Inhibitors of ash1l and methods of use thereof

Abstract

Provided herein are small molecules that bind to ASH1L and inhibit ASH1L activity, and methods of use thereof for the treatment of cancer.

Description

[0001] INHIBITORS OF ASH1L AND METHODS OF USE THEREOF

[0002] CROSS-REFERENCE

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 673,385, filed July 19, 2024; which is incorporated by reference herein in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under CA244254 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] Provided herein are small molecules that bind to ASH1L and inhibit ASH1L activity, and methods of use thereof for the treatment of cancer.

[0008] BACKGROUND

[0009] The absent, small, or homeotic-like 1 (ASH1L) protein is a histone lysine methyltransferase encoded by the ASH IL gene located on the long arm of human chromosome 1 (lq21). The gene transcribes a 10.5-kb mRNA, which translates into a 2962-residue protein. ASH1L comprises several domains, including four AT hook motifs, three reader domains (bromodomain, BAH, and PHD), and a catalytic SET domain responsible for methyl group transfer from the S-adenosyl methionine (SAM) cofactor to the lysine substrate. ASH1L is classified as a member of the trithorax group (TrxG) proteins, functioning as transcriptional activators in gene expression regulation. It can mono / dimethylate Lysine36 on histone H3 (H3K36) to regulate HOX genes by counteracting Polycomb silencing.

[0010] In mammals, ASH IL shares a conserved functional relationship with another TrxG protein, mixed lineage leukemia 1 (MLL1). Both ASH1L and MLL1 play critical roles in supporting the self-renewal potential of hematopoietic stem cells. Notably, ASHIL's involvement in the pathogenesis of acute leukemia is underscored by chromosomal translocations involving the MLL1 gene (also known as MLL or KMT2A), which occur in 5-10% of acute leukemia cases and are associated with a very poor prognosis, with only approximately 35% 5 -year survival rate. Knockdown of ASH IL in MLL leukemia cells leads to growth arrest, apoptosis, differentiation, and downregulation of HOXA9 genes, which are pivotal in leukemogenesis. Moreover, in vivo studies demonstrate that ASH1L knockdown substantially suppresses the progression of tumor growth and significantly improves the survival rate. Beyond leukemia, the epigenetic protein ASH1L is found to be overexpressed in various solid tumors, including breast, liver, and thyroid cancers. SUMMARY Provided herein are small molecules that bind to ASH1L and inhibit ASH1L activity, and methods of use thereof for the treatment of cancer. In one aspect, the disclosure provides a compound of formula (I): or a pharmaceutically acceptable salt thereof, wherein: E and A are a spirocyclic ring system; Z is CH or N; Y is N or O; if Y is O then R1is absent; if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; R2is selected from -C(S)NH2, -C(O)NH2, -C(Se)NH2, oxirane, thiirane, aziridine, -CF3, and -CN; R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and wherein R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, -halogen (e.g., -F), etc.); and R7is selected from H, halogen, CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2- OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3. In some embodiments, R8is selected from H, CH3, CF3, -C(O)CH3, - CH2NHC(O)OC(CH3)3, In some embodiments, R6 is selected from:

[0011] In some embodiments, R6is –(CH2)2– R9, and wherein R9is selected from: In some embodiments, R6is -(CH2)2NHC(O)-R10, wherein R10is selected from: In some embodiments, E, A, Z, Y, R1, R2, R6, and R7are independently selected from the E, A, Z, Y, R1, R2R6, and R7functional groups of compounds 3-4, 23-25, 33-43, 50-66, 66s, 77- 78, 123-127, 143-145, 160-167, and 168-194. In some embodiments, the compound of formula (I) is selected from one of compounds 3-4, 23-25, 33-43, 50-66, 66s, 77-78, 123-127, 143-145, 160-167, and 168-194. In a second aspect, the disclosure provides a compound of formula (Ia):

[0012] or a pharmaceutically acceptable salt thereof, wherein: E and A are a spirocyclic ring system; Z is CH or N; Y is N or O; if Y is O then R1is absent; if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; X is O, Se, or S; R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and wherein R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, -halogen (e.g., -F), etc.); and R7is selected from H, halogen, CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2- OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3. In some embodiments, R8is selected from H, CH3, CF3, -C(O)CH3, -CH2NHC(O)OC(CH3)3,

[0013] In some embodiments, R6is selected from: In some embodiments, R6is –(CH2)2– R9, and wherein R9is selected from: In some embodiments, R6is -(CH2)2NHC(O)-R10, wherein R10is selected from: , In some embodiments, E, A, Z, Y, R1, R6, and R7are independently selected from the E, A, Z, Y, R1, R2R6, and R7functional groups of compounds 3-4, 23-25, 33-43, 50-66, 66s, 77-78, 143-145, 160-167, 168-194. In some embodiments, the compound of formula (Ia) is selected from one of compounds 3-4, 23-25, 33-43, 50-66, 66s, 77-78, 143-145, 160-167, 168-194. In a third aspect, the disclosure provides a compound of formula (Ib): or a pharmaceutically acceptable salt thereof, wherein: Z is CH or N; Y is N or O; if Y is O then R1is absent; if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; X is O, Se, or S; R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, -halogen; and R7is selected from H, halogen, CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2- OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3. In some embodiments, R8is selected from H, CH3, CF3, -C(O)CH3, - CH2NHC(O)OC(CH3)3, In some embodiments, R6is selected from:

[0014] In some embodiments, R6is –(CH2)2– R9, and wherein R9is selected from: In some embodiments, R6is -(CH2)2NHC(O)-R10, wherein R10is selected from: , In some embodiments, Z, Y, R1, R6, and R7are independently selected from the Z, Y, R1, R2R6, and R7functional groups of compounds 4, 23-25, 160-162, and 168-169. In some embodiments, the compound of formula (Ib) is selected from one of compounds 4, 23-25, 160-162, and 168-169. In a fourth aspect, the disclosure provides a compound of formula (Ic):

[0015] or a pharmaceutically acceptable salt thereof, wherein: Z is CH or N; Y is N or O; if Y is O then R1is absent; if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; X is O, Se, or S; R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, -halogen (e.g., -F), etc.); and R7is selected from H, halogen, CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2- OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3. In some embodiments, R8is selected from H, CH3, CF3, -C(O)CH3, - CH2NHC(O)OC(CH3)3, In some embodiments, R6is selected from: In some embodiments, R6is –(CH2)2– R9, and wherein R9is selected from: In some embodiments, R6is -(CH2)2NHC(O)-R10, wherein R10is selected from: , In some embodiments, Z, Y, R1, R6, and R7are independently selected from the Z, Y, R1, R2R6, and R7functional groups of compounds 3, 33-43, 50-66, 66s, 77-78, 143-145, 163- 167, and 170-194. In some embodiments, the compound of formula (Ic) is selected from one of compounds 3, 33-43, 50-66, 66s, 77-78, 143-145, 163-167, and 170-194. In a fifth aspect, the disclosure provides a compound of formula (II): or a pharmaceutically acceptable salt thereof, wherein: Z is CH or N; Y is N or O; if Y is O then R1is absent; if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; X is O, Se, or S; and R7is selected from H, halogen, CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2- OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3. In some embodiments, Z, Y, R1, and R7are independently selected from the Z, Y, R1, R2R6, and R7functional groups of compounds 139-142 and 158-159. In some embodiments, the compound of formula (II) is selected from one of compounds 139-142 and 158-159. In a fifth aspect, the disclosure provides a compound of formula (III): or a pharmaceutically acceptable salt thereof, wherein: R1is selected from NH2and (CH2)NHC(O)CH3; and R2is selected from -C(S)NH2, -C(O)NH2, -C(Se)NH2, oxirane, thiirane, aziridine, -CF3, In some embodiments, R1and R2are independently selected from the R1and R2functional groups of compounds 100-112, 115-122, 128-135, and 146-153. In some embodiments, the compound of formula (III) is selected from one of compounds 100-112, 115-122, 128-135, and 146-153. In some embodiments, provided herein is a compound selected from the compounds of tables 1-4 and A-D. In some embodiments, provided herein is a compound selected from one of compounds 2-4, 22-25, 33-39, 40-44, 50-66, 66s, 77-79, and 100-194. In some embodiments, provided herein is a pharmaceutical composition comprising a compound described herein (e.g., of one of formulas (I), (Ia), (Ib), (Ic), (II), or (III), or of one of tables 1-4 or A-D), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated for parenteral administration. In some embodiments, provided herein a method of inhibiting ASH1L activity in a sample, comprising contacting the sample with an effective amount of a compound described herein (e.g., of one of formulas (I), (Ia), (Ib), (Ic), (II), or (III), or of one of tables 1-4 or A-D) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of a compound described herein (e.g., of one of formulas (I), (Ia), (Ib), (Ic), (II), or (III), or of one of tables 1-4 or A-D). In some embodiments, provided herein a method of reducing proliferation of cancer cells in a sample, comprising contacting the sample with an effective amount of a compound described herein (e.g., of one of formulas (I), (Ia), (Ib), (Ic), (II), or (III), or of one of tables 1-4 or A-D) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of a compound described herein (e.g., of one of formulas (I), (Ia), (Ib), (Ic), (II), or (III), or of one of tables 1-4 or A-D). In some embodiments, provided herein a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., of one of formulas (I), (Ia), (Ib), (Ic), (II), or (III), or of one of tables 1-4 or A-D) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of a compound described herein (e.g., of one of formulas (I), (Ia), (Ib), (Ic), (II), or (III), or of one of tables 1-4 or A-D). In some embodiments, the cancer is selected from leukemia, hematologic malignancy, solid tumor cancer, breast cancer, prostate cancer, ovarian cancer, liver cancer and thyroid cancer. In some embodiments, the cancer is selected from AML, ALL, Mixed Lineage Leukemia or a leukemia with Partial Tandem Duplication of MLL. In some embodiments, the method further comprises administering an additional chemotherapeutic agent to the subject. In some embodiments, the subject is a human. In some embodiments, provided herein is a use of a compound described herein (e.g., of one of formulas (I), (Ia), (Ib), (Ic), (II), or (III), or of one of tables 1-4 or A-D) or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of a compound described herein (e.g., of one of formulas (I), (Ia), (Ib), (Ic), (II), or (III), or of one of tables 1-4 or A-D), for the treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A-B. Known ASH1L inhibitors. A. Chemical structures and activities of known ASH1L inhibitors. IC50values were measured using HMT assay and Kd values were assessed using ITC

[0015] . B. Binding mode of AS-85 to ASH1L based on the crystal structure of the complex (6WZW in PDB). Color coding: carbon atoms are in gray (for ASH1L) or green (for AS-85), nitrogen atoms are in blue, oxygens in red, sulfur in yellow and fluorine atoms in light blue. Figure 2A-C. Development of FP assay for ASH1L. A. Chemical structure of fluorescein-labeled probe 3. B. Titration curve for Kd determination for binding of 3 to ASH1L SET using FP assay. C. Titration curve and IC50value for AS-99 from FP assay with ASH1L SET. Figure 3A-B. LMI pocket on ASH1L. A. Crystal structure of ASH1L-AS-85 reveals LMI pocket on ASH1L (marked in salmon color). ASH1L is shown in surface representation. AS-85 is shown in stick representation (green carbons) and the N-terminal fragment of ASH1L SET crystallization construct forming the crystal contacts with LMI pocket is also shown (carbons, residues A(-3), M(-4) and A(-5)). Coordinates from 6WZW structure in PDB. B. Crystal structure of ASH1L-AS-85 complex (6WZW in PDB) showing LMI pocket is stick representation (carbon atoms of residues forming the LMI pocket are shown). Figure 4A-D. Inhibition of ASH1L HMT activity. A, B. Titration curves and IC50values from the HMT assay for compounds 66s (A) and 65 (B). Data are mean ± SD. from two independent experiments. C. Selectivity of 66s (at 5 µM) against a panel of histone methyltransferases. Data represents two independent experiments each performed in duplicates. D. Binding isotherm from the ITC experiment performed for the binding of 66s to ASH1L. Data are mean ± SD from two independent experiments. A representative binding isotherm is shown. Figure 5A-D. Activity of 66s in leukemia cells. A. Assessment of epigenetic marks by WB in MV4;11 and KOPN8 cells treated with 66s for 8 d. Representative gel of two independent experiments is shown. B. Titration curves from the MTT cell viability assay performed at different time points (days 4, 7, 11 and 14) for 66s in MV4;11 leukemia cell line. n = 4. C. Titration curves from the MTT cell viability assay performed after 14 days of treatment of human MLL1 rearranged (MLL1-r) leukemia cell lines (MV4;11, MOLM13, KOPN8) and control leukemia cell line (K562) with 66s, n = 4. D. Flow cytometry analysis of apoptosis induced by 66s in MV4;11 and KOPN8 cells after 10 days of treatment. Mean ± SD, n = 3 biological replicates. Representative graphs are shown from 2–3 independent experiments in panels B-D. P values were calculated using unpaired 2-tailed t test. Figure 6A-C. 66s induces differentiation of MLL1-r leukemia cells. A, B. Quantification of CD11B (A) or CD14 (B) expression in human leukemia cells treated for 10 days with 66s, detected by flow cytometry; mean ± SD, n = 3 biological replicates. Two independent experiments were performed for each cell line in triplicates. Representative graphs are shown. P values were calculated using unpaired 2-tailed t test. C. Wright-Giemsa–stained cytospins for MV4;11 and KOPN8 cells after 10 days of treatment with DMSO or 66s. Figure 7A-B. ASH1L inhibitor GH-397 (compound 186) reduces leukemia progression in mouse models of leukemia. A. Bioimaging of mice. B. Quantification of bioluminescence in control and GH-397-treated mice. DEFINITIONS Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments described herein, some preferred methods, compositions, devices, and materials are described herein. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular molecules, compositions, methodologies or protocols herein described, as these may vary in accordance with routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the embodiments described herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, in case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply. As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “an ASH1L inhibitor” is a reference to one or more ASH1L inhibitors, and so forth.

[0016] As used herein, the term “comprise” and linguistic variations thereof denote the presence of recited feature(s), element(s), method step(s), etc. without the exclusion of the presence of additional feature(s), element(s), method step(s), etc. Conversely, the term “consisting of" and linguistic variations thereof, denotes the presence of recited feature(s), element(s), method step(s), etc. and excludes any unrecited feature(s), element(s), method step(s), etc., except for ordinarily-associated impurities. The phrase “consisting essentially of’ denotes the recited feature(s), element(s), method step(s), etc. and any additional feature(s), element(s), method step(s), etc. that do not materially affect the basic nature of the composition, system, or method. Many embodiments herein are described using open “comprising” language. Such embodiments encompass multiple closed “consisting of’ and / or “consisting essentially of’ embodiments, which may alternatively be claimed or described using such language.

[0017] All chemical names of substituents should be interpreted in light of IUPAC and / or the modified nomenclature and with reference to the chemical structures depicted and / or described herein. For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, and such that the selections and substitutions result in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0018] In accordance with a convention used in the art, the group: is used in structural formulae herein to depict the bond that is the point of attachment of the moiety or substituent to the core or backbone structure.

[0019] As used herein, the term “subject” broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, poultry, fish, crustaceans, etc.). As used herein, the term “patient” typically refers to a subject that is being treated for a disease or condition.

[0020] As used herein, the term “subject at risk for cancer” refers to a subject with one or more risk factors for developing cancer. Risk factors may include, but are not limited to, gender, age, genetic predisposition, environmental exposures, infections, and previous incidents of diseases, lifestyle, etc. As used herein, the term “effective amount” refers to the amount of a compound or composition sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route. As used herein, the terms “administration” and “administering” refer to the act of giving a drug, prodrug, or other agent, or therapeutic treatment to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like. As used herein, the terms “co-administration” and “co-administering” refer to the administration of at least two agent(s) (e.g., an ASH1L inhibitor and one or more additional therapeutics) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s), and / or when co-administration of two or more agents results in sensitization of a subject to beneficial effects of one of the agents via co-administration of the other agent. As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for therapeutic use in vitro, in vivo or ex vivo. The terms “pharmaceutically acceptable” or “pharmacologically acceptable,” as used herein, refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject. As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers including, but not limited to, phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. (1975), incorporated herein by reference in its entirety. As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present invention which, upon administration to a subject, is capable of providing a compound of this invention or an active metabolite or residue thereof. As is known to those of skill in the art, “salts” of the compounds of the present invention may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2- sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts. Examples of bases include, but are not limited to, alkali metals (e.g., sodium) hydroxides, alkaline earth metals (e.g., magnesium), hydroxides, ammonia, and compounds of formula NR4+, wherein each R is independently C1-4 alkyl, and the like. Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present invention compounded with a suitable cation such as Na+, NH4+, and NR4+(wherein each R is independently a C1-4alkyl group), and the like. For therapeutic use, salts of the compounds herein are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound. As used herein, the term “instructions for administering said compound to a subject,” and grammatical equivalents thereof, includes instructions for using the compositions contained in a kit for the treatment of conditions (e.g., providing dosing, route of administration, decision trees for treating physicians for correlating patient-specific characteristics with therapeutic courses of action). “Amino” refers to a -NH2moiety. “Carbonyl” refers to a moiety of formula -C(=O)-. “Carboxy” or “carboxyl” refers to the -CO2H moiety. “Cyano” refers to the -CN moiety. “Hydroxy” or “hydroxyl” refers to the -OH moiety. “Imino” refers to the =NH moiety. Unless stated otherwise specifically in the specification, an imino group is optionally substituted. “Nitro” refers to the -NO2moiety. “Oxo” refers to the =O moiety. “Thioxo” refers to the =S moiety. “Acyl” refers to the group -C(=O)R, where R is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, and heteroalkyl. Unless stated otherwise specifically in the specification, an acyl group is optionally substituted. “Alkyl” refers to a straight or branched saturated hydrocarbon chain having from 1 to thirty carbon atoms, for example from 1 to 16 carbon atoms (C1-C16alkyl), 1 to 12 carbon atoms (C1-C12alkyl), 1 to 8 carbon atoms (C1-C8alkyl), 1 to 6 carbon atoms (C1-C6alkyl), or 1 to 4 carbon atoms (C1-C4alkyl), e.g., methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3- dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl, and the like. Unless stated otherwise specifically in the specification, an alkyl group is optionally substituted. “Alkenyl” refers to a straight or branched refers to a straight or branched hydrocarbon chain containing from 2 to 30 carbon atoms, for example from 2 to 16 carbon atoms (C2-C16alkenyl), 2 to 12 carbon atoms (C2-C12alkenyl), 2 to 8 carbon atoms (C2-C8alkenyl), 2 to 6 carbon atoms (C2-C6alkenyl), or 2 to 4 carbon atoms (C2-C4alkenyl), and containing at least one carbon-carbon double bond. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 1,4-pentadienyl, 5-hexenyl, 2- heptenyl, 2-methyl-1-heptenyl, and 3-decenyl. Unless stated otherwise specifically in the specification, an alkenyl group is optionally substituted. “Alkynyl” refers to a straight or branched hydrocarbon chain containing from 2 to 30 carbon atoms, for example from 2 to 16 carbon atoms (C2-C16alkynyl), 2 to 12 carbon atoms (C2-C12alkynyl), 2 to 8 carbon atoms (C2-C8alkynyl), 2 to 6 carbon atoms (C2-C6alkynyl), or 2 to 4 carbon atoms (C2-C4alkynyl), and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl. Unless stated otherwise specifically in the specification, an alkynyl group is optionally substituted. “Alkylene” refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 30 carbon atoms (C1-C30alkylene), for example, of 1 to 6 carbon atoms (C1- C6alkylene). Representative examples of alkylene include, but are not limited to, -CH2-, - CH2CH2-, -CH(CH3)-, -CH2CH2CH2-, -CH2CH(CH3)-, -CH2CH2CH2CH2-, -CH2CH(CH3)CH2-, - CH2CH2CH(CH3)-, -CH2CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, -CH(CH3)CH2CH2CH2-, - CH2CH2CH2CH2CH2CH2-, -CH2CH2CH(CH3)CH2CH2-, -CH2CH(CH3)CH2CH2CH2-, and - CH(CH3)CH2CH2CH2CH2-. Unless stated otherwise specifically in the specification, an alkylene group is optionally substituted. “Alkoxy” refers to a moiety of the formula -OR where R is an alkyl group as defined herein, e.g., an alkyl group containing 1 to 12 carbon atoms. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy and tert-butoxy. Unless stated otherwise specifically in the specification, an alkoxy group is optionally substituted. “Alkenyloxy” refers to a moiety of the formula -OR where R is an alkenyl group as defined herein, e.g., an alkenyl group containing 2 to 12 carbon atoms. Unless stated otherwise specifically in the specification, an alkenyloxy group is optionally substituted. “Alkynyloxy” refers to a moiety of the formula -OR where R is an alkynyl group as defined herein, e.g., an alkynyl group containing 2 to 12 carbon atoms. Unless stated otherwise specifically in the specification, an alkynyloxy group is optionally substituted. “Alkylamino” refers to a moiety of the formula -NHR where R is an alkyl group as defined herein. Unless stated otherwise specifically in the specification, an alkylamino or dialkylamino group is optionally substituted. “Alkylaminoalkyl” refers to an alkyl moiety comprising at least one alkylamino substituent. Unless stated otherwise specifically in the specification, an alkylaminoalkyl group is optionally substituted. “Amide” or “amido” refers to a moiety with formula -C(=O)NRR’ or -NRC(=O)R’, where R and R’ are each independently selected from the group consisting of hydrogen, alkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl (bonded through a ring carbon), heteroarylalkyl, heterocyclyl, and heterocyclylalkyl. When the amido moiety is -C(=O)NRR’, R and R’ may optionally be taken together with the nitrogen to which they are attached to form a 4- , 5-, 6-, or 7-membered ring. Unless stated otherwise specifically in the specification, an amido group is optionally substituted. “Amidoalkyl” refers to an alkyl moiety, as defined herein, in which at least one hydrogen atom is replaced with an amido group, as defined herein. Unless stated otherwise specifically in the specification, an amidoalkyl group is optionally substituted. “Aminoalkyl” refers to an alkyl moiety, as defined herein, in which at least one hydrogen atom is replaced with an amino group, as defined herein. The amino group can be substituted on a tertiary, secondary or primary carbon. Unless stated otherwise specifically in the specification, an aminoalkyl group is optionally substituted. “Aryl” refers to an aromatic carbocyclic ring system having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic) including fused ring systems, and zero heteroatoms. As used herein, aryl contains 6-20 carbon atoms (C6-C20aryl), 6 to 14 ring carbon atoms (C6-C14 aryl), 6 to 12 ring carbon atoms (C6-C12aryl), or 6 to 10 ring carbon atoms (C6-C10aryl). Representative examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, and phenanthrenyl. Unless stated otherwise specifically in the specification, the term “aryl” is meant to include aryl groups that are optionally substituted. “Arylalkyl” refers to an alkyl group, as defined herein, wherein at least one hydrogen atom is replaced with an aryl group, as defined herein. Exemplary arylalkyl groups include, but are not limited to, benzyl and phenethyl. Unless stated otherwise specifically in the specification, the term “arylalkyl” is meant to include groups that are optionally substituted on the aryl moiety and / or on the alkyl moiety. “Arylene” refers to a divalent aryl group (e.g., phenylene). Unless stated specifically otherwise, an arylene is optionally substituted. “Aryloxy” refers to an -O-aryl moiety. Unless stated otherwise specifically in the specification, an aryloxy is optionally substituted. “Arylamino” refers to a -NRa-aryl moiety, where Rais H or alkyl. Unless stated otherwise specifically in the specification, an arylamino is optionally substituted. “Cycloalkyl” refers to a saturated carbocyclic ring system containing three to ten carbon atoms per ring. The cycloalkyl may be monocyclic, bicyclic, tricyclic, bridged, fused, and / or spirocyclic. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl. Unless stated otherwise specifically in the specification, the term “cycloalkyl” is meant to include cycloalkyl groups that are optionally substituted. “Cycloalkenyl” refers to a non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl. Unless stated otherwise specifically in the specification, the term “cycloalkenyl” is meant to include cycloalkenyl groups that are optionally substituted. “Cycloalkylalkyl” refers to an alkyl group, as defined herein, wherein at least one hydrogen atom is replaced with a cycloalkyl group, as defined herein. Unless stated otherwise specifically in the specification, the term “cycloalkylalkyl” is meant to include groups that are optionally substituted on the cycloalkyl moiety and / or on the alkyl moiety. “Cycloalkylalkylamino” refers to a cycloalkylalkyl-NRa- moiety, where Rais H or alkyl and where the cycloalkylalkyl moiety is attached via a carbon atom to nitrogen, wherein the nitrogen functions as a linker to attach the moiety to the remainder of the molecule. Unless stated otherwise specifically in the specification, a cycloalkylalkylamino is optionally substituted. “Cycloalkylalkyloxy” refers to a -O-cycloalkylalkyl moiety, where the cycloalkylalkyl moiety is attached via a carbon atom to oxygen, wherein the oxygen functions as a linker to attach the moiety to the remainder of the molecule. Unless stated otherwise specifically in the specification, a cycloalkylalkyloxy is optionally substituted. “Cycloalkylamino” refers to a -NRa-cycloalkyl moiety, where Ra is H or alkyl. Unless stated otherwise specifically in the specification, a cycloalkylamino is optionally substituted. “Cycloalkyloxy” refers to an -O-cycloalkyl moiety. Unless stated otherwise specifically in the specification, a cycloalkyloxy is optionally substituted. “Spirocyclic” refers to a ring system in which two connected rings share at a single atom, such that the rings have a single atom in common. “Dialkylamino” refers to a moiety of the formula -NRR’ where R and R’ are each independently an alkyl group as defined herein. Unless stated otherwise specifically in the specification, an alkylamino or dialkylamino group is optionally substituted. “Dialkylaminoalkyl” refers to an alkyl moiety comprising at least one dialkylamino substituent. Unless stated otherwise specifically in the specification, an alkylaminoalkyl group is optionally substituted. “Halo” or “halogen” refers to fluoro, chloro, bromo, or iodo. “Haloalkyl” refers to an alkyl group, as defined herein, that is substituted by one or more halo atoms, as defined herein, e.g., trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, -CH2CF3, -CH2CHF2, -CH2CH2F, -CHFCF3, -CHFCHF2, -CHFCH2F, - CHFCH3, -CF2CF3, -CF2CHF2, -CF2CH2F, -CF2CH3, -CH2CF2CH3, -CH2CHFCH3, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like. Unless stated otherwise specifically in the specification, a haloalkyl group is optionally substituted. “Haloalkoxy” refers to an alkoxy group, as defined herein, that is substituted with one or more halo atoms, as defined herein. As used herein, the term “heteroatom” or “ring heteroatom” is meant to include any element other than carbon or hydrogen. Suitable heteroatoms are oxygen (O), nitrogen (N), sulfur (S), and phosphorus (P). “Heteroalkyl” means an alkyl group, as defined herein, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group such as -NR-, -O-, -S-, -S(O)-, -S(O)2-, and the like, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl or heterocyclyl, each of which may be optionally substituted. By way of example, 1, 2 or 3 carbon atoms may be independently replaced with the same or different heteroatomic group. Examples of heteroalkyl groups include, but are not limited to, - OCH3, -CH2OCH3, -SCH3, -CH2SCH3, -NRCH3, and -CH2NRCH3, where R is hydrogen, alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl, each of which may be optionally substituted. Heteroalkyl also includes groups in which a carbon atom of the alkyl is oxidized (i.e., is -C(O)-). “Heteroalkylene” refers to an alkylene group, as defined herein, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced with a heteroatom group such as -NR-, -O-, -S-, -S(O)-, -S(O)2-, and the like, where R is H, alkyl, aryl, cycloalkyl, heteroalkyl, heteroaryl or heterocyclyl, each of which may be optionally substituted. By way of example, 1, 2 or 3 carbon atoms may be independently replaced with the same or different heteroatomic group. Heteroalkylene also includes groups in which a carbon atom of the alkyl is oxidized (i.e., is -C(O)-). Examples of heteroalkylene groups include, but are not limited to, -CH2-O-CH2-, -CH2-S-CH2-, -CH2-NR-CH2-, -CH2-NH-C(O)-CH2-, and the like, as well as polyethylene oxide chains, polypropylene oxide chains, and polyethyleneimine chains. “Heteroaryl” refers to an aromatic group having a single ring (monocyclic) or multiple rings (bicyclic or tricyclic), having one or more ring heteroatoms independently selected from O, N, and S. The aromatic monocyclic rings are five- or six-membered rings containing at least one heteroatom independently selected from O, N, and S (e.g.1, 2, 3, or 4 heteroatoms independently selected from O, N, and S). The five-membered aromatic monocyclic rings have two double bonds, and the six- membered aromatic monocyclic rings have three double bonds. The bicyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring appended fused to a monocyclic aryl group, as defined herein, or a monocyclic heteroaryl group, as defined herein. The tricyclic heteroaryl groups are exemplified by a monocyclic heteroaryl ring fused to two rings independently selected from a monocyclic aryl group, as defined herein, and a monocyclic heteroaryl group as defined herein. Representative examples of monocyclic heteroaryl include, but are not limited to, pyridinyl (including pyridin-2-yl, pyridin-3-yl, pyridin-4-yl), pyrimidinyl, pyrazinyl, pyridazinyl, pyrrolyl, benzopyrazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, 1,2,4- thiadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, imidazolyl, thiazolyl, isothiazolyl, thienyl, furanyl, oxazolyl, isoxazolyl, 1,2,4-triazinyl, and 1,3,5-triazinyl. Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzodioxolyl, benzofuranyl, benzooxadiazolyl, benzopyrazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl, chromenyl, imidazopyridine, imidazothiazolyl, indazolyl, indolyl, isobenzofuranyl, isoindolyl, isoquinolinyl, naphthyridinyl, purinyl, pyridoimidazolyl, quinazolinyl, quinolinyl, quinoxalinyl, thiazolopyridinyl, thiazolopyrimidinyl, thienopyrrolyl, and thienothienyl. Representative examples of tricyclic heteroaryl include, but are not limited to, dibenzofuranyl and dibenzothienyl. The monocyclic, bicyclic, and tricyclic heteroaryls are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the rings. Unless stated otherwise specifically in the specification, a heteroaryl group is optionally substituted. “Heteroarylalkyl” refers to an alkyl group, as defined herein, wherein at least one hydrogen atom is replaced with a heteroaryl group, as defined herein. Unless stated otherwise specifically in the specification, a heteroarylalkyl group is optionally substituted. “Heteroarylalkylamino” refers to a heteroarylalkyl-NRa- moiety, where Ra is H or alkyl. Unless stated otherwise specifically in the specification, an heteroarylalkylamino is optionally substituted. “Heteroarylalkyloxy” refers to an heteroarylalkyl-O- moiety. Unless stated otherwise specifically in the specification, a heteroarylalkyloxy is optionally substituted. “Heteroarylamino” refers to a -NRa-heteroaryl moiety, where Rais H or alkyl. Unless stated otherwise specifically in the specification, a heteroarylamino is optionally substituted. “Heteroaryloxy” refers to an -O-heteroaryl moiety. Unless stated otherwise specifically in the specification, an heteroaryloxy is optionally substituted. “Heteroarylene” refers to a divalent heteroaryl group. Unless stated specifically otherwise, a heteroarylene is optionally substituted. “Heterocycle” or “heterocyclic” refers to a saturated or partially unsaturated non- aromatic cyclic group having one or more ring heteroatoms independently selected from O, N, and S. means a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle. The monocyclic heterocycle is a three-, four-, five-, six-, seven-, or cight-mcmbcrcd ring containing at least one heteroatom independently selected from O, N, and S. The three- or four-membered ring contains zero or one double bond, and one heteroatom selected from O, N, and S. The fivemembered ring contains zero or one double bond and one, two or three heteroatoms selected from O, N and S. The six-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from O, N, and S. The seven- and eight-membered rings contains zero, one, two, or three double bonds and one, two, or three heteroatoms selected from O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3- dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, 1,2-thiazinanyl, 1,3-thiazinanyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1- dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl. The bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a spiro heterocycle group, or a bridged monocyclic heterocycle ring system in which two non-adjacent atoms of the ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, benzopyranyl, benzothiopyranyl, chromanyl, 2,3- dihydrobenzofuranyl, 2,3 -dihydrobenzothienyl, 2,3-dihydroisoquinoline, 2-azaspiro[3.3]heptan- 2-yl, azabicyclo[2.2.1]heptyl (including 2-azabicyclo[2.2.1]hept-2-yl), 2,3-dihydro-lH-indolyl, isoindolinyl, octahydrocyclopenta[c]pyrrolyl, octahydropyrrolopyridinyl, and tetrahydroisoquinolinyl. Tricyclic heterocycles are exemplified by a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are linked by an alkylene bridge of 1, 2, 3, or 4 carbon atoms, or an alkenylene bridge of two, three, or four carbon atoms. Examples of tricyclic heterocycles include, but are not limited to, octahydro-2,5-epoxypentalene, hexahydro-2H-2,5-methanocyclopenta[b]furan, hexahydro-1H- 1,4-methanocyclopenta[c]furan, aza-adamantane (1-azatricyclo[3.3.1.13,7]decane), and oxa- adamantane (2-oxatricyclo[3.3.1.13,7]decane). The monocyclic, bicyclic, and tricyclic heterocycles are connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the rings. Unless stated otherwise specifically in the specification, a heterocyclyl group is optionally substituted. “Heterocyclylalkyl” refers to an alkyl group, as defined herein, wherein at least one hydrogen atom is replaced with a heterocyclyl group, as defined herein. Unless stated otherwise specifically in the specification, a heterocyclylalkyl group is optionally substituted. “Heterocyclylalkylamino” refers to a heterocyclylalkyl-NRa- moiety, where Rais H or alkyl and where the heterocyclylalkyl moiety is attached via a carbon atom to nitrogen, wherein the nitrogen functions as a linker to attach the moiety to the remainder of the molecule. Unless stated otherwise specifically in the specification, a heterocyclylalkylamino is optionally substituted. “Heterocyclylalkyloxy” refers to a -O-heterocycloalkyl moiety, where the heterocyclylalkyl moiety is attached via a carbon atom to oxygen, wherein the oxygen functions as a linker to attach the moiety to the remainder of the molecule. Unless stated otherwise specifically in the specification, a heterocyclylalkyloxy is optionally substituted. “Heterocyclylamino” refers to a -NRa-heterocyclyl moiety, where Rais H or alkyl and where the heterocyclyl moiety is attached via a carbon atom to nitrogen, wherein the nitrogen functions as a linker to attach the moiety to the remainder of the molecule. Unless stated otherwise specifically in the specification, a heterocyclylamino is optionally substituted. “Heterocyclyloxy” refers to an -O-heterocyclyl moiety, where the heterocyclyl moiety is attached via a carbon atom to oxygen, wherein the oxygen functions as a linker to attach the moiety to the remainder of the molecule. Unless stated otherwise specifically in the specification, a heterocyclyloxy is optionally substituted. “Hydroxyalkyl” refers to an alkyl group comprising at least one hydroxyl substituent. The -OH substituent may be on a primary, secondary, or tertiary carbon. Unless stated otherwise specifically in the specification, a hydroxylalkyl group is optionally substituted. “Sulfonamido” refers to a moiety of the formula -SO2NRR’, wherein where R and R’ are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocyclyl, heterocyclylalkyl, and heteroalkyl. R and R’ may optionally be taken together with the nitrogen to which they are attached to form a 4-, 5-, 6-, or 7 -membered ring. Unless stated otherwise specifically in the specification, a sulfonamido group is optionally substituted.

