherent to the solvent of preparation or crystallization, or foreign to such solvent.

[0170] Wetting agents, emulsifiers 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 compositions.

[0171] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0172] IV. Definitions

[0173] For the purposes of the present invention, the following definitions will apply (unless expressly stated otherwise):

[0174] As used herein, the term "acyl" is art-recognized and refers to a group represented by the general formula hydrocarbylC(O)-, preferably alkylC(O)-.

[0175] 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.

[0176] 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 referring to an alkenyl moiety having a substituent replacing a hydrogen on one or more carbons of the alkenyl. Such substituents may appear on one or more carbons that are included or not included in one or more double bonds. In addition, such substituents include all those expected for alkyl, as discussed below, unless stability is prohibited. For example, it is expected that alkenyl is substituted with one or more alkyl, carbocyclyl, aryl, heterocyclyl or heteroaryl.

[0177] As used herein, "alkyl" or "alkane" is a fully saturated straight or branched chain non-aromatic hydrocarbon. Typically, unless otherwise defined, a straight or branched chain alkyl has 1 to about 20 carbon atoms, preferably 1 to about 10 carbon atoms. Examples of straight and branched chain alkyls include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, amyl, and octyl. C1-C6 straight or branched chain alkyls are also referred to as "lower alkyls".

[0178] In addition, 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 referring to an alkyl moiety having a substituent replacing a hydrogen on one or more carbons of the hydrocarbon backbone. If not otherwise specified, such substituents may include, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinite, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, or aromatic or heteroaromatic moieties. It will be appreciated by those skilled in the art that the moiety substituted on the hydrocarbon chain may itself be substituted, if appropriate. For example, the substituent of the substituted alkyl can include amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinite), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate) and silyl, and ether, alkylthio, carbonyl (including ketone, aldehyde, carboxylate and ester), -CF , -CN etc. substituted and unsubstituted forms. Exemplary substituted alkyl is as described below. Cycloalkyl can be further substituted by alkyl, alkenyl, alkoxy, alkylthio, aminoalkyl, carbonyl substituted alkyl, -CF , -CN etc.

[0179] As used herein, the term “C x-y " when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is intended to include groups containing from x to y carbons in the chain. For example, the term "C x-y "Alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight and branched chain alkyl groups containing from x to y carbons in the chain, including halogenated alkyl groups such as trifluoromethyl and 2,2,2-trifluoroethyl. C0 alkyl indicates hydrogen, where the group is in the terminal position, or a bond if internal. The term "C 2-y "Alkenyl" and "C 2-y "Alkynyl" refers to a substituted or unsubstituted unsaturated aliphatic group analogous in length and possible substitution to the alkyl groups described above, but containing at least one double or triple bond, respectively.

[0180] As used herein, the term "alkynyl" refers to an aliphatic group containing at least one triple bond, and is intended to include both "unsubstituted alkynyls" and "substituted alkynyls," wherein the latter refers to alkynyl moieties having substituents replacing hydrogen on one or more carbons of the alkynyl. Such substituents may appear on one or more carbons that are included or not included in one or more triple bonds. In addition, such substituents include all those contemplated for alkyl groups, as discussed above, unless stability is prohibited. For example, substitution of an alkynyl group with one or more alkyl, carbocyclyl, aryl, heterocyclyl, or heteroaryl groups is contemplated.

[0181] As used herein, the term "amide" refers to a group

[0182]

[0183] Each R 10 independently represent hydrogen or a hydrocarbon group, or two R 10 Together with the N atom to which they are attached, the heterocycles have 4 to 8 atoms in the ring structure.

[0184] As used herein, the terms "amine" and "amino" are art-recognized and refer to both unsubstituted and substituted amines and salts thereof, for example, a moiety represented by

[0185]

[0186] Each R 10 independently represent hydrogen or a hydrocarbon group, or two R 10 Together with the N atom to which they are attached, the heterocycles have 4 to 8 atoms in the ring structure.

[0187] As used herein, the term "aryl" includes substituted or unsubstituted monocyclic aromatic groups, wherein 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 a polycyclic system having two or more rings, wherein two or more carbons are shared by two adjacent rings, wherein at least one ring is an aromatic ring, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclic. Aryl includes benzene, naphthalene, phenanthrene, phenol, aniline, etc.

[0188] As used herein, the terms "carbocycle" and "carbocyclic" refer to saturated or unsaturated rings, wherein each atom of the ring is carbon. The term carbocycle includes both aromatic carbocycles and non-aromatic carbocycles. Non-aromatic carbocycles include cycloalkane rings in which all carbon atoms are saturated and cycloolefin rings containing at least one double bond. "Carbocycle" includes 3-7 membered monocycles and 8-12 membered bicyclic rings. Each ring of the bicyclic carbocycle can be selected from saturated, unsaturated and aromatic rings. Carbocycles include bicyclic molecules, wherein one, two or three or more atoms are shared between two rings. The term "fused carbocycle" refers to a bicyclic carbocycle, wherein each ring shares two adjacent atoms with another ring. Each ring of the fused carbocycle can be selected from saturated, unsaturated and aromatic rings. In an exemplary embodiment, an aromatic ring such as a phenyl ring can be fused to a saturated or unsaturated ring, such as cyclohexane, cyclopentane or cyclohexene. As long as the valence permits, any combination of saturated, unsaturated and aromatic bicyclic rings is included in the definition of carbocycle. 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. "Carbocycles" may be substituted at any one or more positions capable of carrying a hydrogen atom.

[0189] As used herein, "cycloalkyl" is a fully saturated cyclic hydrocarbon. "Cycloalkyl" includes monocyclic and bicyclic rings. Typically, unless otherwise defined, monocyclic cycloalkyl has 3 to about 10 carbon atoms, more typically 3 to 8 carbon atoms. 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 ring shares two adjacent atoms with another ring. The second ring of a fused bicyclic cycloalkyl can be selected from saturated, unsaturated and aromatic rings. "Cycloalkenyl" is a cyclic hydrocarbon containing one or more double bonds.

[0190] As used herein, the terms "halo" and "halogen" mean halogen and include chlorine, fluorine, bromine and iodine.

[0191] As used herein, the terms "hetaralkyl" and "heteroaralkyl" refer to an alkyl group substituted with a heteroaryl group.

[0192] As used herein, the term "heteroalkyl" refers to a saturated or unsaturated chain of carbon atoms and at least one heteroatom, wherein no two heteroatoms are adjacent.

