Tetrahydroquinolino derivatives for the treatment of metastatic and chemotherapy-resistant cancers - Patents.com

Abstract

Disclosed herein are compounds of structural formula I. The compounds of structural formula I inhibit aldehyde dehydrogenase isoform Ia3 (ALDHIa3) and are useful for treating cancer, e.g., metastatic or chemoresistant cancer, such as chemotherapy-resistant metastatic cancer. Also disclosed herein are compositions comprising the compounds of structural formula I and uses of the compounds of structural formula I for treating cancer, e.g., metastatic or chemoresistant cancer. [Selection diagram] None

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 712,434, filed July 31, 2018. The entire teachings of the above application(s) are incorporated herein by reference. [Background technology]

[0002] background Current therapies for treating aggressive and / or chemotherapy-resistant metastatic cancers have limited effectiveness. Thus, there is a need for therapeutic agents with improved efficacy for the treatment of these cancers.

[0003] overview Provided herein are compounds, compositions and methods for treating cancer, e.g., metastatic cancer (e.g., metastatic breast cancer, such as basal-like breast cancer, HER-2 positive breast cancer, etc.), chemotherapy resistant cancer (e.g., paclitaxel and / or doxorubicin resistant cancer), in a subject in need thereof.

[0004] One embodiment is a compound of formula II: [ka] or a pharma- ceutically acceptable salt thereof, wherein the variables (e.g., R 1 , R 2 , R 3 , R 4 , R 5 , R 9 , L, n) are as described herein.

[0005] Another embodiment is a compound of formula I: [ka] or a pharma- ceutical acceptable salt thereof, wherein the variables (e.g., R1 , R 2 , R 3 , R 4 , R 5 , R 9 , L, X 1 , X 2 , X 3 , X 4 , X 5 ) are as described herein. Also provided is a compound represented by structural formula I, or a pharma- ceutically acceptable salt thereof, for use in treating metastatic or chemotherapy-resistant cancer. Also provided is the use of a compound of structural formula I, or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of metastatic or chemotherapy-resistant cancer.

[0006] Yet another embodiment is a method of inhibiting the proliferation of metastatic or chemotherapy-resistant cancer cells, comprising administering to said cells an effective amount of a compound of structural formula I, or a pharma- ceutically acceptable salt thereof. Also provided is a compound for inhibiting the proliferation of metastatic or chemotherapy-resistant cancer cells, the compound being represented by structural formula I, or a pharma- ceutically acceptable salt thereof. Also provided is the use of a compound of structural formula I, or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for inhibiting the proliferation of metastatic or chemotherapy-resistant cancer cells.

[0007] Another embodiment is a method of treating type 2 diabetes in a subject in need thereof, comprising administering to the subject a compound of structural formula I, or a pharma- ceutically acceptable salt thereof. Also provided is a compound represented by structural formula I, or a pharma- ceutically acceptable salt thereof, for use in treating type 2 diabetes. Also provided is the use of a compound of structural formula I, or a pharma- ceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of type 2 diabetes.

[0008] The compounds, compositions and methods described herein can be used to inhibit an enzyme target (ALDH1a3) that is important for metastasis and / or resistance to chemotherapy and is widely expressed in many tumor types. Many of the compounds described herein have low nanomolar affinity for ALDH1a3, and the compound MBE1 has been shown to reduce metastatic lesions in mice without toxicity.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0010] The foregoing will be apparent from the following more particular description of exemplary embodiments. [Brief description of the drawings]