[0021] “Sulfonamidoalkyl” refers to an alkyl group, as defined herein, wherein at least one hydrogen atom is replaced with a sulfonamido group, as defined herein. Unless stated otherwise specifically in the specification, a sulfonamidoalkyl group is optionally substituted.

[0022] “Thioalkyl” refers to a moiety of the formula -SR where R is an alkyl moiety as defined herein containing one to twelve carbon atoms. Unless stated otherwise specifically in the specification, a thioalkyl group is optionally substituted.

[0023] “Thiourea” refers to a moiety of the formula -NH-C(S)-NHR where R is selected from hydrogen, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl, each of which may be optionally substituted.

[0024] “Thioureaalkyl” refers to an alkyl group, as defined herein, wherein at least one hydrogen atom is replaced with a thiourea group, as defined herein. Unless stated otherwise specifically in the specification, a thioureaalkyl group is optionally substituted.

[0025] “Urea” refers to a moiety of the formula -NH-C(O)-NHR where R is selected from hydrogen, alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl, each of which may be optionally substituted.

[0026] “Ureaalkyl” refers to an alkyl group, as defined herein, wherein at least one hydrogen atom is replaced with a urea group, as defined herein. Unless stated otherwise specifically in the specification, a ureaalkyl group is optionally substituted.

[0027] The term “substituted” used herein refers to replacement of at least one hydrogen atom with any of the above groups (e.g., amino, carboxy, hydroxy, imino, acyl, alkyl, alkoxy, alkylamino, alkylaminoalkyl, amido, aminoalkyl, aminocarbonyl, aryl, arylalkyl, arylalkylamino, arylalkyloxy, arylamino, aryloxy, carboxyalkyl, cyano, cyanoalkyl, cycloalkyl, cycloalkyl, cycloalkylamino, cycloalkylalkyloxy, cycloalkylamino, cycloalkyloxy, halo, haloalkyl, heteroatom, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylalkylamino, heteroarylalkyloxy, heteroarylamino, heteroaryloxy, heterobicycloalkyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkylamino, heterocyclylalkyloxy, heterocyclylamino, heterocyclyloxy, hydroxyalkyl, thioalkyl, alkylene, alkylenecarbonyl, alkenylene, alkenylenecarbonyl, arylene, heteroalkylene, heteroalkylenecarbonyl, heteroarylene, heteroarylenecarbonyl, heterocyclylalkylene, and / or heterocyclylalkylenecarbonyl), wherein the at least one hydrogen atom is replaced by a bond to a non-hydrogen atom such as, but not limited to: a halogen atom such as F, Cl, Br, and I; an oxygen atom in groups such as hydroxyl groups, alkoxy groups, and ester groups; a sulfur atom in groups such as thiol groups, thioalkyl groups, sulfone groups such as alkyl sulfone groups, sulfonyl groups such as sulfonamide groups and sulfonylalkyl groups such as sulfonylmethane, and sulfoxide groups such as alkyl sulfoxide groups; a nitrogen atom in groups such as amino, amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; a silicon atom in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; a phosphorus atom in groups such as dialkylphosphine oxide groups; and other heteroatoms in various other groups. “Substituted” also means any of the above groups in which one or more hydrogen atoms are replaced by a higher-order bond (e.g., a double- or triple-bond) to a carbon atom or a heteroatom such as oxygen in oxo, carbonyl, carboxyl, and ester groups; and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. “Substituted” includes any of the above groups in which one or more hydrogen atoms are replaced with -NRgRh, -NRgC(=O)Rh, -NRgC(=O)NRgRh, -NRgC(=O)ORh, -NRgSO2Rh, -OC(=O)NRgRh, - ORg, -SRg, -SORg, -SO2Rg, -OSO2Rg, -SO2ORg, =NSO2Rg, -SO2NRgRh, -C(=O)Rg, -C(=O)ORg, -C(=O)NRgRh, -CH2SO2Rg, or -CH2SO2NRgRh, where Rgand Rhare independently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl, arylalkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heteroalkyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and / or heteroarylalkyl. “Substituted” further means any of the above groups in which one or more hydrogen atoms are replaced by a bond to an amino, carbonyl, carboxy, cyano, hydroxyl, imino, nitro, oxo, thioxo, acyl, alkyl, alkoxy, alkylamino, alkylaminoalkyl, amide, aminoalkyl, aminocarbonyl, aryl, arylalkyl, arylalkylamino, arylalkyloxy, arylamino, aryloxy, bicycloalkyl, carboxyalkyl, cyanoalkyl, cycloalkyl, cycloalkylalkyl, cycloalkylamino, cycloalkyloxy, cycloalkylamino, cycloalkyloxy, halo, haloalkyl, heteroatom, heteroalkyl, heteroaryl, heteroarylalkyl, heteroarylalkylamino, heteroarylalkyloxy, heteroarylamino, heteroaryloxy, heterobicycloalkyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkylamino, heterocyclylalkyloxy, heterocyclylamino, heterocyclyloxy, hydroxyalkyl, N-heteroaryl, N-heterocyclyl, thioalkyl, alkylene, alkylenecarbonyl, alkenylene, alkenylenecarbonyl, arylene, heteroalkylene, heteroalkylenecarbonyl, heteroarylene, heteroarylenecarbonyl, heterocyclylalkylene, heterocyclylalkylenecarbonyl, methylidene, trimethylsilanyl, dialkylphosphine oxide, -OR, -SR, -OC(O)-R, -N(R)2, -C(O)R, -C(O)OR, -C(O)N(R)2, -N(R)C(O)OR, -N(R)C(O)R, -N(R)S(O)tR (where t is 1 or 2), -S(O)tOR (where t is 1 or 2), -S(O)tN(R)2(where t is 1 or 2), -PO(R)2, or -PO(OR)2group, where each R is independently hydrogen, alkyl, haloalkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl group. In addition, each of the foregoing substituents is optionally substituted with one or more of the above substituents. The term “optionally substituted,” as used herein, means that the referenced group (e.g., alkyl, cycloalkyl, etc.) may or may not be substituted with one or more substituents. DETAILED DESCRIPTION Provided herein are small molecules that bind to ASH1L and inhibit ASH1L activity, and methods of use thereof for the treatment of cancer. Modifications of chromatin, including histone lysine methylation, play important roles in physiological and disease states. The ASH1L (absent, small, or homeotic-like 1) protein belongs to the family of histone lysine methyltransferases and catalyzes the mono- and demethylation of histone 3 lysine 36 (H3K36), which represents an activating chromatin mark. ASH1L is a large protein (>3,000 amino acids) that contains multiple structural domains, including catalytic SET domain and three chromatin reader domains (bromodomain, PHD and BAH domains) in addition to a long unstructured region at the N-terminal portion of the protein. The SET domain of ASH1L catalyzes the transfer of a methyl group from S-adenosyl methionine (SAM) cofactor to the lysine substrate (H3K36). ASH1L was shown to regulate the expression of HOXA genes and plays an important role in development of acute leukemia with translocations of the Mixed Lineage Leukemia 1 (MLL1) gene (also known as KMT2A). MLL1 translocations are found in ~5-10% of acute leukemia patients, leading to poor clinical outcome with only 35% 5-year survival, supporting the need for new therapeutics. ASH1L knockdown was shown to induce cell growth arrest, apoptosis, and differentiation, associated with downregulation of HOXA9 genes in leukemia cells with MLL1 translocations, and it also abrogates development of leukemia in mice. It has been demonstrated that the catalytic SET domain of ASH1L plays an important role in leukemogenesis mediated by MLL1 fusion proteins, supporting that small molecule inhibitors of the ASH1L SET domain could result in new anti-leukemic agents. Furthermore, ASH1L overexpression was found in different solid tumors, including thyroid, breast, and liver cancers, and was linked to cancer cell growth and aggressive disease, supporting further ASH1L as an attractive therapeutic target in hematologic and solid tumors. First-in-class small molecule inhibitors targeting the catalytic SET domain of ASH1L have been described (See, e.g., U.S. Pat. No.9,855,302; incorporated by reference in its entirety), which represent the only ASH1L inhibitors reported to date. These compounds, represented by AS-85 (compound 1) and AS-99 (compound 2), bind to the SET domain of ASH1L with sub-micromolar binding affinities and selectively inhibit its catalytic activity with the IC50values of 0.6 µM and 0.79 µM, respectively (Figure 1A), without affecting other histone methyltransferases (HMTs). AS-99 also demonstrated anti-leukemic activity in leukemic cells with MLL1 translocations, supporting that blocking the catalytic SET domain of ASH1L leads to anti-leukemic effects. Provided herein is the structure-based development of ‘next- generation’ of ASH1L inhibitors derived from AS-85 and AS-99 to occupy an additional pocket on ASH1L. Development of a new fluorescence polarization (FP) assay to effectively assess the activity of these new ASH1L inhibitors and systematic structure-activity relationship (SAR) for these compounds is presented herein, leading to compounds with substantially improved inhibitory activity over the previous generation of ASH1L inhibitors. When tested in leukemia cells with MLL1 translocations, the most potent of the ASH1L inhibitors herein (e.g.,66s (AS- 254s)) demonstrated more pronounced anti-leukemic activity in blocking proliferation and inducing apoptosis and differentiation that previous ASH1L inhibitors. The significance of ASH1L in acute leukemia and other cancers strongly supports its potential as a promising therapeutic target in oncology. In this context, provided herein are ASH1L inhibitors with improved inhibitory activity (e.g., IC50< 0.2 µM) and enhanced cellular efficacy in leukemia cells (e.g., GI50 < 1 µM, strong differentiation, downregulation of leukemia relevant target genes) as compared to existing ASH1L inhibitors. The ASH1L inhibitors herein also demonstrate in vivo activity in xenograft models of MLL1-rearranged leukemia. In some embodiments, the compounds described herein find use in the treatment or prevention of cancer (e.g., leukemias) and / or the alleviation of symptoms associated therewith. In some embodiments, provided herein are pharmaceutical compositions comprising a compound described and / or within the scope herein. In some embodiments, pharmaceutical compositions comprising a compound described and / or within the scope herein are administered to a subject to treat cancer (e.g., leukemias). Formula (I) In some embodiments, provided herein are compounds of formula (I): or a pharmaceutically acceptable salt thereof; wherein E and A are a spirocyclic ring system; wherein Z is CH or N; wherein Y is N or O; and wherein if R1, R2, R6, and R7are selected from functional groups described herein and / or provided in the exemplary compounds of, for example Tables 1-4 and A-D. In some embodiments, if Y is O then R1is absent. In some embodiments, if Y is N then R1is SO2-alkyl, wherein alkyl is selected from - CH3, -CH2CH3, -(CH2)2CH3, and –CH(CH3)2. In some embodiments, if Y is N then R1is SO2CH3. In some embodiments, R2is selected from -C(S)NH2, -C(O)NH2, -C(Se)NH2, oxirane, thiirane, aziridine, -CF3, and -CN. In some embodiments, R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and wherein R8is selected from any of the R8groups of the compounds of Tables 1-4. In some embodiments, R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, -halogen (e.g., -F), etc.). In some embodiments, R8is selected from H, CH3, CF3, - C(O)CH3, -CH2NHC(O)OC(CH3)3, In some embodiments, R7is selected from H and halogen (e.g., Cl). In other embodiments, R7is selected from H, halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2-OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3. In some embodiments, the EA spirocyclic ring system is selected from: . Formula (Ia) In some embodiments, provided herein are compounds of formula (Ia): wherein E and A are a spirocyclic ring system; wherein Z is CH or N; wherein Y is N or O; wherein if Y is O then R1is absent; wherein if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; wherein X is O, Se, or S; In some embodiments, R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and wherein R8is selected from any of the R8groups of the compounds of Tables 1-4 or described herein. In some embodiments, R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, -halogen (e.g., -F), etc.). In some embodiments, R8is selected from H, CH3, CF3, -C(O)CH3, -CH2NHC(O)OC(CH3)3, In some embodiments, R7is selected from H and halogen (e.g., Cl). In other embodiments, R7is selected from H, halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2-OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3. In some embodiments, the EA spirocyclic ring system is selected from: . Formula (Ib) In some embodiments, provided herein are compounds of formula (Ib):

[0028] wherein Z is CH or N; wherein Y is N or O; wherein if Y is O then R1is absent; wherein if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; wherein X is O, Se, or S; wherein R7is selected from H, halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2-OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3; and wherein R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and wherein R8is selected from any of the R8groups of the compounds of Tables 1-4 or described herein. In some embodiments, R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, - halogen (e.g., -F), etc.). In some embodiments, R8is selected from H, CH3, CF3, -C(O)CH3, - CH2NHC(O)OC(CH3)3, In some embodiments, R7is selected from H and halogen (e.g., Cl). Formula (Ic) In some embodiments, provided herein are compounds of formula (Ic): wherein Z is CH or N; wherein Y is N or O; wherein if Y is O then R1is absent; wherein if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; wherein X is O, Se, or S; wherein R7is selected from H, halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2-OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3; and wherein R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and wherein R8is selected from any of the R8groups of the compounds of Tables 1-4 or described herein. In some embodiments, R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, - halogen (e.g., -F), etc.). In some embodiments, R8is selected from H, CH3, CF3, -C(O)CH3, - CH2NHC(O)OC(CH3)3, In some embodiments, R7is selected from H and halogen (e.g., Cl). Formula (II) In some embodiments, provided herein are compounds of formula (II): wherein Z is CH or N; wherein Y is N or O; wherein if Y is O then R1is absent; wherein if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; wherein X is O, Se, or S; and wherein R7is selected from H, halogen (e.g., Cl, F, Br, I), CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2-OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3.Formula (III) In some embodiments, provided herein are compounds of formula (III):

[0029] wherein R1 is selected from NH2and (CH2)NHC(O)CH3; and wherein R2is selected from -C(S)NH2, -C(O)NH2, -C(Se)NH2, oxirane, thiirane, aziridine, -CF3, Exemplary compounds have been synthesized using the techniques and procedures described herein. The exemplary compounds of Tables 1-4 have been synthesized, had their molecular weight confirmed by HR-MS, and affinity for ASH1L measured by fluorescence polarization. Table 1. (IC50= 20-500 μM) Table 2. (IC50= 2- 20 μM) Table 3. (IC50= 0.2- 2 μM) Table 4. (IC50< 0.2 μM) The compounds and intermediates may be isolated and purified by methods well-known to those skilled in the art of organic synthesis. Examples of conventional methods for isolating and purifying compounds can include, but are not limited to, chromatography on solid supports such as silica gel, alumina, or silica derivatized with alkylsilane groups, by recrystallization at high or low temperature with an optional pretreatment with activated carbon, thin-layer chromatography, distillation at various pressures, sublimation under vacuum, and trituration, as described for instance in “Vogel's Textbook of Practical Organic Chemistry” 5th edition (1989), by Furniss, Hannaford, Smith, and Tatchell, pub. Longman Scientific & Technical, Essex CM20 2JE, England. Reaction conditions and reaction times for each individual step can vary depending on the particular reactants employed and substituents present in the reactants used. Reactions can be worked up in the conventional manner, e.g. by eliminating the solvent from the residue and further purified according to methodologies generally known in the art such as, but not limited to, crystallization, distillation, extraction, trituration and chromatography. Unless otherwise described, the starting materials and reagents arc cither commercially available or can be prepared by one skilled in the art from commercially available materials using methods described in the chemical literature. Starting materials, if not commercially available, can be prepared by procedures selected from standard organic chemical techniques, techniques that are analogous to the synthesis of known, structurally similar compounds, or techniques that are analogous to the above described schemes or the procedures described in the synthetic examples section.

[0030] Routine experimentations, including appropriate manipulation of the reaction conditions, reagents and sequence of the synthetic route, protection of any chemical functionality that cannot be compatible with the reaction conditions, and deprotection at a suitable point in the reaction sequence of the method are included in the scope of the disclosure. Suitable protecting groups and the methods for protecting and deprotecting different substituents using such suitable protecting groups are well known to those skilled in the art; examples of which can be found in P G M Wuts and T W Greene, in Greene's book titled Protective Groups in Organic Synthesis (4thed.), John Wiley & Sons, NY (2006), which is incorporated herein by reference in its entirety. Synthesis of the compounds of the disclosure can be accomplished by methods analogous to those described in the synthetic schemes described above and in specific examples described below.

[0031] The compounds described herein may in some cases exist as diastereomers, enantiomers, or other stereoisomeric forms. The compounds presented herein include all diastereomeric, enantiomeric, and stereoisomeric forms as well as the appropriate mixtures thereof. Separation of stereoisomers may be performed by chromatography or by the forming diastereomeric and separation by recrystallization, or chromatography, or any combination thereof. (Jean Jacques, Andre Collet, Samuel H. Wilen, “Enantiomers, Racemates and Resolutions”, John Wiley And Sons, Inc., 1981, herein incorporated by reference for this disclosure). Stereoisomers may also be obtained by stereoselective synthesis.

[0032] In some embodiments, compounds may exist as tautomers. All tautomers are included within the formulas described herein.

[0033] Unless specified otherwise, divalent variables or groups described herein may be attached in the orientation in which they are depicted or they may be attached in the reverse orientation. The methods and compositions described herein include the use of amorphous forms as well as crystalline forms (also known as polymorphs). The compounds described herein may be in the form of pharmaceutically acceptable salts. As well, active metabolites of these compounds having the same type of activity are included in the scope of the present disclosure. In addition, the compounds described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, etc. The solvated forms of the compounds presented herein are also considered to be disclosed herein.

[0034] In some embodiments, compounds or salts described herein may be prodrugs. A “prodrug” refers to an agent that is converted into the parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. An example, without limitation, of a prodrug would be a compound described herein, which is administered as an ester (the “prodrug”) to facilitate transmittal across a cell membrane where water solubility is detrimental to mobility but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water-solubility is beneficial. A further example of a prodrug might be a short peptide (polyaminoacid) bonded to an acid group where the peptide is metabolized to reveal the active moiety. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In certain embodiments, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.

[0035] To produce a prodrug, a pharmaceutically active compound is modified such that the active compound will be regenerated upon in vivo administration. The prodrug can be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. In some embodiments, by virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, once a pharmaceutically active compound is determined, prodrugs of the compound are designed, (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392; Silverman (1992), The Organic Chemistry of Drug Design and Drug Action, Academic Press, Inc., San Diego, pages 352-401, Saulnier et al., (1994), Bioorganic and Medicinal Chemistry Letters, Vol.4, p.1985; Rooseboom et al., Pharmacological Reviews, 56:53–102, 2004; Miller et al., J. Med. Chem. Vol.46, no.24, 5097-5116, 2003; Aesop Cho, “Recent Advances in Oral Prodrug Discovery”, Annual Reports in Medicinal Chemistry, Vol.41, 395-407, 2006). The compounds described herein may be labeled isotopically (e.g. with a radioisotope) or by other means, including, but not limited to, the use of chromophores or fluorescent moieties, bioluminescent labels, photoactivatable or chemiluminescent labels, affinity labels (e.g. biotin), etc. Compounds and salts described herein include isotopically-labeled compounds. In general, isotopically-labeled compounds are identical to those recited in the various formulae and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number most common in nature. Examples of isotopes that can be incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, for example,2H,3H,13C,14C,15N,18O,17O,35S,18F, or36Cl. Certain isotopically-labeled compounds described herein, for example those into which radioactive isotopes such as3H and14C are incorporated, are useful in drug and / or substrate tissue distribution assays. Further, substitution with isotopes such as deuterium, i.e.,2H, can afford certain therapeutic advantages resulting from greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements. In additional or further embodiments, the compounds described herein are metabolized upon administration to an organism in need to produce a metabolite that is then used to produce a desired effect, including a desired therapeutic effect. Compounds described herein may be formed as, and / or used as, pharmaceutically acceptable salts. The type of pharmaceutical acceptable salts, include, but are not limited to: (1) acid addition salts, formed by reacting the free base form of the compound with a pharmaceutically acceptable: inorganic acid, such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, metaphosphoric acid, and the like; or with an organic acid, such as, for example, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, cthancsulfonic acid, 1,2-cthancdisulfonic acid, 2-hydroxycthancsulfonic acid, bcnzcncsulfonic acid, toluenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-l- carboxylic acid, glucoheptonic acid, 4,4’-methylenebis-(3-hydroxy-2-ene-l-carboxylic acid), 3- phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, butyric acid, phenylacetic acid, phenylbutyric acid, valproic acid, and the like; (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion (e.g. lithium, sodium, potassium), an alkaline earth ion (e.g. magnesium, or calcium), or an aluminum ion. In some cases, compounds described herein may coordinate with an organic base, such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, dicyclohexylamine, tris(hydroxymethyl)methylamine. In other cases, compounds described herein may form salts with amino acids such as, but not limited to, arginine, lysine, and the like. Acceptable inorganic bases used to form salts with compounds that include an acidic proton, include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

[0036] It should be understood that a reference to a pharmaceutically acceptable salt includes the solvent addition forms or crystal forms thereof, particularly solvates or polymorphs. Solvates contain either stoichiometric or non- stoichiometric amounts of a solvent, and may be formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is alcohol. Solvates of compounds described herein can be conveniently prepared or formed during the processes described herein. In addition, the compounds provided herein can exist in unsolvated as well as solvated forms. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the compounds and methods provided herein.

[0037] In some embodiments, compounds described herein are in various forms, including but not limited to, amorphous forms, milled forms and nano-particulate forms. In addition, compounds described herein include crystalline forms, also known as polymorphs. Polymorphs include the different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, melting points, density, hardness, crystal shape, optical properties, stability, and solubility. Various factors such as the recrystallization solvent, rate of crystallization, and storage temperature may cause a single crystal form to dominate.

[0038] The screening and characterization of the pharmaceutically acceptable salts, polymorphs and / or solvates may be accomplished using a variety of techniques including, but not limited to, thermal analysis, x-ray diffraction, spectroscopy, vapor sorption, and microscopy. Thermal analysis methods address thermo chemical degradation or thermo physical processes including, but not limited to, polymorphic transitions, and such methods are used to analyze the relationships between polymorphic forms, determine weight loss, to find the glass transition temperature, or for excipient compatibility studies. Such methods include, but are not limited to, Differential scanning calorimetry (DSC), Modulated Differential Scanning Calorimetry (MDCS), Thermogravimetric analysis (TGA), and Thermogravi-metric and Infrared analysis (TG / IR). X- ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron sources. The various spectroscopic techniques used include, but are not limited to, Raman, FTIR, UV-VIS, and NMR (liquid and solid state). The various microscopy techniques include, but are not limited to, polarized light microscopy, Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX), Environmental Scanning Electron Microscopy with EDX (in gas or water vapor atmosphere), IR microscopy, and Raman microscopy.

[0039] Pharmaceutical Compositions

[0040] In certain embodiments, a compound disclosed herein are ASH1L inhibitors (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof, is combined with one or more additional agents to form a pharmaceutical composition. Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries that facilitate processing of the active compound into a preparation, which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Additional details about suitable excipients for pharmaceutical compositions described herein may be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), herein incorporated by reference for such disclosure.

[0041] A pharmaceutical composition, as used herein, refers to a mixture of a compound disclosed herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof, with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the compound to a subject. In practicing the methods of treatment or use provided herein, therapeutically effective amounts of compounds described herein are administered in a pharmaceutical composition to a subject having a disease, disorder, or condition to be treated (e.g., cancer). In some embodiments, the subject is a human. A therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. The compound or pharmaceutically acceptable salt thereof, can be used singly or in combination with one or more therapeutic agents as components of mixtures (as in combination therapy).

[0042] The pharmaceutical formulations described herein can be administered to a subject by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration routes. Moreover, the pharmaceutical compositions described herein, which include a compound disclosed herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof, can be formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, aerosols, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, and capsules.

[0043] One may administer the compounds and / or compositions in a local rather than systemic manner, for example, via injection of the compound directly into an organ or tissue, often in a depot preparation or sustained release formulation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with organ-specific antibody. The liposomes will be targeted to and taken up selectively by the organ. In addition, the drug may be provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate release formulation.

[0044] Pharmaceutical compositions including a compound described herein may be manufactured in a conventional manner, such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0045] In certain embodiments, compositions provided herein may also include one or more preservatives to inhibit microbial activity. Suitable preservatives include quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0046] Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipients with one or more of the compounds disclosed herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets, pills, or capsules. Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents may be added, such as the cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0047] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, scaled capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

[0048] In some embodiments, the solid dosage forms disclosed herein may be in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapiddisintegration tablet, an effervescent tablet, or a caplet), a pill, a powder (including a sterile packaged powder, a dispensable powder, or an effervescent powder), a capsule (including both soft or hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”), solid dispersion, solid solution, bioerodible dosage form, multiparticulate dosage forms, pellets, granules, or an aerosol. In other embodiments, the pharmaceutical formulation is in the form of a powder. In still other embodiments, the pharmaceutical formulation is in the form of a tablet, including but not limited to, a fast-melt tablet. Additionally, pharmaceutical formulations of the compounds described herein may be administered as a single capsule or in multiple capsule dosage form. In some embodiments, the pharmaceutical formulation is administered in two, or three, or four, capsules or tablets.

[0049] In some embodiments, solid dosage forms, e.g., tablets, effervescent tablets, and capsules, are prepared by mixing particles of a compound disclosed herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof, with one or more pharmaceutical excipients to form a bulk blend composition. When referring to these bulk blend compositions as homogeneous, it is meant that the particles of the compound are dispersed evenly throughout the composition so that the composition may be subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. The individual unit dosages may also include film coatings, which disintegrate upon oral ingestion or upon contact with diluent. These formulations can be manufactured by conventional pharmacological techniques.

[0050] The pharmaceutical solid dosage forms described herein can include a compound disclosed herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof. In still other aspects, using standard coating procedures, such as those described in Remington's Pharmaceutical Sciences, 20th Edition (2000), a film coating is provided around the formulation of the compound described herein. In one embodiment, some or all of the particles of the compound described herein are coated. In another embodiment, some or all of the particles of the compound described herein are microencapsulated. In still another embodiment, the particles of the compound described herein are not microencapsulated and are uncoated.

[0051] Suitable carriers for use in the solid dosage forms described herein include, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodium chloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, sucrose, microcrystalline cellulose, lactose, mannitol and the like.

[0052] Suitable filling agents for use in the solid dosage forms described herein include, but are not limited to, lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, hydroxypropylmethycellulose (HPMC), hydroxypropylmethycellulose phthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS), sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, and the like.

[0053] In order to release the compound from a solid dosage form matrix as efficiently as possible, disintegrants are often used in the formulation, especially when the dosage forms are compressed with binder. Disintegrants help rupturing the dosage form matrix by swelling or capillary action when moisture is absorbed into the dosage form. Suitable disintegrants for use in the solid dosage forms described herein include, but are not limited to, natural starch such as com starch or potato starch, a pregelatinized starch such as National 1551 or Amijel®, or sodium starch glycolate such as Promogel®or Explotab®, a cellulose such as a wood product, methylcrystalline cellulose, e.g., Avicel®, Avicel®PH101, Avicel®PH102, Avicel®PH105, Elcema®P100, Emcocel®, Vivacel®, Ming Tia®, and Solka-Floc®, methylcellulose, croscarmellose, or a cross-linked cellulose, such as cross-linked sodium carboxymethylcellulose (Ac-Di-Sol®), cross-linked carboxymethylcellulose, or cross-linked croscarmellose, a cross- linked starch such as sodium starch glycolate, a cross-linked polymer such as crospovidone, a cross-linked polyvinylpyrrolidone, alginate such as alginic acid or a salt of alginic acid such as sodium alginate, a clay such as Veegum®HV (magnesium aluminum silicate), a gum such as agar, guar, locust bean, Karaya, pectin, or tragacanth, sodium starch glycolate, bentonite, a natural sponge, a surfactant, a resin such as a cation-exchange resin, citrus pulp, sodium lauryl sulfate, sodium lauryl sulfate in combination starch, and the like. Binders impart cohesiveness to solid oral dosage form formulations: for powder filled capsule formulation, they aid in plug formation that can be filled into soft or hard shell capsules and for tablet formulation, they ensure the tablet remaining intact after compression and help assure blend uniformity prior to a compression or fill step. Materials suitable for use as binders in the solid dosage forms described herein include, but are not limited to, carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose (e.g. Hypromellose USP Pharmacoat-603, hydroxypropylmethylcellulose acetate stearate (Aqoate HS- LF and HS), hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g., Avicel®), microcrystalline dextrose, amylose, magnesium aluminum silicate, polysaccharide acids, bentonites, gelatin, polyvinylpyrrolidone / vinyl acetate copolymer, crospovidone, povidone, starch, pregelatinized starch, tragacanth, dextrin, a sugar, such as sucrose (e.g., Dipac®), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g., Xylitab®), lactose, a natural or synthetic gum such as acacia, tragacanth, ghatti gum, mucilage of isapol husks, starch, polyvinylpyrrolidone (e.g., Povidone®CL, Kollidon®CL, Polyplasdone®XL-10, and Povidone®K-12), larch arabogalactan, Veegum®, polyethylene glycol, waxes, sodium alginate, and the like. In general, binder levels of 20-70% are used in powder-filled gelatin capsule formulations. Binder usage level in tablet formulations varies whether direct compression, wet granulation, roller compaction, or usage of other excipients such as fillers which itself can act as moderate binder. In some embodiments, formulators determine the binder level for the formulations, but binder usage level of up to 70% in tablet formulations is common.

[0054] Suitable lubricants or glidants for use in the solid dosage forms described herein include, but are not limited to, stearic acid, calcium hydroxide, talc, com starch, sodium stearyl fumerate, alkali-metal and alkaline earth metal salts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, magnesium stearate, zinc stearate, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, a polyethylene glycol or a methoxypolyethylene glycol such as Carbowax™, PEG 4000, PEG 5000, PEG 6000, propylene glycol, sodium oleate, glyceryl behenate, glyceryl palmitostearate, glyceryl benzoate, magnesium or sodium lauryl sulfate, and the like.

[0055] Suitable diluents for use in the solid dosage forms described herein include, but are not limited to, sugars (including lactose, sucrose, and dextrose), polysaccharides (including dextrates and maltodextrin), polyols (including mannitol, xylitol, and sorbitol), cyclodextrins and the like.

[0056] Suitable wetting agents for use in the solid dosage forms described herein include, for example, oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, quaternary ammonium compounds (e.g., Polyquat 10®), sodium oleate, sodium lauryl sulfate, magnesium stearate, sodium docusate, triacetin, vitamin E TPGS and the like.

[0057] Suitable surfactants for use in the solid dosage forms described herein include, for example, sodium lauryl sulfate, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic®(BASF), and the like.

[0058] Suitable suspending agents for use in the solid dosage forms described here include, but are not limited to, polyvinylpyrrolidone, e.g., polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., the polyethylene glycol can have a molecular weight of about 300 to about 6000, or about 3350 to about 4000, or about 5400 to about 7000, vinyl pyrrolidone / vinyl acetate copolymer (S630), sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, polysorbate- 80, hydroxyethylcellulose, sodium alginate, gums, such as, e.g., gum tragacanth and gum acacia, guar gum, xanthans, including xanthan gum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80, sodium alginate, polycthoxylatcd sorbitan monolauratc, polycthoxylatcd sorbitan monolauratc, povidone and the like.

[0059] Suitable antioxidants for use in the solid dosage forms described herein include, for example, e.g., butylated hydroxytoluene (BHT), sodium ascorbate, and tocopherol.

[0060] There is considerable overlap between additives used in the solid dosage forms described herein. Thus, the above-listed additives should be taken as merely exemplary, and not limiting, of the types of additives that can be included in solid dosage forms of the pharmaceutical compositions described herein.

[0061] In other embodiments, one or more layers of the pharmaceutical formulation are plasticized. Illustratively, a plasticizer is generally a high boiling point solid or liquid. Suitable plasticizers can be added from about 0.01% to about 50% by weight (w / w) of the coating composition. Plasticizers include, but are not limited to, diethyl phthalate, citrate esters, polyethylene glycol, glycerol, acetylated glycerides, triacetin, polypropylene glycol, polyethylene glycol, triethyl citrate, dibutyl sebacate, stearic acid, stearol, stearate, and castor oil.

[0062] Compressed tablets are solid dosage forms prepared by compacting the bulk blend of the formulations described above. In various embodiments, compressed tablets which are designed to dissolve in the mouth will include one or more flavoring agents. In other embodiments, the compressed tablets will include a film surrounding the final compressed tablet. In some embodiments, the film coating aids in patient compliance (e.g., Opadry®coatings or sugar coating). Film coatings including Opadry®typically range from about 1% to about 3% of the tablet weight. In other embodiments, the compressed tablets include one or more excipients.