[0193] As used herein, the terms "heteroaryl" and "hetaryl" include substituted or unsubstituted aromatic monocyclic structures, preferably 5- to 7-membered rings, more preferably 5- to 6-membered rings, whose ring structures contain at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heteroaryl" and "hetaryl" also include polycyclic systems having two or more rings, wherein two or more carbon atoms are shared by two adjacent rings, wherein at least one ring is heteroaromatic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl. Heteroaryl includes, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, etc.

[0194] As used herein, the term "heteroatom" means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen and sulfur.

[0195] As used herein, the terms "heterocyclyl", "heterocycle" and "heterocyclic" refer to substituted or unsubstituted non-aromatic ring structures, preferably 3-10-membered rings, more preferably 3-7-membered rings, whose ring structures contain at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The terms "heterocyclyl" and "heterocyclic" also include polycyclic ring systems having two or more rings, wherein two or more carbons are shared by two adjacent rings, wherein at least one ring is heterocyclic, for example, the other rings may be cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl. Heterocyclyl includes, for example, piperidine, piperazine, pyrrolidine, morpholine, lactone, lactam, etc. Heterocyclyl may also be substituted by oxo groups. For example, "heterocyclyl" encompasses both pyrrolidine and pyrrolidone.

[0196] As used herein, the term "hydrocarbyl" refers to a group bonded by a carbon atom that does not have a =O or =S substituent, and typically has at least one carbon-hydrogen bond and a main chain that is primarily carbon, but may optionally contain heteroatoms. Therefore, for the purposes of this application, groups such as methyl, ethoxyethyl, 2-pyridyl, and trifluoromethyl are considered to be hydrocarbyl, but substituents such as acetyl (which has a =O substituent on the connecting carbon) and ethoxy (which is connected by oxygen rather than carbon) are not considered to be hydrocarbyl. Hydrocarbyl includes, but is not limited to, aryl, heteroaryl, carbocycle, heterocyclyl, alkyl, alkenyl, alkynyl, and combinations thereof.

[0197] As used herein, the term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group.

[0198] As used herein, the term "lower" when used in conjunction with a chemical moiety such as acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy, is intended to include groups in which there are ten or less, preferably six or less non-hydrogen atoms in the substituent." Lower alkyl" for example refers to an alkyl group containing ten or less, preferably six or less carbon atoms. In certain embodiments, the acyl, acyloxy, alkyl, alkenyl, alkynyl or alkoxy substituents defined herein are respectively lower acyl, lower acyloxy, lower alkyl, lower alkenyl, lower alkynyl or lower alkoxy, whether they occur alone or in combination with other substituents, such as in describing hydroxyalkyl and aralkyl (in this case, for example, when counting the carbon atoms in the alkyl substituent, the atoms in the aryl are not counted).

[0199] As used herein, the term "oxo" refers to a carbonyl group. When an oxo substituent is present on an otherwise saturated group, such as in the case of 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 with an "oxo" group, this may mean that a carbonyl moiety (i.e., -C(=O)-) replaces a methylene unit (i.e., -CH2-).

[0200] As used herein, the terms "polycyclic group", "polycyclic" and "polycyclic" refer to two or more rings (e.g., cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl and / or heterocyclyl) wherein two or more atoms are common to two adjacent rings, e.g., the rings are "fused rings". Each ring of the polycyclic ring may be substituted or unsubstituted. In certain embodiments, each ring of the polycyclic ring contains 3 to 10 atoms, preferably 5 to 7 atoms, in the ring.

[0201] As used herein, the term "substituted" refers to the part of the substituent having a hydrogen on one or more carbons of the replacement main chain. It should be understood that "replacement" or "substituted with ... " includes implicit conditions, i.e., such substitution is based on the allowed valence of the substituted atom and the substituent, and the substitution produces a stable compound, for example, the compound will not spontaneously undergo transformation, such as by rearrangement, cyclization, elimination, etc. As used herein, the term "substituted" is expected to include all permissible substituents of organic compounds. In a broad sense, permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. For appropriate organic compounds, permissible substituents can be one or more and are the same or different. For purposes of the present invention, heteroatoms such as nitrogen can have hydrogen substituents and / or any permissible substituents of organic compounds described herein that satisfy the valence of heteroatoms. Substituents can include any substituent described herein, for example, halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl or acyl), thiocarbonyl (such as thioester, thioacetate or thioformate), alkoxy, phosphoryl, phosphate, phosphonate, phosphinite, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclic, aralkyl or aromatic or heteroaromatic moiety. It will be appreciated by those skilled in the art that, if appropriate, the substituent itself can be substituted. Unless specifically stated as "unsubstituted", reference to chemical moieties herein should be understood to include substituted variants. For example, reference to "aryl" or moieties implicitly includes both substituted variants and unsubstituted variants.

[0202] As used herein, the term "administering" means the actual physical introduction of a composition into or onto a subject, as the case may be. Any and all methods of introducing a composition into a subject are contemplated according to the present invention; the method is not dependent on any particular means of introduction and should not be so construed. Means of introduction are well known to those skilled in the art and are also exemplified herein.

[0203] As used herein, the terms "effective amount," "effective dose," "sufficient amount," "amount effective to," "therapeutically effective amount," or their grammatical equivalents, mean a dose sufficient to produce the desired result, relieve or in some way lessen the symptoms or stop or reverse the progression of a condition and provide subjective relief of symptoms or an objectively identifiable improvement as noted by a clinician or other qualified observer. Relief of symptoms of a particular condition by administration of a pharmaceutical composition described herein refers to any relief, whether permanent or temporary, long-lasting or transient, that may be associated with the administration of the pharmaceutical composition.

[0204] As used herein, the term "prodrug" is intended to encompass compounds that are converted to therapeutic agents of the present invention under physiological conditions. A common method for preparing a prodrug is to include one or more selected moieties that are hydrolyzed under physiological conditions to reveal the desired molecule. In other embodiments, the prodrug is converted by the enzymatic 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 may be substituted with corresponding suitable prodrugs, for example, wherein the hydroxyl group in the parent compound exists as an ester, or the carbonic acid or carboxylic acid present in the parent compound exists as an ester.

[0205] As used herein, the term "pharmaceutically acceptable" refers to a composition that is physiologically tolerable and does not typically produce allergic or similar adverse reactions when administered to a subject, preferably a human subject. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals and more particularly in humans.

[0206] As used herein, the phrase "pharmaceutically acceptable carrier" means a pharmaceutically 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 injurious to the patient. Some examples of materials that can serve as pharmaceutically 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, ethyl cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, , safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols such as glycerol, 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) ethanol; (20) phosphate buffer solution; and (21) other nontoxic compatible substances used in pharmaceutical preparations.

[0207] As used herein, a therapeutic agent that "prevents" a disorder or condition refers to a compound that, in a statistical sample, reduces the occurrence of the disorder or condition in a treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample.