[0011] [Figure 1A] 1A is a bar graph of the percentage of ALDEFLUOR™ positive cells in the presence of various compounds described herein and shows the percentage of SUM159-M1a-Aldh1a3 cells above background fluorescence levels as detected by flow cytometry after incubation using the standard ALDEFLUOR™ protocol described herein with 100 nanomolar concentrations of compound. Gating for background fluorescence was performed using 1 millimolar N,N-diethylaminobenzaldehyde (DEAB) as a negative control. [Figure 1B]1B is a line graph of the percentage of ALDEFLUOR™ positive cells in the presence of various concentrations of MBE1 or MBE1.5, and shows the percentage of SUM159-M1a-Aldh1a3 cells above background fluorescence levels as detected by flow cytometry following incubation according to the standard ALDEFLUOR™ protocol described herein in combination with dose titration of MBE1 or MBE1.5. The [inh-min] threshold was set at the lower limit of two standard deviations of the control samples, while the IC50 threshold was set at 50% of the mean of the control samples. [Figure 1C] FIG. 1C is a line graph of high ALDH percentage in the presence of various concentrations of MBE1 and MBE1.5, and shows the percentage of SUM159-M1a-Aldh1a3 cells with high fluorescence levels as detected by flow cytometry after incubation according to the standard ALDEFLUOR™ protocol described herein in combination with dose titration of MBE1 or MBE1.5. High activity was gated on cells at the 99th percentile at 10 nanomolar MBE1.5 concentration. The [inh-min] threshold was set at the lower limit of two standard deviations of the control sample. [Figure 2A] FIG. 2A is a histogram of MCF7 cells stably transduced with human Aldh1a1, Aldh1a3, or Aldh3a1 and analyzed using the ALDEFLUOR™ protocol by flow cytometry in combination with the listed concentrations of DMSO or MBE1, and shows the effect of DMSO or MBE1 on ALDEFLUOR™ activity induced by each enzyme. [Figure 2B] FIG. 2B is a histogram of 293T cells transiently transfected with human Aldh1a2 or Aldh1a3 and analyzed using the ALDEFLUOR™ protocol by flow cytometry in combination with the listed concentrations of DMSO or MBE1.5, and shows the effect of either DMSO or MBE1.5 on ALDEFLUOR™ activity induced by each enzyme. [Figure 3A] FIG. 3A is a bar graph of ALDEFLUOR™ positive cells in various cancer types in the presence of DEAB or MBE1 and shows that ALDH1a3 activity is observed in many cancer types in addition to Mla (breast cancer) and MDA-468 (breast cancer) and is inhibited by MBE1 in many cancer types. [Figure 3B] FIG. 3B is a bar graph of ALDEFLUOR™ activity in various cancer types in the presence of 1 mM DEAB (a pan-ALDH inhibitor) or 100 nM MBE1.5 (a specific ALDH1a3 inhibitor) and shows that the majority of human cancer cell lines exhibit Aldh1a3 activity. [Figure 4A] FIG. 4A is a flow cytometry spectrum and shows that CD45 positive bone marrow cells are ALDEFLUOR™ positive but are not affected by MBE1.5. [Figure 4B] FIG. 4B is a flow cytometry spectrum and shows that CD11c positive bone marrow cells are ALDEFLUOR™ positive but are not affected by MBE1.5. [Figure 5A] FIG. 5A is a diagram of the dosing strategy used to administer MBE1 and paclitaxel to mice injected with M1a-Aldh1a3 cells via intravenous tail-vein injection, and shows the design of an in vivo experiment designed to test the efficacy of MBE1 in treating metastatic cancer. [Figure 5B] Figure 5B is a line graph of lung metastases versus time (days) measured using bioluminescence imaging (BLI) and compares lung metastases in the presence and absence of MBE1 in mice from the experiment outlined in Figure 5A. Student's t-test, two-tailed, unequal variances assumed. [Figure 6A]FIG. 6A is a diagram of the dosing strategy used to administer MBE1 and paclitaxel to mice injected with M1a-Aldh1a3 cells via intracardiac injection, and shows the design of an in vivo experiment designed to test the efficacy of MBE1 in treating metastatic cancer. [Figure 6B] Figure 6B is a line graph of bone metastasis versus time (days) measured using BLI and compares bone metastasis in the presence and absence of MBE1 in mice from the experiment outlined in Figure 6A. Student's t-test, one-tailed, unequal variances assumed. [Figure 7A] FIG. 7A is a flow cytometry spectrum and shows that genetic knockout of ALDH1a3 in MDA-MB-468 breast cancer cells (middle and rightmost spectra) substantially reduces ALDEFLUOR™ activity compared to control MDA-MB-468 cells (leftmost spectrum). [Figure 7B] FIG. 7B is a line graph of tumor volume (mm3) versus time (days) and shows that genetic knockout of ALDH1a3 in MDA-MB-468 breast cancer cells slows primary tumor growth and sensitizes tumors to paclitaxel. [Figure 7C] FIG. 7C is a bar graph of tumor mass (g) versus gene knockout and shows that gene knockout of ALDH1a3 in MDA-MB-468 breast cancer cells slows primary tumor growth and sensitizes tumors to paclitaxel. [Figure 8A] FIG. 8A is a line graph of bioluminescence (ph / s) versus time (days) and shows the development of lung metastases in mice injected with SUM159-Mlb cells transduced with vectors encoding the three ALDH enzymes, ALDH1a1, ALDH1a3, and ALDH3a1. [Figure 8B] Figure 8B is a plot of lung nodes counted ex vivo at the end point of the experiment described in Figure 8A. Student's t-test, two-tailed, unequal variances assumed. [Figure 9A]Figure 9A is a flow cytometry spectrum and shows that genetic knockout of ALDH1a3 in Suml59-M1a breast cancer cells nearly abolishes ALDEFLUOR™ activity in the cells, and that ALDEFLUOR™ activity can be rescued by transducing the cells with a rescue vector. [Figure 9B] FIG. 9B is a line graph of bone metastases versus time (days) as measured by bioluminescence (ph / s) and shows that knockout of ALDH1a3 in Suml59-M1a breast cancer cells slows bone metastasis growth. [Figure 9C] FIG. 9C is a Kaplan-Meier plot of bone metastasis-free survival over time and shows that knockout of ALDH1a3 in Suml59-M1a breast cancer cells significantly increases survival time. [Figure 10A] FIG. 10A is a patient survival curve stratified by high (red) and low (black) expression of Aldh1a3 based on the data analysis tools provided at kmplot.com, and shows distant metastasis-free survival of patients with renal clear cell carcinoma as a function of ALDH1a3 expression levels. [Figure 10B] FIG. 10B is a patient survival curve stratified by high (red) and low (black) expression of Aldh1a3 based on the data analysis tools provided at kmplot.com, and shows the overall survival of triple-negative breast cancer patients as a function of ALDH1a3 expression levels. [Figure 10C] FIG. 10C is a patient survival curve stratified by high (red) and low (black) expression of Aldh1a3 based on the data analysis tools provided at kmplot.com, and shows the overall survival of gastric cancer patients as a function of ALDH1a3 expression levels. [Figure 10D] FIG. 10D is a patient survival curve stratified by high (red) and low (black) expression of Aldh1a3 based on the data analysis tools provided at kmplot.com, and shows the overall survival of bladder cancer patients as a function of ALDH1a3 expression levels. [Figure 10E]FIG. 10E is a patient survival curve stratified by high (red) and low (black) expression of Aldh1a3 based on the data analysis tools provided at kmplot.com, and shows the overall survival of ovarian cancer patients as a function of ALDH1a3 expression levels. [Figure 10F] FIG. 10F is a patient survival curve stratified by high (red) and low (black) expression of Aldh1a3 based on the data analysis tools provided at kmplot.com, and shows the overall survival of patients with lung squamous cell carcinoma as a function of ALDH1a3 expression levels. [Figure 10G] FIG. 10G shows patient survival curves stratified by high (red) and low (blue) expression of Aldh1a3 based on survival time series data and patient-level RNA expression data from The Cancer Genome Atlas, and shows overall survival of colorectal cancer patients as a function of ALDH1a3 expression levels. [Figure 10H] FIG. 10H shows patient survival curves stratified by high (red) and low (blue) expression of Aldh1a3 based on survival time series data and patient-level RNA expression data from The Cancer Genome Atlas, and shows overall survival of low-grade glioma patients as a function of ALDH1a3 expression levels. [Figure 11A] FIG. 11A is a graph of Aldh1a3 mRNA expression from the METABRIC clinical breast cancer dataset and shows expression of Aldh1a3 by breast cancer subtype and chemotherapy history. [Figure 11B] FIG. 11B is a series of survival curves based on the Erasmus Medical Center-Memorial Sloan-Kettering (EMC-MSK) dataset and shows survival of breast cancer patients by subtype and stratified by median ALDH1a3 expression level. [Figure 12] FIG. 12 is a graph of ALDEFLUOR™ activity versus concentration and shows the ALDEFLUOR™ activity of several compounds described herein at concentrations of 10 nM and 100 nM. [Figure 13A]FIG. 13A is a Western blot and shows the expression of various ALDH isoforms, including 1a1, 1a2, 1a3 and 3a1, in MCF7 and SEIM 159 cells. [Figure 13B] FIG. 13B is a line graph of the percentage of ALDEFLUOR™-positive MCF7 cells expressing the indicated ALDH isoforms versus the log of MBE 1.5 concentration, and shows that MBE 1.5 specifically inhibits ALDH1a3 at concentrations less than 10 μM. [Figure 13C] FIG. 13C is a line graph of the percentage of ALDEFLUOR™-positive SEIM159 cells expressing the indicated ALDH isoforms versus the log of MBE 1.5 concentration, and shows that MBE 1.5 specifically inhibits ALDH1a3 at concentrations less than 10 pM. [Figure 14] Figure 14 is a line graph of lung metastases measured by bioluminescence imaging (BLI) versus time (days) and shows that three doses of 50 mg / kg MBE1.5 in combination with 25 mg / kg paclitaxel administered on days 17, 19 and 21 caused regression of established metastatic disease in a mouse xenograft model. Student's t-test, two-tailed, unequal variances assumed. [Figure 15A] FIG. 15A is a line graph of body weight (g) versus time (days) and shows that there was no significant toxicity associated with MBE1.5 treatment in this experiment. [Figure 15B] Figure 15B is a line graph of tumor volume (mm3) versus time (days) and shows that treatment with MBE1.5 for 12 days caused regression of MDA-MB-468 primary breast tumors. Statistics by Student's t-test. *p < 0.05. [Figure 16A] Figure 16A is a line graph of lung metastasis bioluminescence versus time (days) and shows the progression of lung metastases before and after treatment with MBE1.5 or vehicle. Statistics by Student's t-test. *p<0.05. [Figure 16B]Figure 16B is a Kaplan-Meier plot of mouse survival over time as a function of treatment group and shows that treatment with MBE1.5 for 12 days extended survival in mice with late-stage established breast cancer lung metastases. Statistics from the Cox proportional hazards model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description The compounds, compositions and methods are based, in part, on the discovery that aldehyde dehydrogenase (Aldh, ALDH), and in particular ALDH isoform 1a3 (ALDH1a3), are involved in many types of cancer and other ALDH1a3-mediated diseases and disorders (e.g., type 2 diabetes). Accordingly, the compounds, compositions and methods provided herein relate to modulating (eg, inhibiting) ALDH, such as ALDH1a3.

[0013] A description of an exemplary embodiment follows.

[0014] definition The compounds described herein include those generally described and are further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of the present invention, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook ofChemistry and Physics, 75th Ed. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University ScienceBooks, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, MB and March, J.,John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0015] Unless otherwise specified herein, the nomenclature used herein generally follows the examples and rules set forth in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979, which is incorporated herein by reference for its chemical structure names and rules for naming chemical structures. Optionally, the names of compounds may be generated using a chemical naming program (e.g., CHEMDRAW®, version 17.0.0.206, PerkinElmer Informatics, Inc.).

[0016] The compounds of the present invention may have chiral centers, chiral axes, and chiral planes (e.g., those described in EL Eliel and SH Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190) and occur as racemates, racemic mixtures, and individual diastereomers or enantiomers, with all possible isomers and mixtures thereof (including optical isomers) being included in the present invention.

[0017] "Alkyl" refers to a saturated aliphatic branched or straight-chain monovalent hydrocarbon radical having the specified number of carbon atoms. Thus, "(C 1 -C 3 "(C )alkyl" means a group having from 1 to 3 carbon atoms in a straight or branched arrangement. Alkyl includes methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, and the like. 1 -C 3 )Alkyl" includes methyl, ethyl, propyl and isopropyl.

[0018] "Amino" means -NH 2 means...

[0019] "Alkylamino" means (alkyl)(H)-N-, where the alkyl group is as described herein. In one aspect, alkylamino is (C 1 -C 3 ) alkylamino. Particular alkylamino groups include methylamino, ethylamino, propylamino and isopropylamino.

[0020] "Dialkylamino" means (alkyl) 2 -N-, where the alkyl groups may be the same or different and are as described herein. Particular dialkylamino groups include ((C 1 -C 3 )Alkyl) 2-N-, where the alkyl groups can be the same or different. Dialkylamino groups include dimethylamino, diethylamino and methylethylamino.

[0021] "Halogen" and "halo" are used interchangeably herein and refer to fluorine, chlorine, bromine, or iodine, respectively. In some embodiments, halogen is selected from fluoro or chloro.

[0022] "Chloro" means --Cl.

[0023] "Fluoro" means -F.

[0024] "Cyano" means --CN.

[0025] "Nitro" means -NO 2 means...

[0026] "Hydroxy" means --OH.

[0027] "Sulfhydryl" means --SH.

[0028] "Alkoxy" refers to an alkyl radical attached through an oxygen linking atom, where "alkyl" is as defined herein. 1 -C 6 )Alkoxy" includes methoxy, ethoxy, propoxy, butoxy, pentoxy and hexoxy.

[0029] "Thioalkoxy" refers to an alkyl radical attached through a sulfur linking atom where "alkyl" is as defined herein.

[0030] "Haloalkyl" includes mono-, poly-, and perhaloalkyl groups, where each halogen is independently selected from fluorine, chlorine, and bromine, and alkyl is as described herein. In one aspect, the haloalkyl is a perhaloalkyl (e.g., perfluoroalkyl). Haloalkyl groups include trifluoromethyl and pentafluoroethyl.

[0031] "Haloalkoxy" refers to a haloalkyl radical attached through an oxygen linking atom, where "haloalkyl" is as described herein.