[0063] A capsule may be prepared, for example, by placing the bulk blend of the formulation of the compound described above, inside of a capsule. In some embodiments, the formulations (non-aqueous suspensions and solutions) are placed in a soft gelatin capsule. In other embodiments, the formulations are placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC. In other embodiments, the formulation is placed in a sprinkle capsule, wherein the capsule may be swallowed whole or the capsule may be opened and the contents sprinkled on food prior to eating. In some embodiments, the therapeutic dose is split into multiple (e.g., two, three, or four) capsules. In some embodiments, the entire dose of the formulation is delivered in a capsule form. In various embodiments, the particles of the compound disclosed herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof), and one or more excipients are dry blended and compressed into a mass, such as a tablet, having a hardness sufficient to provide a pharmaceutical composition that substantially disintegrates within less than about 30 minutes, less than about 35 minutes, less than about 40 minutes, less than about 45 minutes, less than about 50 minutes, less than about 55 minutes, or less than about 60 minutes, after oral administration, thereby releasing the formulation into the gastrointestinal fluid.

[0064] In another aspect, dosage forms may include microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulation material. Exemplary materials include, but are not limited to, pH modifiers, erosion facilitators, anti-foaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0065] Materials useful for the microencapsulation described herein include materials compatible with compounds described herein, which sufficiently isolate the compound from other non-compatible excipients.

[0066] In still other embodiments, effervescent powders are also prepared in accordance with the present disclosure. Effervescent salts have been used to disperse medicines in water for oral administration. Effervescent salts are granules or coarse powders containing a medicinal agent in a dry mixture, usually composed of sodium bicarbonate, citric acid and / or tartaric acid. When such salts are added to water, the acids and the base react to liberate carbon dioxide gas, thereby causing “effervescence.” Examples of effervescent salts include, e.g., the following ingredients: sodium bicarbonate or a mixture of sodium bicarbonate and sodium carbonate, citric acid and / or tartaric acid. Any acid-base combination that results in the liberation of carbon dioxide can be used in place of the combination of sodium bicarbonate and citric and tartaric acids, as long as the ingredients were suitable for pharmaceutical use and result in a pH of about 6.0 or higher.

[0067] In other embodiments, the formulations described herein are solid dispersions. Methods of producing such solid dispersions include, but are not limited to, for example, U.S. Pat. Nos. 4,343,789, 5,340,591, 5,456,923, 5,700,485, 5,723,269, and U.S. patent publication no. 2004 / 0013734. In still other embodiments, the formulations described herein are solid solutions. Solid solutions incorporate a substance together with the active agent and other excipients such that heating the mixture results in dissolution of the drug and the resulting composition is then cooled to provide a solid blend which can be further formulated or directly added to a capsule or compressed into a tablet. Methods of producing such solid solutions include, but are not limited to, for example, U.S. Pat. Nos. 4,151,273, 5,281,420, and 6,083,518.

[0068] In some embodiments, pharmaceutical formulations are provided that include particles of the compound disclosed herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof, and at least one dispersing agent or suspending agent for oral administration to a subject. The formulations may be a powder and / or granules for suspension, and upon admixture with water, a substantially uniform suspension is obtained.

[0069] Liquid formulation dosage forms for oral administration can be aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. See, e.g., Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002).

[0070] The aqueous suspensions and dispersions described herein can remain in a homogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005 edition, chapter 905), for at least 4 hours. The homogeneity should be determined by a sampling method consistent with regard to determining homogeneity of the entire composition. In one embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 1 minute. In another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 45 seconds. In yet another embodiment, an aqueous suspension can be re-suspended into a homogenous suspension by physical agitation lasting less than 30 seconds. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion.

[0071] The pharmaceutical compositions described herein may include sweetening agents such as, but not limited to, acacia syrup, acesulfame K, alitame, anise, apple, aspartame, banana, Bavarian cream, berry, black currant, butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream, chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream, cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate, cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger, glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey, isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate (MagnaSweet®), maltol, mannitol, maple, marshmallow, menthol, mint cream, mixed berry, neohesperidine DC, ncotamc, orange, pear, peach, peppermint, peppermint cream, Prosweet®Powder, raspberry, root beer, rum, saccharin, safrole, sorbitol, spearmint, spearmint cream, strawberry, strawberry cream, stevia, sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfame potassium, mannitol, talin, sucralose, sorbitol, swiss cream, tagatose, tangerine, thaumatin, tutti fruitti, vanilla, walnut, watermelon, wild cherry, Wintergreen, xylitol, or any combination of these flavoring ingredients, e.g., anise-menthol, cherry-anise, cinnamon-orange, cherry-cinnamon, chocolate-mint, honey-lemon, lemon-lime, lemon-mint, menthol-eucalyptus, orange-cream, vanilla-mint, and mixtures thereof.

[0072] In some embodiments, the pharmaceutical formulations described herein can be selfemulsifying drug delivery systems (SEDDS). Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions are created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase can be added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. SEDDS may provide improvements in the bioavailability of hydrophobic active ingredients. Methods of producing self-emulsifying dosage forms include, but are not limited to, for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and 6,960,563.

[0073] There is overlap between the above-listed additives used in the aqueous dispersions or suspensions described herein, since a given additive is often classified differently by different practitioners in the field, or is commonly used for any of several different functions. Thus, the above-listed additives should be taken as merely exemplary, and not limiting, of the types of additives that can be included in formulations described herein.

[0074] Potential excipients for intranasal formulations include, for example, U.S. Pat. Nos.

[0075] 4,476,116, 5,116,817 and 6,391,452. Formulations solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizing or dispersing agents. See, for example, Ansel, H. C. et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, Sixth Ed. (1995). Preferably these compositions and formulations are prepared with suitable nontoxic pharmaceutically acceptable ingredients.. The choice of suitable carriers is highly dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents may also be present. Preferably, the nasal dosage form should be isotonic with nasal secretions.

[0076] For administration by inhalation, the compounds described herein may be in a form as an aerosol, a mist or a powder. Pharmaceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, such as, by way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound described herein and a suitable powder base such as lactose or starch.

[0077] Buccal formulations that include compounds described herein may be administered using a variety of formulations which include, but are not limited to, U.S. Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739,136. In addition, the buccal dosage forms described herein can further include a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal mucosa. The buccal dosage form is fabricated so as to erode gradually over a predetermined time period, wherein the delivery of the compound is provided essentially throughout. Buccal drug delivery avoids the disadvantages encountered with oral drug administration, e.g., slow absorption, degradation of the active agent by fluids present in the gastrointestinal tract and / or first-pass inactivation in the liver. With regard to the bioerodible (hydrolysable) polymeric carrier, virtually any such carrier can be used, so long as the desired drug release profile is not compromised, and the carrier is compatible with the compounds described herein, and any other components that may be present in the buccal dosage unit. Generally, the polymeric carrier comprises hydrophilic (water-soluble and water-swellable) polymers that adhere to the wet surface of the buccal mucosa. Examples of polymeric carriers useful herein include acrylic acid polymers and co, e.g., those known as “carbomers” (Carbopol®, which may be obtained from B.F. Goodrich, is one such polymer). Other components may also be incorporated into the buccal dosage forms described herein include, but are not limited to, disintegrants, diluents, binders, lubricants, flavoring, colorants, preservatives, and the like. For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner. Transdermal formulations described herein may be administered using a variety of devices including but not limited to, U.S. Pat. Nos.3,598,122, 3,598,123, 3,710,795, 3,731,683, 3,742,951, 3,814,097, 3,921,636, 3,972,995, 3,993,072, 3,993,073, 3,996,934, 4,031,894, 4,060,084, 4,069,307, 4,077,407, 4,201,211, 4,230,105, 4,292,299, 4,292,303, 5,336,168, 5,665,378, 5,837,280, 5,869,090, 6,923,983, 6,929,801 and 6,946,144. The transdermal dosage forms described herein may incorporate certain pharmaceutically acceptable excipients which are conventional in the art. In one embodiment, the transdermal formulations described herein include at least three components: (1) a formulation of a compound disclosed herein (e.g., a compound of formula (I), (Ia), (Ib), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof; (2) a penetration enhancer; and (3) an aqueous adjuvant. In addition, transdermal formulations can include additional components such as, but not limited to, gelling agents, creams and ointment bases, and the like. In some embodiments, the transdermal formulation can further include a woven or non-woven backing material to enhance absorption and prevent the removal of the transdermal formulation from the skin. In other embodiments, the transdermal formulations described herein can maintain a saturated or supersaturated state to promote diffusion into the skin. Formulations suitable for transdermal administration of compounds described herein may employ transdermal delivery devices and transdermal delivery patches and can be lipophilic emulsions or buffered, aqueous solutions, dissolved and / or dispersed in a polymer or an adhesive. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. Still further, transdermal delivery of the compounds described herein can be accomplished by means of iontophoretic patches and the like. Additionally, transdermal patches can provide controlled delivery of the compounds described herein. The rate of absorption can be slowed by using rate-controlling membranes or by trapping the compound within a polymer matrix or gel. Conversely, absorption enhancers can be used to increase absorption. An absorption enhancer or carrier can include absorbable pharmaceutically acceptable solvents to assist passage through the skin. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.

[0078] Formulations suitable for intramuscular, subcutaneous, or intravenous injection may include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles including water, ethanol, polyols (propyleneglycol, polyethylene-glycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Formulations suitable for subcutaneous injection may also contain additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, such as aluminum monostearate and gelatin.

[0079] For intravenous injections, compounds described herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally recognized in the field. For other parenteral injections, appropriate formulations may include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are generally recognized in the field.

[0080] Parenteral injections may involve bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The pharmaceutical composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0081] In certain embodiments, delivery systems for pharmaceutical compounds may be employed, such as, for example, liposomes and emulsions. In certain embodiments, compositions provided herein also include an mucoadhesive polymer, selected from among, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate and dextran.

[0082] In some embodiments, the compounds described herein may be administered topically and are formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments. Such pharmaceutical compounds can contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0083] The compounds described herein may also be formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycerides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like. In suppository forms of the compositions, a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter is first melted.

[0084] Generally, an agent, such as a compound disclosed herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)) or a pharmaceutically acceptable salt thereof), is administered in an amount effective for amelioration of, or prevention of the development of symptoms of, the disease or disorder (i.e., a therapeutically effective amount). Thus, a therapeutically effective amount can be an amount that is capable of at least partially preventing or reversing a disease or disorder. The dose required to obtain an effective amount may vary depending on the agent, formulation, disease or disorder, and individual to whom the agent is administered.

[0085] Determination of effective amounts may also involve in vitro assays in which varying doses of agent are administered to cells in culture and the concentration of agent effective for ameliorating some or all symptoms is determined in order to calculate the concentration required in vivo. Effective amounts may also be based in in vivo animal studies.

[0086] An agent can be administered prior to, concurrently with and subsequent to the appearance of symptoms of a disease or disorder. In some embodiments, an agent is administered to a subject with a family history of the disease or disorder, or who has a phenotype that may indicate a predisposition to a disease or disorder, or who has a genotype which predisposes the subject to the disease or disorder.

[0087] In some embodiments, the compositions described herein are provided as pharmaceutical and / or therapeutic compositions. The pharmaceutical and / or therapeutic compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional carriers; aqueous, powder, or oily bases; thickeners; and the like can be necessary or desirable. Compositions and formulations for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. Compositions and formulations for parenteral, intrathecal or intraventricular administration can include sterile aqueous solutions that can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients. Pharmaceutical and / or therapeutic compositions of the present invention include, but arc not limited to, solutions, emulsions, and liposome containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids.

[0088] The pharmaceutical and / or therapeutic formulations, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical / nutriceutical industries. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carriers) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous, oil-based, or mixed media. Suspensions can further contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension can also contain stabilizers. In one embodiment of the present invention the pharmaceutical compositions can be formulated and used as foams. Pharmaceutical foams include formulations such as, but not limited to, emulsions, microemulsions, creams, jellies and liposomes. While basically similar in nature these formulations vary in the components and the consistency of the final product.

[0089] The pharmaceutical composition described herein may be in unit dosage forms suitable for single administration of precise dosages. In unit dosage form, the formulation is divided into unit doses containing appropriate quantities of one or more compound. The unit dosage may be in the form of a package containing discrete quantities of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. Aqueous suspension compositions can be packaged in single-dose non-reclosable containers. Alternatively, multipledose reclosable containers can be used, in which case it is typical to include a preservative in the composition. By way of example only, formulations for parenteral injection may be presented in unit dosage form, which include, but are not limited to ampoules, or in multi-dose containers, with an added preservative. Dosing and administration regimes are tailored by the clinician, or others skilled in the pharmacological arts, based upon well-known pharmacological and therapeutic considerations including, but not limited to, the desired level of therapeutic effect, and the practical level of therapeutic effect obtainable. Generally, it is advisable to follow well-known pharmacological principles for administrating chemotherapeutic agents (e.g., it is generally advisable to not change dosages by more than 50% at time and no more than every 3-4 agent half-lives). For compositions that have relatively little or no dose-related toxicity considerations, and where maximum efficacy is desired, doses in excess of the average required dose are not uncommon. This approach to dosing is commonly referred to as the “maximal dose” strategy. In certain embodiments, the compounds are administered to a subject at a dose of about 0.01 mg / kg to about 200 mg / kg, more preferably at about 0.1 mg / kg to about 100 mg / kg, even more preferably at about 0.5 mg / kg to about 50 mg / kg. When the compounds described herein are coadministered with another agent (e.g., as sensitizing agents), the effective amount may be less than when the agent is used alone. Dosing may be once per day or multiple times per day for one or more consecutive days.

[0090] Methods of Use / Treatment

[0091] The present disclosure provides methods of using the compounds and compositions described herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or pharmaceutically acceptable salts thereof). The methods include methods of inhibiting ASH1L and methods of treating diseases such as cancer.

[0092] In certain embodiments, the disclosure provides a method of inhibiting ASH1L activity in a sample, comprising contacting the sample with an effective amount a compound described herein or a pharmaceutically acceptable salt thereof (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof). The sample may be an in vitro or ex vivo sample (e.g., a sample comprising cells, tissue, or an organ).

[0093] In some embodiments, the disclosure provides a method of inhibiting ASH IL activity by contacting the ASH1L with an effective amount of a compound described herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof, , e.g., by contacting a cell, tissue, or organ that expresses ASH1L with the compound or the salt thereof. In some embodiments, the disclosure provides a method of inhibiting ASH1L activity in subject (including but not limited to rodents and mammals, e.g., humans), by administering into the subject an effective amount of a compound described herein or a pharmaceutically acceptable salt thereof (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)). In some embodiments, the percentage inhibition exceeds 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0094] In some embodiments, the disclosure provides methods of inhibiting ASH IL activity in a cell, comprising contacting the cell with an amount of a compound described herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)) sufficient to inhibit the activity. In some embodiments, the disclosure provides methods of inhibiting ASH IL activity in a tissue by contacting the tissue with an amount of a compound described herein or a pharmaceutically acceptable salt thereof (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), sufficient to inhibit the ASH1L activity in the tissue. In some embodiments, the disclosure provides methods of inhibiting ASH1L activity in an organism (e.g., mammal, human, etc.) by contacting the organism with an amount of a compound described herein or a pharmaceutically acceptable salt thereof (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), sufficient to inhibit ASH1L activity in the organism.

[0095] Inhibition of ASH IL activity may be assessed and demonstrated by a wide variety of ways known in the art. Non-limiting examples include measure (a) a direct decrease in ASH IL activity; (b) a decrease in cell proliferation and / or cell viability; (c) an increase in cell differentiation; (d) a decrease in the levels of downstream targets of ASH1L activity; and (e) decrease in tumor volume and / or tumor volume growth rate. Kits and commercially available assays can be utilized for determining one or more of the above.

[0096] The disclosure also provides methods for treating cancer in a subject in need thereof (e.g., a subject suffering from cancer), comprising administering a compound or pharmaceutical composition described herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutical composition comprising a compound of (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)) to the subject. In certain embodiments, the cancer is associated with ASH1L expression (e.g., aberrant expression, overexpression, etc.) and / or activity. In certain embodiments, the cancer is brain cancer (e.g., an astrocytoma or glioblastoma), a sarcoma, colorectal cancer, lung cancer (e.g., non-small cell lung cancer), or gastric cancer. In certain embodiments, the disclosure provides a method of treating cancer in a subject, wherein the method comprises determining if the subject has a ASHIL-mediated cancer, and administering to the subject a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (in)).

[0097] Determining whether a tumor or cancer expresses (e.g., overexpresses, aberrantly expresses, etc.) ASH1L can be undertaken by assessing the nucleotide sequence encoding ASH1L or by assessing the amino acid sequence of ASH1L. Methods for detecting a ASH1L nucleotide sequence are known by those of skill in the art. These methods include, but are not limited to, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) assays, real-time PCR assays, PCR sequencing, mutant allele-specific PCR amplification (MASA) assays, direct sequencing, primer extension reactions, electrophoresis, oligonucleotide ligation assays, hybridization assays, TaqMan assays, SNP genotyping assays, high resolution melting assays, and microarray analyses. Methods for detecting a ASH1L protein are known by those of skill in the art. These methods include, but are not limited to, detection using a binding agent, e.g., an antibody specific for ASH1L, protein electrophoresis and Western blotting, and direct peptide sequencing.

[0098] Methods for determining whether a tumor or cancer expresses (e.g., overexpresses, aberrantly expresses, etc.) ASH1L or is mediated by ASH1L activity can use a variety of samples. In some embodiments, the sample is taken from a subject having a cancer or tumor. In some embodiments, the sample is a fresh tumor / cancer sample. In some embodiments, the sample is a frozen tumor / cancer sample. In some embodiments, the sample is a formalin-fixed paraffin-embedded sample. In some embodiments, the sample is processed to a cell lysate. In some embodiments, the sample is processed to DNA or RNA.

[0099] The disclosure also relates to a method of treating a hyperproliferative disorder in a mammal that comprises administering to the mammal a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)). In some embodiments, the method relates to the treatment of cancer such as acute myeloid leukemia, cancer in adolescents, adrenocortical carcinoma childhood, AIDS-related cancers, e.g., Lymphoma and Kaposi's Sarcoma, anal cancer, angiosarcoma, appendix cancer, astrocytomas, atypical teratoid rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, chondrosarcoma, embryonal tumors, germ cell tumor, primary lymphoma, cervical cancer, childhood cancers, chordoma, cardiac tumors, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myleoproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic ductal carcinoma in situ (DCIS), embryonal tumors, CNS cancer, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fibrous histiocytoma of bone, gall bladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumor, gestational trophoblastic tumor, glioblastoma, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, laryngeal cancer, leiomyosarcoma, lip and oral cavity cancer, liposarcoma, liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma, metastatic squamous neck cancer with occult primary, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms, multiple myeloma, merkel cell carcinoma, malignant mesothelioma, malignant fibrous histiocytoma of bone and osteosarcoma, myxofibrosarcoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer (NSCLC), oral cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach (gastric) cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, synovial sarcoma, T-Cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, unusual cancers of childhood, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Viral-Induced cancer. In some embodiments, the method relates to the treatment of a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin, c.g., psoriasis, restenosis, or prostate, c.g., benign prostatic hypertrophy (BPH). In some embodiments, the method relates to the treatment of brain cancer (e.g., astrocytoma or glioblastoma), a sarcoma, colorectal cancer, lung cancer (e.g., nonsmall cell lung cancer), or gastric cancer.

[0100] Subjects that can be treated with compounds of the disclosure according to the methods of this disclosure include, for example, subjects that have been diagnosed as having acute myeloid leukemia, cancer in adolescents, adrenocortical carcinoma childhood, AIDS-related cancers, e.g., Lymphoma and Kaposi's Sarcoma, anal cancer, angiosarcoma, appendix cancer, astrocytomas, atypical teratoid rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain stem glioma, brain tumor, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, chondrosarcoma, embryonal tumors, germ cell tumor, primary lymphoma, cervical cancer, childhood cancers, chordoma, cardiac tumors, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myleoproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, extrahepatic ductal carcinoma in situ (DCIS), embryonal tumors, CNS cancer, endometrial cancer, ependymoma, epithelioid sarcoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fibrous histiocytoma of bone, gall bladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (GIST), germ cell tumor, gestational trophoblastic tumor, glioblastoma, hairy cell leukemia, head and neck cancer, heart cancer, liver cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumors, pancreatic neuroendocrine tumors, kidney cancer, laryngeal cancer, leiomyosarcoma, lip and oral cavity cancer, liposarcoma, liver cancer, lobular carcinoma in situ (LCIS), lung cancer, lymphoma, metastatic squamous neck cancer with occult primary, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia syndromes, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms, multiple myeloma, merkel cell carcinoma, malignant mesothelioma, malignant fibrous histiocytoma of bone and osteosarcoma, myxofibrosarcoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer (NSCLC), oral cancer, lip and oral cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, plcuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach (gastric) cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, synovial sarcoma, T-Cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, trophoblastic tumor, unusual cancers of childhood, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, or Viral-Induced cancer. In some embodiments, the method relates to the treatment of a non-cancerous hyperproliferative disorder such as benign hyperplasia of the skin, e.g., psoriasis, restenosis, or prostate, e.g., benign prostatic hypertrophy (BPH). In some embodiments, the subject has been diagnosed with brain cancer (e.g., astrocytoma or glioblastoma), a sarcoma, colorectal cancer, lung cancer (e.g., nonsmall cell lung cancer), or gastric cancer

[0101] The compositions containing the compounds or salts thereof described herein can be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the compounds or compositions are administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially arrest the symptoms of the disease. Amounts effective for this use will depend on the severity and course of the disease, previous therapy, the patient's health status, weight, and response to the drugs, and the judgment of the treating clinician.

[0102] In prophylactic applications, compositions containing the compounds or salts thereof described herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined to be a “prophylactically effective amount or dose.” In this use, the precise amounts also depend on the patient’s state of health, weight, and the like. When used in a patient, effective amounts for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient’s health status and response to the drugs, and the judgment of the treating clinician.

[0103] In the case wherein the patient’s condition does not improve, upon the clinician’s discretion the administration of the compounds may be administered chronically, that is, for an extended period of time, including throughout the duration of the patient’s life in order to ameliorate or otherwise control or limit the symptoms of the patient’s disease. In the case wherein the patient’s status does improve, upon the clinician’s discretion the administration of the compounds may be given continuously; alternatively, the dose of drug being administered may be temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”). The length of the drug holiday can vary between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday may be from about 10% to about 100%, including, by way of example only, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0104] Once improvement of the patient’s conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained. Patients can, however, require intermittent treatment on a long-term basis upon any recurrence of symptoms.

[0105] The amount of a given agent that will correspond to such an amount will vary depending upon factors such as the particular compound, disease and its severity, the identity (e.g., weight) of the subject or host in need of treatment, but can nevertheless be determined in a manner recognized in the field according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated. In general, however, doses employed for adult human treatment will typically be in the range of about 0.02 - about 5000 mg per day, in some embodiments, about 1 - about 1500 mg per day. The desired dose may conveniently be presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.

[0106] Toxicity and therapeutic efficacy of such therapeutic regimens can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50(the dose lethal to 50% of the population) and the ED50(the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50and ED50. Compounds exhibiting high therapeutic indices arc preferred. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50with minimal toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.

[0107] Combination Therapies

[0108] Provided herein are methods for combination therapies in which an agent known to modulate other pathways, or other components of the same pathway, or even overlapping sets of target enzymes are used in combination with a compound described herein or a pharmaceutically acceptable salt thereof (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)). In one aspect, such therapy includes but is not limited to the combination of one or more compounds of the disclosure with chemotherapeutic agents, targeted agents, therapeutic antibodies, and / or radiation treatment, to provide a synergistic or additive therapeutic effect.

[0109] In general, the compounds and compositions described herein and, in embodiments where combinational therapy is employed, other agents do not have to be administered in the same pharmaceutical composition, and may, because of different physical and chemical characteristics, have to be administered by different routes. The determination of the mode of administration and the advisability of administration, where possible, in the same pharmaceutical composition, is well within the knowledge of the clinician. The initial administration can be made according to established protocols recognized in the field, and then, based upon the observed effects, the dosage, modes of administration and times of administration can be modified by the clinician.

[0110] In certain instances, it may be appropriate to administer at least one compound described herein in combination with another therapeutic agent. By way of example only, if one of the side effects experienced by a patient upon receiving a compound described herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), is nausea, then it may be appropriate to administer an anti-nausea agent in combination with the initial therapeutic agent. Or, by way of example only, the therapeutic effectiveness of one of the compounds described herein may be enhanced by administration of an adjuvant (i.e., by itself the adjuvant may have minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced). Or, by way of example only, the benefit experienced by a patient may be increased by administering one of the compounds described herein with another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit. In any case, regardless of the disease, disorder or condition being treated, the overall benefit experienced by the patient may simply be additive of the two therapeutic agents or the patient may experience a synergistic benefit.

[0111] The particular choice of compounds used will depend upon the diagnosis and judgment of the condition of the patient and the appropriate treatment protocol. The compounds may be administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the disease, disorder, or condition, the condition of the patient, and the actual choice of compounds used. The determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol, is well within the knowledge of the clinician after evaluation of the disease being treated and the condition of the patient.

[0112] Therapeutically-effective dosages can vary when the drugs are used in treatment combinations. Methods for experimentally determining therapeutically-effective dosages of drugs and other agents for use in combination treatment regimens are described in the literature. For example, the use of metronomic dosing, i.e., providing more frequent, lower doses in order to minimize toxic side effects, has been described extensively in the literature. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.

[0113] For combination therapies described herein, dosages of the co-administered compounds will of course vary depending on the type of co-drug employed, on the specific drug employed, on the disease being treated and so forth. In addition, when co-administered with one or more biologically active agents, the compound provided herein may be administered either simultaneously with the biologically active agent(s), or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering protein in combination with the biologically active agent(s).

[0114] In any case, the multiple therapeutic agents (one of which is a compound described herein or a pharmaceutically acceptable salt thereof (e.g., a compound of formula (I), (la), (lb), (Ic), (IT), or (ITT)), may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents; the use of multiple therapeutic combinations are also envisioned.

[0115] It is understood that the dosage regimen to treat, prevent, or ameliorate the condition(s) for which relief is sought, can be modified in accordance with a variety of factors. These factors include the disorder or condition from which the subject suffers, as well as the age, weight, sex, diet, and medical condition of the subject. Thus, the dosage regimen actually employed can vary widely and therefore can deviate from the dosage regimens set forth herein.

[0116] The pharmaceutical agents which make up the combination therapy disclosed herein may be a combined dosage form or in separate dosage forms intended for substantially simultaneous administration. The pharmaceutical agents that make up the combination therapy may also be administered sequentially, with either therapeutic compound being administered by a regimen calling for two-step administration. The two-step administration regimen may call for sequential administration of the active agents or spaced-apart administration of the separate active agents. The time period between the multiple administration steps may range from, a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the pharmaceutical agent. Circadian variation of the target molecule concentration may also determine the optimal dose interval.

[0117] In addition, the compounds described herein also may be used in combination with procedures that may provide additional or synergistic benefit to the patient. By way of example only, patients are expected to find therapeutic and / or prophylactic benefit in the methods described herein, wherein pharmaceutical composition of a compound disclosed herein and / or combinations with other therapeutics are combined with genetic testing to determine whether that individual is a carrier of a mutant gene that is known to be correlated with certain diseases or conditions. The compounds described herein and combination therapies can be administered before, during or after the occurrence of a disease, and the timing of administering the composition containing a compound can vary. Thus, for example, the compounds can be used as a prophylactic and can be administered continuously to subjects with a propensity to develop conditions or diseases in order to prevent the occurrence of the disease. The compounds and compositions can be administered to a subject during or as soon as possible after the onset of the symptoms. The administration of the compounds can be initiated within the first 48 hours of the onset of the symptoms, preferably within the first 48 hours of the onset of the symptoms, more preferably within the first 6 hours of the onset of the symptoms, and most preferably within 3 hours of the onset of the symptoms. The initial administration can be via any route practical, such as, for example, an intravenous injection, a bolus injection, infusion over about 5 minutes to about 5 hours, a pill, a capsule, transdermal patch, buccal delivery, and the like, or combination thereof. A compound is preferably administered as soon as is practicable after the onset of a disease is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from 1 day to about 3 months. The length of treatment can vary for each subject, and the length can be determined using the known criteria. For example, the compound or a formulation containing the compound can be administered for at least 2 weeks, preferably about 1 month to about 5 years.

[0118] Compounds and pharmaceutical compositions disclosed herein may be co-administered with one or more chemotherapeutics. Many chemotherapeutics are presently known in the art and can be used in combination with the compounds herein. In some embodiments, the chemotherapeutic is selected from the group consisting of mitotic inhibitors, alkylating agents, anti-metabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inhibitors, enzyme inhibitors, topoisomerase inhibitors, protein-protein interaction inhibitors, biological response modifiers, anti-hormones, angiogenesis inhibitors, and anti-androgens.

[0119] Non-limiting examples are chemotherapeutic agents, cytotoxic agents, and non-peptide small molecules such as Gleevec® (Imatinib Mesylate), Velcade® (bortezomib), Casodex (bicalutamide), Iressa® (gefitinib), and Adriamycin as well as a host of chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXANTM); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamine; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, CasodexTM, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6- azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide; procarbazine; PSK.RTM.; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes, e.g., paclitaxel (TAXOLTM, Bristol-Myers Squibb Oncology, Princeton, N.J.) and docetaxel (TAXOTERETM, Rhone-Poulenc Rorer, Antony, France); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included as suitable chemotherapeutic cell conditioners are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, (NolvadexTM), raloxifene, aromatase inhibiting 4(5)-imidazoles, 4- hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; chlorambucil; gemcitabine; 6-thioguaninc; mcrcaptopurinc; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; camptothecin-11 (CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO). Where desired, the compounds or pharmaceutical composition of the present invention can be used in combination with commonly prescribed anticancer drugs such as Herceptin®, Avastin®, Erbitux®, Rituxan®, Taxol®, Arimidex®, Taxotere®, ABVD, AVICINE, Abagovomab, Acridine carboxamide, Adecatumumab, 17-N- Allylamino-17-demethoxygeldanamycin, Alpharadin, Alvocidib, 3-Aminopyridine-2- carboxaldehyde thiosemicarbazone, Amonafide, Anthracenedione, Anti-CD22 immunotoxins, Antineoplastic, Antitumorigenic herbs, Apaziquone, Atiprimod, Azathioprine, Belotecan, Bendamustine, BIBW 2992, Biricodar, Brostallicin, Bryostatin, Buthionine sulfoximine, CBV (chemotherapy), Calyculin, cell-cycle nonspecific antineoplastic agents, Dichloroacetic acid, Discodermolide, Elsamitrucin, Enocitabine, Epothilone, Eribulin, Everolimus, Exatecan, Exisulind, Ferruginol, Forodesine, Fosfestrol, ICE chemotherapy regimen, IT-101, Imexon, Imiquimod, Indolocarbazole, Irofulven, Laniquidar, Larotaxel, Lenalidomide, Lucanthone, Lurtotecan, Mafosfamide, Mitozolomide, Nafoxidine, Nedaplatin, Olaparib, Ortataxel, PAC-1, Pawpaw, Pixantrone, Proteasome inhibitor, Rebeccamycin, Resiquimod, Rubitecan, SN-38, Salinosporamide A, Sapacitabine, Stanford V, Swainsonine, Talaporfin, Tariquidar, Tegafur- uracil, Temodar, Tesetaxel, Triplatin tetranitrate, Tris(2-chloroethyl)amine, Troxacitabine, Uramustine, Vadimezan, Vinflunine, ZD6126 or Zosuquidar.

[0120] Embodiments herein further relate to methods for using a compound disclosed herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutical composition provided herein, in combination with radiation therapy for inhibiting abnormal cell growth or treating the hyperproliferative disorder in the mammal. Techniques for administering radiation therapy are known in the art, and these techniques can be used in the combination therapy described herein. The administration of the compound of the invention in this combination therapy can be determined as described herein.