[0208] As used herein, "subject" means a human or an animal (in the case of an animal, more typically a mammal). In one aspect, the subject is a human.

[0209] As used herein, the term "treating" is art-recognized and includes administering one or more of the subject compositions to a host, e.g., to reduce, alleviate, or stabilize an existing undesirable condition or its side effects. All definitions, as defined and used herein, should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or the ordinary meaning of the defined terms.

[0210] Example

[0211] Example 1: Synthesis of Compound 1.

[0212] Solution 1

[0213]

[0214] like Figure 1 As shown in Figure 2, compound 1 has an IC in an in vitro NNMT enzyme inhibition assay. 50 =105nM.

[0215] Example 2: Synthesis of compound 11.

[0216] Solution 2

[0217]

[0218] Compound 2

[0219] At -78 ° C under N2 atmosphere, n-butyl lithium solution (2.5M in hexane, 18.3 mL, 45.80 mmol, 1.20 equiv) was added dropwise to a stirred mixture of 4-chloro-2-fluoro-pyridine (5.0 g, 38.17 mmol, 1.00 equiv) in THF (20.0 mL). The resulting mixture was stirred at this temperature for 0.5 h. At -78 ° C, a solution of CuI (290.0 mg, 1.53 mmol, 0.04 equiv) and tert-butyl 2,2-dioxothiazolidine-3-carboxylate (8.1 g, 34.4 mmol, 0.90 equiv) in THF (20.0 mL) was added thereto, 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 in vacuo. 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)-1-methyl-ethyl]carbamate (4.8 g, 17.5%) as a white solid. 1 HNMR (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) + .

[0220] Compound 2 has an IC of 50 =53-150nM.

[0221] Compound 3

[0222] A mixture of tert-butyl N-[rac-(1R)-2-(4-chloro-6-fluoro-3,4-dihydropyridin-5-yl)-1-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)-1-(4-chloro-2-fluoropyridin-3-yl)propan-2-amine 2,2,2-trifluoroacetate (crude, 4.2 g) as a light yellow oil, which was used in the next step without further purification. MS (ESI, m / z): 189, 191 (M+H) + .

[0223] Compound 4

[0224] A mixture of (R)-1-(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 in vacuo. The residue was purified by reverse phase flash chromatography on C18 gel (0 to 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 light 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) + .

[0225] Compound 5

[0226] 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 3 times with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo. 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 HNMR(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) + .

[0227] Compound 6

[0228] 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 under N2 atmosphere for 8 h. The resulting mixture was diluted with water and extracted 3 times with EtOAc. The organic layers were combined, washed with brine, and concentrated in vacuo. The residue was purified by reverse phase flash chromatography on C18 gel (0 to 80% acetonitrile in water with 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. 1 H 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) + .

[0229] Compound 7

[0230] 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 and basified to pH 10 with NaOH (1 M) 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 product, 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) + .

[0231] Compound 11

[0232] 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) was added ammonium formate (71.8 mg, 1.14 mmol, 3.00 equiv) at ambient temperature. The resulting mixture was stirred at 70 ° C for 3 h and then filtered through a celite pad. The filtrate was concentrated in vacuo. 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) + .

[0233] Example 3: Synthesis of additional compounds.

[0234] Compound 2 was prepared via the synthetic route outlined in Scheme 3:

[0235] Solution 3

[0236]

[0237] Compound 3 was prepared via the synthetic route outlined in Scheme 4:

[0238] Solution 4

[0239]

[0240] Compound 5 was prepared via the synthetic route outlined in Scheme 5:

[0241] Solution 5

[0242]

[0243] Compound 6 was prepared via the synthetic route outlined in Scheme 6:

[0244] Solution 6

[0245]

[0246] Compound 7 was prepared via the synthetic route outlined in Scheme 7:

[0247] Solution 7

[0248]

[0249] Compound 8 was prepared via the synthetic route outlined in Scheme 8:

[0250] Solution 8

[0251]

[0252] Compound 15 was prepared via the synthetic route outlined in Scheme 9:

[0253] Solution 9

[0254]

[0255] Compound 16 was prepared via the synthetic route outlined in Scheme 10:

[0256] Solution 10

[0257]

[0258] Compound 18 was prepared via the synthetic route outlined in Scheme 11:

[0259] Solution 11

[0260]

[0261] The enantiomers of compound 18, i.e., compounds 19A and 19B, were separated using chiral chromatography. 24 mg of compound 18 were chirally separated by HPLC under basic conditions to obtain 5.9 mg of the first enantiomer identified by the first peak (peak-1: rt=2.134 min, ee=100%, chemical purity=99.62%) and 4.8 mg of the second enantiomer identified by the second peak (peak-2: 2.933 min, ee=99.65%, chemical purity=99.60%). The preparative HPLC conditions are provided below.

[0262]

[0263]

[0264] Compound 21 was prepared via the synthetic route outlined in Scheme 12:

[0265] Solution 12

[0266]

[0267] Compound 24 was prepared via the synthetic route outlined in Scheme 13:

[0268] Solution 13

[0269]

[0270] Compound 28 was prepared via the synthetic route outlined in Scheme 14:

[0271] Solution 14

[0272]

[0273] Compound 26B was prepared by the synthetic route outlined in Scheme 15:

[0274] Solution 15

[0275]

[0276] Example 4: Biological Data

[0277] 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 1X 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 allowed to proceed for 20 minutes at room temperature. MTase-Glo reagent and MTase-Glo detection solution were added according to the manufacturer's instructions, and the luminescent signal was measured with a SpectraMax i3 plate reader, and the data was analyzed with GraphPad Prism version 9. Scheme 16 depicts a visual summary of the Promega MTase-Glo methyltransferase assay for evaluating the biochemical activity of the novel inhibitor.

[0278] Compound 11 (IC 50 =35-42nM), 18 (IC 50 =848nM), 19A (IC 50 =28-43 nM) and 19B (IC 50 =5.1 μM). The NNMT biochemical activity of compound 26A was also evaluated (IC 50 =20 nM).

[0279] Scheme 16

[0280]

[0281] After incubating the compound overnight at different concentrations, the cell potency in K562 (ATCC) cells was measured by measuring the level of (1-methylnicotinamide) 1-MNA. The cells were plated at 1 million / well and incubated for 24h with the compound at the specified concentration. The cells were collected by pipetting and extracted with acetonitrile containing 10ng / ml d4-1-MNA of the internal deuterated standard 200ul. Samples were analyzed on Agilent 64-60Triple Quad LC / MS and Agilent 1290Infinity HPLC using Restek Allure 5um PPFP column 150mm x 2.1. Mobile phase A was an aqueous solution of 2.5mM ammonium formate, and mobile phase B was methanol containing 0.1% formic acid, delivered at 0.25mL / min. The capillary voltage was set to 2400V and the nozzle voltage was set to 300V. 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. The mass transitions were m / z 137.1 to 94.1 for 1-MNA and m / z 141.1 to 98.1 for d4-MNA.