[0032] It is understood that the substituents on the compounds of the invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and can be readily synthesized by techniques known in the art and the methods described below. Combinations of substituents envisioned by the invention (e.g., R 1 -R 9 , m, n, L, X 1 -X 5 ) are preferably those that result in the production of stable or chemically feasible compounds. As used herein, the term "stable" refers to compounds that do not substantially change when subjected to conditions that permit their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.

[0033] As used herein, the term "pharmaceutical acceptable salt" refers to a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutical acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the compounds described herein include salts derived from appropriate inorganic and organic acids and bases compatible with the treatment of a subject.

[0034] Examples of pharma- ceutically acceptable, non-toxic acid addition salts are salts of amino groups formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric, and perchloric acids, or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic, or malonic acids, or by using other methods used in the art, such as ion exchange. Other pharma-ceutically acceptable acid addition salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, hydrogensulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, and the like. These include phonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate salts.

[0035] In some embodiments, exemplary inorganic acids that form suitable salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, as well as acid metal salts (e.g., sodium monohydrogen orthophosphate and potassium hydrogen sulfate, etc.). Exemplary organic acids that form suitable salts include monocarboxylic acids, dicarboxylic acids, and tricarboxylic acids. Examples of such acids are, for example, acetic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, benzoic acid, hydroxybenzoic acid, phenylacetic acid, cinnamic acid, salicylic acid, 2-phenoxybenzoic acid, p-toluenesulfonic acid, and other sulfonic acids such as methanesulfonic acid and 2-hydroxyethanesulfonic acid. Either mono- or di-acid salts can be formed, and such salts can exist in either hydrated, solvated, or substantially anhydrous form. In general, the acid addition salts of these compounds are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms.

[0036] In some embodiments, acid addition salts are most suitably formed from pharma- ceutically acceptable acids, and include, for example, those formed with inorganic acids (e.g., hydrochloric acid, sulfuric acid, or phosphoric acid) and organic acids (e.g., succinic acid, maleic acid, acetic acid, or fumaric acid).

[0037] Exemplary inorganic bases that form suitable salts include, but are not limited to, lithium hydroxide, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or barium hydroxide. Exemplary organic bases that form suitable salts include aliphatic, cycloaliphatic, or aromatic organic amines (such as methylamine, trimethylamine, and picoline), or ammonia. The criteria for selecting an appropriate salt will be known to those skilled in the art.

[0038] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + (C 1 -C4 )Alkyl) 4 Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. Pharmaceutically acceptable salts further include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxyls, sulfates, phosphates, nitrates, lower alkylsulfonates and arylsulfonates.

[0039] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, by the replacement of hydrogen with deuterium or tritium, or of carbon with 13 C- or 14 Compounds produced by substituting C-enriched carbons are within the scope of the invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents according to the invention.

[0040] When introducing elements disclosed herein, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "having," and "including" are intended to be open-ended and mean that there may be additional elements other than the listed elements.

[0041] compound A first embodiment is a compound represented by structural formula I: [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 and R 5 are each independently hydrogen or (C 1 -C 6 ) alkyl; R 2 , R 3 and R 4 are each independently hydrogen, halo, halo(C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkoxy, (C 1 -C 3 ) thioalkoxy, halo(C 1 -C 3 ) alkoxy, amino, (C 1 -C 3 ) alkylamino, (C 1 -C 3 ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl; L is -C(O)-, -C(R 6 )(R 7 )-, -C(CH 2 CH 2 )-, -C(OCH 2 )- or -C(N(H)CH 2 )- and; R 6 and R 7 are each independently hydrogen, hydroxy, sulfhydryl, halo, (C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkoxy, (C 1 -C 3 ) thioalkoxy or halo(C 1 -C 3 ) alkoxy; X 1 , X 2 , X 3 , X 4 and X 5 are each independently -C(R 8 )- or -N-; and Each R 8 are independently hydrogen, halo, halo(C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl, (C 1 -C6 ) alkoxy, (C 1 -C 6 ) thioalkoxy, halo(C 1 -C 6 ) alkoxy, amino, (C 1 -C 6 ) alkylamino, (C 1 -C 6 ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl.

[0042] In a first aspect of the first embodiment, the compound is not MBE1, MBE2, MBE3.1, MBE3.2, MBE3.3, MBE3.4, MBE3.5 or MBE3.6, or a pharma- ceutically acceptable salt of any of the foregoing. The values ​​of the variables are as described in the first embodiment.

[0043] In a second aspect of the first embodiment, R 2 and R 4 are each hydrogen. The values ​​of the remaining variables are as described in the first embodiment or first aspect thereof.

[0044] In a third aspect of the first embodiment, R 1 is hydrogen. The values ​​of the remaining variables are as described in the first embodiment, or the first or second aspect thereof.

[0045] In a fourth aspect of the first embodiment, R 5 is hydrogen. The values ​​of the remaining variables are as described in the first embodiment, or the first to third aspects thereof.

[0046] In a fifth aspect of the first embodiment, R 3 is hydrogen or halo (e.g., halo). The values ​​of the remaining variables are as described in the first embodiment or its first to fourth aspects.

[0047] In a sixth aspect of the first embodiment, R 3is halo (eg, fluoro). The values ​​of the remaining variables are as described in the first embodiment, or the first to fifth aspects thereof.

[0048] In a seventh aspect of the first embodiment, L is -C(O)- or -C(R 6 )(R 7 The values ​​of the remaining variables are as described in the first embodiment or its first to sixth aspects.

[0049] In an eighth aspect of the first embodiment, L is -C(O)-. The values ​​of the remaining variables are as described in the first embodiment, or aspects 1 to 7 thereof.

[0050] In a ninth aspect of the first embodiment, L is -CH 2 The values ​​of the remaining variables are as described in the first embodiment, or its first to eighth aspects.

[0051] In a tenth aspect of the first embodiment, X 1 , X 2 , X 3 , X 4 and X 5 is -N-. The values ​​of the remaining variables are as described in the first embodiment or its first to ninth aspects.

[0052] In an eleventh aspect of the first embodiment, X 3 is -N-; and X 1 , X 2 , X 4 and X 5 are each independently -C(R 8 The values ​​of the remaining variables are as described in the first embodiment or its first to tenth aspects.

[0053] In a twelfth aspect of the first embodiment, each R 8 are independently hydrogen, halo, halo(C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkyl, (C1 -C 3 ) alkoxy, (C 1 -C 3 ) thioalkoxy, halo(C 1 -C 3 ) alkoxy, amino, (C 1 -C 3 ) alkylamino, (C 1 -C 3 ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl. The values ​​of the remaining variables are as described in the first embodiment, or aspects 1 to 11 thereof.

[0054] In a thirteenth aspect of the first embodiment, each R 8 are independently hydrogen, halo or (C 1 -C 6 ) alkyl (e.g., hydrogen or (C 1 -C 6 ) alkyl). The values ​​of the remaining variables are as described in the first embodiment, or aspects 1 to 12 thereof.

[0055] In a fourteenth aspect of the first embodiment, R 6 and R 7 are each independently hydrogen, hydroxy, halo, (C 1 -C 3 ) alkyl or (C 1 -C 3 ) alkoxy, for example, R 6 and R 7 are each independently hydrogen, hydroxy or halo, e.g., R 6 and R 7 are each hydrogen. The values ​​of the remaining variables are as described in the first embodiment, or the first to thirteenth aspects thereof.

[0056] Compounds of structural formula I include MBE1, MBE1.2, MBE1.3, MBE1.5, MBE1.6, MBE2, MBE3.1, MBE3.2, MBE3.3, MBE3.4, MBE3.5 and MBE3.6, or a pharma- ceutically acceptable salt of any of the foregoing. Compounds of structural formula I also include MBE1.5A, MBE1.5B, MBE1.5C and MBE1.5D, or a pharma- ceutically acceptable salt of any of the foregoing.

[0057] In a second embodiment, the compound is a compound represented by structural formula II: [ka] or a pharma- ceutically acceptable salt thereof, wherein: R 1 and R 5 are each independently hydrogen or (C 1 -C 6 ) alkyl; R 2 and R 4 are each independently hydrogen, halo, halo(C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkoxy, (C 1 -C 3 ) thioalkoxy, halo(C 1 -C 3 ) alkoxy, amino, (C 1 -C 3 ) alkylamino, (C 1 -C 3 ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl; R 3 Ha, halo, halo (C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkoxy, (C 1 -C 3 ) thioalkoxy, halo(C1 -C 3 ) alkoxy, amino, (C 1 -C 3 ) alkylamino, (C 1 -C 3 ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl; L is -C(O)-, -C(R 6 )(R 7 )-, -C(CH 2 CH 2 )-, -C(OCH 2 )- or -C(N(H)CH 2 )- and; R 6 and R 7 are each independently hydrogen, hydroxy, sulfhydryl, halo, (C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkoxy, (C 1 -C 3 ) thioalkoxy or halo(C 1 -C 3 ) alkoxy; Each R 9 are independently halo, halo(C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkoxy, (C 1 -C 6 ) thioalkoxy, halo(C 1 -C 6 ) alkoxy, amino, (C 1 -C 6 ) alkylamino, (C 1 -C 6 ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl; and n is 0, 1, 2, 3, 4, or 5. Alternative values ​​for the variables are as described in the first embodiment, or any aspect thereof.