[0121] Radiation therapy can be administered through one of several methods, or a combination of methods, including without limitation external-beam therapy, internal radiation therapy, implant radiation, stereotactic radiosurgery, systemic radiation therapy, radiotherapy and permanent or temporary interstitial brachytherapy. The term “brachytherapy,” as used herein, refers to radiation therapy delivered by a spatially confined radioactive material inserted into the body at or near a tumor or other proliferative tissue disease site. The term is intended without limitation to include exposure to radioactive isotopes (e.g., At-211, I-131, I-125, Y-90, Re-186, Re-188, Sm-153, Bi-212, P-32, and radioactive isotopes of Lu). Suitable radiation sources for use as a cell conditioner of the present invention include both solids and liquids. By way of non- limiting example, the radiation source can be a radionuclide, such as I-125, I-131, Yb-169, Ir- 192 as a solid source, I-125 as a solid source, or other radionuclides that emit photons, beta particles, gamma radiation, or other therapeutic rays. The radioactive material can also be a fluid made from any solution of radionuclide(s), e.g., a solution of I-125 or I-131, or a radioactive fluid can be produced using a slurry of a suitable fluid containing small particles of solid radionuclides, such as Au-198, Y-90. Moreover, the radionuclide(s) can be embodied in a gel or radioactive micro spheres. The compounds or pharmaceutical compositions herein are also used in combination with an amount of one or more substances selected from anti-angiogenesis agents, signal transduction inhibitors, antiproliferative agents, glycolysis inhibitors, or autophagy inhibitors. Anti-angiogenesis agents, such as MMP-2 (matrix-metalloproteinase 2) inhibitors, MMP- 9 (matrix-metalloprotienase 9) inhibitors, and COX-11 (cyclooxygenase 11) inhibitors, can be used in conjunction with a compound of the disclosure and pharmaceutical compositions described herein. Anti-angiogenesis agents include, for example, rapamycin, temsirolimus (CCI- 779), everolimus (RAD001), sorafenib, sunitinib, and bevacizumab. Examples of useful COX-II inhibitors include CELEBREXTM(alecoxib), valdecoxib, and rofecoxib. Examples of useful matrix metalloproteinase inhibitors are described in WO 96 / 33172 (published October 24,1996), WO 96 / 27583 (published March 7,1996), European Patent Application No.97304971.1 (filed July 8,1997), European Patent Application No.99308617.2 (filed October 29, 1999), WO 98 / 07697 (published February 26,1998), WO 98 / 03516 (published January 29,1998), WO 98 / 34918 (published August 13,1998), WO 98 / 34915 (published August 13,1998), WO 98 / 33768 (published August 6,1998), WO 98 / 30566 (published July 16, 1998), European Patent Publication 606,046 (published July 13,1994), European Patent Publication 931, 788 (published July 28,1999), WO 90 / 05719 (published May 31,1990), WO 99 / 52910 (published October 21,1999), WO 99 / 52889 (published October 21, 1999), WO 99 / 29667 (published June 17,1999), PCT International Application No. PCT / IB98 / 01113 (filed July 21,1998), European Patent Application No.99302232.1 (filed March 25,1999), Great Britain Patent Application No. 9912961.1 (filed June 3, 1999), United States Provisional Application No.60 / 148,464 (filed August 12,1999), United States Patent 5,863, 949 (issued January 26,1999), United States Patent 5,861, 510 (issued January 19,1999), and European Patent Publication 780,386 (published June 25, 1997), all of which are incorporated herein in their entireties by reference. Preferred MMP-2 and MMP-9 inhibitors are those that have little or no activity inhibiting MMP-1. More preferred, are those that selectively inhibit MMP-2 and / or AMP-9 relative to the other matrix- metalloproteinases (e.g., MAP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP- 7, MMP-8, MMP- 10, MMP-ll, MMP-12, andMMP-13). Some specific examples of MMP inhibitors useful in the invention are AG-3340, RO 32-3555, and RS 13-0830. Autophagy inhibitors include, but are not limited to chloroquine, 3-methyladenine, hydroxychloroquine (Plaquenil™), bafilomycin A1, 5-amino-4-imidazole carboxamide riboside (AICAR), okadaic acid, autophagy-suppressive algal toxins which inhibit protein phosphatases of type 2A or type 1, analogues of cAMP, and drugs which elevate cAMP levels such as adenosine, LY204002, N6-mercaptopurine riboside, and vinblastine. In addition, antisense or siRNA that inhibits expression of proteins including but not limited to ATG5 (which are implicated in autophagy), may also be used. In some embodiments, the compounds described herein are formulated or administered in conjunction with liquid or solid tissue barriers also known as lubricants. Examples of tissue barriers include, but are not limited to, polysaccharides, polyglycans, seprafilm, interceed and hyaluronic acid. In some embodiments, medicaments which are administered in conjunction with the compounds described herein include any suitable drugs usefully delivered by inhalation for example, analgesics, e.g., codeine, dihydromorphine, ergotamine, fentanyl or morphine; anginal preparations, e.g., diltiazem; antiallergics, e.g., cromoglycate, ketotifen or nedocromil; anti- infectives, e.g., cephalosporins, penicillins, streptomycin, sulphonamides, tetracyclines or pentamidine; antihistamines, e.g., methapyrilene; anti-inflammatories, e.g., beclomethasone, flunisolide, budesonide, tipredane, triamcinolone acetonide or fluticasone; antitussives, e.g., noscapine; bronchodilators, e.g., ephedrine, adrenaline, fenoterol, formoterol, isoprenaline, metaproterenol, phenylephrine, phenylpropanolamine, pirbuterol, reproterol, rimiterol, salbutamol, salmeterol, terbutalin, isoetharine, tulobuterol, orciprenaline or (-)-4-amino-3,5- dichloro-α-[[[6-[2-(2-pyridinyl)ethoxy]hexyl]-amino]methyl]benzenemethanol; diuretics, e.g., amiloride; anticholinergics e.g., ipratropium, atropine or oxitropium; hormones, e.g., cortisone, hydrocortisone or prednisolone; xanthines e.g., aminophylline, choline theophyllinate, lysine theophyllinate or theophylline; and therapeutic proteins and peptides, e.g., insulin or glucagon. It will be clear to a person skilled in the art that, where appropriate, the medicaments are used in the form of salts (e.g., as alkali metal or amine salts or as acid addition salts) or as esters (e.g., lower alkyl esters) or as solvates (e.g., hydrates) to optimize the activity and / or stability of the medicament. Other exemplary therapeutic agents useful for a combination therapy include but are not limited to agents as described above, radiation therapy, hormone antagonists, hormones and their releasing factors, thyroid and antithyroid drugs, estrogens and progestins, androgens, adrenocorticotropic hormone; adrenocortical steroids and their synthetic analogs; inhibitors of the synthesis and actions of adrenocortical hormones, insulin, oral hypoglycemic agents, and the pharmacology of the endocrine pancreas, agents affecting calcification and bone turnover: calcium, phosphate, parathyroid hormone, vitamin D, calcitonin, vitamins such as water-soluble vitamins, vitamin B complex, ascorbic acid, fat-soluble vitamins, vitamins A, K, and E, growth factors, cytokines, chemokines, muscarinic receptor agonists and antagonists; anticholinesterase agents; agents acting at the neuromuscular junction and / or autonomic ganglia; catecholamines, sympathomimetic drugs, and adrenergic receptor agonists or antagonists; and 5- hydroxytryptamine (5-HT, serotonin) receptor agonists and antagonists. Other suitable therapeutic agents for coadministration with compounds herein also include agents for pain and inflammation such as histamine and histamine antagonists, bradykinin and bradykinin antagonists, 5-hydroxytryptamine (serotonin), lipid substances that are generated by biotransformation of the products of the selective hydrolysis of membrane phospholipids, eicosanoids, prostaglandins, thromboxanes, leukotrienes, aspirin, nonsteroidal anti-inflammatory agents, analgesic-antipyretic agents, agents that inhibit the synthesis of prostaglandins and thromboxanes, selective inhibitors of the inducible cyclooxygenase, selective inhibitors of the inducible cyclooxygenase-2, autacoids, paracrine hormones, somatostatin, gastrin, cytokines that mediate interactions involved in humoral and cellular immune responses, lipid-derived autacoids, eicosanoids, 0-adrenergic agonists, ipratropium, glucocorticoids, mcthylxanthincs, sodium channel blockers, opioid receptor agonists, calcium channel blockers, membrane stabilizers and leukotriene inhibitors.

[0122] Additional therapeutic agents contemplated for co- administration with compounds and compositions herein include diuretics, vasopressin, agents affecting the renal conservation of water, rennin, angiotensin, agents useful in the treatment of myocardial ischemia, antihypertensive agents, angiotensin converting enzyme inhibitors, β-adrenergic receptor antagonists, agents for the treatment of hypercholesterolemia, and agents for the treatment of dyslipidemia.

[0123] Other therapeutic agents contemplated for co-administration with compounds and compositions herein include drugs used for control of gastric acidity, agents for the treatment of peptic ulcers, agents for the treatment of gastroesophageal reflux disease, prokinetic agents, antiemetics, agents used in irritable bowel syndrome, agents used for diarrhea, agents used for constipation, agents used for inflammatory bowel disease, agents used for biliary disease, agents used for pancreatic disease. Therapeutic agents used to treat protozoan infections, drugs used to treat Malaria, Amebiasis, Giardiasis, Trichomoniasis, Trypanosomiasis, and / or Leishmaniasis, and / or drugs used in the chemotherapy of helminthiasis. Other therapeutic agents include antimicrobial agents, sulfonamides, trimethoprim- sulfamethoxazole quinolones, and agents for urinary tract infections, penicillins, cephalosporins, and other, P-lactam antibiotics, an agent comprising an aminoglycoside, protein synthesis inhibitors, drugs used in the chemotherapy of tuberculosis, mycobacterium avium complex disease, and leprosy, antifungal agents, antiviral agents including nonretro viral agents and antiretroviral agents.

[0124] Examples of therapeutic antibodies that can be combined with a compound herein include but are not limited to anti-receptor tyrosine kinase antibodies (cetuximab, panitumumab, trastuzumab), anti CD20 antibodies (rituximab, tositumomab), and other antibodies such as alemtuzumab, bevacizumab, and gemtuzumab.

[0125] Moreover, therapeutic agents used for immunomodulation, such as immunomodulators, immunosuppressive agents, tolerogens, and immunostimulants are contemplated by the methods herein. In addition, therapeutic agents acting on the blood and the blood-forming organs, hematopoietic agents, growth factors, minerals, and vitamins, anticoagulant, thrombolytic, and antiplatelet drugs. Further therapeutic agents that can be combined with a compound herein are found in Goodman and Gilman’s “The Pharmacological Basis of Therapeutics” Tenth Edition edited by Hardman, Limbird and Gilman or the Physician’s Desk Reference, both of which are incorporated herein by reference in their entirety.

[0126] In some embodiments, a compound described herein is co-administered with another therapeutic agent effective in treating brain cancer, such as glioblastoma or astrocytoma. In some embodiments, the other therapeutic agent may be bevacizumab, carmustine (e.g., carmustine wafer), cisplatin, everolimus, lomustine, procarbazine, temozolomide, vincristine, or any combination thereof (e.g., a combination of procarbazine hydrochloride, lomustine, and vincristine sulfate).

[0127] In some embodiments, a compound described herein is co-administered with one or more therapeutic agents approved for the treatment of a sarcoma, such as adriamycin, bevacizumab, carboplatin, cisplatin, cyclophosphamide, dacarbazine, dactinomycin, docetaxel, doxorubicin (e.g., doxorubicin hydrochloride liposome), epirubicin, eribulin, etoposide, gemcitabine, ifosfamide, imatinib, ixabepilone, methotrexate, paclitaxel, pazopanib, pomalidomide, recombinant interferon alfa-2b, tazemetostat, temozolomide, topotecan, trabectedin, vinblastine, vincristine, vinorelbine, or any combination thereof.

[0128] In some embodiments, a compound described herein is co-administered with one or more therapeutic agents approved for the treatment of colorectal cancer, such as 5-fluorouracil, bevacizumab, capecitabine, cetuximab, ipilmumab, irinotecan, leucovorin, nivolumab, oxaliplatin, panitumumab, pembrolizumab, ramucirumab, regorafenib, tipiracil, trifluridine, ziv- afibercept, or any combination thereof.

[0129] In some embodiments, a compound described herein is co-administered with one or more therapeutic agents approved for the treatment of lung cancer, such as non- small cell lung cancer. In such embodiments, the other therapeutic agent may be afatinib, alectinib, atezolizumab, bevacizumab, brigatinib, capmatinib, carboplatin, ceritinib, cisplatin, crizotinib, dabrafenib, dacomitinib, docetaxel, doxorubicin, durvalumab, entrectinib, erlotinib, everolimus, gefitinib, gemcitabine, ipilimumab, lorlatinib, mechlorethamine, methotrexate, necitumumab, nivolumab, osimertinib, paclitaxel, pembrolizumab, pemetrexed, ramucirumab, selpercatinib, trametinib, vinorelbine, or any combination thereof. In some embodiments, a compound described herein is co-administered with one or more therapeutic agents approved for the treatment of gastric cancer, such as 5-fluorouracil, capecitabine, carboplatin, cisplatin, docetaxel, epirubicin, irinotecan, oxaliplatin, paclitaxel, trifluridine, tipiracil, trastuzumab, or any combination thereof.

[0130] In some embodiments, a compound described herein is co-administered with one or more alkylating agents (e.g., for the treatment of cancer) selected from, for example, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, thiotepa, ranimustine, nimustine, temozolomide, altretamine, apaziquone, brostallicin, bendamustine, carmustine, estramustine, fotemustine, glufosfamide, mafosfamide, bendamustin, mitolactol, cisplatin, carboplatin, eptaplatin, lobaplatin, nedaplatin, oxaliplatin, and satraplatin.

[0131] In some embodiments, a compound described herein is co-administered with one or more anti-metabolites (e.g., for the treatment of cancer) selected from, for example, methotrexate, 6- mercaptopurineriboside, mercaptopurine, 5-fluorouracil, tegafur, doxifluridine, carmofur, cytarabine, cytarabine oefosfate, enocitabine, gemcitabine, fludarabin, 5-azacitidine, capecitabine, cladribine, clofarabine, decitabine, eflomithine, ethynylcytidine, cytosine arabinoside, hydroxyurea, melphalan, nelarabine, nolatrexed, ocfosf[iota]te, disodium premetrexed, pentostatin, pelitrexol, raltitrexed, triapine, trimetrexate, vidarabine, vincristine, and vinorelbine;

[0132] In some embodiments, a compound described herein is co-administered with one or more hormonal therapy agents (e.g., for the treatment of cancer) selected from, for example, exemestane, Lupron, anastrozole, doxercalciferol, fadrozole, formestane, abiraterone acetate, finasteride, epristeride, tamoxifen citrate, fulvestrant, Trelstar, toremifene, raloxifene, lasofoxifene, letrozole, sagopilone, ixabepilone, epothilone B, vinblastine, vinflunine, docetaxel, and paclitaxel;

[0133] In some embodiments, a compound described herein is co-administered with one or more cytotoxic topoisomerase inhibiting agents (e.g., for the treatment of cancer) selected from, for example, aclarubicin, doxorubicin, amonafide, belotecan, camptothecin, 10- hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, irinotecan, topotecan, edotecarin, epimbicin, etoposide, exatecan, gimatecan, lurtotecan, mitoxantrone, pirambicin, pixantrone, rubitecan, sobuzoxane, tafluposide, etc. In some embodiments, a compound described herein is co-administered with one or more anti-angiogenic compounds (e.g., for the treatment of cancer) selected from, for example, acitretin, aflibercept, angiostatin, aplidine, asentar, axitinib, recentin, bevacizumab, brivanib alaninat, cilengtide, combretastatin, DAST, endostatin, fenretinide, halofuginone, pazopanib, ranibizumab, rebimastat, removab, revlimid, sorafenib, vatalanib, squalamine, sunitinib, telatinib, thalidomide, ukrain, and vitaxin.

[0134] In some embodiments, a compound described herein is co-administered with one or more antibodies (e.g., for the treatment of cancer) selected from, for example, trastuzumab, cetuximab, bevacizumab, rituximab, ticilimumab, ipilimumab, lumiliximab, catumaxomab, atacicept, oregovomab, and alemtuzumab.

[0135] In some embodiments, a compound described herein is co-administered with one or more VEGF inhibitors (e.g., for the treatment of cancer) selected from, for example, sorafenib, DAST, bevacizumab, sunitinib, recentin, axitinib, aflibercept, telatinib, brivanib alaninate, vatalanib, pazopanib, and ranibizumab.

[0136] In some embodiments, a compound described herein is co-administered with one or more EGFR inhibitors (e.g., for the treatment of cancer) selected from, for example, cetuximab, panitumumab, vectibix, gefitinib, erlotinib, and Zactima.

[0137] In some embodiments, a compound described herein is co-administered with one or more HER2 inhibitors (e.g., for the treatment of cancer) selected from, for example, lapatinib, tratuzumab, and pertuzumab; CDK inhibitor is selected from roscovitine and flavopiridol;

[0138] In some embodiments, a compound described herein is co-administered with one or more proteasome inhibitors (e.g., for the treatment of cancer) selected from, for example, bortezomib and carfilzomib.

[0139] In some embodiments, a compound described herein is co-administered with one or more serine / threonine kinase inhibitors (e.g., for the treatment of cancer), for example, MEK inhibitors and Raf inhibitors such as sorafenib.

[0140] In some embodiments, a compound described herein is co-administered with one or more tyrosine kinase inhibitors (e.g., for the treatment of cancer) selected from, for example, dasatinib, nilotibib, DAST, bosutinib, sorafenib, bevacizumab, sunitinib, AZD2171, axitinib, aflibercept, telatinib, imatinib mesylate, brivanib alaninate, pazopanib, ranibizumab, vatalanib, cetuximab, panitumumab, vectibix, gefitinib, erlotinib, lapatinib, tratuzumab and pertuzumab. In some embodiments, a compound described herein is co-administered with one or more androgen receptor antagonists (e.g., for the treatment of cancer) selected from, for example, nandrolone decanoate, fluoxymesterone, Android, Prostaid, andromustine, bicalutamide, flutamide, apocyproterone, apoflutamide, chlormadinone acetate, Androcur, Tabi, cyproterone acetate, and nilutamide.

[0141] In some embodiments, a compound described herein is co-administered with one or more aromatase inhibitors (e.g., for the treatment of cancer) selected from, for example, anastrozole, letrozole, testolactone, exemestane, aminoglutethimide, and formestane.

[0142] In some embodiments, a compound described herein is co-administered with one or more other anti-cancer agents including, e.g., alitretinoin, ampligen, atrasentan bexarotene, borte- zomib, bosentan, calcitriol, exisulind, fotemustine, ibandronic acid, miltefosine, mitoxantrone, 1- asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin, tazaroten, velcade, gallium nitrate, canfosfamide, darinaparsin, and tretinoin. In a preferred embodiment, the compounds of the present disclosure may be used in combination with chemotherapy (e.g., cytotoxic agents), anti-hormones and / or targeted therapies such as other kinase inhibitors, mTOR inhibitors and angiogenesis inhibitors.

[0143] In embodiments in which the compounds and pharmaceutical compositions herein are used for the treatment or prevention of non-cancer diseases and / or conditions, the compounds and pharmaceutical compositions herein may be co-administered with therapeutics and / or therapies known in the field to be appropriate for the treatment of such diseases and / or conditions.

[0144] Kits

[0145] For use in the therapeutic applications described herein, kits and articles of manufacture are also provided, which include a compound or pharmaceutical composition described herein (e.g., a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof ). In some embodiments, such kits comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers are formed from a variety of materials such as glass or plastic. The articles of manufacture provided herein contain packaging materials. Packaging materials for use in packaging pharmaceutical products include those found in, c.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. For example, in some embodiments the containers) includes a compound of formula (I), (la), (lb), (Ic), (II), or (III)), or a pharmaceutically acceptable salt thereof, optionally in a composition or in combination with another agent as disclosed herein. The container(s) optionally have a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprising a compound with an identifying description or label or instructions relating to its use in the methods described herein.