[0282] Compound 11 (IC 50 =38nM), 19A (IC 50 =12 nM) for cell potency assay. Compound 26A was also tested for cell potency (IC 50 =114nM).

[0283] Example 5: Microsome Stability

[0284] Table 3 summarizes the results of metabolic stability assays performed with (R)-11 in pooled human and male mouse liver microsomes.

[0285] Table 3

[0286]

[0287] Prepare stock solutions according to the table below.

[0288] Reagents Stock solution concentration volume Final concentration Phosphate buffered saline 200mM 200μL 100mM <![CDATA[Ultra-pure H2O]]> - 106μL - <![CDATA[MgCl2 solution]]> 50mM 40μL 5mM Microsomes 20mg / mL 10 μL 0.5mg / mL

[0289] Add 40 μL of 10 mM NADPH solution to each well. The final concentration of NADPH is 1 mM. Preheat the mixture at 37 °C for 5 minutes. Prepare a negative control sample by replacing the NADPH solution with 40 μL of ultrapure H2O. The negative control is used to exclude misleading factors caused by the instability of the chemical itself. Prepare samples with NADPH in duplicate. Prepare negative controls in single copies.

[0290] The reaction was started by adding 4 μL of 200 μM control compound or test compound solution. Verapamil was used as a positive control in this study. The final concentration of the test compound or control compound was 2 μM. Aliquots of 50 μL were taken from the reaction solution at 0, 15, 30, 45 and 60 minutes. The reaction was terminated by adding 4 volumes of cold acetonitrile with IS (100 nM alprazolam, 200 nM imipramine, 200 nM labetalol and 2 μM ketoprofen). The sample was centrifuged at 3,220 g for 40 minutes. An aliquot of 90 μL of the supernatant was mixed with 90 μL of ultrapure H2O and then used for LC-MS / MS analysis.

[0291] For metabolite standard samples: 1 μL of 400 μM working solution was added to 89 μL of stock solution, followed by 400 μL of cold acetonitrile with internal standard (IS) (100 nM alprazolam, 200 nM imipramine, 200 nM labetalol, and 2 μM ketoprofen). Then, 10 μL of 10 mM NADPH solution or ultrapure H2O was added to the mixture. The samples were centrifuged at 3,220 g for 40 minutes to precipitate the proteins. An aliquot of 90 μL of supernatant diluted with 90 μL of water was used for LC-MS / MS analysis.

[0292] Example 6: Permeability

[0293] Table 4 summarizes the results of the permeability assays performed with 11 in Caco-2 cells. Table 5 summarizes the results of the bidirectional assays performed with 11 in Caco-2 cells.

[0294] Table 4

[0295]

[0296] Table 5

[0297]

[0298] Preparation of Caco-2 cells: Add 50 μL and 25 mL of cell culture medium to each well of the Transwell chamber and reservoir, respectively. Then incubate the HTS transwell plate at 37°C, 5% CO2 for 1 hour before cell seeding. Dilute Caco-2 cells to 6.86x10 5 Cells / mL, and 50 μL of cell suspension was dispensed into the filter wells of a 96-well HTS Transwell plate. Cells were cultured in a cell culture incubator at 37°C, 5% CO2, 95% relative humidity for 14-18 days. Cell culture medium was changed every two days, starting no later than 24 hours after initial plating.

[0299] 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.

[0300] Evaluation of cell monolayer integrity: The medium was removed from the reservoir and each Transwell chamber and replaced with pre-warmed fresh medium. The trans-epithelial electrical resistance (TEER) on the monolayer was measured using the Millicell Epithelial Volt-Ohm measurement system (Millipore, USA). Once the measurement was completed, the plate was returned to the incubator.

[0301] Assay procedure: The Caco-2 plate was removed from the incubator and washed twice with preheated HBSS (10mM HEPES, pH 7.4) and then incubated at 37°C for 30 minutes. The stock solutions of the control and test compounds were diluted in DMSO to obtain a 1mM solution and then diluted with HBSS (10mM HEPES, pH 7.4) to obtain a 5μM working solution. The final concentration of DMSO in the incubation system was 0.5%. In order to determine the rate at which the drug is transported in the apical to basolateral direction. 125μL of the test compound was added to the Transwell chamber (apical compartment) and 50μL of the sample (D0 sample) was immediately transferred from the apical compartment to a new 96-well plate. The wells in the receiver plate (basolateral compartment) were filled with 235μL HBSS (10mM HEPES, pH 7.4). In order to determine the rate at which the drug is transported in the basolateral to apical direction. 285 μL of test compound was added to the receiver plate well (basolateral compartment), and 50 μL of sample (D0 sample) was immediately transferred from the basolateral compartment to a new 96-well plate. The Transwell chamber (apical compartment) was filled with 75 μL of HBSS (10 mM HEPES, pH 7.4). The 0 time sample was prepared by transferring 50 μL of 5 μM working solution to the wells of a 96-deep well plate, followed by the addition of 200 μL of cold acetonitrile containing 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 hours. At the end of the incubation, 50 μL samples from the donor side (apical compartment for Ap→Bl flux, and basolateral compartment for Bl→Ap) and the receiver side (basolateral compartment for Ap→Bl flux, and apical compartment for Bl→Ap) were transferred to the wells of a new 96-well plate, followed by the addition of 4 volumes of cold methanol containing appropriate internal standards (100 nM alprazolam, 200 nM labetalol, 200 nM caffeine, and 200 nM diclofenac). The samples were vortexed for 5 minutes and then centrifuged at 3,220 g for 40 minutes. An aliquot of 100 μL of the supernatant was mixed with 100 μL of ultrapure water for LC-MS / MS analysis. To determine the leakage of Lucifer Yellow after a 2-hour 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 chamber (apical compartment), followed by filling the wells in the receiver plate (basolateral compartment) with 300 μL HBSS (10 mM HEPES, pH 7.4). The plate was incubated at 37°C for 30 minutes. 80 μL of sample was removed directly from the apical and basolateral wells (using the basolateral access wells) and transferred to the wells of a new 96-well plate.Lucifer Yellow fluorescence (to monitor monolayer integrity) signal was measured at 485 nM excitation and 530 nM emission in a fluorescence plate reader.

[0302] Example 7: Pharmacokinetic and pharmacodynamic studies

[0303] Table 6A summarizes the results of the mouse PK study with 11.