[0058] In a first aspect of the second embodiment, each R 9are independently halo, halo(C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkoxy, (C 1 -C 3 ) thioalkoxy, halo(C 1 -C 3 ) alkoxy, amino, (C 1 -C 3 ) alkylamino, (C 1 -C 3 ) dialkylamino, cyano, nitro, hydroxy, or sulfhydryl. The values ​​of the remaining variables are as described in the first embodiment, or any aspect thereof, or the second embodiment.

[0059] In a second aspect of the second embodiment, each R 9 are independently halo or (C 1 -C 6 ) alkyl (e.g., halo or (C 1 -C 3 ) alkyl). The values ​​of the remaining variables are as described in the first embodiment, or any aspect thereof, or the second embodiment, or first aspect thereof.

[0060] In a third aspect of the second embodiment, n is 0, 1 or 2 (e.g., n is 0, n is 1 or n is 2). The values ​​of the remaining variables are as described in the first embodiment, or any aspect thereof, or the second embodiment, or the first or second aspect thereof.

[0061] In a fourth aspect of the second embodiment, each R 9 are independently (C 1 -C 6 ) alkyl. Values ​​for the remaining variables are as described in the first embodiment, or any aspect thereof, or the second embodiment, or first through third aspects thereof.

[0062] In a fifth aspect of the second embodiment, n is 1 or 2 (e.g., 1). The values ​​of the remaining variables are as described in the first embodiment, or any aspect thereof, or the second embodiment, or the first to fourth aspects thereof.

[0063] Compounds of formula II include MBE1.5, or a pharma- ceutically acceptable salt thereof. Compounds of formula II also include MBE1.5A, MBE1.5B, MBE1.5C, and MBE1.5D, or a pharma- ceutically acceptable salt thereof.

[0064] A third embodiment is a compound represented by structural formula III: [ka] or a pharma- ceutically acceptable salt thereof, wherein the values ​​of the variables are as described in the first or second embodiment, or any aspect preceding this one.

[0065] A fourth embodiment is a compound represented by structural formula IV: [ka] or a pharma- ceutically acceptable salt thereof, wherein: Each R 9 are independently halo, halo(C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkoxy, (C 1 -C 6 ) thioalkoxy, halo(C 1 -C 6 ) alkoxy, amino, (C 1 -C 6 ) alkylamino, (C 1 -C 6 ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl; and m is 0, 1, 2, 3 or 4. Variable R 9and the values ​​of the remaining variables are as described in the first or second embodiment, or any of the aspects previously described.

[0066] In a first aspect of the fourth embodiment, X 3 is -C(R 8 )-. The values ​​of the remaining variables are as described in the first or second embodiment or any of the previous aspects, or the fourth embodiment.

[0067] In a second aspect of the fourth embodiment, X 3 is -N-. The values ​​of the remaining variables are as described in the first or second embodiment or any of the preceding aspects, or the fourth embodiment, or its first aspect.

[0068] In a third aspect of the fourth embodiment, m is 0, 1 or 2. The values ​​of the remaining variables are as described in the first or second embodiment, or any of the preceding aspects, or the fourth embodiment, or its first or second aspects.

[0069] In a fourth aspect of the fourth embodiment, R 3 Ha, halo, halo (C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkyl, (C 1 -C 3 ) alkoxy, (C 1 -C 3 ) thioalkyloxy, halo(C 1 -C 3 ) alkoxy, amino, (C 1 -C 3 ) alkylamino, (C 1 -C 3 ) dialkylamino, cyano, nitro, hydroxy, or sulfhydryl. The values ​​of the remaining variables are as described in the first or second embodiment, or any of the preceding aspects, or the fourth embodiment, or first through third aspects thereof.

[0070] In a fifth aspect of the fourth embodiment, the compound is not MBE1 or MBE3.6, or a pharma- ceutically acceptable salt thereof. The values ​​of the remaining variables are as described in the first or second embodiment, or any of the preceding aspects, or the fourth embodiment, or any of the first through fourth aspects thereof.

[0071] A fifth embodiment is a compound represented by structural formula V: [ka] or a pharma- ceutically acceptable salt thereof. The values ​​of the variables are as described in the first, second or fourth embodiment, or any of the preceding aspects.

[0072] Compounds of formula V include MBE1.2, MBE1.3 and MBE1.6, or pharma- ceutically acceptable salts of the foregoing.

[0073] A sixth embodiment is a compound represented by structural formula VI: [ka] or a pharma- ceutically acceptable salt thereof, wherein R 8’ is hydrogen, halo, halo(C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkoxy, (C 1 -C 6 ) thioalkoxy, halo(C 1 -C 6 ) alkoxy, amino, (C 1 -C 6 ) alkylamino, (C 1 -C 6 ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl (e.g., hydrogen or (C 1 -C 6 ) alkyl). The values ​​of the remaining variables are as described in the first, second or fourth embodiment, or any aspect preceding this embodiment.

[0074] In a first aspect of the sixth embodiment, R 8 is (C 1 -C 6 ) alkyl. The values ​​of the remaining variables are as described in the first, second or fourth embodiment, or any of the preceding aspects, or the sixth embodiment.

[0075] A seventh embodiment is a compound represented by structural formula VII: [ka] or a pharma- ceutically acceptable salt thereof, wherein R 8’ is hydrogen, halo, halo(C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkyl, (C 1 -C 6 ) alkoxy, (C 1 -C 6 ) thioalkoxy, halo(C 1 -C 6 ) alkoxy, amino, (C 1 -C 6 ) alkylamino, (C 1 -C 6 ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl (e.g., hydrogen or (C 1 -C 6 ) alkyl). The values ​​of the remaining variables are as described in the first, second, fourth or sixth embodiment, or any aspect preceding this embodiment.

[0076] Compositions and Kits Provided herein are compositions (e.g., pharma- ceutically acceptable compositions) that include an agent that inhibits ALDH1a3 (e.g., a compound of any of structural formulas I-V, or a pharma- ceutically acceptable salt of the foregoing), and a pharma- ceutically acceptable carrier or excipient. In certain embodiments, the compositions of the invention are formulated for administration to a subject (e.g., a patient) in need of the composition. In some embodiments, the compositions of the invention are formulated for oral, intravenous, subcutaneous, intraperitoneal, or dermatological administration to a subject in need thereof.

[0077] The phrase "pharmaceutically acceptable carrier or excipient" refers to a non-toxic carrier or excipient that does not destroy the pharmacological activity of the drug with which it is formulated and is non-toxic when administered in a sufficient dose to deliver a therapeutic amount of the drug.The pharmaceutically acceptable carrier or excipient that may be used in the compositions described herein includes, but is not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

[0078] The compositions described herein may be administered orally, parenterally (including subcutaneously, intramuscularly, intravenously and intradermally), by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. In some embodiments, provided compounds or compositions can be administered intravenously and / or intraperitoneally.

[0079] The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraocular, intravitreal, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intrahepatic, intraperitoneal, intralesional and intracranial injection or infusion techniques. Preferably, the compositions are administered orally, subcutaneously, intraperitoneally or intravenously.

[0080] The compositions provided herein can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, dispersions and solutions.For tablets for oral use, commonly used carriers include lactose and cornstarch.Lubricants such as magnesium stearate are also typically added.For oral administration in capsule form, useful diluents include lactose and dried cornstarch.When aqueous suspensions and / or emulsions are required for oral use, the active ingredient can be suspended or dissolved in an oil phase and combined with emulsifying and / or suspending agents.If desired, certain sweeteners, flavorings or colorings can be added.

[0081] In some embodiments, the oral formulations are formulated for immediate or sustained / delayed release.

[0082] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharma- ceutically acceptable excipient or carrier (e.g., sodium citrate or dicalcium phosphate) and / or (a) fillers or extenders (e.g., starch, lactose, sucrose, glucose, mannitol, and silicic acid), (b) binders (e.g., carboxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidone, sucrose, and acacia), (c) humectants (e.g., glycerol), (d) disintegrants (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), (e) solution retarders (e.g., glycerol), (f) disintegrants (e.g., agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), (g) dissolution inhibitors (e.g., glycerol), (h) disintegrants (e.g., glycerol), (i) disintegrants (e.g., glycerol), (j ... agents (e.g., paraffin), (f) absorption enhancers (e.g., quaternary ammonium salts), (g) wetting agents (e.g., acetyl alcohol and glycerol monostearate), (h) absorbents (e.g., kaolin and bentonite clay), and (i) lubricants (e.g., talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof). In the case of capsules, tablets, and pills, the dosage forms may also contain buffering agents.

[0083] Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, where the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.

[0084] Solid compositions of a similar type may also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills and granules may be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents, and may also be of a composition that optionally releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract in a delayed manner. Examples of embedding compositions that may be used include polymeric substances and waxes.