[0146] For example, a kit typically includes one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and / or devices) desirable from a commercial and user standpoint for use of a compound described herein. Nonlimiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included. A label is optionally on or associated with the container. For example, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In addition, a label is used to indicate that the contents are to be used for a specific therapeutic application. In addition, the label indicates directions for use of the contents, such as in the methods described herein. In certain embodiments, the pharmaceutical composition is presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. Or, the pack or dispenser device is accompanied by instructions for administration. Or, the pack or dispenser is accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. In some embodiments, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. EXPERIMENTAL Example 1 Materials and methods General Chemistry All reagents were commercially available and used without further purification. Nuclear magnetic resonance spectra were recorded in CDCl3, CD3OD and DMSO solutions.1H and13C NMR were recorded on Bruker spectrometers operating at 600 MHz. Data are reported as follows: chemical shift (δ), multiplicity, integrated intensity, and coupling constant (J) in hertz. Low-resolution mass spectroscopy (LRMS) images were obtained on Shimadzu LC-2020 system (DUIS-ESI). High-performance liquid chromatography (HPLC) analysis was performed on Shimadzu Prominence system (20 series: binary pump, UV / vis at 254 nm, heated column compartment 28 °C) using a Restek Ultra C18 column (150 × 4.6 mm, 5 μm) at room temperature with a gradient elution using the mobile phase (A) nanopure water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. HPLC condition: 30% of B at 0- 3.99 min, 30-85% of B at 3.99-5.50 min, 85-30% of B at 5.50-6.50 min, and 30% of B at 6.50- 8.50 min and a flow rate of 0.9 mL / min. The purity of all final compounds was > 95%. General procedure A for synthesis of spiro amides A mixture of appropriate carboxylic acid (0.52 mmol, 1.3 eq.), DMAP (0.2 mmol, 0.5 eq.), relevant primary amine (0.4 mmol, 1 eq.), and EDCI (1.0 mmol, 2.5 eq.) in DCM (10 mL) was stirred overnight at room temperature. Upon completion, the reaction was quenched with water and extracted with DCM. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography. General procedure B for thioamidation reaction To a stirred solution of appropriate benzonitrile (0.1 mmol) in DMF (2.5 mL) was added sodium hydrosulfide hydrate (222.2 mg, 3.0 mmol) and magnesium chloride (238 mg, 2.5 mmol). The mixture was stirred overnight at room temperature. Upon completion, the reaction was quenched with saturated NH4Cl aqueous solution and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography. General procedure C for deprotection of N-Boc protecting group A stirred solution of relevant N-Boc-amine (0.037 mmol) in DCM (2 mL) was cooled to 0 °C. TFA (0.4 mL) was then added dropwise, and the reaction mixture was allowed to warm to room temperature and stirred for 1 h. Upon completion, the solvent was removed under reduced pressure. If not indicated otherwise, the N-Boc deprotected product was directly used in the next step. General procedure D for reductive amination To a stirred solution of appropriate amine (N-Boc deprotected product) (0.037 mmol) and triethylamine (0.102 mmol) in DCE (3 mL) was added sodium triacetoxyborohydride (54 mg, 0.255 mmol) and relevant aldehyde (0.765 mmol) at 0 °C. The reaction mixture was then allowed to warm to room temperature and stirred for 4 h. Upon completion, saturated NH4Cl aqueous solution was added to quench the reaction. The organic layer was separated. The aqueous layer was extracted with DCM. The combined organic layers were dried over sodium sulfate, filtrated, and concentrated in vacuo. The residue was purified by reverse-phase flash chromatography and then lyophilized to give the corresponding target compound. Tert-butyl 6-(((3-(3-cyanophenyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (6) The title compound was synthesized from 2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptane- 6-carboxylic acid and primary amine 5 according to the method described for procedure A. It was obtained as a white solid (66.0% yield).1H NMR (600 MHz, CDCl3) δ 7.89 (t, J = 1.5 Hz, 1H), 7.84 – 7.81 (m, 2H), 7.56 – 7.51 (m, 3H), 7.39 (s, 1H), 7.36 (s, 1H), 7.14 – 7.12 (m, 1H), 4.56 (d, J = 5.8 Hz, 2H), 4.43 – 4.40 (m, 1H), 4.09 – 4.06 (m, 2H), 3.92 (s, 2H), 3.89 (s, 2H), 3.06 – 3.01 (m, 1H), 3.00 – 2.97 (m, 2H), 2.90 (s, 3H), 2.49 – 2.45 (m, 4H), 2.26 – 2.20 (m, 4H), 1.42 (s, 9H). LCMS (ESI): m / z 632 [M+H]+. Tert-butyl 6-(((3-(3-carbamothioylphenyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (7) The title compound was synthesized from benzonitrile 6 according to the method described for procedure B. It was obtained as a yellow solid (96.6% yield).1H NMR (600 MHz, CD3OD) δ 8.24 (s, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 7.0 Hz, 1H), 7.75 (d, J = 7.4 Hz, 1H), 7.68 (s, 1H), 7.47 – 7.44 (m, 2H), 7.11 (d, J = 7.9 Hz, 1H), 4.56 – 4.52 (m, 1H), 4.50 (s, 2H), 3.96 – 3.92 (m, 4H), 3.87 (s, 2H), 3.09 – 3.04 (m, 2H), 3.02 – 2.97 (m, 1H), 2.94 (s, 3H), 2.45 – 2.37 (m, 4H), 2.22 – 2.18 (m, 4H), 1.43 (s, 9H);13C NMR (150 MHz, CD3OD) δ 202.9, 175.4, 156.7, 140.4, 136.7, 132.6, 129.5, 128.1, 125.9, 125.3, 123.8, 122.8, 120.1, 119.5, 116.1, 108.9, 79.5, 60.1, 52.6, 45.3, 43.4, 35.3, 34.3, 33.9, 33.6, 31.6, 27.2. LCMS (ESI): m / z 666 [M+H]+. N-((3-(3-Carbamothioylphenyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-6-yl) methyl)-2-azaspiro[3.3]heptane-6-carboxamide (4) The title compound was synthesized according to the modified procedure C. To a solution of compound 7 (45 mg, 0.068 mmol) in DCM (2 mL) was added TFA (400 µL, 4.18 mmol) at 0 °C. The mixture was allowed to warm to room temperature and was stirred for 1 h. Upon completion, the solvent was removed. To a suspension solution of the residue in DCM (2 mL) was then added Et3N (30 µL, 0.22 mmol). The mixture was stirred at room temperature for another 1 h, then diluted with saturated NaHCO3aqueous solution. The organic layer was separated, and the aqueous layer was extracted with DCM. The combined organic extract was dried over sodium sulfate, filtered, and evaporated under reduced pressure. The residue was purified by reverse-phase flash chromatography (0~60% MeOH in H2O) and then lyophilized to give compound 4 (7.2 mg, 18.7% yield) as a white solid.1H NMR (600 MHz, CD3OD) δ 8.14 (t, J = 1.6 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.69 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H), 7.58 (s, 1H), 7.37 (s, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.00 (d, J = 8.1 Hz, 1H), 4.47 – 4.43 (m, 1H), 4.40 (s, 2H), 4.00 (s, 2H), 3.99 (s, 2H), 3.86 (d, J = 12.1 Hz, 2H), 2.97 (t, J = 11.3 Hz, 2H), 2.93 – 2.89 (m, 1H), 2.85 (s, 3H), 2.47 – 2.43 (m, 2H), 2.40 – 2.35 (m, 2H), 2.18 – 2.14 (m, 2H), 2.11 – 2.07 (m, 2H);13C NMR (150 MHz, CD3OD) δ 202.9, 175.4, 140.4, 136.5, 135.7, 132.4, 129.5, 128.1, 125.5, 123.5, 123.4, 119.9, 119.6, 116.0, 109.0, 57.7, 57.2, 53.2, 48.2, 45.4, 43.3, 38.0, 34.7, 33.8, 33.5, 31.5. HPLC analysis: retention time = 2.349 min; peak area, 99.2 % (λ = 254 nm). LCMS (ESI): m / z 566 [M+H]+. 7-Chloro-1H-indole-6-carboxylic acid (9) To a stirred solution of 2-chloro-3-nitrobenzoic acid (5.0 g, 24.8 mmol) in dry THF (60 mL) at -45 °C was added cold Vinyl Grignard reagent (141.7 mL, 99.2 mmol) dropwise. Then 100 mL of THF was added to the reaction mixture. The reaction was allowed to slowly warm to room temperature overnight. The flask was then cooled to 0 °C with an ice bath and saturated NH4Cl aqueous solution was added. The mixture was stirred for 1 h and then the solvents were removed. The residue was acidified to pH = 2 with 2M HCl. The precipitate was collected by filtration and washed with water and hexane. The solid obtained was then dried to give the compound 9 (3.86 g, 79.6%) as a tan powder that was used without further purification.1H NMR (600 MHz, CD3OD) δ 7.65 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 7.48 (d, J = 3.1 Hz, 1H), 6.59 (d, J = 3.1 Hz, 1H). Methyl 7-chloro-1H-indole-6-carboxylate (10) To a stirred solution of lH-indole-6-carboxylic acid 9 (3.3 g, 16.9 mmol) and potassium carbonate (2.34 g, 16.9 mmol) in DMF (25 mL) was dropwise added methyl iodide (1.16 mL, 18.6 mmol) at room temperature. The mixture was stirred for 2 h. Upon completion, the reaction was quenched with water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (0~20% ethyl acetate in hexane) to afford the desired compound 10 (2.18 g, 61.6% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 8.70 (s, 1H), 7.62 (d, J = 8.3 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.29 – 7.28 (m, 1H), 6.51 – 6.50 (m, 1H), 3.86 (s, 3H). LCMS (ESI): m / z 210 [M+H]+. Methyl 7-chloro-3-iodo-1H-indole-6-carboxylate (11) To a solution of indole 10 (2.18 g, 10.4 mmol) and KOH (1.46 g, 26.0 mmol) in DMF (15.0 mL) was added I2 (2.69 g, 10.6 mmol) in portion at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 3 h. Upon completion, the reaction was quenched with water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (0~17.5% ethyl acetate in hexane) to afford the desired compound 11 (2.79 g, 79.9% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 8.76 (s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 3.89 (s, 3H);13C NMR (150 MHz, CDCl3) δ 166.3, 133.7, 133.2, 131.8, 123.6, 122.6, 119.2, 118.0, 58.1, 52.3. LCMS (ESI): m / z 336 [M+H]+. 1-(Tert-butyl) 6-methyl 7-chloro-3-iodo-1H-indole-1,6-dicarboxylate (12) To a solution of indole 11 (3.32 g, 9.89 mmol), DMAP (0.12 g, 0.99 mmol) and triethylamine (1.9 mL, 13.8 mmol) in DCM (60 mL) was added di-tert-butyl dicarbonate (2.60 g, 11.9 mmol) at 0 °C in portion wise. The reaction mixture was allowed to warm to room temperature and stirred overnight. Upon completion, the reaction was quenched with 1.0 M HCl solution and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (0~16.5% ethyl acetate in hexane) to give the desired compound 12 (3.79 g, 87.7% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 7.65 (s, 1H), 7.64 (d, J = 8.2 Hz, 1H), 7.24 (d, J = 8.2 Hz, 1H), 3.86 (s, 3H), 1.55 (s, 9H). LCMS (ESI): m / z 436 [M+H]+. 1-(Tert-butyl) 6-methyl 7-chloro-3-(3-cyanophenyl)-1H-indole-1,6-dicarboxylate (13) The mixture of 1-(tert-butyl) 6-methyl 7-chloro-3-iodo-1H-indole-1,6-dicarboxylate (12) (2.47 g, 5.67 mmol), Na2CO3(0.91 g, 8.59 mmol), Pd(dppf)Cl2.DCM (0.46 g, 0.56 mmol) and (3-cyanophenyl)boronic acid (0.83 g, 5.67 mmol) in THF / H2O (45 mL / 15 mL) was heated to 65 °C for 12 h under an Argon atmosphere. Upon completion, the resulting mixture was cooled to room temperature, filtered through celite, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (0~20% ethyl acetate in hexane) to give the compound 13 (1.72 g, 73.8%) as a white solid.1H NMR (600 MHz, CDCl3) δ 7.78 (t, J = 1.4 Hz, 1H), 7.73 (dt, J = 7.8, 1.4 Hz, 1H), 7.69 (s, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.58 (dt, J = 7.8, 1.4 Hz, 1H), 7.56 (d, J = 6.0 Hz, 1H), 7.50 (t, J = 7.7 Hz, 1H), 3.89 (s, 3H), 1.60 (s, 9H). Methyl 7-chloro-3-(3-cyanophenyl)-1H-indole-6-carboxylate (14) The title compound was synthesized according to the modified procedure C. To a stirred solution of compound 13 (1.72 g, 4.19 mmol) in DCM (40 mL) was added TFA (8.8 mL, 91.7 mmol) at 0 °C. The mixture was allowed to warm to room temperature and was stirred for 3 h. Upon completion, the reaction was quenched with saturated sodium bicarbonate aqueous solution and extracted with DCM. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by the flash column chromatography (0~25% ethyl acetate in hexane) to afford the desired compound 14 (1.13 g, 86.9% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 8.82 (s, 1H), 7.82 (t, J = 1.4 Hz, 1H), 7.78 (dt, J = 7.7, 1.5 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 8.4 Hz, 1H), 7.54 – 7.52 (m, 2H), 7.49 (t, J = 7.7 Hz, 1H), 3.91 (s, 3H);13C NMR (150 MHz, CDCl3) δ 166.2, 135.8, 134.8, 131.6, 130.7, 130.0, 129.8, 128.6, 125.6, 123.9, 122.5, 118.8, 118.7, 117.6, 117.3, 113.2, 52.3. LCMS (ESI): m / z 311 [M+1]+. Methyl 7-chloro-3-(3-cyanophenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indole-6-carboxylate (16) The mixture of compound 14 (596.2 mg, 1.92 mmol) and cesium carbonate (3753.4 mg, 11.52 mmol) in dry DMF (6 mL) was stirred at room temperature for 30 minutes. Then a solution of tetrahydro-2H-pyran-4-yl methanesulfonate (1384.1 mg, 7.68 mmol) in DMF (2.0 mL) was added. The reaction mixture was stirred at 100 °C for 18 h. The reaction was then quenched with water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by the flash column chromatography (0~28% ethyl acetate in hexane) to give the desired compound 16 (422.9 mg, 55.8% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 7.83 (s, 1H), 7.80 – 7.76 (m, 2H), 7.75 (d, J = 8.4 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.66 (t, J = 8.1 Hz, 1H), 5.68 – 5.63 (m, 1H), 4.12 – 4.09 (m, 2H), 3.91 (s, 3H), 3.59 – 3.56 (m, 2H), 2.16 – 2.13 (m, 2H), 2.04 – 2.01 (m, 2H); LCMS (ESI): m / z 395 [M+H]+. 3-(7-Chloro-6-(hydroxymethyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl) benzonitrile (17) The mixture of compound 16 (800 mg, 2.03 mmol) and LiBH4(446.6 mg, 20.3 mmol, 10 eq.) in THF / MeOH (35 mL / 0.5 mL) was heated to 65 °C under an argon atmosphere. and stirred for 25 minutes. Upon completion, the reaction was cooled at room temperature and quenched with saturated NH4Cl aqueous solution. The resultant mixture was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by the flash column chromatography (0~45% ethyl acetate in hexane) to give the desired compound 17 (493.7 mg, 66.3% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 7.78 (t, J = 1.4 Hz, 1H), 7.74 (dt, J = 7.6, 1.5 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.50 (dt, J = 7.7, 1.4 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.39 (s, 1H), 7.25 (d, J = 8.1 Hz, 1H), 5.58 (tt, J = 11.7, 3.9 Hz, 1H), 4.87 (d, J = 5.8 Hz, 2H), 4.10 (dd, J = 11.6, 4.3 Hz, 2H), 3.57 (td, J = 11.9, 1.7 Hz, 2H), 2.14 – 2.10 (m, 2H), 2.05 – 1.98 (m, 2H). LCMS (ESI): m / z 349 [M-18+H]+. 3-(6-(Azidomethyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl) benzonitrile (18) To a stirred solution of alcohol 17 (366.6 mg, 1.0 mmol) in anhydrous THF (10 mL) was added DPPA (560 µL, 2.6 mmol) at 0 °C. After 10 minutes, DBU (456.7 µL, 13.0 mmol) was slowly added. The resulting mixture was warmed to room temperature and stirred overnight. Upon completion, the reaction was quenched with saturated NaHCO3aqueous solution. The resultant mixture was extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (0~23% ethyl acetate in hexane) to give the title compound 18 (214.6 mg, 74.0% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 7.78 (t, J = 1.4 Hz, 1H), 7.73 (dt, J = 7.6, 1.5 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.51 (dt, J = 7.7, 1.4 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.42 (s, 1H), 7.15 (d, J = 8.1 Hz, 1H), 5.61 – 5.55 (m, 1H), 4.57 (s, 2H), 4.10 (dd, J = 11.6, 4.3 Hz, 2H), 3.58 (td, J = 11.9, 1.6 Hz, 2H), 2.15 – 2.11 (m, 2H), 2.06 – 1.99 (m, 2H). 3-(6-(Aminomethyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl) benzonitrile (19) To a solution of azide 18 (410 mg, 1.05 mmol) in THF-H2O (11 mL, V / V =10:1) added PPh3(786.9 mg, 3 mmol). The reaction mixture was stirred at room temperature for 16 h. Upon completion, the solvents were removed. The residue was purified by flash column chromatography (0~7% MeOH in DCM) to give the title compound 19 (261.3 mg, 68% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 7.78 (t, J = 1.3 Hz, 1H), 7.73 (dt, J = 7.6, 1.5 Hz, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.50 (dt, J = 7.7, 1.4 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.38 (s, 1H), 7.15 (d, J = 8.1 Hz, 1H), 5.59 (tt, J = 11.8, 3.9 Hz, 1H), 4.10 (dd, J = 11.5, 4.2 Hz, 2H), 4.04 (s, 2H), 3.58 (td, J = 11.8, 1.6 Hz, 2H), 2.15 – 2.10 (m, 2H), 2.05 – 1.98 (m, 2H). LCMS (ESI): m / z 349 [M-17+H]+. Tert-butyl 6-(((7-chloro-3-(3-cyanophenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (20) Compound 20 was prepared according to the method described for procedure A. It was obtained as a white solid (92.6% yield).1H NMR (600 MHz, CDCl3) δ 7.76 (t, J = 1.6 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.39 (s, 1H), 7.14 (d, J = 8.2 Hz, 1H), 5.68 (t, J = 5.5 Hz, 1H), 5.58 – 5.53 (m, 1H), 4.61 (d, J = 5.7 Hz, 2H), 4.10 (dd, J = 11.5, 4.1 Hz, 2H), 3.84 (s, 2H), 3.81 (s, 2H), 3.59 – 3.55 (m, 2H), 2.97 – 2.92 (m, 1H), 2.42 – 2.38 (m, 4H), 2.28 – 2.25 (m, 2H), 2.13 – 2.09 (m, 2H), 1.35 (s, 9H). LCMS (ESI): m / z 589 [M+H]+. Tert-butyl 6-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H- indol-6-yl)methyl)carbamoyl)-2-azaspiro[3.3]heptane-2-carboxylate (21) Compound 21 was prepared according to the method described for procedure A. It was obtained as a yellow oil (96.6% yield).1H NMR (600 MHz, CD3OD) δ 8.18 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.81 – 7.79 (m, 1H), 7.79 (s, 1H), 7.77 – 7.76 (m, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 5.73 – 5.67 (m, 1H), 4.62 (s, 2H), 4.16 – 4.12 (m, 2H), 3.94 (s, 2H), 3.87 (s, 2H), 3.71 – 3.66 (m, 2H), 3.05 – 3.02 (m, 1H), 2.46 – 2.37 (m, 4H), 2.19 – 2.15 (m, 4H), 1.44 (s, 9H); LCMS (ESI): m / z 623 [M+H]+. N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-2-azaspiro[3.3]heptane-6-carboxamide (22) Compound 22 was prepared using a similar procedure for the synthesis of compound 4. It was obtained as a white solid (12.5 mg, 42.7% yield).1H NMR (600 MHz, DMSO-d6) δ 9.91 (s, 1H), 9.55 (s, 1H), 8.16 (s, 1H), 8.11 (s, 1H), 8.01 (s, 1H), 7.83 (d, J = 8.2 Hz, 1H), 7.82 – 7.78 (m, 2H), 7.49 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 5.59 – 5.55 (m, 1H), 4.47 (d, J = 5.7 Hz, 2H), 4.05 (dd, J = 10.9, 3.8 Hz, 2H), 3.60 – 3.56 (m, 2H), 3.48 (s, 2H), 3.36 (s, 2H), 2.94 – 2.89 (m, 1H), 2.24 – 2.21 (m, 3H), 2.16 – 2.04 (m, 5H);13C NMR (150 MHz, DMSO-d6) δ 200.8, 174.3, 140.7, 134.6, 132.1, 131.5, 130.0, 128.9, 128.3, 126.3, 125.8, 125.7, 121.7, 118.4, 116.1, 115.7, 67.0, 59.6, 58.7, 53.9, 40.9, 40.6, 36.3, 34.8, 33.8. HPLC analysis: retention time = 2.077 min; peak area, 96.764% (λ = 254 nm). LCMS (ESI): m / z 523 [M+H]+. 2-(2-Butoxyethyl)-N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)- 1H-indol-6-yl)methyl)-2-azaspiro[3.3]heptane-6-carboxamide (23) Compound 23 was prepared from aldehyde 30a by following the general procedure D. It was obtained as a white solid (31.4% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.18 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.78 (s, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 5.72 – 5.67 (m, 1H), 4.61 (s, 2H), 4.15 – 4.12 (m, 2H), 3.70 – 3.65 (m, 2H), 3.45 – 3.43 (m, 2H), 3.42 (s, 2H), 3.35 (s, 2H), 3.01 (q, J = 7.8 Hz, 1H), 2.68 (t, J = 5.4 Hz, 2H), 2.43 – 2.33 (m, 4H), 2.20 – 2.14 (m, 4H), 1.59 – 1.52 (m, 3H), 1.43 – 1.36 (m, 3H), 0.94 (t, J = 7.4 Hz, 3H);13C NMR (150 MHz, CD3OD) δ 202.7, 175.6, 140.4, 134.8, 132.2, 130.3, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 70.6, 68.3, 67.1, 66.8, 66.2, 57.7, 53.6, 41.2, 35.8, 35.1, 34.4, 31.5, 18.9, 12.8. HPLC analysis: retention time = 2.873 min; peak area, 97.384% (λ = 254 nm). LCMS (ESI): m / z 623 [M+H]+. N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-2-(2-(cyclobutylmethoxy)ethyl)-2-azaspiro[3.3]heptane-6-carboxamide (24) Compound 24 was prepared from aldehyde 30b by following the general procedure D. It was obtained as a white solid (36.9% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 – 7.79 (m, 1H), 7.78 (s, 1H), 7.76 (dt, J = 7.7, 1.1 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 5.72 – 5.67 (m, 1H), 4.61 (s, 2H), 4.16 – 4.12 (m, 2H), 3.70 – 3.65 (m, 2H), 3.45 (t, J = 5.6 Hz, 2H), 3.43 (s, 2H), 3.39 (d, J = 6.6 Hz, 2H), 3.36 (s, 2H), 3.01 (q, J = 8.0 Hz, 1H), 2.69 (t, J = 5.5 Hz, 2H), 2.59 – 2.54 (m, 1H), 2.42 – 2.34 (m, 4H), 2.18 – 2.14 (m, 4H), 2.07 – 2.03 (m, 2H), 1.96 – 1.88 (m, 2H), 1.81 – 1.74 (m, 2H).13C NMR (150 MHz, CD3OD) δ 202.7, 175.6, 140.4, 134.8, 132.2, 130.3, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 75.4, 68.6, 67.1, 66.8, 66.2, 53.6, 41.2, 35.8, 35.1, 34.4, 33.9, 24.6, 18.0. HPLC analysis: retention time = 3.50 min; peak area, 96.8% (λ = 254 nm). LCMS (ESI): m / z 635 [M+H]+. N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-2-(2-(thiazol-5-ylmethoxy)ethyl)-2-azaspiro[3.3]heptane-6-carboxamide (25) Compound 25 was prepared from aldehyde 30i by following the general procedure D. It was obtained as a white solid (45.4% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.97 (s, 1H), 8.19 (t, J = 1.7 Hz, 1H), 7.82 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.76 (s, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.15 (d, J = 8.2 Hz, 1H), 5.70 – 5.64 (m, 1H), 4.74 (s, 2H), 4.60 (s, 2H), 4.14 – 4.11 (m, 2H), 3.68 – 3.64 (m, 2H), 3.52 (t, J = 5.4 Hz, 2H), 3.41 (s, 2H), 3.34 (s, 2H), 3.01 (q, J = 8.3 Hz, 1H), 2.71 (t, J = 5.3 Hz, 2H), 2.40 – 2.32 (m, 4H), 2.16 – 2.12 (m, 4H).13C NMR (150 MHz, CD3OD) δ 202.7, 175.6, 154.8, 141.1, 140.4, 136.3, 134.8, 131.2, 130.3, 130.0, 128.8, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.6, 115.9, 67.8, 67.1, 66.7, 66.2, 64.3, 57.5, 53.6, 41.2, 35.8, 35.1, 34.4, 34.0. HPLC analysis: retention time = 2.764 min; peak area, 96.7% (λ = 254 nm). LCMS (ESI): m / z 664 [M+H]+. Tert-butyl 2-(((7-chloro-3-(3-cyanophenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonane-7-carboxylate (31) Compound 31 was prepared from 7-(tert-butoxycarbonyl)-7-azaspiro[3.5]nonane-2- carboxylic acid and primary amine 19 by following the general procedure A. It was obtained as a white solid (88% yield).1H NMR (600 MHz, CDCl3) δ 7.85 (t, J = 1.3 Hz, 1H), 7.81 (dt, J = 7.6, 1.5 Hz, 1H), 7.71 (d, J = 8.1 Hz, 1H), 7.60 (dt, J = 7.7, 1.4 Hz, 1H), 7.56 (t, J = 7.7 Hz, 1H), 7.48 (s, 1H), 7.29 (s, 1H), 7.25 (d, J = 8.2 Hz, 1H), 5.79 (t, J = 5.6 Hz, 1H), 5.65 (tt, J = 11.7, 3.9 Hz, 1H), 4.72 (d, J = 5.8 Hz, 2H), 4.19 (dd, J = 11.5, 4.2 Hz, 2H), 3.66 (t, J = 11.0 Hz, 2H), 3.37 – 3.35 (m, 2H), 3.30 – 3.28 (m, 2H), 2.23 – 2.19 (m, 2H), 2.14 – 2.09 (m, 4H), 2.06 – 2.02 (m, 2H), 1.60 – 1.55 (m, 4H), 1.46 (s, 9H). LCMS (ESI): m / z 617 [M+H]+. Tert-butyl 2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H- indol-6-yl)methyl)carbamoyl)-7-azaspiro[3.5]nonane-7-carboxylate (32) Compound 32 was prepared from benzonitrile 31 according to the method described for procedure B. It was obtained as a yellow solid (93% yield).1H NMR (600 MHz, CDCl3) δ 8.03 (t, J = 1.6 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.64 (s, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.43 (s, 1H), 7.40 (brs, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.10 (d, J = 8.2 Hz, 1H), 5.74 (t, J = 5.4 Hz, 1H), 5.55 – 5.51 (m, 1H), 4.59 (d, J = 5.6 Hz, 2H), 4.10 – 4.07 (m, 2H), 3.55 (t, J = 11.0 Hz, 2H), 3.25 – 3.23 (m, 2H), 3.18 – 3.16 (m, 2H), 2.10 – 2.07 (m, 2H), 2.04 – 1.98 (m, 4H), 1.94 – 1.90 (m, 2H), 1.48 – 1.46 (m, 2H), 1.45 – 1.43 (m, 2H), 1.36 (s, 9H). LCMS (ESI): m / z 651 [M+1]+. N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-azaspiro[3.5]nonane-2-carboxamide (33) Compound 33 was prepared from N-Boc amine 32 using a similar procedure for the synthesis of compound 4. It was obtained as a white solid (44.1% yield).1H NMR (600 MHz, CD3OD) δ 8.08 (t, J = 1.7 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.68 – 7.66 (m, 2H), 7.65 (dt, J = 7.7, 1.1 Hz, 1H), 7.37 (t, J = 7.7 Hz, 1H), 7.07 (d, J = 8.2 Hz, 1H), 5.61 – 5.57 (m, 1H), 4.52 (s, 2H), 4.04 – 4.01 (m, 2H), 3.59 – 3.54 (m, 2H), 3.07 – 3.03 (m, 1H), 2.95 – 2.92 (m, 2H), 2.88 – 2.85 (m, 2H), 2.08 – 2.04 (m, 4H), 2.01 – 1.97 (m, 4H), 1.72 (t, J = 5.3 Hz, 2H), 1.65 (t, J = 5.5 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.0, 140.4, 134.8, 132.2, 130.3, 130.0, 128.9, 128.2, 126.5, 124.6, 124.2, 121.4, 117.9, 116.7, 115.9, 67.1, 53.7, 41.3, 41.2, 41.1, 35.3, 34.4, 34.1, 33.8, 32.8, 32.5. HPLC analysis: retention time = 2.7021 min; peak area, 95.781% (λ = 254 nm). LCMS (ESI): m / z 551 [M+H]+. 7-(2-Butoxyethyl)-N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)- 1H-indol-6-yl)methyl)-7-azaspiro[3.5]nonane-2-carboxamide (34) The title compound 34 was synthesized from 2-butoxyacetaldehyde 30a according to the method described for procedure D. It was obtained as a white solid (25.4% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.81 – 7.78 (m, 2H), 7.77 (d, J = 7.9 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.69 (m, 1H), 4.64 (s, 2H), 4.16 – 4.13 (m, 2H), 3.71 – 3.67 (m, 2H), 3.59 (t, J = 5.7 Hz, 2H), 3.46 (t, J = 6.5 Hz, 2H), 3.14 – 3.10 (m, 1H), 2.65 – 2.61 (m, 2H), 2.58 – 2.52 (m, 2H), 2.20 – 2.16 (m, 4H), 2.07 – 2.02 (m, 4H), 1.74 (t, J = 5.3 Hz, 2H), 1.67 (t, J = 5.6 Hz, 2H), 1.56 (dt, J = 14.4, 6.4 Hz, 3H), 1.39 (dt, J = 14.7, 7.5 Hz, 3H), 0.94 (t, J = 7.4 Hz, 3H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 117.0, 115.9, 70.6, 67.1, 57.4, 53.7, 50.4, 50.1, 48.2, 41.2, 37.2, 35.4, 34.4, 33.2, 32.8, 31.4, 19.0, 12.8. HPLC analysis: retention time = 3.469 min; peak area, 95.831% (λ = 254 nm). LCMS (ESI): m / z 651 [M+H]+. N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(cyclobutylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (35) The title compound 35 was synthesized from 2-(cyclobutylmethoxy)acetaldehyde 30b according to the method described for procedure D. It was obtained as a white solid (38.8% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.09 (t, J = 1.6 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.67 (s, 1H), 7.67 – 7.63 (m, 2H), 7.36 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 5.60 – 5.55 (m, 1H), 4.52 (s, 2H), 4.04 – 4.00 (m, 2H), 3.64 – 3.62 (m, 2H), 3.58 – 3.54 (m, 2H), 3.38 (d, J = 6.8 Hz, 2H), 3.25 – 3.22 (m, 1H), 3.17 – 3.14 (m, 2H), 3.05 (p, J = 8.6 Hz, 2H), 2.51 (dt, J = 14.9, 7.5 Hz, 1H), 2.10 – 1.91 (m, 12H), 1.88 – 1.75 (m, 6H), 1.69 – 1.64 (m, 2H).13C NMR (150 MHz, CD3OD) δ 202.7, 175.8, 140.4, 134.8, 132.2, 130.3, 130.0, 128.9, 128.2, 126.6, 124.7, 124.1, 121.4, 117.9, 116.7, 115.9, 75.6, 67.1, 53.7, 49.9, 49.6, 41.2, 34.8, 34.4, 33.1, 32.3, 32.1, 24.6, 18.0. HPLC analysis: retention time = 3.418 min; peak area, 97.518% (λ = 254 nm). LCMS (ESI): m / z 663 [M+H]+. N-((3-(3-Carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-cyclobutoxyethyl)-7-azaspiro[3.5]nonane-2-carboxamide (36) The title compound 36 was synthesized from 2-cyclobutylacetaldehyde 30c according to the method described for procedure D. It was obtained as a white solid (30.6% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.81 – 7.78 (m, 2H), 7.78 – 7.76 (m, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.63 (s, 2H), 4.16 – 4.12 (m, 2H), 3.99 – 3.94 (m, 1H), 3.71 – 3.66 (m, 2H), 3.53 (t, J = 5.5 Hz, 2H), 3.13 (p, J = 8.6 Hz, 1H), 2.77 – 2.55 (m, 6H), 2.24 – 2.20 (m, 2H), 2.18 – 2.15 (m, 4H), 2.09 – 2.03 (m, 4H), 1.96 – 1.90 (m, 2H), 1.80 – 1.76 (m, 2H), 1.74 – 1.69 (m, 3H), 1.59 – 1.53 (m, 1H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.3, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.2, 121.4, 117.9, 116.7, 115.9, 73.4, 67.1, 57.2, 53.7, 50.3, 50.0, 48.2, 41.2, 36.7, 34.9, 34.4, 34.2, 33.0, 32.7, 29.8, 11.9. HPLC analysis: retention time = 3.248 min; peak area, 99.9% (λ = 254 nm). LCMS (ESI): m / z 649 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(2-(1-methylcyclopropyl)ethoxy)ethyl)-7-azaspiro[3.5]nonane-2- carboxamide (37) The title compound 37 was synthesized from 2-(2-(1- methylcyclopropyl)ethoxy)acetaldehyde 30d according to the method described for procedure D. It was obtained as a white solid (35.5% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.20 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 4.9 Hz, 2H), 7.77 (d, J = 8.0 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.69 (m, 1H), 4.64 (s, 2H), 4.15 (dt, J = 10.6, 3.3 Hz, 2H), 3.70 – 3.65 (m, 4H), 3.56 – 3.53 (m, 2H), 3.16 – 3.12 (m, 1H), 2.87 – 2.82 (m, 2H), 2.20 – 2.16 (m, 4H), 2.11 – 2.05 (m, 4H), 1.81 (t, J = 9.6 Hz, 2H), 1.75 (t, J = 9.6 Hz, 2H), 1.65 (s, 2H), 1.60 – 1.58 (m, 3H), 1.56 – 1.50 (m, 3H), 0.92 (s, 3H), 0.33 (t, J = 4.7 Hz, 1H), 0.25 (t, J = 4.8 Hz, 1H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.2, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.2, 121.4, 117.9, 116.7, 115.9, 69.6, 67.1, 56.9, 53.7, 50.3, 49.9, 48.2, 41.2, 39.1, 38.5, 34.4, 32.7, 31.2, 29.3, 25.1, 22.5, 22.0, 14.4, 12.1. HPLC analysis: retention time = 3.822 min; peak area, 95.498% (λ = 254 nm). LCMS (ESI): m / z 677 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(thiophen-2-ylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (38) The title compound 38 was synthesized from 2-(thiophen-2-ylmethoxy)acetaldehyde 30e according to the method described for procedure D. It was obtained as a white solid (28.9% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 6.4 Hz, 2H), 7.77 (d, J = 7.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.39 – 7.38 (m, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.06 (d, J = 3.1 Hz, 1H), 6.99 (d, J = 1.4 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.71 (s, 2H), 4.63 (s, 2H), 4.16 – 4.13 (m, 2H), 3.71 – 3.68 (m, 2H), 3.66 (t, J = 5.5 Hz, 2H), 3.14 – 3.10 (m, 1H), 2.78 – 2.73 (m, 2H), 2.63 – 2.46 (m, 4H), 2.20 – 2.14 (m, 4H), 2.09 – 2.01 (m, 4H), 1.75 (t, J = 5.3 Hz, 2H), 1.69 (t, J = 5.5 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.3, 140.5, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.6, 126.4, 126.3, 125.7, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 66.9, 65.6, 56.7, 53.7, 50.2, 50.0, 48.2, 41.2, 36.7, 34.9, 34.4, 33.0, 32.7. HPLC analysis: retention time = 3.427 min; peak area, 95.329% (λ = 254 nm). LCMS (ESI): m / z 691 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(furan-2-ylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (39) The title compound 39 was synthesized from 2-(furan-2-ylmethoxy)acetaldehyde 30f according to the method described for procedure D. It was obtained as a white solid (28.9% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.18 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.81 – 7.79 (m, 1H), 7.79 (s, 1H), 7.78 – 7.75 (m, 1H), 7.50 – 7.46 (m, 2H), 7.18 (d, J = 8.2 Hz, 1H), 6.39 – 6.37 (m, 2H), 5.73 – 5.68 (m, 1H), 4.63 (s, 2H), 4.46 (s, 2H), 4.16 – 4.12 (m, 2H), 3.71 – 3.66 (m, 2H), 3.61 (t, J = 5.7 Hz, 2H), 3.09 (q, J = 8.7 Hz, 1H), 2.57 (t, J = 5.6 Hz, 2H), 2.52 – 2.36 (m, 4H), 2.19 – 2.15 (m, 4H), 2.06 – 1.99 (m, 4H), 1.70 (t, J = 5.3 Hz, 2H), 1.62 (t, J = 5.5 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 151.8, 142.6, 140.4, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.3, 124.6, 124.3, 121.4, 117.9, 116.7, 109.9, 109.1, 67.1, 66.6, 64.2, 57.3, 53.7, 50.3, 50.0, 48.2, 41.2, 37.4, 35.5, 34.4, 33.3, 32.9. HPLC analysis: retention time = 3.313 min; peak area, 96.999% (λ = 254 nm). LCMS (ESI): m / z 675 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(pyridin-4-ylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (40) The title compound 40 was synthesized from 2-(pyridin-4-ylmethoxy)acetaldehyde 30g according to the method described for procedure D. It was obtained as a white solid (53.2% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.50 (d, J = 6.0 Hz, 2H), 8.19 (t, J = 4.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (s, 1H), 7.79 (s, 1H), 7.77 (d, J = 7.7 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.43 (d, J = 5.5 Hz, 2H), 7.18 (d, J = 8.2 Hz, 1H), 5.72 – 5.68 (m, 1H), 4.63 (s, 2H), 4.60 (s, 2H), 4.16 – 4.13 (m, 2H), 3.70 (t, J = 5.6 Hz, 2H), 3.69 – 3.65 (m, 2H), 3.13 – 3.09 (m, 1H), 2.67 (t, J = 5.4 Hz, 2H), 2.54 – 2.46 (m, 4H), 2.19 – 2.15 (m, 4H), 2.07 – 2.02 (m, 4H), 1.73 (t, J = 5.3 Hz, 2H), 1.66 (t, J = 5.5 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 149.3, 148.6, 140.4, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.4, 124.6, 124.3, 122.1, 121.4, 117.9, 116.7, 115.9, 70.7, 68.0, 67.1, 57.5, 53.7, 50.5, 50.1, 48.2, 41.2, 37.5, 35.6, 34.4, 33.3, 32.9. HPLC analysis: retention time = 1.848 min; peak area, 95.336% (λ = 254 nm). LCMS (ESI): m / z 686 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(pyridin-2-ylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (41) The title compound 41 was synthesized from 2-(pyridin-3-ylmethoxy)acetaldehyde 30h according to the method described for procedure D. It was obtained as a white solid (64.6% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.50 (d, J = 4.6 Hz, 1H), 8.20 – 8.18 (m, 1H), 7.88 – 7.84 (m, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.81 – 7.76 (m, 3H), 7.55 (d, J = 7.8 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.37 – 7.34 (m, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.64 (s, 2H), 4.54 (s, 2H), 4.16 – 4.12 (m, 2H), 3.79 – 3.73 (m, 2H), 3.73 – 3.67 (m, 2H), 3.15 – 3.10 (m, 1H), 2.78 (s, 2H), 2.70 – 2.53 (m, 4H), 2.19 – 2.14 (m, 4H), 2.09 – 1.99 (m, 4H), 1.77 (t, J = 5.3 Hz, 2H), 1.70 (t, J = 5.5 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.3, 157.8, 148.2, 140.4, 137.5, 134.5, 132.2, 130.4, 130.0, 128.8, 128.2, 126.4, 124.6, 124.3, 122.8, 122.0, 121.4, 117.9, 116.7, 115.9, 72.9, 67.1, 57.2, 53.6, 50.3, 50.0, 48.2, 41.2, 37.0, 35.1, 34.4, 33.1, 32.8. HPLC analysis: retention time = 2.614 min; peak area, 96.063% (λ = 254 nm). LCMS (ESI): m / z 686 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(thiazol-5-ylmethoxy)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (42) The title compound 42 was synthesized from 2-(thiazol-5-ylmethoxy)acetaldehyde 30i according to the method described for procedure D. It was obtained as a white solid (42.6% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.88 (s, 1H), 8.07 (t, J = 1.6 Hz, 1H), 7.74 (s, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.67 (s, 1H), 7.66 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 5.59 – 5.54 (m, 1H), 4.70 (s, 2H), 4.50 (s, 2H), 4.03 – 3.99 (m, 2H), 3.61 (t, J = 5.3 Hz, 2H), 3.57 – 3.53 (m, 2H), 3.03 – 2.98 (m, 1H), 2.84 – 2.79 (m, 2H), 2.76 – 2.57 (m, 4H), 2.05 – 2.01 (m, 4H), 1.98 – 1.92 (m, 4H), 1.72 – 1.68 (m, 2H), 1.65 – 1.60 (m, 2H).13C NMR (150 MHz, CD3OD) δ 202.7, 176.1, 155.0, 141.5, 140.4, 135.8, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.4, 124.6, 124.2, 121.4, 117.9, 116.7, 115.9, 67.1, 64.2, 56.5, 53.7, 49.8, 41.2, 36.0, 34.4, 34.0, 32.7, 32.6. HPLC analysis: retention time = 2.925 min; peak area, 96.017% (λ = 254 nm). LCMS (ESI): m / z 692 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl) methyl)-7-(2-(thiazol-5-yl)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (43) The title compound 43 was synthesized from aldehyde 46 according to the method described for procedure D. It was obtained as a white solid (30.5% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.86 (s, 1H), 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 6.1 Hz, 2H), 7.78 – 7.76 (m, 1H), 7.70 (s, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.72 – 5.69 (m, 1H), 4.64 (s, 2H), 4.16 – 4.13 (m, 2H), 3.70 – 3.67 (m, 2H), 3.16 – 3.11 (m, 3H), 2.79 – 2.75 (m, 2H), 2.69 – 2.56 (m, 4H), 2.18 – 2.15 (m, 4H), 2.09 – 2.05 (m, 4H), 1.80 (t, J = 5.5 Hz, 2H), 1.72 (t, J = 5.5 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.3, 153.2, 140.4, 140.0, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.2, 121.4, 117.9, 116.7, 115.9, 67.1, 53.7, 53.4, 49.9, 49.6, 48.2, 41.2, 35.4, 34.4, 33.2, 32.8. HPLC analysis: retention time = 2.851 min; peak area, 95.13% (λ = 254 nm). LCMS (ESI): m / z 662 [M+H]+. 7-(2-Acetamidoethyl)-N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4- yl)-1H-indol-6-yl)methyl)-7-azaspiro[3.5]nonane-2-carboxamide (50) The title compound 50 was synthesized from aldehyde 49a according to the method described for procedure D. It was obtained as a white solid (43% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.19 (t, J = 1.5 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.78 (s, 1H), 7.76 (d, J = 7.9 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.17 (d, J = 8.2 Hz, 1H), 5.72 – 5.67 (m, 1H), 4.63 (s, 2H), 4.15 – 4.12 (m, 2H), 3.70 – 3.66 (m, 2H), 3.35 (t, J = 6.9 Hz, 2H), 3.11 (q, J = 8.6 Hz, 1H), 2.57 – 2.50 (m, 4H), 2.48 – 2.37 (m, 2H), 2.18 – 2.14 (m, 4H), 2.07 – 2.01 (m, 4H), 1.95 (s, 3H), 1.73 (t, J = 5.0 Hz, 2H), 1.66 (t, J = 5.4 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.7, 176.3, 172.0, 140.4, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 57.0, 53.7, 50.0, 49.8, 48.2, 41.2, 37.4, 36.0, 35.5, 34.4, 33.3, 32.8, 21.2. HPLC analysis: retention time = 2.453 min; peak area, 97.398% (λ = 254 nm). LCMS (ESI): m / z 636 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(2,2,2-trifluoroacetamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (51) The title compound 51 was synthesized from aldehyde 49b according to the method described for procedure D. It was obtained as a white solid (78.3% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.07 (t, J = 1.6 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.67 (dd, J = 8.2, 1.7 Hz, 2H), 7.65 (d, J = 7.7 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 5.61 – 5.56 (m, 1H), 4.51 (s, 2H), 4.04 – 4.00 (m, 2H), 3.59 – 3.54 (m, 2H), 3.32 (t, J = 6.9 Hz, 1H), 3.02 – 2.97 (m, 1H), 2.86 (t, J = 6.1 Hz, 1H), 2.44 – 2.39 (m, 2H), 2.37 – 2.25 (m, 4H), 2.08 – 2.02 (m, 4H), 1.96 – 1.89 (m, 4H), 1.60 (t, J = 5.4 Hz, 2H), 1.53 (t, J = 5.5 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 157.6 (q, J = 36.0 Hz), 140.4, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.3, 124.6, 124.3, 121.4, 117.9, 117.1, 116.7, 116.5 (q, J = 285.0 Hz), 67.1, 56.3, 53.7, 50.0, 49.8, 48.2, 41.2, 37.7, 36.4, 35.8, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.838 min; peak area, 99.1% (λ = 254 nm). LCMS (ESI): m / z 690 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(3,3,3-trifluoropropanamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (52) The title compound 52 was synthesized from aldehyde 49c according to the method described for procedure D. It was obtained as a white solid (78.3% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.07 (t, J = 1.6 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.66 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.05 (d, J = 8.2 Hz, 1H), 5.60 – 5.55 (m, 1H), 4.51 (s, 2H), 4.03 – 4.00 (m, 2H), 3.58 – 3.54 (m, 2H), 3.25 (t, J = 6.8 Hz, 2H), 3.07 – 3.01 (m, 2H), 2.98 (d, J = 8.7 Hz, 1H), 2.41 – 2.22 (m, 6H), 2.06 – 2.02 (m, 4H), 1.95 – 1.88 (m, 4H), 1.60 (t, J = 5.2 Hz, 2H), 1.52 (t, J = 5.4 Hz, 2H);13C NMR (150 MHz, CD3OD) δ 202.7, 176.4, 160.3, 140.4, 134.8, 130.4, 130.0, 128.8, 128.2, 126.4, 124.6, 124.4 (q, J = 274.5 Hz), 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 56.7, 53.7, 50.0, 49.8, 48.2, 41.2, 40.0 (q, J = 29.1 Hz), 37.6, 36.3, 35.6, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.667 min; peak area, 99.348% (λ = 254 nm). LCMS (ESI): m / z 704 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(3-propoxypropanamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (53) To a stirred solution of compound 68 (12 mg, 0.0173 mmol) in DCM (2.0 mL) was added TFA (0.4 mL) at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1 h. Upon completion, the solvent was removed and concentrated to dryness under vacuum. The crude product was directly used for the next step. The mixture of trifluoroacetate (0.0173 mmol, 1 eq), 3-propoxypropanoic acid (2.97 mg, 0.0225 mmol, 1.3 eq), HATU (13.2 mg, 0.0346 mmol, 2 eq) and DIPEA (9.0 µL, 6.71 mg, 0.0519 mmol, 3 eq) in DMF (0.6 mL) were stirred at 0 °C. The reaction mixture was warmed to room temperature and stirred for 1.5 h. The reaction completion was monitored by TLC, and the analysis indicated that the reaction was complete. The reaction mixture was then quenched with water and extracted with ethyl acetate. The combined organic layer was dried over sodium sulfate, filtrated, and evaporated. The residue was purified by reverse-phase flash chromatography and then lyophilized to give compound 53 (5.1 mg, 41.5% yield for two steps) as a white solid.1H NMR (600 MHz, CD3OD) δ 8.19 (s, 1H), 7.83 – 7.75 (m, 4H), 7.49 – 7.46 (m, 1H), 7.17 (s, 1H), 5.71 – 5.67 (m, 1H), 4.62 (s, 2H), 4.15 – 4.11 (m, 2H), 3.70 – 3.65 (m, 4H), 3.43 – 3.40 (m, 2H), 3.37 – 3.34 (m, 2H), 3.13 – 3.08 (m, 1H), 2.50 – 2.41 (m, 6H), 2.39 – 2.31 (m, 2H), 2.18 – 2.14 (m, 4H), 2.06 – 1.99 (m, 4H), 1.72 – 1.68 (m, 2H), 1.65 – 1.61 (m, 2H), 1.60 – 1.54 (m, 2H), 0.95 – 0.91 (m, 3H).13C NMR (150 MHz, CD3OD) δ 202.7, 176.4, 172.6, 140.4, 134.8, 132.2, 130.0, 128.8, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 72.3, 67.1, 66.4, 57.0, 53.7, 50.1, 49.8, 48.2, 41.2, 37.7, 36.4, 36.2, 35.8, 34.4, 33.4, 32.9, 22.5, 9.5. HPLC analysis: retention time = 3.093 min; peak area, 100.00% (λ = 254 nm). LCMS (ESI): m / z 708 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(cyclopentanecarboxamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (54) The title compound 54 was synthesized from cyclopentanecarboxylic acid according to the method described for synthesis of compound 53. It was obtained as a white solid (43.4% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.18 (t, J = 1.6 Hz, 1H), 7.83 – 7.75 (m, 4H), 7.49 – 7.46 (m, 1H), 7.19 – 7.15 (m, 1H), 5.72 – 5.67 (m, 1H), 4.63 (s, 2H), 4.15 – 4.11 (m, 2H), 3.71 – 3.65 (m, 2H), 3.13 – 3.08 (m, 1H), 2.62 – 2.58 (m, 1H), 2.50 – 2.30 (m, 6H), 2.18 – 2.13 (m, 4H), 2.06 – 1.99 (m, 4H), 1.88 – 1.82 (m, 2H), 1.77 – 1.68 (m, 6H), 1.65 – 1.57 (m, 4H).13C NMR (150 MHz, CD3OD) δ 202.8, 177.8, 176.4, 140.4, 134.8, 132.2, 130.4, 130.0, 128.8, 128.2, 126.3, 124.6, 124.3, 121.4, 116.7, 115.9, 67.1, 57.1, 53.7, 50.0, 49.8, 48.2, 45.2, 41.2, 37.7, 36.1, 35.8, 34.4, 33.4, 32.9, 30.1, 25.5. HPLC analysis: retention time = 3.197 min; peak area, 100% (λ = 254 nm). LCMS (ESI): m / z 690 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(tetrahydrofuran-2-carboxamido)ethyl)-7-azaspiro[3.5]nonane-2- carboxamide (55) The title compound 55 was synthesized from tetrahydrofuran-2-carboxylic acid according to the method described for synthesis of compound 53. It was obtained as a white solid (36.1% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.84 – 7.76 (m, 4H), 7.50 – 7.47 (m, 1H), 7.18 (d, J = 7.4 Hz, 1H), 5.74 – 5.67 (m, 1H), 4.63 (s, 2H), 4.31 – 4.27 (m, 1H), 4.16 – 4.12 (m, 2H), 4.01 – 3.97 (m, 1H), 3.90 – 3.85 (m, 1H), 3.72 – 3.66 (m, 2H), 3.38 – 3.35 (m, 2H), 3.15 – 3.08 (m, 1H), 2.49 – 2.44 (m, 3H), 2.41 – 2.24 (m, 4H), 2.20 – 2.14 (m, 4H), 2.07 – 2.00 (m, 4H), 1.97 – 1.87 (m, 3H), 1.71 (t, J = 5.4 Hz, 2H), 1.63 (t, J = 5.4 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 174.7, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 78.0, 69.0, 67.1, 56.9, 53.7, 50.0, 49.8, 48.2, 41.2, 37.8, 35.9, 35.6, 34.4, 33.5, 32.9, 30.1, 24.9. HPLC analysis: retention time = 2.765 min; peak area, 100% (λ = 254 nm). LCMS (ESI): m / z 692 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl) methyl)-7-(2-(nicotinamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (56) The title compound 56 was synthesized from aldehyde 49d according to the method described for procedure D. It was obtained as a white solid (75.8% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.99 (d, J = 1.7 Hz, 1H), 8.69 (dd, J = 4.9, 1.4 Hz, 1H), 8.26 (dt, J = 8.0, 1.8 Hz, 1H), 8.19 (s, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (s, 1H), 7.79 (s, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.55 (dd, J = 7.9, 4.9 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.64 (s, 2H), 4.16 – 4.13 (m, 2H), 3.71 – 3.66 (m, 2H), 3.59 (t, J = 6.7 Hz, 2H), 3.15 – 3.10 (m, 1H), 2.67 (t, J = 6.4 Hz, 2H), 2.61 – 2.50 (m, 4H), 2.19 – 2.15 (m, 4H), 2.08 – 2.03 (m, 4H), 1.76 (t, J = 4.9 Hz, 2H), 1.69 (t, J = 5.3 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 166.5, 151.3, 147.7, 140.4, 135.6, 134.8, 132.2, 130.5, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 123.7, 121.4, 117.9, 116.7, 115.9, 67.1, 56.9, 53.7, 50.1, 49.8, 48.2, 41.2, 37.4, 36.5, 35.5, 34.4, 33.3, 32.8. HPLC analysis: retention time = 2.306 min; peak area, 99.225% (λ = 254 nm). LCMS (ESI): m / z 699 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl) methyl)-7-(2-(isonicotinamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (57) The title compound 57 was synthesized from aldehyde 49e according to the method described for procedure D. It was obtained as a white solid (84.5% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.70 (d, J = 5.9 Hz, 2H), 8.19 (s, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 3.7 Hz, 4H), 7.77 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.71 (q, J = 6.6 Hz, 1H), 4.63 (s, 2H), 4.16 – 4.12 (m, 2H), 3.70 – 3.66 (m, 2H), 3.57 (t, J = 6.8 Hz, 2H), 3.15 – 3.10 (m, 1H), 2.60 (t, J = 6.8 Hz, 2H), 2.54 – 2.38 (m, 4H), 2.19 – 2.14 (m, 4H), 2.07 – 2.01 (m, 4H), 1.73 (t, J = 4.8 Hz, 2H), 1.66 (t, J = 5.2 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 166.3, 149.6, 142.5, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 56.9, 53.7, 50.1, 49.8, 48.2, 41.2, 37.7, 36.8, 35.8, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.253 min; peak area, 99.384% (λ = 254 nm). LCMS (ESI): m / z 699 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(2-fluoroisonicotinamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (58) The title compound 58 was synthesized from aldehyde 49f according to the method described for procedure D. It was obtained as a white solid (80.1% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.35 (d, J = 5.2 Hz, 1H), 8.19 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 6.7 Hz, 2H), 7.77 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 5.2 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.44 (s, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.69 (m, 1H), 4.64 (s, 2H), 4.16 – 4.13 (m, 2H), 3.71 – 3.67 (m, 2H), 3.57 (t, J = 6.8 Hz, 2H), 3.15 – 3.10 (m, 1H), 2.62 (t, J = 6.7 Hz, 2H), 2.54 – 2.43 (m, 4H), 2.19 – 2.15 (m, 4H), 2.08 – 2.02 (m, 4H), 1.74 (t, J = 5.2 Hz, 2H), 1.67 (t, J = 5.4 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 165.1, 164.1 (d, J = 237.0 Hz), 148.1 (d, J = 15.0 Hz), 147.8 (d, J = 7.5 Hz), 140.5, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 119.2 (d, J = 4.5 Hz), 117.9, 115.9, 107.4 (d, J = 39.0 Hz), 67.1, 56.8, 53.7, 50.1, 49.8, 48.2, 41.2, 37.6, 36.7, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.664 min; peak area, 98.681% (λ = 254 nm). LCMS (ESI): m / z 717 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(4-fluorobenzamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (59) The title compound 59 was synthesized from aldehyde 49g according to the method described for procedure D. It was obtained as a white solid (80.1% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.89 (dd, J = 8.8, 5.3 Hz, 2H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 8.2 Hz, 1H), 7.78 (s, 1H), 7.76 (d, J = 8.2 Hz, H), 7.48 (t, J = 7.7 Hz, 1H), 7.21 – 7.17 (m, 3H), 5.72 – 5.68 (m, 1H), 4.63 (s, 2H), 4.15 – 4.12 (m, 2H), 3.70 – 3.64 (m, 2H), 3.54 (t, J = 6.8 Hz, 2H), 3.14 – 3.09 (m, 1H), 2.60 (t, J = 6.8 Hz, 2H), 2.57 – 2.42 (m, 4H), 2.18 – 2.14 (m, 4H), 2.08 – 2.01 (m, 4H), 1.74 (t, J = 5.1 Hz, 2H), 1.66 (t, J = 5.4 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 167.6, 164.8 (d, J = 249.0 Hz), 140.4, 134.8, 132.2, 130.6, 130.4, 130.0, 129.4 (d, J = 9.0 Hz), 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 114.9 (d, J = 21.0 Hz), 67.1, 57.0, 53.7, 50.1, 49.8, 48.2, 41.2, 37.5, 36.6, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.919 min; peak area, 99.284% (λ = 254 nm). LCMS (ESI): m / z 716 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(3-((4-fluorophenyl)amino)-3-oxopropyl)-7-azaspiro[3.5]nonane-2- carboxamide (60) A mixture of N-Boc deprotected product 33 (0.0385 mmol), K2CO3(8 mg, 0.058 mmol), Et3N (10.7 µL, 0.077 mmol), and KI (3.2 mg, 0.019 mmol) in DMF (1.5 mL) was stirred at room termperature for 30 min. Then (3-bromo-N-(4-fluorophenyl)propanamide (18.9 mg, 0.077 mmol) was added, and the reaction mixture was stirred at 60 °C for 8 h. The reaction was quenched with water and extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered, and evaporated. The residue was purified by reverse-phase flash chromatography and then lyophilized to give the desired compound (6.2 mg, 22.5% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.07 (t, J = 1.7 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.67 (d, J = 9.1 Hz, 2H), 7.66 – 7.64 (m, 1H), 7.42 (dd, J = 9.1, 4.9 Hz, 2H), 7.36 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 6.93 (t, J = 8.8 Hz, 2H), 5.61 – 5.56 (m, 1H), 4.51 (s, 2H), 4.04 – 4.01 (m, 2H), 3.59 – 3.55 (m, 2H), 3.03 – 2.98 (m, 1H), 2.61 (t, J = 7.1 Hz, 2H), 2.44 (t, J = 7.1 Hz, 2H), 2.40 – 2.24 (m, 4H), 2.07 – 2.02 (m, 4H), 1.97 – 1.90 (m, 4H), 1.62 (t, J = 5.1 Hz, 2H), 1.55 (t, J = 5.4 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 171.4, 159.2 (d, J = 241.5 Hz), 140.5, 134.8, 134.7, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.6 (d, J = 9.0 Hz), 121.4, 117.9, 116.7, 115.9, 114.8 (d, J = 22.5 Hz), 67.1, 53.8, 53.7, 49.8, 49.4, 48.2, 41.2, 37.8, 35.9, 34.4, 33.5, 33.2, 32.9. HPLC analysis: retention time = 3.141 min; peak area, 97.877% (λ = 254 nm). LCMS (ESI): m / z 716 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(furan-2-carboxamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (61) The title compound 61 was synthesized from aldehyde 49h according to the method described for procedure D. It was obtained as a white solid (64.7% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.78 (s, 1H), 7.76 (J = 8.2 Hz, 1H), 7.66 (s, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.10 (d, J = 3.4 Hz, 1H), 6.58 (dd, J = 3.4, 1.7 Hz, 1H), 5.72 – 5.67 (m, 1H), 4.63 (s, 2H), 4.15 – 4.12 (m, 2H), 3.70 – 3.66 (m, 2H), 3.51 (t, J = 6.8 Hz, 2H), 3.12 (q, J = 8.7 Hz, 1H), 2.58 (t, J = 6.7 Hz, 2H), 2.53 – 2.38 (m, 4H), 2.18 – 2.14 (m, 4H), 2.06 – 2.01 (m, 4H), 1.73 (t, J = 5.0 Hz, 2H), 1.66 (t, J = 5.3 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 159.5, 147.7, 144.8, 140.4, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 124.6, 124.3, 121.4, 117.9, 115.9, 113.8, 111.5, 67.1, 57.0, 53.7, 50.1, 49.8, 48.2, 41.2, 37.6, 35.8, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.641 min; peak area, 99.3% (λ = 254 nm). LCMS (ESI): m / z 688 [M+H]+. N-(2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)oxazole-5-carboxamide (62) The title compound 62 was synthesized from aldehyde 49i according to the method described for procedure D. It was obtained as a white solid (49.5% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.32 (s, 1H), 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.77 (d, J = 7.7 Hz, 1H), 7.71 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.69 (m, 1H), 4.63 (s, 2H), 4.16 – 4.13 (M, 2H), 3.71 – 3.66 (m, 2H), 3.53 (t, J = 6.8 Hz, 2H), 3.14 – 3.09 (m, 1H), 2.58 (t, J = 6.8 Hz, 2H), 2.53 – 2.42 (m, 4H), 2.19 – 2.15 (m, 4H), 2.06 – 2.01 (m, 4H), 1.73 (t, J = 5.2 Hz, 2H), 1.66 (t, J = 5.4 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 157.7, 153.0, 145.7, 140.4, 134.8, 132.2, 130.0, 128.9, 128.2, 126.4, 124.6, 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 56.9, 53.7, 50.1, 49.8, 48.2, 41.2, 37.6, 35.9, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.316 min; peak area, 99.8% (λ = 254 nm). LCMS (ESI): m / z 689 [M+H]+. N-((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)-7-(2-(thiophene-2-carboxamido)ethyl)-7-azaspiro[3.5]nonane-2-carboxamide (63) The title compound 63 was synthesized from aldehyde 49j according to the method described for procedure D. It was obtained as a white solid (46.2% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (d, J = 11.0 Hz, 2H), 7.77 (d, J = 7.8 Hz, 1H), 7.66 (dd, J = 15.2, 4.8 Hz, 2H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 7.13 (dd, J = 4.9, 3.8 Hz, 1H), 5.71 (q, J = 7.6, 6.8 Hz, 1H), 4.63 (s, 2H), 4.16 – 4.12 (m, 2H), 3.68 (dd, J = 13.2, 6.6 Hz, 2H), 3.52 (t, J = 6.9 Hz, 2H), 3.15 – 3.09 (m, 1H), 2.57 (t, J = 6.9 Hz, 2H), 2.52 – 2.38 (m, 4H), 2.19 – 2.14 (m, 4H), 2.07 – 2.01 (m, 4H), 1.73 (t, J = 5.2 Hz, 2H), 1.66 (t, J = 5.4 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 163.0, 140.4, 138.8, 134.8, 132.2, 130.1, 130.0, 128.9, 128.2, 128.1, 127.3, 126.4, 124.6, 124.3, 121.4, 57.1, 53.7, 50.1, 49.8, 48.2, 48.0, 41.2, 37.7, 36.5, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.995 min; peak area, 95.850% (λ = 254 nm). LCMS (ESI): m / z 704 [M+H]+. N-(2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)thiazole-5-carboxamide (64) The title compound 64 was synthesized from aldehyde 49k according to the method described for procedure D. It was obtained as a white solid (48.9% yield for two steps).1H NMR (600 MHz, CD3OD) δ 9.00 (s, 1H), 8.27 (s, 1H), 8.07 (t, J = 1.6 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 7.67 (s, 2H), 7.65 (d, J = 7.8 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.06 (d, J = 8.2 Hz, 1H), 5.62 – 5.57 (m, 1H), 4.51 (s, 2H), 4.04 – 4.01 (m, 2H), 3.59 – 3.54 (m, 2H), 3.42 (t, J = 6.8 Hz, 2H), 3.03 – 2.97 (m, 1H), 2.48 (t, J = 6.8 Hz, 2H), 2.42 – 2.30 (m, 4H), 2.07 – 2.03 (m, 4H), 1.96 – 1.90 (m, 4H), 1.62 (t, J = 5.2 Hz, 2H), 1.54 (t, J = 5.4 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 161.2, 157.5, 143.2, 140.4, 135.1, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.4, 124.6, 121.4, 117.9, 116.7, 115.9, 67.1, 57.0, 53.7, 50.1, 49.8, 48.2, 41.2, 37.6, 36.6, 35.7, 34.4, 33.4, 32.9. HPLC analysis: retention time = 2.741 min; peak area, 95.726% (λ = 254 nm). LCMS (ESI): m / z 705 [M+H]+. N-(2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)thiazole-4-carboxamide (65) The title compound 65 was synthesized from aldehyde 49l according to the method described for procedure D. It was obtained as a white solid (43.4% yield for two steps).1H NMR (600 MHz, CD3OD) δ 9.00 (d, J = 2.0 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.19 (t, J = 1.7 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.79 (s, 1H), 7.79 (s, 1H), 7.78 – 7.76 (m, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.63 (s, 2H), 4.16 – 4.12 (m, 2H), 3.71 – 3.66 (m, 2H), 3.56 (t, J = 6.7 Hz, 2H), 3.15 – 3.09 (m, 1H), 2.59 (t, J = 6.7 Hz, 2H), 2.54 – 2.40 (m, 4H), 2.19 – 2.15 (m, 4H), 2.08 – 2.01 (m, 4H), 1.73 (t, J = 5.3 Hz, 2H), 1.66 (t, J = 5.5 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 162.1, 153.9, 150.6, 140.5, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.3, 124.6, 123.3, 121.4, 117.9, 116.7, 115.9, 67.1, 57.0, 53.7, 50.1, 