[0304] Table 6A

[0305]

[0306]

[0307] a The formulation is 0.2% Tween 80, 10% PEG300, b The formulation is 0.2% Tween 80, 20% PEG300

[0308] Table 6B summarizes the results of additional mouse PK studies conducted with 11.

[0309] Table 6B

[0310]

[0311] a The formulation is 0.2% Tween 80, 10% PEG300, b The formulation is 0.2% Tween 80, 20% PEG300

[0312] Table 7 summarizes the results of additional mouse PK studies conducted with 11.

[0313] Table 7.

[0314]

[0315] Table 8 summarizes the PK parameters of 1-MNA in mice after dosing with 11.

[0316] Table 8

[0317]

[0318] a The a formulation is 0.2% Tween 80, 10% PEG300, and the b formulation is 0.2% Tween 80, 20% PEG300

[0319] Doses were prepared fresh on the day of dosing. The vehicle compositions for each dose are listed below. For IV, the appropriate amount of the test article was dissolved in the vehicle while vortexing and / or sonicating to obtain a solution with the desired concentration level. For PO, the appropriate amount of the test article was dissolved in an appropriate vehicle; vortexing and / or sonicating may be used to obtain a solution formulation with the desired concentration level.

[0320] IV (compound): 0.5% Tween 80 in saline

[0321] 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

[0322] Study groups and dosing information are shown in the table below.

[0323] Dosage and route of administration

[0324]

[0325]

[0326] After dosing, 0.03 mL blood samples were collected from the dorsal plantar vein at the following time points (0.2 mL was collected via cardiac puncture at the last time point):

[0327] IV: before administration, 0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, 24 hours after administration

[0328] PO: before administration, 0.25, 0.5, 1, 2, 4, 6, 8, 24 hours after administration

[0329] Blood from each sample was transferred to a plastic microcentrifuge tube with EDTAK2 as 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.

[0330] The samples were analyzed on an AB Sciex Triple Quad 5500 LC / MS / MS instrument using a Gemini 5 μm C18 110A 150 x 3 mm column (in the case of JBS NF-000265, XBridge BEH C18 2.5 μm 4.6 x 75 mm) with the following equipment installed.

[0331] Prominence Degasser DGU-20A5R(C), serial number L20705414138IX; Liquid Chromatograph LC-30AD, serial numbers L20555408197AE and L20555408195AE; Communication Bus Module CBM-20A, serial number L20235429486CD; Automatic Sampler SIL-30AC, serial number L20565403814AE; Rack Changer II: L20585400900SS

[0332] 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.

[0333] Sample preparation: The required series of concentrations of the working solution was achieved by diluting the stock solution of the analyte with 50% acetonitrile in water. 5 μL of the working solution (1, 2, 3, 5, 10, 50, 100, 500, 1000 ng / mL) was added to 5 μL of blank C57Bl6 / N mouse plasma to obtain calibration standards ranging 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 of plasma were prepared independently of those samples used for the calibration curve. These QC samples were prepared in the same manner as the calibration standards on the day of analysis.

[0334] 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 D4-1-MNA 50 ng / mL) to precipitate the protein. Then, the sample was vortexed for 30 s. After centrifugation at 4 ° C, 4,000 rpm for 15 min, the supernatant was diluted 3 times with water. 10 μL of the diluted supernatant (20 μL in the case of JBS NF-000265) was injected into the LC / MS / MS system for quantitative analysis.

[0335] Sample preparation: 1-Methylnicotinamide (1-MNA): The desired concentration series of working solutions was achieved 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 ranging 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 of water were prepared independently of those samples used for the calibration curve. These QC samples were prepared in the same manner as the calibration standards on the day of analysis.

[0336] 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 D4-1-MNA 100 ng / mL) to precipitate the protein. Then, the sample was vortexed for 30 seconds. After centrifugation at 4°C and 4000 rpm for 15 minutes, 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.

[0337] Recovery: 5 μL of working solution (10, 100, 800 ng / mL) was added to 5 μL of blank C57Bl6 / N mouse plasma to obtain three samples of 10 ng / mL, 100 ng / mL, and 800 ng / mL. 30 μL of each sample was added to 100 μL of acetonitrile containing IS mixture to precipitate the protein. Then, the sample was vortexed for 30 s. After centrifugation at 4°C and 4000 rpm for 15 min, 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.

[0338] Example 8: pH Dependent Solubility

[0339] pH-dependent solubility of 11 ( Figure 6 ) is analyzed according to the following procedure:

[0340] Preparation of stock solutions: Prepare stock solutions of test and control compounds at a concentration of 30 mM (50 mM: R-NA47) in DMSO. Dilute the 50 mM DMSO compound stock solution to 30 mM with DMSO. Prepare a stock solution of control compound at a concentration of 30 mM in DMSO. Diclofenac was used as a positive control in this assay.

[0341] Procedure for solubility determination: 10 μL of each compound stock solution was placed in their appropriate 96-well holders in turn, followed by 990 μL of PBS at pH 1.5, pH 3.0, pH 4.5, pH 6.8, pH 7.4 and pH 9.0 added to each vial of the uncapped solubility sample plate. This study was performed in duplicate. A stirring bar was added to each vial, and the vials were then sealed using molded PTDE / SIL 96-well plate covers. The solubility sample plate was transferred to a comfortable constant temperature mixer plate oscillator and incubated at 1100 rpm shaking for 2 hours at RT. After 2 hours of incubation, the stirring bar was removed using a large magnet, and all samples from the solubility sample plate were transferred to a filtration plate. All samples were filtered using a vacuum manifold. The filtered samples were diluted with methanol. The dilution factor can be changed according to the solubility value and LC / MS signal response.

[0342] Preparation of 0.3 μM Standard (STD): 30 mM DMSO compound stock was diluted to 300 μM with DMSO and then diluted with methanol to obtain 0.3 μM STD.

[0343] 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 relative to standards of known concentration in DMSO using LC combined with mass spectrometry peak identification and quantification. Solubility values ​​for test compounds were calculated as follows:

[0344]

[0345] DF = dilution factor

[0346] Example 9: hERG Safety Assessment

[0347] The hERG safety of compound 11 was evaluated according to the following protocol: Figure 8 ).

[0348] Cell lines and cell culture: The hERG stably expressing HEK 293 cell line (Catalog No. K1236) was purchased from Invitrogen. The cells were 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. TrypLE TM Express cells were split approximately three times per week and maintained at approximately 40% to 80% confluence. Prior to the assay, cells were plated at 5x10 5 Cells / 6 cm cell culture dish were plated on coverslips and induced with doxycycline at 1 μg / mL for 48 hours.