[0085] The drug may also be in the form of microencapsulation with one or more excipients as described above.In such solid dosage forms, the drug may be mixed with at least one inert diluent such as sucrose, lactose or starch.Such dosage forms may also contain additional substances other than the inert diluent, such as tableting lubricants and other tableting aids, as is common practice, such as magnesium stearate and microcrystalline cellulose.

[0086] Compositions for oral administration may be designed to protect the active ingredient against degradation as it passes through the digestive tract, for example by an outer coating of the formulation on a tablet or capsule.

[0087] In another embodiment, the drug may be provided in an extended (or "delayed" or "sustained") release composition. This delayed release composition includes the drug in combination with a delayed release component. Such a composition allows for targeted release of the provided drug to the lower gastrointestinal tract, e.g., the small intestine, large intestine, colon, and / or rectum. In certain embodiments, the delayed release composition further includes an enteric or pH-dependent coating, such as cellulose acetate phthalate and other phthalates (e.g., polyvinyl acetate phthalate, methacrylates (Eudragits)). Alternatively, the delayed release composition provides sustained release to the small intestine and / or colon by providing a pH-sensitive methacrylate coating, pH-sensitive polymeric microspheres, or a polymer that undergoes hydrolytic degradation. The delayed release composition may be formulated with hydrophobic excipients or coatings or gelling excipients or coatings. Colonic delivery may further be provided by coatings that are digested by bacterial enzymes such as amylose or pectin, by pH-dependent polymers, by hydrogel plugs that swell over time (Pulsincap), by time-dependent hydrogel coatings, and / or by acrylic acid bound to an azoaromatic binding coating.

[0088] The compositions described herein can also be administered in the form of suppositories for rectal administration.These can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycol.

[0089] The compositions described herein may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0090] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topical-transdermal patches may also be used.

[0091] For other topical applications, the composition may be formulated in a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of drugs include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water and penetration enhancers. Alternatively, the composition may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more pharma- ceutically acceptable carriers. Alternatively, the composition may be formulated in a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with a suitable emulsifier. In some embodiments, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. In other embodiments, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water and penetration enhancers.

[0092] For ophthalmic use, the composition may be formulated as a micronized suspension in isotonic, pH-adjusted, sterile saline, with or without a preservative such as benzylalkonium chloride, or, preferably, as a solution in isotonic, pH-adjusted, sterile saline. Alternatively, for ophthalmic use, the composition may be formulated in an ointment such as petrolatum.

[0093] The compositions may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0094] The amount of drug that can be combined with the carrier materials to produce a composition in a single dosage form will vary depending on the host being treated, the particular mode of administration, and the activity of the drug being used. Preferably, the composition should be formulated so that a dosage of about 0.01 mg / kg to about 100 mg / kg body weight / day of drug can be administered to a subject receiving the composition.

[0095] It should also be understood that the specific administration and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific agent used, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the judgment of the treating physician, and the severity of the particular disease being treated. The amount of agent in the composition will also depend on the particular agent in the composition.

[0096] Other pharma- ceutically acceptable carriers, adjuvants and solvents that may be used in the compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS), such as da-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms, such as Tween or other similar polymeric delivery matrices, serum proteins, such as human serum albumin, buffer substances, such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat. Cyclodextrins, such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives, such as hydroxyalkyl cyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrin, or other solubilized derivatives, may also be advantageously used to enhance delivery of the agents described herein.

[0097] The composition may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation may be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable solvents and solvents that may be used are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are commonly used as a solvent or suspending medium. For this purpose, any bland, fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharma- ceutically acceptable oils such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersing agents, which are commonly used in the formulation of pharma- ceutically acceptable dosage forms such as emulsions and / or suspensions. Other commonly used surfactants, such as Tween or Span and / or other similar emulsifying agents or bioavailability enhancers, which are commonly used in the manufacture of pharma- ceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0098] In some embodiments, a composition comprising an agent that inhibits ALDH1a3 (e.g., a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof) can also contain one or more other therapeutic agents (e.g., chemotherapeutic agents, e.g., paclitaxel, doxorubicin, 5-fluorouracil, tamoxifen, octreotide). When the compositions of the present invention comprise a combination of an agent that inhibits ALDH1a3 with one or more other therapeutic agents, the agents should be present at a dosage level of about 1-100%, and more preferably about 5%-95% of the dosage normally administered in a monotherapy regimen. The additional agent(s) can be administered separately from the agent that inhibits ALDH1a3 as part of a multiple dose regimen. Alternatively, the additional agent(s) can be part of a single dosage form, mixed together with the agent that inhibits ALDH1a3 in a single composition.

[0099] The compositions described herein may be administered, for example, by injection, intravenously, intraarterially, intraocularly, intravitreously, subcutaneously, orally, buccally, nasally, transmucosally, topically, in ophthalmic solutions, or by inhalation, at doses ranging from about 0.5 mg / kg to about 100 mg / kg body weight, or alternatively, at doses ranging from about 1 mg / dose to about 1000 mg / dose, every 4 to 120 hours, or according to the requirements of the particular drug. Typically, the compositions will be administered about 1 to about 6 times per day, or alternatively, as a continuous infusion. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending on the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w / w). Alternatively, the preparation may contain from about 20% to about 80% active compound (w / w).

[0100] Doses lower or higher than those mentioned above may be required. The specific administration and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the particular drug used, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, severity and course of disease, condition or symptom, predisposition of the patient to the disease, condition or symptom, and the judgment of the treating physician.

[0101] Once the subject's condition has improved, a maintenance dose of the agent (e.g., any compound of structural formula I-VII, or a pharma- ceutically acceptable salt thereof), composition, or combination of the present invention may be administered as needed. Thereafter, the dose or frequency of administration, or both, may be reduced as a function of symptoms, to a level at which the improved condition is maintained when symptoms are alleviated to a desired level. However, the subject may require intermittent treatment over a long period of time upon recurrence of disease symptoms.

[0102] Also provided herein are kits comprising an agent described herein (e.g., a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt of the foregoing) and an additional agent(s). In one embodiment, the kit comprises an effective amount of an agent described herein to treat a disease, disorder, or condition described herein (e.g., cancer, type 2 diabetes), and an effective amount of an additional agent(s) to treat the disease, disorder, or condition. In some embodiments, the kit further comprises instructions for administering the agent and additional agent(s) to a subject to treat a disease, disorder, or condition described herein (e.g., cancer, type 2 diabetes).

[0103] method Aldehyde dehydrogenases (ALDHs) belong to a superfamily of NAD(P+)-dependent enzymes that play a role in aldehyde metabolism by irreversibly catalyzing the oxidation of both endogenously and exogenously generated aldehydes to their respective carboxylic acids. ALDHs have a wide range of biological activities, including retinoic acid (RA) biosynthesis and alcohol metabolism.

[0104] The ALDH family of enzymes includes 19 members with diverse functions. Enzymes within this family irreversibly catalyze the oxidation of aldehydes to the corresponding carboxylic acids while reducing NAD+ / NADP+ to NADH / NADPH. These enzymes are found in several cellular compartments, but most are localized in the cytosol or mitochondria. Some ALDH enzymes participate in global metabolism through expression in the liver, where they function to detoxify acetylaldehydes formed from alcohol dehydrogenase, biosynthesize vitamin A from retinal stereoisomers, or detoxify other reactive aldehydes. In contrast, most ALDH enzymes are expressed in a cell- or disease-specific manner and regulate cellular biochemistry, although the mechanism of action is often unclear.

[0105] Aldehyde dehydrogenase isoform 1a3 (ALDH1a3) is a cell- and disease-specific isoform / isoenzyme of the ALDH1a subfamily that is crucial in the control of RA biosynthesis and RA signaling. ALDH1a3 was known as ALDH6 before 2000 and as Raldh3 from 2000 to 2007. Under normal conditions, ALDH1a3 is only required during embryonic development and is dispensable in healthy adult mice. In contrast to its minor role in normal physiology, ALDH1a3 has recently been shown to be a major determinant of ALDEFLUOR™ responsiveness across most cancer types. Despite excessive ignorance about whether / how ALDEFLUOR™ activity affects tumor progression, ALDEFLUOR™ activity has long been used as a marker to distinguish aggressive cancer cells from bulk tumors.

[0106] It has been found that ALDEFLUOR™ activity driven by ALDH1a3 is a functional driver of cancer invasiveness and is crucial for tumor progression, metastasis, and resistance to chemotherapy. Thus, human ALDH1a3 (UniProtKB Accession Number: P47895) is a functional driver of chemotherapy resistance and metastatic phenotype in cancer, including breast cancer. Thus, ALDH1a3 represents a potential therapeutic target in multiple disease conditions, and targeting ALDH1a3 may overcome the current barriers in the treatment of stage 3 / 4 patients whose tumors are resistant to conventional therapies.