49.8, 48.2, 41.2, 37.7, 36.1, 35.8, 34.4, 33.5, 32.9. HPLC analysis: retention time = 2.494 min; peak area, 98.862% (λ = 254 nm). LCMS (ESI): m / z 705 [M+H]+. N-(2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)thiazole-2-carboxamide (66) The title compound 66 was synthesized from aldehyde 49m according to the method described for procedure D. It was obtained as a white solid (43.4% yield for two steps). NMR (600 MHz, CD3OD) δ 8.19 (t, J = 1.6 Hz, 1H), 7.95 (d, J = 3.1 Hz, 1H), 7.84 – 7.81 (m, 2H), 7.80 (d, J = 9.3 Hz, 2H), 7.78 – 7.76 (m, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 8.2 Hz, 1H), 5.73 – 5.68 (m, 1H), 4.63 (s, 2H), 4.16 – 4.13 (m, 2H), 3.71 – 3.67 (m, 2H), 3.56 (t, J = 6.8 Hz, 2H), 3.15 – 3.09 (m, 1H), 2.59 (t, J = 6.8 Hz, 2H), 2.54 – 2.40 (m, 4H), 2.19 – 2.16 (m, 4H), 2.07 – 2.01 (m, 4H), 1.73 (t, J = 5.3 Hz, 2H), 1.65 (t, J = 5.5 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 202.8, 176.4, 163.3, 160.3, 143.6, 140.5, 134.8, 132.2, 130.4, 130.0, 128.9, 128.2, 126.3, 124.6, 124.5, 124.3, 121.4, 117.9, 116.7, 115.9, 67.1, 56.9, 53.7, 50.1, 49.8, 48.2, 41.2, 37.8, 36.3, 35.8, 34.4, 33.5, 32.9. HPLC analysis: retention time = 2.797 min; peak area, 98.58% (λ = 254 nm). LCMS (ESI): m / z 705 [M+H]+. N-(2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)thiazole-2-carboxamide hydrocholoride (66s) To a vial were charged compound 66 (25 mg, 0.035 mmol), acetonitrile (2 mL), and 0.2 M hydrochloride acid (230 µL, 0.046 mmol). The reaction mixture was stirred at room temperature for 4 h. The solvent was removed. The residue was purified by reverse-phase flash chromatography and then lyophilized to give the desired compound (23.9 mg, 91.9% yield) as a white solid.1H NMR (600 MHz, CD3OD) δ 8.21 (s, 1H), 7.99 (s, 1H), 7.89 (s, 1H), 7.82 (d, J = 7.1 Hz, 1H), 7.79 (s, 1H), 7.77 (d, J = 9.7 Hz, 2H), 7.48 (t, J = 7.7 Hz, 1H), 7.19 (d, J = 8.2 Hz, 1H), 5.72 – 5.67 (m, 1H), 4.64 (s, 2H), 4.16 – 4.12 (m, 2H), 3.81 (t, J = 6.8 Hz, 2H), 3.70 – 3.63 (m, 3H), 3.61 – 3.56 (m, 1H), 3.39 – 3.36 (m, 2H), 3.21 – 3.15 (m, 1H), 3.10 – 3.06 (m, 1H), 3.02 – 2.98 (m, 1H), 2.24 – 2.14 (m, 7H), 2.12 – 2.04 (m, 3H), 1.89 –1.79 (m, 2H).13C NMR (150 MHz, CD3OD) δ 202.7, 175.8, 162.4, 161.6, 143.8, 140.4, 134.8, 132.2, 130.3, 130.0, 128.9, 128.2, 126.7, 125.2, 124.7, 124.1, 121.4, 118.0, 116.7, 115.9, 67.1, 56.0, 53.7, 50.0, 49.7, 48.2, 41.2, 34.4, 34.2, 33.1, 32.6, 32.3, 32.1. HPLC analysis: retention time = 2.617 min; peak area, 99.444%. LCMS (ESI): m / z 705 [M+H]+. N-(2-(2-(((3-(3-carbamothioylphenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)thiazole-2-carboxamide (77) The title compound 77 was synthesized from aldehyde 49m according to the method described for procedure D. It was obtained as a white solid (58% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.24 (s, 1H), 7.95 (s, 1H), 7.90 (t, J = 6.1 Hz, 1H), 7.85 – 7.81 (m, 2H), 7.75 (t, J = 7.7 Hz, 1H), 7.71 (s, 1H), 7.50 (d, J = 5.5 Hz, 1H), 7.48 – 7.44 (m, 1H), 7.14 – 7.11 (m, 1H), 4.68 – 4.64 (m, 1H), 4.53 (s, 2H), 4.17 – 4.13 (m, 2H), 3.72 (t, J = 11.7 Hz, 2H), 3.58 – 3.53 (m, 2H), 3.13 – 3.08 (m, 1H), 2.59 (t, J = 5.5 Hz, 2H), 2.54 – 2.40 (m, 4H), 2.23 – 2.17 (m, 2H), 2.11 – 2.02 (m, 6H), 1.74 (t, J = 7.7 Hz, 2H), 1.66 (t, J = 7.6 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 203.0, 176.4, 163.3, 160.3, 143.6, 140.4, 136.7, 135.8, 132.6, 129.5, 128.1, 125.8, 125.3, 124.5, 123.7, 122.7, 120.0, 119.5, 115.9, 108.8, 67.0, 56.9, 52.2, 50.1, 49.8, 48.2, 43.4, 37.8, 36.3, 35.8, 34.5, 33.5, 33.0. HPLC analysis: retention time = 2.453 min; peak area, 97.262% (λ = 254 nm). LCMS (ESI): m / z 671 [M+H]+. N-(2-(2-(((3-(6-carbamothioylpyridin-2-yl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)thiazole-2-carboxamide (78) The title compound 78 was synthesized from aldehyde 49m according to the method described for procedure D. It was obtained as a white solid (58% yield for two steps).1H NMR (600 MHz, CD3OD) δ 8.42 (d, J = 7.6 Hz, 1H), 8.24 (d, J = 8.7 Hz, 2H), 7.99 – 7.94 (m, 2H), 7.90 – 7.86 (m, 1H), 7.83 (s, 1H), 7.52 (s, 1H), 7.18 (d, J = 7.5 Hz, 1H), 4.71 – 4.66 (m, 1H), 4.53 (s, 2H), 4.18 – 4.14 (m, 2H), 3.73 (t, J = 11.7 Hz, 2H), 3.55 (t, J = 11.7 Hz, 2H), 3.14 – 3.08 (m, 1H), 2.59 (t, J = 7.4 Hz, 2H), 2.52 – 2.38 (m, 4H), 2.27 – 2.21 (m, 2H), 2.12 – 2.08 (m, 2H), 2.06 – 2.03 (m, 4H), 1.73 (t, J = 7.4 Hz, 2H), 1.66 (t, J = 7.4 Hz, 2H).13C NMR (150 MHz, CD3OD) δ 196.7, 176.4, 163.3, 160.3, 153.4, 151.1, 143.6, 137.0, 132.9, 126.0, 125.1, 124.6, 122.3, 120.8, 120.6, 115.1, 108.9, 67.0, 56.9, 52.4, 50.1, 49.8, 48.2, 43.3, 37.8, 36.3, 35.8, 34.5, 33.0. HPLC analysis: retention time = 2.791 min; peak area, 96.931% (λ = 254 nm). LCMS (ESI): m / z 672 [M+H]+. General procedure E for synthesis of tert-butyl acetates 27a-i To a stirred solution of appropriate alcohol (6.75 mmol, 1 eq.) in toluene (5 mL) was added tert-butyl 2-bromoacetate (1.39 mL, 9.45 mmol, 1.4 eq.), tetrabutylammonium bromide (1.1 g, 3.38 mmol, 0.5 eq.) and 40% aq. NaOH (5 mL). The reaction mixture was stirred at room temperature for 16 h. The resultant mixture was diluted with cold water and extracted with ethyl acetate. The combined organic layers were dried over sodium sulfate, filtered, and concentrated under reduced pressure. Tert-butyl 2-butoxyacetate (27a) Compound 27a was prepared from n-butan-1-ol (26a) using a general procedure E. It was obtained as a colorless oil (66.9% yield).1H NMR (600 MHz, CDCl3) δ 4.02 (s, 1H), 3.87 (s, 1H), 3.44 (t, J = 6.6 Hz, 2H), 1.55 – 1.51 (m, 2H), 1.41 (s, 9H), 1.35 – 1.31 (m, 2H), 0.86 (t, J = 7.4 Hz, 3H). Tert-butyl 2-(cyclobutylmethoxy)acetate (27b) Compound 27b was prepared from cyclobutylmethanol (26b) using a general procedure E. It was obtained as a yellow oil (74.8% yield).1H NMR (600 MHz, CDCl3) δ 3.95 (p, J = 7.4, 6.9 Hz, 1H), 3.80 (s, 2H), 2.17 – 2.11 (m, 2H), 1.97 – 1.89 (m, 2H), 1.65 – 1.60 (m, 1H), 1.45 – 1.42 (m, 1H), 1.40 (s, 9H). Tert-butyl 2-cyclobutoxyacetate (27c) Compound 27c was prepared from cyclobutanol (26c) using a general procedure E. It was obtained as a colorless oil (75.3% yield).1H NMR (600 MHz, CDCl3) δ 3.95 (p, J = 7.4, 6.9 Hz, 1H), 3.80 (s, 2H), 2.17 – 2.11 (m, 2H), 1.97 – 1.89 (m, 2H), 1.65 – 1.60 (m, 1H), 1.45 – 1.42 (m, 1H), 1.40 (s, 9H). Tert-butyl 2-(2-(1-methylcyclopropyl)ethoxy)acetate (27d) Compound 27d was prepared from 2-(1-methylcyclopropyl)ethan-1-ol (26d) using a general procedure E. It was purified by flash column chromatography and obtained as a colorless oil (36.1% yield). NMR (600 MHz, CDCl3) δ 3.85 (s, 2H), 3.51 (t, J = 7.3 Hz, 2H), 1.48 (t, J = 7.3 Hz, 2H), 1.38 (s, 9H), 0.94 (s, 3H), 0.22 – 0.20 (m, 2H), 0.14 – 0.13 (m, 2H);13C NMR (150 MHz, CDCl3) δ 169.8, 81.4, 70.4, 68.9, 38.8, 23.1, 13.1, 12.8. Tert-butyl 2-(thiophen-2-ylmethoxy)acetate (27e) Compound 27e was prepared from thiophen-2-ylmethanol (26e) using a general procedure E. It was purified by flash column chromatography and obtained as a colorless oil (34.5% yield).1H NMR (600 MHz, CDCl3) δ 7.32 (dd, J = 5.1, 1.1 Hz, 1H), 7.05 (d, J = 2.8 Hz, 1H), 7.00 (dd, J = 5.0, 3.5 Hz, 1H), 4.82 (s, 2H), 4.00 (s, 2H), 1.51 (s, 9H);13C NMR (150 MHz, CDCl3) δ 169.4, 139.8, 127.1, 126.7, 126.3, 81.7, 67.3. Tert-butyl 2-(furan-2-ylmethoxy)acetate (27f) Compound 27f was prepared from furan-2-ylmethanol (26f) using a general procedure E. It was obtained as a yellow oil (82.1% yield).1H NMR (600 MHz, CDCl3) δ 7.43 (dd, J = 1.7, 0.8 Hz, 1H), 6.37 – 6.35 (m, 2H), 4.60 (s, 2H), 3.99 (s, 2H), 1.49 (s, 9H);13C NMR (150 MHz, CDCl3) δ 169.4, 151.0, 143.0, 110.3, 110.0, 81.6, 67.3, 64.7, 28.1. Tert-butyl 4-(pyridin-2-ylmethoxy)acetate (27g) Compound 27g was prepared from pyridin-2-ylmethanol (26g) using a general procedure E. It was purified by flash column chromatography and obtained as a colorless oil (55.2% yield).1H NMR (600 MHz, CDCl3) δ 8.49 (d, J = 5.9 Hz, 2H), 7.22 (d, J = 5.7 Hz, 2H), 4.55 (s, 2H), 3.95 (s, 2H), 1.40 (s, 9H).13C NMR (150 MHz, CDCl3) δ 169.1, 149.6, 147.0, 121.9, 82.0, 71.4, 68.4. Tert-butyl 2-(pyridin-2-ylmethoxy)acetate (27h) Compound 27h was prepared from pyridin-2-ylmethanol (26h) using a general procedure E. It was obtained as a yellow oil (77.8% yield).1H NMR (600 MHz, CDCl3) δ 8.36 (d, J = 4.8 Hz, 1H), 7.53 (td, J = 7.7, 1.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 7.03 – 7.00 (m, 1H), 4.56 (s, 2H), 3.92 (s, 2H), 1.30 (s, 9H);13C NMR (150 MHz, CDCl3) δ 169.3, 157.7, 148.9, 136.8, 122.5, 121.7, 81.8, 74.0, 68.6, 28.1. Tert-butyl 2-(thiazol-5-ylmethoxy)acetate (27i) Compound 27i was prepared from thiazol-5-ylmethanol (26i) using a general procedure E.1H NMR (600 MHz, CDCl3) δ 8.83 (s, 1H), 7.83 (s, 1H), 4.87 (s, 2H), 4.00 (s, 2H), 1.49 (s, 9H). General procedure F for synthesis of carboxylic acids 28a-i To a stirred solution of appropriate tert-butyl acetate (2.66 mmol) in DCM (4 mL) was added TFA (1 mL) at 0 °C. The mixture was allowed to warm to room temperature and was stirred for 8 h. Upon completion, the solvent was removed to give the corresponding carboxylic acid which was directly used in the next step. General procedure G for synthesis of Weinreb amides 29a-i To a stirred solution of appropriate carboxylic acid in DCM (20 mL) and Et3N (0.74 mL, 5.32 mmol) added EDCI (1017 mg, 5.32 mmol) and hydroxybenzotriazole (HOBt, 359 mg, 2.66 mmol) at 0 °C. The mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched with water and then diluted with DCM. The water phase was extracted with DCM. The organic layers were combined, washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography. 2-Butoxy-N-methoxy-N-methylacetamide (29a) Compound 29a was prepared from carboxylic acid 28a using a general procedure G. It was obtained as a colorless oil (79.4% yield).1H NMR (600 MHz, CDCl3) δ 4.20 (s, 2H), 3.65 (s, 3H), 3.50 (t, J = 6.7 Hz, 2H), 3.14 (s, 3H), 1.58 (dt, J = 14.5, 6.8 Hz, 2H), 1.35 (dt, J = 14.7, 7.5 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H). LCMS (ESI): m / z 176 [M+H]+. 2-(Cyclobutylmethoxy)-N-methoxy-N-methylacetamide (29b) Compound 29b was prepared from carboxylic acid 28b using a general procedure G. It was obtained as a colorless oil (31.5% yield).1H NMR (600 MHz, CDCl3) δ 4.21 (s, 2H), 3.66 (s, 3H), 3.51 (d, J = 6.9 Hz, 2H), 3.15 (s, 3H), 2.65 – 2.57 (m, 1H), 2.07 – 2.01 (m, 2H), 1.92 – 1.81 (m, 2H), 1.78 – 1.72 (m, 2H). LCMS (ESI): m / z = 188 [M+1]+. 2-Cyclobutoxy-N-methoxy-N-methylacetamide (29c) Compound 29c was prepared from carboxylic acid 28c using a general procedure G. It was obtained as a colorless oil (46% yield).1H NMR (600 MHz, CDCl3) δ 3.92 (s, 2H), 3.84 – 3.79 (m, 1H), 3.44 (s, 3H), 2.94 (s, 3H), 2.01 – 1.99 (m, 2H), 1.80 – 1.74 (m, 2H), 1.49 – 1.43 (m, 1H), 1.28 – 1.20 (m, 1H). LCMS (ESI): m / z = 174 [M+1]+. N-methoxy-N-methyl-2-(2-(1-methylcyclopropyl)ethoxy)acetamide (29d) Compound 29d was prepared from carboxylic acid 28d using a general procedure G. It was obtained as a colorless oil (86.6% yield). LCMS (ESI): m / z = 202 [M+1]+. N-methoxy-N-methyl-2-(thiophen-2-ylmethoxy)acetamide (29e) Compound 29e was prepared from carboxylic acid 28e using a general procedure G. It was obtained as a colorless oil (90.2% yield).1H NMR (600 MHz, CDCl3) δ 7.33 (dd, J = 5.1, 1.2 Hz, 1H), 7.07 (d, J = 3.0 Hz, 1H), 7.00 (dd, J = 5.0, 3.5 Hz, 1H), 4.86 (s, 2H), 4.31 (s, 2H), 3.66 (s, 3H), 3.21 (s, 3H). LCMS (ESI): m / z = 216 [M+1]+. 2-(Furan-2-ylmethoxy)-N-methoxy-N-methylacetamide (29f) Compound 29f was prepared from carboxylic acid 28f using a general procedure G. It was obtained as a colorless oil (76.3% yield).1H NMR (600 MHz, CDCl3) δ 7.43 (dd, J = 1.7, 0.7 Hz, 1H), 6.38 – 6.35 (m, 2H), 4.64 (s, 2H), 4.29 (s, 2H), 3.67 (s, 3H), 3.20 (s, 3H). LCMS (ESI): m / z = 200 [M+1]+. N-methoxy-N-methyl-2-(pyridin-4-ylmethoxy)acetamide (29g) Compound 29g was prepared from carboxylic acid 28g using a general procedure G. It was obtained as a colorless oil (31% yield).1H NMR (600 MHz, CDCl3) δ 8.59 – 8.57 (m, 2H), 7.33 (d, J = 5.9 Hz, 2H), 4.70 (s, 2H), 4.36 (s, 2H), 3.67 (s, 3H), 3.21 (s, 3H). LCMS (ESI): m / z = 211 [M+1]+. N-methoxy-N-methyl-2-(pyridin-2-ylmethoxy)acetamide (29h) Compound 29h was prepared from carboxylic acid 28h using a general procedure G. It was obtained as a colorless oil (36.5% yield).1H NMR (600 MHz, CDCl3) δ 8.56 (d, J = 5.4 Hz, 1H), 7.74 – 7.71 (m, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.21 (dd, J = 7.2, 4.7 Hz, 1H), 4.79 (s, 2H), 4.41 (s, 2H), 3.68 (s, 3H), 3.21 (s, 3H). LCMS (ESI): m / z 211 [M+1]+. N-methoxy-N-methyl-2-(thiazol-5-ylmethoxy)acetamide (29i) Compound 29i was prepared from carboxylic acid 28i using a general procedure G. It was obtained as a colorless oil (42.2% yield for 2 steps).1H NMR (600 MHz, CDCl3) δ 8.74 (s, 1H), 7.75 (s, 1H), 4.83 (s, 2H), 4.23 (s, 2H), 3.58 (s, 3H), 3.12 (s, 3H);13C NMR (150 MHz, CDCl3) δ 170.5, 154.3, 142.5, 134.8, 66.7, 64.8, 61.4, 32.3; LCMS (ESI): m / z 217 [M+1]+. General procedure H for reduction of Weinreb amides LiAlH4(116 mg, 3.05 mmol) was added in small portions to a solution of appropriate Weinreb amide (2.03 mmol) in anhydrous diethyl ether (10 mL) at 0 °C. The reaction mixture was stirred at 0 °C for 30 minutes. The reaction was quenched by addition of 1.0 M HCl at 0 °C, warmed to room temperature, and then extracted with diethyl ether. The combined organic layer was washed with brine and dried over sodium sulfate and concentrated to give the corresponding aldehyde without purification. 2-Butoxyacetaldehyde (30a) Compound 30a was prepared from Weinreb amide 29a using a general procedure H. It was obtained as a pale-yellow oil (76.2% yield).1H NMR (600 MHz, CDCl3) δ 9.67 (t, J = 0.8 Hz, 1H), 3.98 (d, J = 0.9 Hz, 2H), 3.47 (J = 6.6 Hz, 2H), 1.58 – 1.56 (m, 2H), 1.37 – 1.32 (m, 2H), 0.86 (J = 7.4 Hz, 3H).13C NMR (150 MHz, CDCl3) δ 201.2, 76.3, 71.9, 31.6, 19.1, 13.8. 2-(Cyclobutylmethoxy)acetaldehyde (30b) Compound 30b was prepared from Weinreb amide 29b using a general procedure H. It was obtained as a pale-yellow oil (84.8% yield).1H NMR (600 MHz, CDCl3) δ 9.67 (s, 1H), 3.98 (s, 2H), 3.46 (d, J = 3.7 Hz, 2H), 2.56 – 2.53 (m, 1H), 2.04 – 2.01 (m, 2H), 1.89 – 1.84 (m, 2H), 1.73 – 1.68 (m, 2H). LCMS (ESI): m / z 129 [M+H]+. 2-Cyclobutoxyacetaldehyde (30c) Compound 30c was prepared from Weinreb amide 29c using a general procedure H. It was obtained as a pale-yellow oil (74.5% yield).1H NMR (600 MHz, CDCl3) δ 9.75 (s, 1H), 4.05 – 4.01 (m, 1H), 4.00 (d, J = 0.8 Hz, 2H), 2.27 – 2.23 (m, 2H), 2.05 – 1.98 (m, 2H), 1.79 – 1.73 (m, 1H), 1.57 – 1.52 (m, 1H). 2-(2-(1-Methylcyclopropyl)ethoxy)acetaldehyde (30d) Compound 30d was prepared from Weinreb amide 29d using a general procedure H. It was obtained as a pale-yellow oil (47.3% yield). LCMS (ESI): m / z 143 [M+H]+. 2-(Thiophen-2-ylmethoxy)acetaldehyde (30e) Compound 30e was prepared from Weinreb amide 29e using a general procedure H. It was obtained as a pale-yellow oil (92.0% yield). LCMS (ESI): m / z 157 [M+H]+. 2-(Furan-2-ylmethoxy)acetaldehyde (30f) Compound 30f was prepared from Weinreb amide 29f using a general procedure H. It was obtained as a pale-yellow oil (91.0% yield). LCMS (ESI): m / z 141 [M+H]+. 2-(Pyridin-4-ylmethoxy)acetaldehyde (30g) Compound 30g was prepared from Weinreb amide 29g using a general procedure H. It was obtained as a pale-yellow oil (64.1% yield). LCMS (ESI): m / z 152 [M+H]+. 2-(Pyridin-2-ylmethoxy)acetaldehyde (30h) Compound 30h was prepared from Weinreb amide 29h using a general procedure H. It was obtained as a pale-yellow oil (36.7% yield). LCMS (ESI): m / z 152 [M+H]+. 2-(Thiazol-5-ylmethoxy)acetaldehyde (30i) Compound 30i was prepared from Weinreb amide 29i using a general procedure H. It was obtained as a pale-yellow oil (64.0% yield).1H NMR (600 MHz, CDCl3) δ 9.64 (s, 1H), 8.77 (s, 1H), 7.76 (s, 1H), 4.80 (s, 2H), 4.07 (s, 2H). LCMS (ESI): m / z 158 [M+H]+. 5-(2-Methoxyvinyl)thiazole (45) To a suspension of methoxymethyltriphenylphosphonium chloride (2.06 g, 6.0 mmol) in anhydrous THF (4.5 mL) cooled to 0 °C, t-BuOK (0.73 g, 6.5 mmol) was added, and the resulting orange solution was stirred at 0 °C for 30 min. A solution of thiazole-5-carbaldehyde (40) (0.56 g, 5.0 mmol) in THF (0.5 mL) was added dropwise, and the resulting mixture was warmed to room temperature and stirred at the same temperature for 6 h. Thereafter the reaction was quenched with water and the aqueous phase was extracted with DCM and the collected organic layers were dried over sodium sulfate. The solvent was evaporated, and the residue was purified by flash column chromatography (0~30% ethyl acetate in hexane) to give the enol ether (333.2 mg, 47.1% yield) as a yellow oil. LCMS (ESI): m / z 142 [M+H]+. The ration of E / Z isomer is 6.67:1. E isomer:1H NMR (600 MHz, CDCl3) δ 8.35 (s, 1H), 7.45 (s, 1H), 6.81 (d, J = 12.8 Hz, 1H), 5.80 (d, J = 12.8 Hz, 1H), 3.69 (s, 1H), 3.54 (s, 3H). 2-(Thiazol-5-yl)acetaldehyde (46) 4 M aqueous hydrogen chloride (1.93 mL) was added dropwise to a solution of 5-(2- methoxyvinyl)thiazole (300 mg, 2.12 mmol) in THF (5 mL) at 0 °C. The resulting solution was then stirred for 3.5 h at 60 °C under an Argon atmosphere. Saturated NaHCO3aqueous solution was added, and the aqueous phase was extracted with DCM, the collected organic layers were dried over sodium sulfate, and the solvent was evaporated. The residue was purified by flash column chromatography (0~82% ethyl acetate in hexane) to give the desired compound (148.0 mg, 54.9% yield).1H NMR (600 MHz, CDCl3) δ 9.71 (s, 1H), 8.72 (s, 1H), 7.68 (s, 1H), 3.92 (s, 2H). LCMS (ESI): m / z 128 [M+1]+. N-(2,2-dimethoxyethyl)acetamide (48a) Acetic anhydride (1.23 g, 12 mmol) and Et3N (1.66 mL, 12 mmol) were added to a solution of 2,2-dimethoxyethanamine (1.05 g, 10 mmol) in dry DCM (16 mL), and the resulting mixture was stirred at room temperature for 2 h. The reaction was quenched with NaHCO3aqueous solution, and the reaction mixture was extracted with DCM. The combined organic phases were washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to afford the desired compound (0.91 g, 62% yield) as a colorless oil without purification.1H NMR (600 MHz, CDCl3) δ 5.64 (s, 1H), 4.31 (t, J = 5.2 Hz, 1H), 3.33 (d, J = 2.3 Hz, 8H), 1.92 (s, 3H). N-(2,2-dimethoxyethyl)-2,2,2-trifluoroacetamide (48b) The title compound 48b was synthesized from 2,2,2-trifluoroacetic anhydride according to the method described for preparation of compound 48a. It was obtained as a colorless oil (89.6% yield).1H NMR (600 MHz, CDCl3) δ 6.57 (s, 1H), 4.45 (td, J = 5.0, 1.5 Hz, 1H), 3.51 (t, J = 5.1 Hz, 2H), 3.44 (s, 6H).13C NMR (150 MHz, CDCl3) δ 157.3 (q, J = 37.5 Hz), 115.8 (q, J = 286.5 Hz), 101.7, 54.7, 41.3. N-(2,2-dimethoxyethyl)-3,3,3-trifluoropropanamide (48c) To a solution of 2,2-dimethoxyethanamine (0.653 g, 6.21 mmol) and Et3N (1.72 mL, 12.42 mmol) in DCM (10 mL), 3,3-trifluoropropanoyl chloride (1.0 g, 6.83 mmol) was added at 0 °C. After stirring for 10 min, the reaction was quenched with saturated aqueous NH4Cl, and the mixture was extracted with DCM. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (35% ethyl acetate in hexane) to afford the desired compound (810 mg, 60.6% yield) as a yellow oil.1H NMR (600 MHz, CDCl3) δ 5.90 (s, 1H), 4.32 (t, J = 5.1 Hz, 1H), 3.38 (d, J = 5.4 Hz, 2H), 3.33 (s, 6H), 3.01 (q, J = 10.6 Hz, 2H).13C NMR (150 MHz, CDCl3) δ 162.7, 124.0 (q, J = 276.0 Hz), 102.3, 54.6, 41.6 (q, J = 30.0 Hz). N-(2,2-dimethoxyethyl)nicotinamide (48d) The title compound 48d was synthesized by following the method described procedure A. Nicotinic acid (387.4 mg, 3 mmol), 2,2-dimethoxyethanamine (412.3 mg, 3.6 mmol), DMAP (73.3 mg, 0.60 mmol), EDCI (745.3 mg, 3.9 mmol), and DCM (10 mL) were used in this condensation reaction. Compound 48d (583 mg, 92.5% yield) was obtained as a colorless oil.1H NMR (600 MHz, CDCl3) δ 9.26 (d, J = 1.6 Hz, 1H), 8.76 (dd, J = 5.0, 1.6 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 7.55 (dd, J = 7.9, 5.0 Hz, 1H), 7.04 (s, 1H), 4.58 (t, J = 5.2 Hz, 1H), 3.66 (t, J = 5.5 Hz, 2H), 3.46 (s, 6H).13C NMR (150 MHz, CDCl3) δ 164.7, 146.4, 124.2, 102.3, 54.4, 41.6. LCMS (ESI): m / z 211 [M+H]+. N-(2,2-dimethoxyethyl)isonicotinamide (48e) The title compound 48e was synthesized from isonicotinic acid according to the method described for preparation of compound 48d. It was obtained as a colorless oil (68.5% yield).1H NMR (600 MHz, CDCl3) δ 8.82 (d, J = 6.1 Hz, 2H), 7.90 (d, J = 6.1 Hz, 2H), 6.98 (s, 1H), 4.55 (t, J = 5.1 Hz, 1H), 3.65 (t, J = 5.4 Hz, 2H), 3.46 (s, 6H).13C NMR (150 MHz, CDCl3) δ 164.6, 147.9, 144.1, 122.3, 102.3, 54.6, 41.7. LCMS (ESI): m / z 211 [M+H]+. N-(2,2-dimethoxyethyl)-2-fluoroisonicotinamide (48f) The title compound 48f was synthesized from 2-fluoroisonicotinic acid according to the method described for preparation of compound 48d. It was obtained as a colorless oil (86.9% yield).1H NMR (600 MHz, CDCl3) δ 8.36 (d, J = 5.1 Hz, 1H), 7.51 (dt, J = 5.1, 1.5 Hz, 1H), 7.30 (s, 1H), 6.46 (s, 1H), 4.51 (t, J = 5.0 Hz, 1H), 3.63 (t, J = 5.0 Hz, 2H), 3.46 (s, 6H).13C NMR (150 MHz, CDCl3) δ 164.3 (d, J = 3.0 Hz), 164.2 (d, J = 238.5 Hz), 148.7 (d, J = 13.5 Hz), 147.1 (d, J = 6.0 Hz), 118.7, 107.8 (d, J = 39.0 Hz), 102.4, 54.7, 41.7. LCMS (ESI): m / z 229 [M+H]+. N-(2,2-dimethoxyethyl)-4-fluorobenzamide (48g) The title compound 48g was synthesized from 4-fluorobenzoic acid according to the method described for preparation of compound 48d. It was obtained as a white solid (83.6% yield).1H NMR (600 MHz, CDCl3) δ 7.82 – 7.79 (m, 2H), 7.14 – 7.10 (m, 2H), 6.34 (s, 1H), 4.50 (t, J = 5.2 Hz, 1H), 3.61 (t, J = 5.5 Hz, 2H), 3.45 (s, 6H).13C NMR (150 MHz, CDCl3) δ 166.5, 164.8 (d, J = 250.5 Hz), 129.3 (d, J = 7.5 Hz), 115.6 (d, J = 22.5 Hz), 102.7, 54.6, 41.6. LCMS (ESI): m / z 228 [M+H]+. N-(2,2-dimethoxyethyl)furan-2-carboxamide (48h) The title compound 48h was synthesized from furan-2-carboxylic acid according to the method described for preparation of compound 48d. It was obtained as a colorless oil (89.0% yield).1H NMR (600 MHz, CDCl3) δ 7.46 (s, 1H), 7.13 (d, J = 3.9 Hz, 1H), 6.51 (dd, J = 3.5, 1.7 Hz, 1H), 4.48 (t, J = 5.3 Hz, 1H), 3.59 (t, J = 5.6 Hz, 2H), 3.45 (s, 6H).13C NMR (150 MHz, CDCl3) δ 158.4, 147.9, 143.9, 114.3, 112.1, 102.7, 54.5, 40.6. LCMS (ESI): m / z 200 [M+H]+. N-(2,2-dimethoxyethyl)oxazole-5-carboxamide (48i) The title compound 48i was synthesized from oxazole-5-carboxylic acid according to the method described for preparation of compound 48d. It was obtained as a colorless oil (78% yield).1H NMR (600 MHz, CDCl3) δ 7.92 (s, 1H), 7.74 (s, 1H), 4.48 (t, J = 5.1 Hz, 1H), 3.60 (t, J = 5.2 Hz, 2H), 3.45 (s, 6H).13C NMR (150 MHz, CDCl3) δ 156.9, 151.3, 145.4, 130.4, 102.5, 54.6, 40.7. LCMS (ESI): m / z 201 [M+H]+. N-(2,2-dimethoxyethyl)thiophene-2-carboxamide (48j) The title compound 48j was synthesized from thiophene-2-carboxylic acid according to the method described for preparation of compound 48d. It was obtained as a colorless oil (87% yield).1H NMR (600 MHz, CDCl3) δ 7.52 (d, J = 3.7 Hz, 1H), 7.48 (d, J = 5.0 Hz, 1H), 7.07 (t, J = 4.3 Hz, 1H), 6.32 (s, 1H), 4.49 (t, J = 5.2 Hz, 1H), 3.58 (t, J = 5.6 Hz, 2H), 3.43 (s, 6H); LCMS (ESI): m / z 216 [M+H]+. N-(2,2-dimethoxyethyl)thiazole-5-carboxamide (48k) The title compound 48k was synthesized from thiazole-5-carboxylic acid according to the method described for preparation of compound 48d. It was obtained as a colorless oil (74% yield).1H NMR (600 MHz, CDCl3) δ 8.93 (s, 1H), 8.31 (s, 1H), 6.34 (s, 1H), 4.51 (t, J = 5.1 Hz, 1H), 3.62 (d, J = 5.6 Hz, 2H), 3.46 (s, 6H). LCMS (ESI): m / z 217 [M+1]+. N-(2,2-dimethoxyethyl)thiazole-4-carboxamide (48l) The title compound 48l was synthesized from thiazole-4-carboxylic acid according to the method described for preparation of compound 48d. It was obtained as a colorless oil (94.8% yield).1H NMR (600 MHz, CDCl3) δ 8.77 (d, J = 2.1 Hz, 1H), 8.18 (d, J = 2.1 Hz, 1H), 7.56 (s, 1H), 4.51 (t, J = 5.3 Hz, 1H), 3.64 – 3.62 (m, 2H), 3.45 (s, 6H).13C NMR (150 MHz, CDCl3) δ 161.0, 152.7, 151.0, 123.2, 102.7, 54.4, 40.9. LCMS (ESI): m / z 217 [M+H]+. N-(2,2-dimethoxyethyl)thiazole-2-carboxamide (48m) The title compound 48m was synthesized from thiazole-2-carboxylic acid according to the method described for preparation of compound 48d. It was obtained as a colorless oil (75.9% yield).1H NMR (600 MHz, CDCl3) δ 7.89 (d, J = 3.1 Hz, 1H), 7.59 (d, J = 3.1 Hz, 1H), 7.48 (s, 1H), 4.52 (t, J = 5.3 Hz, 1H), 3.63 (t, J = 5.3 Hz, 2H), 3.45 (s, 6H).13C NMR (150 MHz, CDCl3) δ 163.6, 159.6, 143.5, 124.5, 102.5, 54.4, 41.1. LCMS (ESI): m / z 217 [M+H]+. General procedure I for synthesis of aldehydes 49a-m To a solution of relevant N-(2,2-dimethoxyethyl) carboxamide (1.91 mol) in acetone / water (4.8 mL, 1:1) was added Amberlyst-15 resin (683 mg). The mixture was stirred overnight at room temperature. The suspension was filtered through celite. The filtrate was dried over anhydrous sodium sulfate, filtered, and evaporated to yield the target compound as a colorless oil, which was then directly used in the next step. Tert-butyl (3-(2-(((7-chloro-3-(3-cyanophenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)propyl)carbamate (67) A stirred solution of compound 32 (133 mg, 0.215 mmol) in DCM (3.0 mL) was treated with TFA (1 mL) at 0 °C. The reaction mixture was then warmed to room temperature and stirred for 1 h. After solvent removal and concentration under vacuum, the crude product was directly used for the next step. In a separate reaction, the N-Boc deprotected product (0.215 mmol) and K2CO3(44.6 mg, 0.323 mmol) in DMF (2.5 mL) were mixed with Et3N (59.6 µL, 0.24 mmol) and stirred for 30 minutes. Then, a solution of tert-butyl (2-bromoethyl)carbamate (144.5 mg, 0.645 mmol) was added. The resulting mixture was stirred at 60 °C for 16 h. Upon completion, the reaction was quenched with water and extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, filtered, and evaporated under vacuum. The residue was purified by flash column chromatography (0~11.5% MeOH in DCM) to afford the desired compound 67 (108.5 mg, 76.4% yield for two steps) as a white solid.1H NMR (600 MHz, CD3OD) δ 7.96 (d, J = 8.4 Hz, 2H), 7.85 (s, 1H), 7.79 – 7.75 (m, 1H), 7.63 (s, 2H), 7.20 (t, J = 8.0 Hz, 1H), 5.74 – 5.68 (m, 1H), 4.63 (s, 2H), 4.16 – 4.12 (m, 2H), 3.71 – 3.66 (m, 2H), 3.38 – 3.35 (m, 2H), 3.24 – 3.19 (m, 2H), 3.14 – 3.09 (m, 1H), 2.53 – 2.42 (m, 4H), 2.21 – 2.13 (m, 4H), 2.08 – 2.00 (m, 4H), 1.74 (t, J = 5.5 Hz, 2H), 1.65 (t, J = 5.5 Hz, 2H), 1.45 (s, 9H). LCMS (m / z): 660 [M+H]+. Tert-butyl (2-(2-(((3-(3-carbamothioylphenyl)-7-chloro-1-(tetrahydro-2H-pyran-4-yl)-1H- indol-6-yl)methyl)carbamoyl)-7-azaspiro[3.5]nonan-7-yl)ethyl)carbamate (68) The title compound 68 was synthesized according to the modified method described for procedure B. Compound 67 (66 mg, 0.1 mmol), sodium hydrosulfide hydrate (444.5 mg, 6.0 mmol), magnesium chloride (476.1 mg, 5.0 mmol), and DMF (3.0 mL) were used. The target compound was obtained as a yellow solid (101.6 mg, 97.6% yield).1H NMR (600 MHz, CD3OD) δ 8.19 (s, 1H), 7.84 – 7.75 (m, 4H), 7.50 – 7.46 (m, 1H), 7.19 – 7.17 (m, 1H), 5.74 – 5.68 (m, 1H), 4.63 (s, 2H), 4.53 (s, 2H), 4.16 – 4.12 (m, 2H), 3.71 – 3.65 (m, 2H), 3.28 – 3.23 (m, 2H), 3.14 – 3.09 (m, 1H), 2.65 – 2.53 (m, 4H), 2.20 – 2.12 (m, 4H), 2.09 – 2.01 (m, 4H), 1.77 (t, J = 5.4 Hz, 2H), 1.70 (t, J = 5.4 Hz, 2H), 1.45 (s, 9H). LCMS (m / z): 694 [M+H]+. 3-Bromo-N-(4-fluorophenyl)propanamide (70) To a solution of 4-fluoroaniline (0.67 g, 6.0 mmol) and Et3N (1.72 mL, 12.42 mmol) in THF (20 mL) was added 3-bromopropanoyl chloride (1.03 g, 6.0 mmol) at 0 °C. After completion of the reaction, it was quenched with saturated NH4Cl aqueous solution. The resulting mixture was then extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (0~35% ethyl acetate in hexane) to afford compound 70 (337 mg, 22.8% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 7.51 (dd, J = 8.9, 4.7 Hz, 2H), 7.31 (s, 1H), 7.05 (t, J = 8.6 Hz, 2H), 3.73 (t, J = 6.5 Hz, 2H), 2.96 (t, J = 6.5 Hz, 2H). LCMS (ESI): m / z 246, 248 [M+H]+. 3-Bromo-1H-indole-6-carbaldehyde (80) To a mixture of 1H-indole-6-carbaldehyde (5 g, 34.4 mmol) in DCM (300 mL) was added NBS (6.15 g, 34.6 mmol) in portion. The reaction mixture was stirred at room temperature for 5 h. Water was then added to quench the reaction. The DCM phase was separated and dried over anhydrous sodium sulfate. After filtration and concentration, the crude product was obtained (6.35 g, 82.4% yield) as a purple solid. 3-(6-Formyl-1H-indol-3-yl)benzonitrile (81) The mixture of compound 80 (2.5 g, 11.16 mmol), (3-cyanophenyl)boronic acid (3.3 g, 22.430 mmol), tris(dibenzylideneacetone) dipalladium (0) (1.54 g, 1.68 mmol), tri-tert- butylphosphonium tetrafluoroborate (0.976 g, 3.36 mmol) and anhydrous KF (2.61 g, 44.92 mmol) in anhydrous THF (100 mL) was stirred at 40 °C overnight under an argon atmosphere. The mixture was cooled down to room temperature, filtered through celite and washed by ethyl acetate. The filtrate was concentrated in vacuo, and the resulting residue was purified by flash column chromatography (0~50% ethyl acetate in hexane) to yield the title compound (1.38 g, 50.2% yield) as a yellow solid.1H NMR (400 MHz, CD3OD) δ 10.0 (s, 1H), 8.02 (m, 4H), 7.91 (m, 1H), 7.72 (dd, J = 8.0, 1.3 Hz, 1H), 7.62 (m, 2H). 3-(6-Formyl-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)benzonitrile (71a) The title compound 71a was synthesized from compound 81 according to the method described for preparation of compound 16. It was obtained as a white solid (94.2% yield).1H NMR (600 MHz, CDCl3) δ 10.04 (s, 1H), 7.97 (s, 1H), 7.89 (d, J = 8.3 Hz, 1H), 7.83 (s, 1H), 7.79 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 9.2 Hz, 1H), 7.56 (s, 1H), 7.52 – 7.47 (m, 2H), 4.61 – 4.55 (m, 1H), 4.15 – 4.11 (m, 2H), 3.61 (t, J = 12.8 Hz, 2H), 2.13 – 2.05 (m, 4H).13C NMR (150 MHz, CDCl3) δ 192.2, 136.1, 136.0, 131.6, 131.3, 130.7, 130.6, 129.8, 126.6, 122.7, 119.9, 118.9, 116.1, 113.2, 111.6, 67.3, 52.6, 33.6. LCMS (ESI): m / z 331 [M+H]+. 3-(6-(Hydroxymethyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)benzonitrile (72a) A mixture of aldehyde 71a (610 mg, 1.85 mmol) and sodium borohydride (349.9 mg, 9.25mmol) in MeOH (24 mL) and MeCN (24 mL) was stirred at room temperature for 3 h. Upon completion, saturated NH4Cl aqueous solution was added to quench the reaction. The solvent was then removed under reduced pressure. The residue was diluted with water and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography (0~63% ethyl acetate in hexane) to provide the title compound (323 mg, 52.5% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 7.94 (s, 1H), 7.91 – 7.88 (m, 2H), 7.57 – 7.54 (m, 3H), 7.46 (s, 1H), 7.29 (s, 1H), 7.24 (d, J = 8.2 Hz, 1H), 4.89 (s, 2H), 4.60 – 4.55 (m, 1H), 4.24 – 4.20 (m, 2H), 3.70 – 3.66 (m, 2H), 2.22 – 2.14 (m, 4H).13C NMR (150 MHz, CDCl3) δ 136.9, 136.6, 135.6, 131.3, 130.5, 129.6, 129.1, 125.4, 122.7, 120.3, 119.8, 119.1, 115.3, 113.0, 108.3, 67.4, 65.9, 52.6, 33.5. LCMS (ESI): m / z 315 [M+H-18]+. 3-(6-(Azidomethyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)benzonitrile (73a) The title compound 73a was synthesized from 72a according to the method described for preparation of compound 18. It was obtained as a white solid (85.1% yield).1H NMR (600 MHz, CDCl3) δ 7.94 (s, 1H), 7.92 – 7.88 (m, 2H), 7.56 (d, J = 7.5 Hz, 2H), 7.49 (s, 1H), 7.45 (s, 1H), 7.21 (d, J = 9.2 Hz, 1H), 4.60 – 4.54 (m, 1H), 4.54 (s, 2H), 4.25 – 4.21 (m, 2H), 3.70 (td, J = 11.7, 2.4 Hz, 2H), 2.22 – 2.14 (m, 4H).13C NMR (150 MHz, CDCl3) δ 136.8, 136.4, 131.4, 130.5, 129.8, 129.6, 129.3, 125.8, 123.1, 121.3, 120.1, 119.1, 115.4, 113.0, 109.7, 67.4, 55.5, 52.7, 33.4. 3-(6-(Aminomethyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)benzonitrile (74a) The title compound 74a was synthesized from 73a according to the method described for preparation of compound 19. It was obtained as a white solid (82.5% yield).1H NMR (600 MHz, CDCl3) δ 8.77 (s, 2H), 7.80 (s, 1H), 7.77 (s, 2H), 7.52 (s, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 6.1 Hz, 1H), 7.40 (s, 1H), 7.18 (d, J = 8.2 Hz, 1H), 4.50 – 4.44 (m, 1H), 4.21 (s, 2H), 4.05 – 4.02 (m, 2H), 3.55 (t, J = 10.7 Hz, 2H), 2.22 – 2.14 (m, 2H), 2.05 – 2.00 (m, 2H). LCMS (ESI): m / z 315 [M-17+H]+. Tert-butyl 2-(((3-(3-cyanophenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6-yl) methyl)carbamoyl)-7-azaspiro[3.5]nonane-7-carboxylate (75a) The title compound 75a was synthesized from 74a according to the method described for general procedure A. It was obtained as a white solid (97.6% yield).1H NMR (600 MHz, CDCl3) δ 7.81 (s, 1H), 7.78 – 7.73 (m, 2H), 7.47 – 7.43 (m, 2H), 7.35 (s, 1H), 7.32 (s, 1H), 7.06 (d, J = 8.2 Hz, 1H), 5.71 (s, 1H), 4.51 (d, J = 4.4 Hz, 2H), 4.45 – 4.41 (m, 1H), 4.13 – 4.09 (m, 2H), 3.58 (t, J = 11.6 Hz, 2H), 3.28 – 3.25 (m, 2H), 3.22 – 3.18 (m, 2H), 2.90 (q, J = 8.5 Hz, 1H), 2.09 – 2.00 (m, 6H), 1.96 – 1.92 (m, 2H), 1.50 –1.46 (m, 4H), 1.37 (s, 9H). LCMS (ESI): m / z 583 [M+H]+. Tert-butyl 2-(((3-(3-carbamothioylphenyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonane-7-carboxylate (76a) The title compound 76a was synthesized from 75a according to the method described for general procedure B. It was obtained as a light yellow solid (94.4% yield).1H NMR (600 MHz, CD3OD) δ 8.24 (s, 1H), 7.90 (d, J = 7.7 Hz, 1H), 7.84 – 7.80 (m, 1H), 7.70 – 7.30 (m, 1H), 7.71 (s, 1H), 7.50 (s, 1H), 7.48 – 7.45 (m, 1H), 7.14 – 7.11 (m, 1H), 4.68 – 4.63 (m, 1H), 4.52 (s, 2H), 4.17 – 4.13 (m, 2H), 3.72 (t, J = 11.6 Hz, 2H), 3.41 – 3.37 (m, 2H), 3.32 – 3.28 (m, 2H), 3.16 – 3.10 (m, 1H), 2.23 – 2.17 (m, 2H), 2.10 – 2.05 (m, 6H), 1.64 – 1.60 (m, 2H), 1.56 – 1.52 (m, 2H), 1.49 – 1.43 (m, 9H). LCMS (ESI): m / z 617 [M+H]+. 3-Bromo-1-(tetrahydro-2H-pyran-4-yl)-1H-indole-6-carbaldehyde (82) The title compound 82 was synthesized according to the method described for preparation of compound 16. It was obtained as a white solid (70.7% yield).1H NMR (600 MHz, CDCl3) δ 10.02 (d, J = 2.3 Hz, 1H), 7.90 (s, 1H), 7.63 – 7.61 (m, 1H), 7.38 (d, J = 2.2 Hz, 1H), 7.18 (d, J = 2.2 Hz, 1H), 4.54 – 4.48 (m, 1H), 4.12 – 4.08 (m, 2H), 3.58 – 3.54 (m, 2H), 2.03 – 1.97 (m, 4H).13C NMR (150 MHz, CDCl3) δ 134.8, 132.1, 131.5, 127.6, 122.2, 111.4, 91.1, 67.2, 53.2, 33.5. LCMS (ESI): m / z 308, 310 [M+H]+. 1-(Tetrahydro-2H-pyran-4-yl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-6- carbaldehyde (83) The mixture of 3-bromo-1-(tetrahydro-2H-pyran-4-yl)-1H-indole-6-carbaldehyde (353.0 mg, 1.15 mmol), KOAc (338.6 mg, 3.45 mmol), Bis(pinacolato)diboron (B2pin2) (380.9 mg, 1.50 mmol) and Pd(dppf)Cl2dichloromethane complex (98.0 mg, 0.12 mmol) in 1,4-dioxane (10 mL) was heated to 90 °C for 5.5 h under an argon atmosphere. After cooling to rt, the reaction mixture was diluted with ethyl acetate and filtered through Celite, and the filter cake was washed with ethyl acetate. The resulting filtration was concentrated to dryness. The residue was purified by flash column chromatography (0~27% ethyl acetatec in hexanes) to give the desired compound 83 (184.4 mg, 45.1% yield) as a colorless oil.1H NMR (600 MHz, CDCl3) δ 10.01 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 7.89 (s, 1H), 7.81 (s, 1H), 7.64 – 7.60 (m, 1H), 4.53 – 4.46 (m, 1H), 4.13 – 4.08 (m, 2H), 3.57 (t, J = 11.5 Hz, 2H), 2.08 – 1.96 (m, 4H), 1.47 (s, 3H), 1.31 (s, 6H), 1.20 (s, 3H). LCMS (ESI): m / z 356 [M+H]+. 6-(6-Formyl-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)picolinonitrile (71b) The mixture of compound 83 (252.9 mg, 0.71 mmol), Na2CO3(150.5 mg, 1.42 mmol), Pd(PPh3)4(82 mg, 0.071 mmol) and 6-bromopicolinonitrile (195.8 mg, 1.07 mmol) in 1.4- dioxane / H2O (8 mL / 0.8 mL) was heated to 90 °C for 3 h under an argon atmosphere. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and filtered, and the filter cake was washed with ethyl acetate. The resulting filtration was concentrated to give a residue. The residue was purified by flash column chromatography (0~55% ethyl acetate in hexane) to give the title compound 71b (84 mg, 35.7% yield) as a green solid. LCMS (ESI): m / z 332 [M+H]+. 6-(6-(Hydroxymethyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)picolinonitrile (72b) The title compound 72b was synthesized from 71b according to the method described for preparation of compound 72a. It was obtained as a white solid (64% yield).1H NMR (600 MHz, CDCl3) δ 8.30 (d, J = 8.2 Hz, 1H), 7.84 (s, 1H), 7.78 (s, 1H), 7.71 (t, J = 7.7 Hz, 1H), 7.44 (s, 1H), 7.39 (d, J = 7.4 Hz, 1H), 7.20 (s, 1H), 4.80 (s, 2H), 4.50 – 4.46 (m, 1H), 4.15 – 4.11 (m, 2H), 3.59 (t, J = 11.8 Hz, 2H), 2.14 – 2.04 (m, 4H). LCMS (ESI): m / z 334 [M+H]+. 6-(6-(Azidomethyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)picolinonitrile (73b) The title compound 73b was synthesized from 72b according to the method described for preparation of compound 18. It was obtained as a white solid (58.5% yield).1H NMR (600 MHz, CDCl3) δ 8.35 (d, J = 8.2 Hz, 1H), 7.85 (s, 1H), 7.78 (d, J = 7.6 Hz, 1H), 7.73 – 7.69 (m, 1H), 7.40 (d, J = 7.4 Hz, 1H), 7.34 (s, 1H), 7.16 (d, J = 8.8 Hz, 1H), 4.50 – 4.46 (m, 1H), 4.45 (s, 2H), 4.16 – 4.12 (m, 2H), 3.63 – 3.58 (m, 2H), 2.15 – 2.05 (m, 4H). LCMS (ESI): m / z 359 [M+H]+. 6-(6-(Aminomethyl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-3-yl)picolinonitrile (74b) The title compound 74b was synthesized from 73b according to the method described for preparation of compound 19. It was obtained as a colorless oil (58.1% yield).1H NMR (600 MHz, CD3OD) δ 8.44 (d, J = 7.6 Hz, 1H), 8.18 (s, 1H), 8.06 (d, J = 7.1 Hz, 1H), 7.88 (t, J = 11.5 Hz, 1H), 7.60 (s, 1H), 7.56 (d, J = 5.7 Hz, 1H), 7.25 (d, J = 7.3 Hz, 1H), 4.74 – 4.69 (m, 1H), 4.19 – 4.14 (m, 2H), 4.00 (s, 2H), 3.74 (t, J = 11.7 Hz, 2H), 2.24 – 2.18 (m, 2H), 2.14 – 2.09 (m, 2H). LCMS (ESI): m / z 316 [M-17+H]+. Tert-butyl 2-(((3-(6-cyanopyridin-2-yl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonane-7-carboxylate (75b) The title compound 75b was synthesized from 74b according to the method described for general procedure A. It was obtained as a white solid (86.8% yield).1H NMR (600 MHz, CDCl3) δ 8.39 (d, J = 6.9 Hz, 1H), 7.94 (s, 1H), 7.88 – 7.85 (m, 1H), 7.80 (t, J = 7.0 Hz, 1H), 7.48 (t, J = 6.9 Hz, 1H), 7.41 (s, 1H), 7.22 (d, J = 7.0 Hz, 1H), 5.76 (s, 1H), 4.61 (s, 2H), 4.57 – 4.52 (m, 1H), 4.24 – 4.20 (m, 2H), 3.68 (t, J = 11.5 Hz, 2H), 3.39 – 3.36 (m, 2H), 3.33 – 3.29 (m, 2H), 3.00 (q, J = 10.3, 7.6 Hz, 1H), 2.22 – 2.17 (m, 2H), 2.15 – 2.10 (m, 4H), 2.08 – 2.03 (m, 2H), 1.62 – 1.57 (m, 4H), 1.47 (s, 9H). LCMS (ESI): m / z 584 [M+1]+. Tert-butyl 2-(((3-(6-carbamothioylpyridin-2-yl)-1-(tetrahydro-2H-pyran-4-yl)-1H-indol-6- yl)methyl)carbamoyl)-7-azaspiro[3.5]nonane-7-carboxylate (76b) The title compound 76b was synthesized from 75b according to the method described for general procedure B. It was obtained as a light yellow solid (93% yield).1H NMR (600 MHz, CD3OD) δ 8.42 (d, J = 7.1 Hz, 1H), 8.25 (d, J = 10.1 Hz, 2H), 7.97 (d, J = 7.1 Hz, 1H), 7.88 (d, J = 6.8 Hz, 1H), 7.53 (s, 1H), 7.19 (d, J = 7.4 Hz, 1H), 4.71 – 4.67 (m, 1H), 4.54 (s, 2H), 4.19 – 4.15 (m, 2H), 3.73 (t, J = 12.0 Hz, 2H), 3.40 – 3.37 (m, 2H), 3.30 – 3.21 (m, 2H), 3.16 – 3.12 (m, 1H), 2.28 – 2.22 (m, 2H), 2.12 – 2.06 (m, 6H), 1.65 – 1.62 (m, 2H), 1.57 – 1.53 (m, 2H), 1.46 (s, 9H). LCMS (ESI): m / z 618 [M+H]+. Methyl 7-chloro-3-(3-cyanophenyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indole-6- carboxylate (85) The title compound 85 was synthesized from methanesulfonate 84 according to the method described for the preparation of compound 16. It was obtained as a white solid (22.1% yield).1H NMR (600 MHz, CDCl3) δ 7.76 (t, J = 1.4 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.65 (d, J = 8.3 Hz, 1H), 7.54 – 7.46 (m, 4H), 5.59 – 5.53 (m, 1H), 4.00 – 3.96 (m, 2H), 3.89 (s, 3H), 2.88 (t, J = 11.6 Hz, 2H), 2.80 (s, 3H), 2.32 – 2.28 (m, 2H), 2.10 – 2.03 (m, 2H). LCMS (ESI): m / z 472 [M+H]+. 3-(7-Chloro-6-(hydroxymethyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-3-yl) Benzonitrile (86) The title compound 86 was synthesized from compound 85 according to the method described for the preparation of compound 17. It was obtained as a white solid (74% yield).1H NMR (600 MHz, CDCl3) δ 7.85 (s, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.58 – 7.53 (m, 2H), 7.44 (s, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.30 – 7.28 (m, 1H), 5.57 – 5.53 (m, 1H), 4.91 (s, 2H), 4.05 – 4.01 (m, 2H), 2.95 (t, J = 12.0 Hz, 2H), 2.88 (s, 3H), 2.63 – 2.59 (m, 2H), 2.15 – 2.10 (m, 2H). LCMS (ESI): m / z: 426 [M+H-18]+. 3-(6-(Azidomethyl)-7-chloro-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-3- yl)benzonitrile (87) The title compound 87 was synthesized from compound 86 according to the method described for the preparation of compound 18. It was obtained as a white solid (74% yield). 3-(6-(Aminomethyl)-7-chloro-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-3-yl) benzonitrile (88) The title compound 88 was synthesized from compound 87 according to the method described for the preparation of compound 19. It was obtained as a white solid (80% yield).1H NMR (600 MHz, CD3OD) δ 7.99 – 7.95 (m, 2H), 7.87 – 7.84 (m, 1H), 7.80 (t, J = 7.4 Hz, 1H), 7.64 (s, 2H), 7.31 – 7.28 (m, 1H), 5.66 – 5.62 (m, 1H), 4.09 (s, 2H), 4.00 – 3.96 (m, 2H), 3.10 – 3.05 (m, 2H), 2.95 (s, 3H), 2.36 – 2.32 (m, 2H), 2.24 – 2.18 (m, 2H). LCMS (ESI): m / z: 426 [M+H-17]+. Tert-butyl (7-(((7-chloro-3-(3-cyanophenyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol- 6-yl)methyl)amino)-7-oxoheptyl)carbamate (89) A mixture of 6-((tert-butoxycarbonyl)amino)hexanoic acid (28 mg, 0.12 mmol, 2 eq.) HATU (45 mg, 0.12 mmol, 2 eq.), primary amine 88 (25 mg, 0.056 mmol, 1 eq), and DIPEA (0.03 mL, 0.18 mmol, 3 eq.) in DMF (2 mL) was stirred overnight at room temperature at under an argon atmosphere. Upon completion, water was added to the reaction mixture and extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by flash column chromatography to afford the desired compound 89 (35 mg, 95% yield) as a white solid.1H NMR (600 MHz, CDCl3) δ 7.82 (s, 1H), 7.77 (d, J = 7.5 Hz, 1H), 7.69 (d, J = 8.2 Hz, 1H), 7.56 (dt, J = 15.2, 7.6 Hz, 2H), 7.42 (s, 1H), 7.23 (d, J = 8.2 Hz, 1H), 5.85 (s, 1H), 5.61 – 5.46 (m, 1H), 4.68 (d, J = 5.6 Hz, 2H), 4.54 (brs, 1H), 4.05 (d, J = 12.6 Hz, 2H), 3.09 (m, 2H), 3.01-2.82 (m, 2H), 2.88 (s, 3H), 2.35 (d, J = 12.5 Hz, 2H), 2.26 – 2.05 (m, 4H), 1.73 – 1.66 (m, 2H), 1.56 – 1.29 (m, 13H). LCMS (ESI): m / z: 556 [M-Boc+H]+. 6-Amino-N-((7-chloro-3-(3-cyanophenyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-6- yl)methyl)hexanamide trifluoroacetate (90) The title compound 90 was synthesized from compound 89 (35 mg, 0.053 mmol) according to the method described for general procedure C. LCMS (ESI): m / z: 556 [M+H]+. N-(6-(((7-chloro-3-(3-cyanophenyl)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol-6- yl)methyl)amino)-6-oxohexyl)-3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'- xanthene]-5-carboxamide (91) To a stirred solution of 5-carboxyfluorescein (65 mg, 0.173 mmol) in DMF (1 mL) were added NHS (26 mg, 0.225 mmol, 1.3 eq.) and DCC (46 mg, 0.223 mmol, 1.3 eq.) under an argon atmosphere. The reaction mixture was stirred for 3 h at room temperature. 0.5 mL of supernatant from in situ prepared activated 5-carboxyfluorescein (0.086 mmol) was added to a stirred solution of 90 (TFA complex) in DMF (0.5 mL). The reaction mixture was stirred overnight at room temperature. Upon completion, the resulting solution was purified by reverse-phase flash chromatography and then lyophilized to give the corresponding target compound 91 (30 mg, 62% yield for two steps). LCMS (ESI): m / z: 914 [M+H]+. N-(6-(((3-(3-carbamothioylphenyl)-7-chloro-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-indol- 6-yl)methyl)amino)-6-oxohexyl)-3',6'-dihydroxy-3-oxo-3H-spiro [isobenzofuran-1,9'-xanthene]-5-carboxamide (3) The title compound 3 was synthesized from compound 91 according to the method described for general procedure B. It was obtained as a yellow solid (64% yield).1H NMR (600 MHz, CD3OD) δ 8.54 (s, 1H), 8.14 (t, J = 1.9 Hz, 1H), 7.94 (dd, J = 7.8, 1.9 Hz, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.76 (dt, J = 7.7, 1.4 Hz, 1H), 7.73 (s, 1H), 7.70 (dt, J = 7.8, 1.3 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.19 (dd, J = 13.0, 8.0 Hz, 2H), 6.89 (d, J = 9.3 Hz, 2H), 6.50 (d, J = 2.1 Hz, 2H), 6.44 (dd, J = 9.3, 2.2 Hz, 2H), 5.57 (tt, J = 11.9, 3.6 Hz, 1H), 4.62 (s, 2H), 3.94 (d, J = 12.2 Hz, 2H), 3.45 (t, J = 6.9 Hz, 2H), 3.04 (t, J = 11.9 Hz, 2H), 2.92 (s, 3H), 2.34-2.29 (m, 4H), 2.15 (qd, J = 12.2, 4.1 Hz, 2H), 1.75 (p, J = 7.3 Hz, 2H), 1.71 (p, J = 7.3 Hz, 2H) 1.49 (p, J = 7.6, 7.2 Hz, 2H). LCMS (ESI): m / z: 948 [M+H]+. Protein expression and purification ASH1L SET domain (amino acids 2046-2330) for FP and HMT assays was expressed and purified as described before. Fluorescence Polarization (FP) assay To determine Kd value, the fluorescein (FLSN) labeled compound 3 at 25 nM, was titrated with a range of ASH1L SET concentrations in the FP buffer (50 mM TRIS, pH = 7.5, 100 mM NaCl, 1mM TCEP). In the competition experiments, the fluorescein-labeled compound 3 (at 25 nM), ASH1L (at 150 nM) and varying concentrations of ASH1L inhibitors (5% final DMSO concentration) in the FP buffer were used for IC50determination. After a 1h incubation of the ASH1L-compound complexes, the fluorescein-labeled compound 3 was added at 25 nM to each sample and changes in fluorescence polarization were monitored at 525 nm after excitation at 495 nm using PHERAstar microplate reader (BMG). Results were used to assess binding or inhibition for the specific compounds with the BMG Labtech MARS Data Analysis Software Program (Version 2.0.0). Histone Methyltransferase (HMT) assay HMT assay was carried out in a similar way as described before (Rogawski et al 2021, Nat. Commun.; incorporated by reference in its entirety).50 nM of ASH1L (amino acids 2046- 2330) in HMT buffer (50 mM Tris pH = 8.5, 2 mM MgCl2, 1 mM TCEP and 0.01% Triton X- 100) was titrated by a range of compound concentrations (2% final DMSO concentration). The mixtures of ASH1L and compounds were incubated at the room temperature for 1h. In the next step, 250 nM chicken nucleosomes (Reaction Biology, HMT-35-179), 1 μM3H-labelled S- adenosyl methionine (Perkin Elmer, NET155V250UC) and 2 μM unlabeled S-adenosyl methionine were added to initiate the HMT reactions, which were continued for 1h at room temperature followed by quenching by addition of 10% Trichloroacetic acid (TCA). The solutions were then transferred to 96-well filter plates (Millipore sigma, MSFBN6B), washed twice with 10% TCA and with 200-proof ethanol. After extensive drying, 70 µL of Microscint-O scintillant (PerkinElmer) was added and counts per minute (CPM) were measured using a MicroBeta22450 Microplate counter (PerkinElmer). Titration curves were plotted using Prism (GraphPad) and IC50values were calculated. HMT selectivity profiling The effect of 66s on the inhibition of a panel of histone methyltransferases was assessed by mixing 5 µM of the compound with ASH1L, SUV39H2, EZH2, PRDM9, G9a, GLP, PRMT1, PRMT4, PRMT6, SMYD1, SMYD2, DOT1L, SETD2, NSD1, NSD2, or NSD3 (final DMSO concentration of 2% using a similar approach as before (Rogawski et al 2021, Nat. Commun.; incorporated by reference in its entirety). Briefly, the mixtures of proteins and 66s were incubated at room temperature for 1 h. The HMT reactions were initiated by addition of 250 nM chicken nucleosomes (Reaction Biology, HMT-35-179) to ASH1L, DOT1L, SETD2, NSD1-3, or 0.05 mg / mL core histone (Millipore sigma, H9250) to other HMTs and 1 μM3H-labeled S-adenosyl methionine (Revvity, NET155V250UC). For HMTs that require core histone as a substrate, a buffer containing 50 mM Tris, pH = 8.5, 25 mM NaCl, 2 mM MgCl2, 1 mM DTT and 0.01% Triton X-100 was used. For HMTs that require nucleosome as a substrate, a buffer containing 50 mM Tris, pH = 8.5, 1.5 mM MgCl2, 1 mM TCEP and 0.01% Triton X-100 was used. The reaction was continued for 1 h at room temperature, then quenched by addition of 10% Trichloroacetic acid (TCA) and then transferred to the 96-well filter plates (Millipore sigma, MSFBN6B), washed twice with 10% TCA and once with 200-proof ethanol. After extensive drying, 50 µL of Microscint-O scintillant (Revvity, 6013611) was added and counts per minute (CPM) were measured using a MicroBeta microplate counter (PerkinElmer). Isothermal titration calorimetry Purified ASH1L (2069-2288) was extensively dialyzed against ITC buffer consisting of 50 mM phosphate (pH = 7.5), 50 mM NaCl, 1 mM TCEP at 4 °C. 66s was dissolved in DMSO and diluted with the ITC buffer to the final concentration of 5 µM in 5% DMSO. ASH1L was diluted with the ITC buffer to the final concentrations of 62-96 µM in 5% DMSO. SAM was introduced to the solutions of ASH1L and 66s to the final concentration of 50 µM to maintain ASH1L stability. All samples were extensively degassed by vacuum aspiration for 20 min prior to measurements. The titrations were performed using a VP-ITC titration calorimetric system (MicroCal) at 25 °C. The titration curves were obtained by injecting 10 µL aliquots of ASH1L solutions into the cell containing 66s at a time intervals of 200 s. All titration data were analyzed with a single-site fitting model using Origin 7.0. Viability assays Human leukemia cells K562 and MV4;11 (purchased from ATCC) and KOPN8, MOLM13 (purchased from DSMZ) were cultured in RPMI 1640 (Invitrogen) supplemented with 10% heat inactivated (h.i.) FBS and 1% penicillin / streptomycin (Invitrogen). Cell viability experiments were carried out as described before (Rogawski et al 2021, Nat. Commun.; incorporated by reference in its entirety). MTT readouts were performed at days 4-14 as indicated using a PHERAstar BMG microplate reader. The experiments were performed 2 times in quadruplicates . Data were analyzed in Prism 9.1.1 to obtain GI50values. Immunoblotting KOPN-8 and MV4;11 cells were plated at 2 x 105 / mL cell density in the same media as in the viability assay and treated with indicated doses of 66s for 7 or 8 days. At day 4, cells were collected and replated at initial cell density with fresh medium and the compound for additional 4 days. On day 8, cells were counted, collected, washed in cold 1X PBS, and whole cell lysate was prepared using RIPA lysis buffer (Thermo Scientific, # 89900) supplemented with 1X protease inhibitor cocktail (Sigma, # P8340) and 1mM DTT (Sigma, # 10197777001). Cell lysate was sonicated for 5 pulses of 10 s each (Bioruptor 300, Diagenode) and quantitated using BCA protein assay kit (Thermo Scientific, # 23225).15 µg of protein per sample was separated using 12% Bis-Tris gels (Invitrogen, #NP0342) for Western blot analysis to detect histone H3K36me2 (Cell Signaling Technology #2901S, clone CZ5H12, dilution 1:1,000), histone H3K36me3 (Cell Signaling Technology, #4909S, clone D5A7, dilution 1:1,000), histone H2AUb (K119) (Cell Signaling Technology #8240S, clone D27C4, dilution 1:50,000). Total H3 (Abcam, #ab1791, dilution 1:40,000) was used as a loading control. Flow cytometry analysis MV4;11 and KOPN8 cells were plated at 1 x 105cell / mL and treated for 10 or 14 days with 66s or DMSO in triplicates, maintaining the final DMSO concentration at 0.25% and were incubated at 37°C in a 5% CO2incubator. Cells were counted at day 3 or 4 using Trypan Blue (Thermo Fisher) and media was changed with compounds resupplied at that time point. Viable cell numbers for DMSO treated samples were restored to the original concentration and the same cell dilution was used for other samples. For Annexin V apoptosis assay, 1 x 105cells per sample were collected at the end of the treatment, washed with PBS and stained using the FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen™) according to the manufacturer’s instructions. In addition, 1 x 105cells per sample were stained with Zombie Aqua™ dye (1:100 dilution) for 15 minutes using the Zombie Aqua™ Fixable Viability Kit (Zombie Aqua™ Fixable Viability Kit, Biolegend®) at 1:50 dilution and then for 30 min with an anti-CD11B-PE antibody (982606, ICRF44, Biolegend®) or anti-CD11B-Pacific Blue (101224, BD BioLegend) antibody at 1:50 dilution, according to the No-wash Sequential Staining Protocol described by the manufacturer (BioLegend, Zombie Aqua Fixable Viability Kit). Cells were washed twice with 500 µL PBS containing 1% FBS and resuspended in 200 µL PBS with 1% FBS. Flow cytometry experiments were performed on FACSCelesta flow cytometer using BD FACSDiva version 8 and all data were analyzed with FlowJo v.10.6.0 software (Tree Star, Inc.). Cytospins and Wrigth-Giemsa staining 0.1 to 1 x 105cells treated with 66s or 0.25% DMSO were collected at days 10-14 for staining. Cytospins were prepared and stained using the PROTOCOL™ Hema 3™ Manual Staining System (22-122911, Fisher Scientific) as described before.0.5 to 1 x105cells in 100 µL PBS were used for cytospins and staining by applying the same procedure as described before. Example 2 Synthesis The synthesis of compound 4 is outlined in Scheme 1. Condensation of amine 5 with 2- (tert-butoxycarbonyl)-2-azaspiro[3.3]heptane-6-carboxylic acid yielded spiroazetidine 6, which was converted to thioamide 7 in the presence of sodium hydrosulfide hydrate and magnesium chloride. Deprotection of N-Boc group in compound 7 yielded target compound 4. Scheme 1. Synthesis of spiroazetidine analog 4α αReagents and conditions: (a) 2-(tert-butoxycarbonyl)-2-azaspiro[3.3]heptane-6-carboxylic acid, EDCI, DMAP, DCM, r.t., overnight; (b) sodium hydrosulfide hydrate, magnesium chloride, DMF, r.t., overnight; (c) TFA, DCM, 1 h. The preparation of compound 22 was commenced from 2-chloro-3-nitrobenzoic acid (8) as illustrated in Scheme 2. The Bartoli indole synthesis involved the reaction of nitroarene 8 with vinyl Grignard reagent, providing access to 7-chloro-1H-indole-6-carboxylic acid (9), which was reacted with CH3I in the presence of K2CO3to afford methyl ester 10. Iodination of compound 10 followed by N-Boc protection furnished 1-(tert-butyl) 6-methyl 7-chloro-3-iodo-1H-indole- 1,6-dicarboxylate (12). Treatment of 12 with (3-cyanophenyl)boronic acid via Suzuki coupling afforded compound 13. The synthesis of compound 16 was achieved by N-Boc deprotection of compound 13 and successive nucleophilic substitution. Subsequent reduction of ester 16 with LiBH4gave rise to alcohol 17, which was transformed into azide 18 in the presence of DPPA and DBU. The Staudinger reduction of azide 18 delivered primary amine 19, which was then functionalized through amidation to provide spiroazetidine analog 20. The target compound 22 was obtained from spiroazetidine derivative 20, following a similar procedure as described for compound 4. Scheme 2. Synthesis of spiroazetidine analog 22α