[0349] Solution preparation: 1) Extracellular solution (in mM): 132 NaCl, 4 KCl, 3 CaCl2, 0.5 MgCl2, 11.1 glucose and 10 HEPES (pH adjusted to 7.35 with NaOH); 2) Intracellular solution (in mM): 140 KCl, 2 MgCl2, 10 EGTA, 10 HEPES and 5 MgATP (pH adjusted to 7.35 with KOH).

[0350] Preparation of working solutions of test compounds: 1) initially prepare the test compounds in DMSO with a final concentration of 10 mM as stock solutions; 2) then serially dilute the stock solution of each compound with DMSO at a ratio of 1:3 to prepare another 3 intermediate solutions including 3.33 mM, 1.11 mM, and 0.37 mM; 3) prior to hERG assay, working solutions were prepared by diluting the 10 mM, 3.33 mM, 1.11 mM, and 0.37 mM intermediate solutions 1000-fold with extracellular solution to give final concentrations of the working solutions of 30 mM, 10 mM, 3.33 mM, 1.11 mM, and 0.37 mM, while the 30 μM working solution was prepared by diluting the 10 mM DMSO stock solution 333.333-fold. The final DMSO concentration was in the range of 0.1%-0.3%; 4) hERG current was measured in the presence of 5 doses including 30 μM, 10 μM, 3.33 μM, 1.11 μM and 0.37 μM for IC50 determination.

[0351] Experimental Procedure: 1) Remove the coverslip from the cell culture dish and place it on the microscope stage in a thermostatic bath; 2) Locate the desired cell using a x10 objective. Position the tip of the electrode under the microscope by focusing above the plane of the cell using the x10 objective. Once the tip is in focus, use the coarse control of the manipulator to advance the electrode down toward the cell while moving the objective to keep the tip in focus; 3) When directly above the cell, switch to the x40 objective and use the fine control of the manipulator to approach the surface of the cell in small steps; 4) Apply gentle suction through the side port of the electrode holder to form a gigaohm seal; 5) Use C 快 The whole-cell configuration is obtained by applying repeated, brief, strong suction until the membrane ruptures; 6) At this point the membrane potential is set to -60 mV to ensure that the hERG channels are not open. The C 慢Eliminate the spike of capacitive current; 7) Set the holding potential to -90mV and hold for 500ms; Record the current at 20kHz and filter at 10kHz. Test the leakage current at -80mV and hold for 500ms; 8) Elicit the hERG current by depolarizing at +30mV for 4.8 seconds, then restore the voltage to -50mV and hold for 5.2 seconds to remove the inactivation and observe the tail current of inactivation. The hERG current amplitude is determined using the maximum amount of the tail current size; 9) Record the current for 120 seconds to evaluate the current stability. Only stable cells with recording parameters above the threshold are applied to drug administration; 10) First, the vehicle control is applied to the cell to establish a baseline. Once the hERG current is found to be stable for 5 minutes, the working solution is applied. The hERG current in the presence of the test compound is recorded for about 5 minutes to reach steady state, and then 5 scans are captured. For dose-response testing, 5 doses of test compounds are cumulatively applied to cells from low concentration to high concentration. After hERG current measurement at the highest concentration of the test compound, the positive control dofetilide at a concentration of 150 nM was also applied to each cell as an internal low control to normalize the inhibition percentage. To ensure good performance and handling of the cultured cells, the same batch of cells was also tested with 5 doses of dofetilide.

[0352] Data Acceptance Criteria: The following criteria were used to determine data acceptability: 1) initial seal resistance >1 GΩ; 2) leakage current <50% of the control peak tail current at any time; 3) peak tail amplitude >250 pA; 4) membrane resistance Rm >500 MΩ; 5) access resistance (Ra) <10 MΩ; 6) apparent decrease in peak current <2.5% / min.

[0353] Data analysis: Data that met the above criteria for hERG current quality were further analyzed according to the following steps: 1) Calculate the hERG current inhibition percentage using the following equation; 2) Plot the dose-response curve of the test compound with the hERG current inhibition percentage versus the concentration of the test compound using Graphpad Prism 8.0, and fit a sigmoidal dose-response curve with a variable slope.

[0354]

[0355] Example 10: CYP inhibition studies

[0356] Inhibition studies of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E, and CYP3A4 were performed according to the following protocol ( Fig. 9 ). Compound 11 did not inhibit any of the CYP isoforms tested.

[0357] Inhibition of 11 in human liver microsomes

[0358] 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-benzyldesmethylmephenytoin, CYP2D6: quinidine, CYP2E1: disulfiram and CYP3A4: ketoconazole) were transferred to the "Compound Plate". The concentrations of the test compounds were 0, 0.6, 2, 6, 20, 60, 2000, 6000 and 20000 μM. The concentrations of the positive control compounds were 0, 0.2, 1, 2, 10, 50, 200, 2000 and 10000 μM.

[0359] 2. Prepare the stock solution according to Tables 9A and 9B and preheat in a 37°C water bath for 5 minutes. Transfer 179 μL of the stock solution to the "Incubation Plate". In the mixing system, the final concentrations of the test compound are 0, 0.003, 0.01, 0.03, 0.1, 0.3, 10, 30 and 100 μM, and the final concentrations of the positive control compound are 0, 0.001, 0.005, 0.01, 0.05, 0.25, 1, 10 and 50 μM. All experiments were performed in duplicate.

[0360] 3. Start the reaction by adding 20 μL of 10 mM NADPH solution, final concentration is 1 mM, and run at 37°C.

[0361] 4. The reaction was terminated 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" at the designated time points listed in Table 9B. The "Incubation Plate" was centrifuged at 3,220 g for 40 minutes to precipitate the protein. An aliquot of 100 μL of the supernatant was diluted with 100 μL ultrapure H2O, and the mixture was used for LC-MS / MS analysis.

[0362] 5. Data Analysis - All calculations were performed using Microsoft Excel. Metabolite formation was analyzed by using LC-MS / MS. The reduction in metabolite formation relative to vehicle control in terms of peak area was used to calculate IC using Excel XLfit. 50 Value (concentration of test compound producing 50% inhibition).

[0363] Table 9A. Preparation of stock solutions

[0364]

[0365]

[0366] Table 9B. Substrate information

[0367] CYP isoforms Substrate Stock solution concentration Final concentration time 1A2 Phenacetin 8mM 40μM 20 minutes 2A6 Coumarin 400μM 2μM 5 minutes 2B6 Bupropion 10mM 50μM 20 minutes 2C8 Paclitaxel 1mM 5μM 10 minutes 2C9 Tolbutamide 40mM 200μM 20 minutes 2C19 Mephenytoin 10mM 50μM 20 minutes 2D6 Dextromethorphan 2mM 10μM 20 minutes 2E1 Chlorzoxazone 10mM 50μM 20 minutes 3A4 Midazolam 1mM 5μM 5 minutes 3A4 Testosterone 10mM 50μM 10 minutes

[0368] Evaluation of the direct and time-dependent inhibition of CYP3A4 by 11

[0369] 1. Prepare stock solutions according to Table 10A. Immediately before use, prepare substrate solutions in a mixture of acetonitrile and ultrapure H2O (1:9, v:v) as listed in Table 10A.