[0107] Thus, provided herein is a method for treating cancer (e.g., metastatic cancer, chemotherapy-resistant cancer) in a subject in need thereof, comprising administering to the subject an effective amount of an agent that inhibits ALDH1a3 (e.g., a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof, or a composition comprising a compound of any of structural formulas I-VII or a pharma- ceutically acceptable salt thereof). Thus, in some embodiments, provided is a method for treating cancer (e.g., metastatic cancer, chemotherapy-resistant cancer) in a subject in need thereof, comprising administering to the subject an effective amount of a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof, or a composition comprising a compound of any of structural formulas I-VII or a pharma- ceutically acceptable salt thereof. In a further embodiment, there is provided a method of treating cancer (e.g., metastatic cancer, chemotherapy resistant cancer) in a subject in need thereof comprising administering to the subject an effective amount of a compound of Table 1 (e.g., MBE1, MBE1.5, MBE1.5A, MBE1.5B, MBE1.5C, MBE1.5D), or a pharma- ceutically acceptable salt thereof, or a composition comprising a compound of Table 1 (e.g., MBE1, MBE1.5, MBE1.5A, MBE1.5B, MBE1.5C, MBE1.5D) or a pharma- ceutically acceptable salt thereof.

[0108] Also provided herein are methods for treating type 2 diabetes in a subject in need thereof, comprising administering to the subject an effective amount of an agent that inhibits ALDH1a3 (e.g., a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof, or a composition comprising a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof). Thus, in some embodiments, there are provided methods for treating type 2 diabetes in a subject in need thereof, comprising administering to the subject an effective amount of a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof, or a composition comprising a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof. In a further embodiment, there is provided a method for treating type 2 diabetes in a subject in need thereof comprising administering to the subject an effective amount of a compound of Table 1 (e.g., MBE1, MBE1.5, MBE1.5A, MBE1.5B, MBE1.5C, MBE1.5D), or a pharma- ceutically acceptable salt thereof, or a composition comprising a compound of Table 1 (e.g., MBE1, MBE1.5, MBE1.5A, MBE1.5B, MBE1.5C, MBE1.5D) or a pharma- ceutically acceptable salt thereof.

[0109] As used herein, the terms "treat," "treating," or "treatment" refer to alleviating a medical condition (e.g., a medical condition associated with cancer) to the extent that the medical condition is improved according to clinically accepted criteria (e.g., a reduction in the number of cancer metastases in a subject).

[0110] In one embodiment, the subject (e.g., a patient) is a mammal (e.g., a human, non-human primate, cow, sheep, goat, horse, dog, cat, rabbit, guinea pig, rat, mouse or other bovine, ovine, equine, canine, feline, or rodent organism). In certain embodiments, the subject is a human. A "subject in need thereof" refers to a subject having or at risk of developing a disease or condition described herein (e.g., cancer, metastatic cancer). A skilled medical professional (e.g., physician) can readily determine whether a subject has or is at risk of developing a disease or condition described herein (e.g., cancer).

[0111] In one embodiment, the subject in need thereof has cancer (e.g., metastatic cancer, chemotherapy-resistant cancer). The cancer may be a solid tumor, leukemia, lymphoma, or myeloma. In certain embodiments, the subject in need thereof has breast cancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer, bone cancer, blood cancer, brain cancer, or liver cancer. In some embodiments, the cancer is breast cancer (e.g., triple-negative breast cancer), renal clear cell carcinoma, gastric cancer, bladder cancer, ovarian cancer, squamous cell lung cancer, colorectal cancer, or glioma (e.g., low-grade glioma).

[0112] Cancers include:Acute Lymphoblastic Leukemia, Adult;Acute Lymphoblastic Leukemia, Childhood;Acute Myeloid Leukemia, Adult;Adrenal Cortical Carcinoma;Adrenal Cortical Carcinoma, Childhood;AIDS-Related Lymphoma;AIDS-Related Malignancies;Anal Cancer;Astrocytoma, Childhood Cerebellar;Astrocytoma, Childhood Cerebral;Choledochocarcinoma, Extrahepatic;Bladder Cancer;Bladder Cancer, Childhood;Bone Cancer, Osteosarcoma / Malignant Fibrous Histiocytoma;Brain Stem Glioma, Childhood;Brain Tumor, Adult;Brain Tumor, Brain Stem Glioma, Childhood;Brain Tumor, Cerebellar Astrocytoma, Childhood;Brain Tumor, Cerebral Astrocytoma / Malignant Glioma, Childhood;Brain Tumor, Ependymoma, Childhood;Brain Tumor, Medulloblastoma, Childhood;Brain Tumor, Supratentorial Primitive Neuroectodermal Tumor Neuroectodermal Tumors), childhood;Brain Tumors, visual pathway gliomas and hypothalamic gliomas, childhood;Brain Tumors, childhood (other);Breast cancer;Breast cancer and pregnancy;Breast cancer, childhood;Breast cancer, male;Bronchial adenoma / carcinoid, childhood;Carcinoid tumors, childhood;Carcinoid tumors, gastrointestinal;Carcinoma, adrenal cortex;Carcinoma, pancreatic islet cell;Carcinoma of unknown primary, central nervous system lymphoma, primary;Cerebellar astrocytoma, childhood;Cerebral astrocytoma / malignant glioma , childhood;cervical cancer;childhood cancer;chronic lymphocytic leukemia;chronic myelogenous leukemia;chronic myeloproliferative disorders;clear cell sarcoma of tendon sheath;colon cancer;colorectal cancer, childhood;cutaneous T-cell lymphoma;endometrial cancer;ependymoma, childhood;epithelial carcinoma, ovary;esophageal cancer;esophageal cancer, childhood;Ewing's tumor;extracranial germ cell tumor, childhood;extragonadal germ cell tumor;extrahepatic bile duct carcinoma;eye cancer, intraocular melanoma;eye cancer, retinoblastoma;gallbladder cancer;gastric (stomach) cancer (Gastric (Stomach) Cancer);Gastric (stomach) cancer, childhood;Gastrointestinal carcinoid;Germ cell tumors, extracranial, childhood;Germ cell tumors, extragonadal;Germ cell tumors, ovarian;Gestational trophoblastic tumor;Glioma, childhoodBrain stem;Glioma, childhoodVisual pathway and hypothalamic;Hairy cell leukemia;Head and neck cancer;Hepatocellular (liver) carcinoma, adult (primary);Hepatocellular (liver) carcinoma, childhood (primary);Hodgkin's lymphoma, adult;Hodgkin's lymphoma, childhood;Hodgkin's lymphoma during pregnancy;Hypopharyngeal carcinoma;Hypothalamic and visual pathway glioma, childhood;Intraocular melanoma;Islet cell carcinoma (endocrine pancreas);Kaposi's sarcoma;Kidney carcinoma;Laryngeal cancer;Laryngeal cancer, childhood;Leukemia, acute lymphoblastic, adult;Leukemia, acute lymphoblastic, childhood;Leukemia, acute myeloid, adult;Leukemia, acute myeloid, childhood;Leukemia, chronic lymphocytic;Leukemia, chronic myeloid;Leukemia, hairy cell;Lip and oral cavity cancer;Liver cancer, adult (primary);Liver cancer, childhood (primary);Lung cancer, non-small cell;Lung cancer, small cell;Lymphoblastic leukemia, adult acute;Lymphoblastic leukemia, childhood acute;Lymphoblastic leukemia, chronic;Lymphoma, AIDS-related;Lymphoma, central nervous system (primary);Lymphoma, cutaneous T cell;Lymphoma, Hodgkin, adult;Lymphoma, Hodgkin, childhood stage;lymphoma, Hodgkin during pregnancy;lymphoma, non-Hodgkin, adult;lymphoma, non-Hodgkin, childhood;lymphoma, non-Hodgkin during pregnancy;lymphoma, primary central nervous system;macroglobulinemia, Waldenstrom type;male breast cancer;malignant mesothelioma, adult;malignant mesothelioma, childhood;malignant thymoma;mantle cell lymphoma;medulloblastoma, childhood;melanoma;melanoma, intraocular;Merkel cell carcinoma;mesothelioma, malignant;occult primary metastatic squamous cell cervical carcinoma;multiple endocrine neoplasia syndrome, childhood;multiple myeloma / plasma cell neoplasm;mycosis fungoides;myelodysplastic syndrome;myeloid leukemia, chronic;myeloid leukemia Hematologic diseases, childhood, acute;Myeloma, multiple;Myeloproliferative disorders, chronic;Nasal and paranasal sinus cancer;Nasopharyngeal cancer;Nasopharyngeal cancer, childhood;Neuroblastoma;Non-Hodgkin's lymphoma, adult;Non-Hodgkin's lymphoma, childhood;Non-Hodgkin's lymphoma during pregnancy;Non-small cell lung cancer;Oral cavity cancer, childhood;Oral cavity and lip cancer;Oropharyngeal cancer;Osteosarcoma / malignant fibrous histiocytoma of bone;Ovarian cancer, childhood;Ovarian epithelial cancer;Ovarian germ cell tumor;Ovarian low malignant potential tumor;Pancreatic cancer;Pancreatic cancer, childhood;Pancreatic islet cell cancer;Sinonasal and nasal cancer;Parathyroid cancer;Penile cancer;Pheochromocytoma;Pineal and supratentorial primitive neuroectodermal tumors, childhood; Pituitary tumors;Plasma cell neoplasms / multiple myeloma;Pleuropulmonary blastoma;Pregnancy and breast cancer;Pregnancy and Hodgkin's lymphoma;Pregnancy and non-Hodgkin's lymphoma;Primary central nervous system lymphoma;Primary liver cancer, adult;Primary liver cancer, childhood;Prostate cancer;Rectal cancer;Renal cell (kidney) cancer;Renal cell carcinoma, childhood;Renal pelvis and ureter, transitional cell carcinoma;Retinoblastoma;Rhabdomyosarcoma, childhood;Salivary gland cancer;Salivary gland cancer, childhood;Sarcoma, Ewing's tumor;Sarcoma, Kaposi;Sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone;Sarcoma, rhabdomyosarcoma, childhood;Sarcoma, soft tissue, adult;Sarcoma, soft tissue, childhood;Sezary syndrome;skin cancer;skin cancer, childhood;skin cancer (melanoma);skin cancer, Merkel cell;small cell lung cancer;small intestine cancer;soft tissue sarcoma, adult;soft tissue sarcoma, childhood;occult primary squamous cell cervical carcinoma, metastatic;stomach (gastric) cancer;stomach (gastric) cancer, childhood;supratentorial primitive neuroectodermal tumor, childhood;T-cell lymphoma, cutaneous;testicular cancer;thymoma, childhood;thymoma, malignant;thyroid cancer;thyroid cancer, childhood;transitional cell carcinoma of the renal pelvis and ureter;trophoblastic tumor, gestational;cancer of unknown primary site, childhood;rare cancer of childhood;ureter and renal pelvis, transitional cell carcinoma;urethral cancer;uterine sarcoma;vaginal cancer;visual pathway glioma and hypothalamic glioma, childhood;vulvar cancer;Waldenstrom macroglobulinemia;and Wilms' tumor.;