[0147] αReagents and conditions: (a) vinylmagnesium bromide, THF, -45 °C to r.t., overnight; (b) K2CO3, CH3I, DMF, r.t., 3 h; (c) KOH, I2, DMF, r.t., 3 h; (d) di-tert-butyl-dicarbonate, Et3N, DMAP, DCM, r.t., overnight; (e) Pd(dppf)Cl2·DCM,(3-cyanophenyl)boronic acid, NaHCO3, THF / H2O, 65 °C, 12 h; (f) TFA, DCM, 3 h; (g) Cs2CO3, tetrahydro-2H-pyran-4-yl methanesulfonate 15, 100 °C, 18 h; (h) LiBH4, THF / MeOH, 65 °C, 25 min; (i) DPPA, DBU, THF, 0 °C to r.t., overnight; (j) PPh3, THF / H2O, r.t., 16 h; (k) 2-(tert-butoxycarbonyl)-2- azaspiro[3.3]heptane-6-carboxylic acid, EDCI, DMAP, DCM, r.t., overnight; (l) sodium hydrosulfide hydrate, magnesium chloride, DMF, r.t., overnight; (m) TFA, DCM, 1 h. To target new hydrophobic pocket, we designed and synthesized spiroazetidines 23-25, in which a flexible ether-containing moiety was tethered to the azetidine motif (Scheme 3). The carboxylic acids 28a-b and 28i were prepared by esterification of alcohols 26a-b and 26i followed by cleavage of the tert-butyl group with TFA. Subsequent condensation of carboxylic acids 28a-b and 28i with N,O-dimethylhydroxylamine yielded Weinreb amides 29a-b and 29i, which underwent LiAlH4reduction to afford aldehydes 30a-b and 30i. Finally, the synthesis of target compounds 23-25 was accomplished by reacting aldehydes 30a-b and 30i with amine 22 via reductive amination. Scheme 3. Synthesis of spiroazetidine analogs 23-25α αReagents and conditions: (a) tert-butyl 2-bromoacetate, 40 % aq. NaOH, tetrabutylammonium bromide, toluene, r.t., 16 h; (b) TFA, DCM, 8 h; (c) N,O-dimethylhydroxylamine, EDCI, DMAP, DCM, r.t., overnight (d) LiAlH4, Et2O, 0 °C, 30 min; (e) 22, sodium triacetoxyborohydride, Et3N, DCE, 4 h. Upon preparing the spiroazetidine analogs, it was found that the azetidine moiety was sensitive to both acid and base, leading to ring-opening products. Of particular note, these byproducts didn’t exert any ASH1L inhibitory activity. Therefore, attention was turned to spiropiperidine moiety. As depicted in Scheme 4, amine 19 was reacted with 7-(tert- butoxycarbonyl)-7-azaspiro[3.5]nonane-2-carboxylic acid under standard conditions to produce spiropiperidine analog 31, which underwent thioamidation to furnish intermediate 32. Afterward, the N-Boc protective group in compound 32 was removed, providing the desired compound 33. The target compounds 34-43 were obtained by reductive amination using aldehydes 30a-i, following procedures similar to those described for compounds 23-25. Thiazole-5-carbaldehyde (44) underwent a Witting reaction with methoxymethyltriphenylphosphonium chloride, resulting in alkene 45 (Scheme 4). Subsequent treatment of alkene 45 with 4N HCl led to the formation of 2-(thiazol-5-yl)acetaldehyde (46). In a similar manner, aldehyde 46 was used to synthesize compound 43 through a reductive amination reaction previously described in Scheme 3. Scheme 4. Synthesis of spiropiperidine analogs 33-43α αReagents and conditions: (a) 7-(tert-butoxycarbonyl)-7-azaspiro[3.5]nonane-2-carboxylic acid, EDCI, DMAP, DCM, r.t., overnight; (b) sodium hydrosulfide hydrate, magnesium chloride, DMF, r.t., overnight; (c) TFA, DCM, 1 h; (d) sodium triacetoxyborohydride, aldehydes 30a-i, Et3N, DCE, r.t., 4 h; (e) t-BuOK, methoxymethyltriphenylphosphonium chloride, 0 °C to r.t., 6 h; (f) 4N HCl, THF, 60 °C, 3.5 h; (g) 33, sodium triacetoxyborohydride, Et3N, DCE, r.t., 4 h. As depicted in Scheme 5, treatment of 2,2-dimethoxyethan-1-amine (47) with appropriate acetic anhydrides, 3,3,3-trifluoropropanoyl chloride or carboxylic acids furnished the aminoacetaldehyde derivatives 48a-b, 48c, and 48d-m, respectively. Cleavage of the acetal protecting group in compounds 48a-m with Amberlyst-15 in acetone / H2O afforded the desired aldehydes 49a-m. The reductive amination reactions were carried out to provide amide- containing spiropiperidine analogs 50-52, 56-59, and 61-66 using corresponding aldehydes 49a- m. Scheme 5. Synthesis of amide-containing spiropiperidine analogs 50-52, 56-59, and 61-66α αReagents and conditions: (a) for 48a: acetic anhydride, Et3N, DCM, r.t., 2 h; for 48b: 2,2,2- trifluoroacetic anhydride, Et3N, DCM, r.t., 2 h; for 48c: 3,3,3-trifluoropropanoyl chloride, Et3N, DCM, 0 °C, 10 min; for 48d-m: carboxylic acids, EDCI, DMAP, DCM, r.t., overnight; (b) Amberlyst-15, acetone / H2O, r.t., overnight; (c) 33, sodium triacetoxyborohydride, Et3N, DCE, r.t., 4 h. An alternative synthetic approach was adopted to prepare spiropiperidine analogs 53-55 and 60 (Scheme 6). Deprotection of Boc group in compound 32 followed by alkylation of the corresponding amine with tert-butyl (2-bromoethyl)carbamate yielded spiropiperidine analog 67, which was subjected to thioamidation with sodium hydrosulfide hydrate to generate compound 68. Cleavage of N-Boc group and successive acylation led to target compounds 53-55. The synthesis of target compound 60 was achieved through the acylation of 4-fluoroaniline (69) followed by N-alkylation (Scheme ...