[0370] 2. Preheat the "stock solution" at 37°C for 5 minutes. Transfer 169 μL of the "pre-incubation solution" and 1 μL of test compounds at various concentrations (0, 0.6, 2, 6, 20, 60, 2000, 6000 and 20000 μM) or 1 μL (0, 0.2, 1, 2, 10, 50, 200, 2000 and 10000 μM) of positive control compounds from the "compound plate" to the "incubation plate".

[0371] 3. For 0 min pre-incubation, add 10 μL of substrate to the incubation plate, then add 20 μL of 10 mM NADPH solution to start the reaction at a final concentration of 1 mM, then incubate for the specified times listed in Table 10B.

[0372] 4. For the 30 min pre-incubation in the absence of NADPH, the incubation plate was pre-incubated for 30 minutes in a 37°C water bath. After the 30-minute 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. The incubation was then continued for the specified time listed in Table 10B.

[0373] 6. Data Analysis - All calculations were performed using Microsoft Excel. Metabolite formation was analyzed by using LC-MS / MS. Three IC values ​​were calculated using the reduction in metabolite formation relative to vehicle control as peak area ratios using Prism 5.0 software (Graphpad). 50 Values ​​(0 min preincubation, 30 min preincubation with NADPH and 30 min preincubation without NADPH). Calculate IC 50 displacement to evaluate the mechanism of inhibition.

[0374] Table 10A. Preparation of stock solutions

[0375] Reagents Stock solution concentration volume Final concentration <![CDATA[MgCl2 solution]]> 50mM 20μL 5mM Phosphate buffered saline 200mM 100 μL 100mM <![CDATA[Ultra-pure H2O]]> - 48μL - Human liver microsomes 20mg / mL 1 μL 0.1mg / mL

[0376] Table 10B. Substrate information

[0377] CYP isoforms Control compound Substrate Working concentration Final concentration Incubation time 3A4 Mifepristone Midazolam 100μM 5μM 5 minutes 3A4 Mifepristone Testosterone 1000μM 50μM 10 minutes

[0378] Example 11: Off-target studies

[0379] A panel screening of off-target safety of 11 was performed. The categories of targets tested included ion channels, GPCRs, transporters, kinases, enzymes, and nuclear hormone receptors. Compound 11 showed no significant off-target activity at 10 μM ( Fig.10 ).

[0380] Example 12: Ames test

[0381] Mini-Ames assays were performed on compound 11 using various bacterial strains. Based on these results, compound 11 is not mutagenic. Fig.11 ).

[0382] 1. The studies evaluated the mutagenic potential of the test articles (or their metabolites) by measuring the ability to induce back mutations at selected sites in the bacteria Salmonella typhimurium (TA98, TA1535, TA1537) and Escherichia coli WP2uvrA (pKM101) in the presence and absence of microsomal enzymes.

[0383] 2. The well diameter of a standard six-well culture plate is approximately 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 for the Salmonella typhimurium strain used, or 0.5 mM D-biotin and 0.5 mM L-tryptophan for the E. coli WP2uvrA (pKM101) used.

[0384] 3. In the Mini-Ames test, 4 strains were selected for testing: Salmonella typhimurium (TA98, TA1535, TA1537) and Escherichia coli WP2uvrA (pKM101). Test strains were prepared from frozen working stocks. 10 μL of frozen working stock added to 5 mL of nutrient broth was incubated at 37 ± 2 ° C with shaking at 220 rpm for 10 hours until an optical density (at 650 nm) of 0.6 to 0.8 was reached. Overnight cultures were used for mutagenesis testing.

[0385] 4. The test article was supplied in powder form and kept at -20°C until use. A 50 mg / mL stock solution was prepared in DMSO (Table 11A). Subdoses were prepared from the stock solution by dilution in DMSO just prior to use. If the test article was insoluble at 50 mg / mL, the highest concentration was reduced to the lowest insoluble concentration.

[0386] 5. Prepare human liver S9 mixture by mixing the following substances in reverse order and keep it 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).

[0387] 6. Control – Negative (DMSO); Positive (Table 11B)

[0388] 7. Six concentrations are required for each test article, in triplicate for each strain. When not limited by solubility or cytotoxicity, the maximum dose level we recommend is 1000 micrograms (μg) / well (or 1 microliter (μL) / well for liquid test substances). If there are solubility issues with the test article, record the precipitate according to the following evaluation: P0: no precipitation in solution or no crystal growth on the plate; P1: 0 to 20% crystal growth; P2: 20% to 60% crystal growth; P3: 60% to 100% crystal growth. The precipitating dose is scored provided that the precipitate does not interfere with the scoring (counting the number of revertants). If the precipitating dose interferes with the scoring (counting the number of revertants), the lowest precipitating dose should be used as the highest dose scored.

[0389] 8.Mini Ames Assay - Melt the top agar using a microwave and maintain ≥47°C in a water bath. Use a heating block set to 45°C ± 2°C for the assay. Set up 12x75mm test tubes in duplicate. Use a heating block set to 45°C ± 2°C for the assay. Set up 12x75mm test tubes in duplicate. For each concentration, add the following to the test tube in order: a. 1600 μL of top agar; b. 80 μL of drug or control; c. 400 μL of S9 mix or PBS buffer; d. 80 μL of overnight culture. Vortex and dispense at 540 μL / well using a disposable pipette. Incubate the plate at 37±2°C for approximately 48 to 72 hours.

[0390] 9. Scoring and Positive Criteria - If F≥2 for strains TA98, WP2uvrA (pKM101), and F≥3 for TA1535, TA1537, and a dose-related increase in mean revertants, the compound was considered mutagenic. Background lawn assessment: 0 means no background lawn growth or complete cytotoxicity; 1 means about 25% growth; 2 means about 50% growth; 3 means about 75% growth; and 4 means complete "100%" growth.

[0391]

[0392] Background lawn is measured by microscope using stereoscope.All background lawn assessments are compared with vehicle control.In full background lawn, the lawn colony is very small and cannot be visualized.However, in the flat board with toxicity, the lawn colony grows into microcolony (small diameter) or bacterium colony (diameter is similar to revertant bacterium colony), which can be visualized without the help of amplification.