[0113] Metastases of the above cancers may also be treated according to the methods described herein. Thus, in some embodiments, the cancer is a metastatic cancer.

[0114] "Metastatic cancer" as used herein refers to a cancer that has spread from its primary site of origin or where it first formed to other areas of the subject's body. Examples of metastatic cancer include metastatic breast cancer, metastatic colon cancer, metastatic lung cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic bone cancer, metastatic blood cancer, metastatic brain cancer, and metastatic liver cancer. In one embodiment, the subject has bone metastasis (e.g., natural bone metastasis) of breast cancer. In another embodiment, the subject has lung metastasis of breast cancer. In yet another embodiment, the subject has bone metastasis.

[0115] In certain embodiments, the subject in need thereof has breast cancer (e.g., metastatic breast cancer). Breast cancer subtypes that are particularly suitable for treatment using the methods described herein include, but are not limited to, basal-like breast cancer and HER2-positive breast cancer.

[0116] "Resistant cancer," as used herein, is a cancer that does not respond to treatment. "Resistant cancer" includes cancers that do not respond to treatment at the start of treatment and cancers that become unresponsive to treatment during treatment. In one embodiment, the cancer is a resistant cancer.

[0117] In one embodiment, a subject in need thereof has a cancer that is resistant to treatment with one or more chemotherapeutic agents, or a chemotherapy-resistant cancer. "Chemoresistant cancer" as used herein refers to cancer that does not respond to treatment with one or more chemotherapeutic agents. "Chemoresistant cancer" includes cancer that does not respond to treatment with one or more therapeutic agents at the beginning of treatment, and cancer that becomes unresponsive to treatment with one or more therapeutic agents during treatment.Chemoresistant cancer that is particularly suitable for treatment using the methods described herein includes, but is not limited to, cancer that is resistant to treatment with paclitaxel and / or doxorubicin.

[0118] In one embodiment, the subject in need thereof has an invasive cancer. As used herein, "invasive cancer" refers to a cancer that progresses (e.g., forms, grows, and / or metastasizes) rapidly or requires more severe or intensive treatment than typical cancer treatment for the same type of cancer. Invasive cancer includes, but is not limited to, lung, breast, pancreas, kidney, colon, rectum, brain, liver, and prostate or blood cancer that does not respond to conventional treatment, such as chemotherapy (including taxanes, anthracyclines, alkylating agents, etc.), radiation therapy (e.g., proton beam radiation) or targeted biological therapy (e.g., Herceptin, Keytruda, etc.), or progresses during treatment with any accepted therapy, or metastasizes at the time of diagnosis (stage III / IV cancer). As a specific example, invasive cancer of the prostate loses response to antiandrogen therapy or radiation and often metastasizes to bone, while invasive melanoma is any tumor that has spread beyond the dermis. All diagnoses of glioblastoma multiforme are considered invasive. Invasive cancers often spread to distant sites and cause significant morbidity due to the lack of targeted therapies.

[0119] In some embodiments, a subject in need thereof has advanced (eg, stage III / IV) cancer.

[0120] In some embodiments, the cancer is a primary cancer (e.g., a primary solid tumor). As used herein, "primary cancer" refers to the site of origin of a cancer (e.g., a tumor) within a subject's body.

[0121] As used herein, an "effective amount" is an amount of an agent that, when administered to a subject, is sufficient to achieve a desired therapeutic effect in the subject under the conditions of administration (e.g., an amount sufficient to inhibit (e.g., reduce, decrease, prevent) tumor formation, tumor growth (e.g., growth, size), tumor angiogenesis, tumor progression (e.g., invasion, metastasis), and / or chemotherapy resistance in a subject (e.g., patient) with cancer). The effectiveness of the treatment (e.g., reduction and / or prevention of tumor metastasis) can be determined by any suitable method known to the skilled artisan (e.g., in situ immunohistochemistry, imaging (e.g., ultrasound, CT scan, MRI, NMR), 3 H-thymidine incorporation).

[0122] The effective amount of the agent to be administered can be determined by a clinician of ordinary skill using the guidance provided herein and other methods known in the art, and depends on several factors, including, for example, the particular agent selected, the subject's age, sensitivity, tolerance to the drug, and overall health. For example, a suitable dose may be about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.01 mg / kg to about 1 mg / kg body weight per treatment. Determining the dosage for a particular agent, subject, and cancer is well within the capabilities of one of ordinary skill in the art. Preferably, the dosage does not cause or minimizes adverse side effects (e.g., immunogenic response, nausea, dizziness, hyperactivity, hyperviscosity syndrome, congestive heart failure, stroke, pulmonary edema).

[0123] The agent that inhibits ALDH1a3 can be administered in a single dose or multiple doses, for example, in a sequence and schedule suitable for achieving the desired therapeutic effect (e.g., inhibiting cancer metastasis).Appropriate doses and regimens of administration can be determined by a clinician of ordinary skill.

[0124] The agent that inhibits ALDH1a3 can also be administered in combination with one or more other therapies or treatments.For administering the agent in combination with one or more other therapies or treatments (such as adjuvant, targeting, cancer treatment-related, etc.), the agent is typically administered as a single dose (e.g., injection, infusion, oral), and then repeatedly administered at specific intervals (e.g., one hour or more) if desired or indicated.

[0125] In one embodiment, the drug can be administered in metronomic dosing regimen, whereby lower doses are administered more frequently compared to maximum tolerated dose.Many preclinical studies have shown that metronomic regimen has superior antitumor activity, strong antiangiogenic effect, and reduced toxicity and side effects (e.g., myelosuppression) compared to maximum tolerated dose (MTD) counterpart (Bocci, et al., Cancer Res,62:6938-6943,(2002); Bocci, et al., Proc. Natl. Acad. Set,100(22): 12917-12922,(2003); and Bertolini, et al., Cancer Res,63(15):4342-4346,(2003)).Metronomic chemotherapy appears to be effective in overcoming some of the drawbacks associated with chemotherapy.

[0126] An agent that inhibits ALDH1a3 can be administered to a subject in need thereof as a primary therapy (e.g., as the primary therapeutic agent in a therapy or treatment regimen); as an adjunct therapy (e.g., as a therapeutic agent used together with another therapeutic agent in a therapy or treatment regimen, where the combination of therapeutic agents provides the desired treatment; "adjunct therapy" is also called "adjunctive therapy"); in combination with adjunct therapy; as adjuvant therapy (e.g., as a therapeutic agent given to a subject in need thereof after the primary therapeutic agent in a therapy or treatment regimen has been given); or in combination with adjuvant therapy. Adjuvant therapies include, for example, chemotherapy (e.g., paclitaxel, doxorubicin, tamoxifen, cisplatin, mitomycin, 5-fluorouracil, sorafenib, octreotide, dacarbazine (DTIC), cisplatin, cimetidine, cyclophosphamide), radiation therapy (e.g., proton beam therapy), hormone therapy (e.g., anti-estrogen therapy, androgen deprivation therapy (ADT), luteinizing hormone releasing hormone (LH-RH) agonists, aromatase inhibitors (AIs, e.g., anastrozole, exemestane, letrozole), estrogen receptor modulators (e.g., tamoxifen, raloxifene, toremifene), or biological therapy. Many other therapies may also be administered during a cancer treatment regime to mitigate the effects of the disease and / or side effects of cancer treatment, including therapies to manage symptoms such as nausea (antiemetic medications), infections (e.g., medications to increase red / white blood cell counts), and the like, all of which will be readily recognized by those of skill in the art.