Claims

CLAIMS 1. A compound of formula (I):or a pharmaceutically acceptable salt thereof, wherein: E and A are a spirocyclic ring system; Z is CH or N; Y is N or O; if Y is O then R1is absent; if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; R2is selected from -C(S)NH2, -C(O)NH2, -C(Se)NH2, oxirane, thiirane, aziridine, -CF3,R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and wherein R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, -halogen (e.g., -F), etc.); and R7is selected from H, halogen, CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2- OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3.

2. The compound of claim 1, wherein R8is selected from H, CH3, CF3, -C(O)CH3, - CH2NHC(O)OC(CH3)3,3. The compound of claim 1, wherein R6is selected from:

4. The compound of claim 1, wherein R6is –(CH2)2– R9, and wherein R9is selected from:

5. The compound of claim 1, wherein R6is -(CH2)2NHC(O)-R10, wherein R10is selected from:

6. The compound of claim 1, wherein E, A, Z, Y, R1, R2, R6, and R7are independently selected from the E, A, Z, Y, R1, R2R6, and R7functional groups of compounds 3- 4, 23-25, 33-43, 50-66, 66s, 77-78, 123-127, 143-145, 160-167, and 168-194.

7. The compound of claim 6, selected from one of compounds 3-4, 23-25, 33-43, 50- 66, 66s, 77-78, 123-127, 143-145, 160-167, and 168-194.

8. A compound of formula (Ia):or a pharmaceutically acceptable salt thereof, wherein: E and A are a spirocyclic ring system; Z is CH or N; Y is N or O; if Y is O then R1is absent; if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; X is O, Se, or S;R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and wherein R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, -halogen (e.g., -F), etc.); and R7is selected from H, halogen, CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2- OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3.

9. The compound of claim 8, R8is selected from H, CH3, CF3, -C(O)CH3, - CH2NHC(O)OC(CH3)3,10. The compound of claim 8, wherein R6is selected from:

11. The compound of claim 8, wherein R6is –(CH2)2– R9, and wherein R9is selected from:

12. The compound of claim 8, wherein R6is -(CH2)2NHC(O)-R10, wherein R10is selected from:

13. The compound of claim 8, wherein E, A, Z, Y, R1, R6, and R7are independently selected from the E, A, Z, Y, R1, R2R6, and R7functional groups of compounds 3-4, 23-25, 33- 43, 50-66, 66s, 77-78, 143-145, 160-167, 168-194.

14. The compound of claim 13, selected from one of compounds 3-4, 23-25, 33-43, 50-66, 66s, 77-78, 143-145, 160-167, 168-194.

15. A compound of formula (Ib):or a pharmaceutically acceptable salt thereof, wherein: Z is CH or N; Y is N or O; if Y is O then R1is absent; if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; X is O, Se, or S;R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, -halogen; and R7is selected from H, halogen, CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2- OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3.

16. The compound of claim 15, wherein R8is selected from H, CH3, CF3, -C(O)CH3, -CH2NHC(O)OC(CH3)3,17. The compound of claim 15, wherein R6is selected from:.

18. The compound of claim 15, wherein R6is –(CH2)2– R9, and wherein R9is selected from:

19. The compound of claim 15, wherein R6is -(CH2)2NHC(O)-R10, wherein R10is selected from:

20. The compound of claim 15, wherein Z, Y, R1, R6, and R7are independently selected from the Z, Y, R1, R2R6, and R7functional groups of compounds 4, 23-25, 160-162, and 168-169.

21. The compound of claim 20, selected from one of compounds 4, 23-25, 160-162, and 168-169.

22. A compound of formula (Ic):or a pharmaceutically acceptable salt thereof, wherein: Z is CH or N; Y is N or O; if Y is O then R1is absent; if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2; X is O, Se, or S;R6is selected from H, -C(O)CH3, and , wherein L is an amide (e.g., -NHC(O)- or -C(O)NH-), O, NH, or -SO2NH-, and R8is selected from H, CH3, CF3, cycloalkyl, heteroalkyl, aryl, or heteroaryl (wherein the cycloalkyl, heteroalkyl, aryl, or heteroaryl may be further substituted by a -CH3, -halogen (e.g., -F), etc.); andR7is selected from H, halogen, CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2- OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3.

23. The compound of claim 22, wherein R8is selected from H, CH3, CF3, -C(O)CH3, -CH2NHC(O)OC(CH3)3,24. The compound of claim 22, wherein R6is selected from:

25. The compound of claim 22, wherein R6is –(CH2)2– R9, and wherein R9is selected from:

26. The compound of claim 22, wherein R6is -(CH2)2NHC(O)-R10, wherein R10is selected from:

27. The compound of claim 22, wherein Z, Y, R1, R6, and R7are independently selected from the Z, Y, R1, R2R6, and R7functional groups of compounds 3, 33-43, 50-66, 66s, 77-78, 143-145, 163-167, and 170-194.

28. The compound of claim 27, selected from one of compounds 3, 33-43, 50-66, 66s, 77-78, 143-145, 163-167, and 170-194.

29. A compound of formula (II):or a pharmaceutically acceptable salt thereof, wherein: Z is CH or N; Y is N or O; if Y is O then R1is absent; if Y is N then R1is SO2-alkyl, wherein alkyl is selected from -CH3, -CH2CH3, - (CH2)2CH3, and –CH(CH3)2. X is O, Se, or S; and R7is selected from H, halogen, CH3, OH, SH, NH2, CN, CF3, CCl3, -CH2-CH3, -CH2- OH, -CH2NH2, CH3SH, CH2Cl, CH2Br, CH2F, CHF2, CH2CN, CH2CF3, and CH2Cl3.

30. The compound of claim 29, wherein Z, Y, R1, and R7are independently selected from the Z, Y, R1, R2R6, and R7functional groups of compounds 139-142 and 158-159.

31. The compound of claim 30, selected from one of compounds 139-142 and 158- 159.

32. A compound of formula (III):or a pharmaceutically acceptable salt thereof, wherein: R1is selected from NH2and (CH2)NHC(O)CH3; and R2is selected from -C(S)NH2, -C(O)NH2, -C(Se)NH2, oxirane, thiirane, aziridine, -CF3,and -CN.

33. The compound of claim 32 wherein R1and R2are independently selected from the R1and R2functional groups of compounds 100-112, 115-122, 128-135, and 146-153.

34. The compound of claim 33, selected from one of compounds 100-112, 115-122, 128-135, and 146-153.

35. A compound selected from one of compounds 2-4, 22-25, 33-39, 40-44, 50-66, 66s, 77-79, and 100-194.

36. A pharmaceutical composition comprising a compound of any one of claims 1-35, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

37. The pharmaceutical composition of claim 35, wherein the pharmaceutical composition is formulated for oral administration.

38. The pharmaceutical composition of claim 35, wherein the pharmaceutical composition is formulated for parenteral administration.

39. A method of inhibiting ASH1L activity in a sample, comprising contacting the sample with an effective amount of a compound of any one of claims 1-35 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 36-38.

40. A method of reducing proliferation of cancer cells in a sample, comprising contacting the sample with an effective amount of a compound of any one of claims 1-35 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 36-38.

41. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound of any one of claims 1-35 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 36-38.

42. The method of claim 41, wherein the cancer is selected from leukemia, hematologic malignancy, solid tumor cancer, breast cancer, prostate cancer, ovarian cancer, liver cancer orand thyroid cancer.

43. The method of claim 42, wherein the cancer is selected from AML, ALL, Mixed Lineage Leukemia or a leukemia with Partial Tandem Duplication of MLL.

44. The method of claim 41, further comprising administering an additional chemotherapeutic agent to the subject.

45. The method of claim 41, wherein the subject is a human.

46. Use of a compound of any one of claims 1-35 or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of any one of claims 36-38, for the treatment of cancer.

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