[0393] Table 11A

[0394]

[0395] Table 11B

[0396]

[0397] Incorporated by Reference

[0398] All publications and patents mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

[0399] Equivalent

[0400] Although specific embodiments of the present invention have been discussed, the above description is illustrative and not restrictive. After reading this specification and the appended claims, many variations of the present invention will become apparent to those skilled in the art. The full scope of the present invention should be determined by reference to the full scope of the claims and their equivalents and the description and these variations.

Claims

1. A compound having a structure of Formula I or a pharmaceutically acceptable salt thereof: in Z 1 Yes O, NR 4 or CHR 2 ; Z 2 O or CHR 3 ; 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 If x is 0, then R 2 and R 3 Together with the carbon atoms to which they are attached, they can form a 3-, 4-, 5-, or 6-membered fused 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; and x is an integer selected from 0 and 1; The condition is Z 2 and Z 3 are not all O; and if x is 0, then Z 1 and Z 2 Not all O and Z 1 and Z 3 Not all O.

2. The compound according to claim 1, wherein Z 1 Yes O, NR 4 or CHR 2 ; Z 2 O or CHR 3 ; 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, alkyl or alkynyl; or If x is 0, then R 2 and R 3 Together with the carbon atoms to which they are attached, they can form a 3-, 4-, 5-, or 6-membered fused carbocyclic ring; R 4 is H, alkyl or acyl; and x is an integer selected from 0 and 1; The condition is Z 2 and Z 3 are not all O; and if x is 0, then Z 1 and Z 2 Not all O and Z 1 and Z 3 Not all O.

3. The compound according to claim 1 or 2, wherein Z 1 and Z 2 Not all O.

4. The compound according to any one of claims 1 to 3, wherein Z 1 For CHR 2 .

5. The compound according to any one of claims 1 to 4, wherein R 2 It's H.

6. The compound according to any one of claims 1 to 5, wherein x is 0.

7. The compound according to any one of claims 1 to 5, wherein x is 1.

8. A compound according to any one of claims 1 to 7, wherein Z 2 For CHR 3 .

9. A compound according to any one of claims 1 to 8, wherein R 3 It's H.

10. A compound according to any one of claims 1 to 8, wherein R 3 It is an alkyl group.

11. A compound according to any one of claims 1 to 8, wherein R 3 It is a lower alkyl group.

12. A compound according to any one of claims 1 to 8, wherein R 3 It's methyl.

13. A compound according to any one of claims 1 to 8, wherein R 3 It is an alkynyl group.

14. A compound according to any one of claims 1 to 8, wherein R 3 It is a lower alkynyl.

15. A compound according to any one of claims 1 to 8, wherein R 3 It is ethynyl.

16. A compound according to any one of claims 1 to 8, wherein R 3 It's cyano.

17. A compound according to any one of claims 1 to 7, wherein Z 2 It's O.

18. A compound according to any one of claims 1 to 8, wherein R 2 and R 3 Together with the carbon atoms to which they are attached they form a 3-membered fused carbocyclic ring.

19. A compound according to any one of claims 1 to 18, wherein Z 3 It's NH.

20. A compound according to any one of claims 1 to 18, wherein Z 3 It's O.

21. A compound according to any one of claims 1 to 20, wherein R 1 It is an alkyl group.

22. A compound according to any one of claims 1 to 20, wherein R 1 It is a lower alkyl group.

23. A compound according to any one of claims 1 to 20, wherein R 1 It's methyl.

24. A compound according to any one of claims 1 to 20, wherein R 1 It is halogenated.

25. A compound according to any one of claims 1 to 20, wherein R 1 It's chlorine.

26. The compound according to claim 1 or 2, having the following structure:

27. The compound according to claim 26, wherein R 1 It is H or CH3.

28. The compound according to claim 26 or 27, wherein R 3 is unsubstituted C 1- C6 alkyl or CH3.

29. The compound according to claim 26 or 27, wherein R 3 is unsubstituted C 2- C6 alkynyl.

30. The compound according to claim 26 or 27, wherein R 3 is replaced by C 2- C6 alkynyl.

31. The compound according to claim 30, wherein the C 2- The C6 alkynyl group is substituted by CF3.

32. The compound according to claim 26 or 27, wherein R 3 is alkyl-OR 5 or alkyl-SR 6 .

33. The compound according to claim 30, wherein R 5 and R 6 Each is independently CHF2, CF3, CH3, CH(CH3)2 or CH(CH2)2.

34. The compound according to claim 1 or 2, having the following structure:

35. The compound according to claim 34, having the following structure:

36. The compound according to claim 34, having the following structure:

37. A compound according to any one of claims 34-36, wherein R 1 It is H or CH3.

38. A compound according to any one of claims 34-37, wherein R 2 is unsubstituted C 1- C6 alkyl.

39. A compound according to any one of claims 34-38, wherein R 3 is unsubstituted C 1- C6 alkyl or CH3.

40. A compound or a pharmaceutically acceptable salt thereof selected from:

41. A compound or a pharmaceutically acceptable salt thereof selected from:

42. A compound or a pharmaceutically acceptable salt thereof having the following structure:

43. A pharmaceutical composition comprising a compound according to any one of claims 1-42; and a pharmaceutically acceptable excipient or carrier.

44. A method for treating or preventing cancer, comprising administering an effective amount of the compound according to any one of claims 1 to 42 or the pharmaceutical composition according to claim 43 to a subject in need thereof, thereby treating or preventing the cancer.

45. The method of claim 44, wherein the cancer is ovarian cancer, colon cancer, or breast cancer.

46. ​​The method of claim 44, wherein the cancer is breast cancer.

47. The method of claim 44, wherein the cancer is colon cancer.

48. The method of claim 44, wherein the cancer is ovarian cancer.

49. The method of claim 46, wherein the cancer is high-grade serous carcinoma (HGSC).

50. The method of claim 44, wherein the cancer is a solid tumor.

51. The method of any one of claims 44-50, further comprising co-administering to the subject an effective amount of one or more additional chemotherapeutic agents.

52. A method of inhibiting tumor growth, comprising administering an effective amount of a compound according to any one of claims 1-42 or a pharmaceutical composition according to claim 43 to a subject in need thereof.

53. The method of claim 53, wherein the tumor is high-grade serous carcinoma (HGSC).

54. The method of claim 53 or 53, further comprising co-administering to the subject an effective amount of one or more additional chemotherapeutic agents.

55. The method of any one of claims 44-54, wherein the subject is a mammal.

56. The method of claim 55, wherein the mammal is a human.

57. The method of claim 55, wherein the mammal is a dog or a cat.

58. 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 1-42.

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