[0127] In some embodiments, the method includes administering an effective amount of an agent that inhibits ALDH1a3 in combination with one or more additional therapies (e.g., chemotherapy, radiation, and / or surgical removal of tumor(s)). When administered in a combination therapy, the agent may be administered before, after, or simultaneously with the other therapy (e.g., administration of a chemotherapeutic agent such as paclitaxel or doxorubicin). When administered simultaneously (e.g., concurrently), the agent and the other therapy may be in separate formulations or the same formulation. Alternatively, the agent and the other therapy may be administered sequentially as separate compositions within an appropriate time frame (e.g., a cancer treatment session / interval, such as about 1.5 to about 5 hours) as determined by a skilled clinician (e.g., a time sufficient to allow overlap of the pharmacological effects of the treatments).

[0128] The agent that inhibits ALDH1a3 can be administered through various administration routes, including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intraarterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous drip and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drip) administration routes, depending on the agent and the specific cancer to be treated.Administration can be local or systemic, as indicated.Preferred administration mode can vary depending on the specific agent selected.

[0129] The actual dosage of therapeutic agents and treatment regimens can be determined by the physician, taking into account, for example, the nature of the cancer (primary or metastatic), the number and size of the tumors, other treatments being given, and the characteristics of the subject.

[0130] Also provided herein is a method of inhibiting the proliferation of a cancer cell (e.g., a metastatic cancer cell, a chemotherapy-resistant cancer cell). The method includes administering (e.g., an effective amount of) an agent that inhibits ALDH1a3 (e.g., a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof, or a composition comprising a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof) to the cell. In certain embodiments, the cancer cell is a breast cancer cell (e.g., a basal-like breast cancer cell or a HER-2 positive breast cancer cell). The cell can be a cultured cell (e.g., a cell line) or a cell in a subject. In certain embodiments, the cell is present in a human subject (e.g., a human subject having cancer).

[0131] Also provided herein are methods of inhibiting ALDH1a3 in a subject in need thereof, the methods including administering to the subject (e.g., an effective amount of) an agent that inhibits ALDH1a3 (e.g., a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof, or a composition comprising a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof).

[0132] Also provided herein is a method for treating a disease or disorder mediated by ALDH1a3 (such as a cancer type described herein) in a subject in need thereof. The method includes administering to the subject an effective amount of an agent that inhibits ALDH1a3 (such as a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof, or a composition comprising a compound of any of structural formulas I-VII, or a pharma- ceutically acceptable salt thereof). ALDH1a3 is also involved in type 2 diabetes. Thus, type 2 diabetes is another example of a disease or disorder mediated by ALDH1a3. EXAMPLES

[0133] Synthesis of N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide [ka]

[0134] 3-Chloro-N-(3-fluorophenyl)propenamide: A 2-L round-bottom flask was charged with a mixture of 3-fluoroaniline (48.0 g, 0.432 mol, 1.00 equiv.) and dichloromethane (440 mL). Pyridine (85.8 mL, 1.08 mol, 2.50 equiv.) and 3-chloropropionyl chloride (49.5 mL, 0.518 mol, 1.20 equiv.) were added dropwise in sequence. The reaction mixture was stirred at room temperature for 3 h until the disappearance of the starting material as shown by TLC analysis. The reaction mixture was then diluted with water (440 mL) and extracted with ethyl acetate (2.2 L x 3). The combined organic layers were washed with brine (1.0 L x 2), dried over anhydrous sodium sulfate, and concentrated in vacuo to give 64.0 g (75%) of 3-chloro-N-(3-fluorophenyl)propanamide as a light brown solid. LC-MS (ES) [M+1] + m / z: 202.1.

[0135] 7-Fluoro-3,4-dihydroquinolin-2(1H)-one: A 1-L round-bottom flask was charged with a mixture of 3-chloro-N-(3-fluorophenyl)propanamide (64.0 g, 0.317 mol, 1.00 equiv.) and aluminum trichloride (148 g, 1.11 mol, 3.50 equiv.). The reaction mixture was stirred at 120°C-125°C for 2 h until the starting material disappeared as shown by TLC analysis. The reaction mixture was poured into ice water (1.2 L) and filtered. The filter cake was collected and washed with water (60 mL x 3) and methyl tert-butyl ether (60 mL x 3). This gave 47.8 g (91%) of 7-fluoro-3,4-dihydroquinolin-2(1H)-one as a light brown solid. LC-MS (ES) [M+1] + m / z: 166.1.

[0136] 7-Fluoro-6-nitro-3,4-dihydroquinolin-2(lH)-one: A 2-L round-bottom flask was charged with a mixture of 7-fluoro-3,4-dihydroquinolin-2(lH)-one (47.8 g, 0.289 mol, 1.00 equiv.) and concentrated sulfuric acid (290 ml). Nitric acid (20.8 mL, 0.304 mol, 1.05 equiv.) was added dropwise. The reaction mixture was stirred at 0° C. for 2 h until the starting material disappeared as shown by TLC analysis. The reaction mixture was poured into ice / water (600 mL) and filtered. The filter was collected and washed with water (60 mL×3) and methyl tert-butyl ether (60 mL×3). This gave 37.0 g (crude) of 7-fluoro-6-nitro-3,4-dihydroquinolin-2(1H)-one as a light brown solid. LC-MS (ES) [M+1] + m / z: 211.1. 1 1 H NMR (DMSO-PBS 3 / 4, 300 MHz) was consistent with a compound having the structure 7-fluoro-6-nitro-3,4-dihydroquinolin-2(1H)-one.

[0137] 6-Amino-7-fluoro-3,4-dihydroquinolin-2(1H)-one: A 3-L round-bottom flask was charged with a mixture of 7-fluoro-6-nitro-3,4-dihydroquinolin-2(1H)-one (37.0 (crude), 0.176 mol, 1.00 equiv.), methanol (1800 ml) and Pd / C (3.70 g). The reaction mixture was stirred at room temperature under hydrogen atmosphere for 4 h until the starting material disappeared as shown by TLC analysis. The reaction mixture was filtered and the filtrate was concentrated. The residue was purified by column chromatography silica gel (DCM / MeOH = 200 / 1). This gave 12.1 g (24% for two steps) of 6-amino-7-fluoro-3,4-dihydroquinolin-2(1H)-one as an orange solid. LC-MS (ES) [M+1] + m / r. 211.1.

[0138] N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide (MBE1.2): A 40-mL vial was charged with a mixture of 6-amino-7-fluoro-3,4-dihydroquinolin-2(lH)-one (180 mg, 1.00 mmol, 1.00 equiv), N,N-dimethylformamide (5 mL), isonicotinic acid (135 mg, 1.10 mmol, 1.10 equiv), N-ethyl-N-isopropylpropan-2-amine (387 mg, 3.00 mmol, 3.00 equiv), and 1-((dimethylamino)(dimethyliminio)methyl)-1H-[1,2,3]triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (V) (570 mg, 1.50 mmol, 1.50 equiv)). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered, and the filtrate was purified by prep-HPLC. This gave 175 mg (61%) of N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide as an off-white solid. LC-MS (ES)[M+1] + m / z 286.0. 1 H NMR (DMSO-r / ^, 300 MHz) was consistent with a compound having the structure N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide.

[0139] Synthesis of 6-((3-ethylpyridin-4-yl)methylamino)-7-fluoro-3,4-dihydroquinolin-2(lH)-one (MBE 1.3) [ka]

[0140] 6-Amino-3,4-dihydroquinolin-2(1H)-one: A 100-mL round-bottom flask was charged with a mixture of 6-nitro-3,4-dihydroquinolin-2(1H)-one (1.92 g, 10.0 mmol, 1.00 equiv), methanol (50 mL) and Pd / C (200 mg). The reaction mixture was stirred at room temperature under a hydrogen atmosphere for 4 h until the starting material disappeared as shown by TLC analysis. The reaction mixture was filtered and the filtrate was concentrated. This gave 1.60 g (99%) of 6-amino-3,4-dihydroquinolin-2(1H)-one as a pale green solid. LC-MS (ES) [M+1] + m / z: 163.1.

[0141] Methyl 3-ethylisonicotinate: In a 1-L three-neck round-bottom flask, add methyl 3-bromoisonicotinate (21.6 g, 100 mmol, 1.00 equiv.), dioxane (300 mL), and Pd(dppf)Cl. 2 (3.66 g, 5.00 mmol, 0.05 equiv.) was added. A solution of diethylzinc in toluene (100 mL, 100 mmol, 1.00 equiv.) was added dropwise at room temperature. The reaction mixture was stirred at 70 °C for 4 h under nitrogen atmosphere. The reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (200 mL × 3). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated under vacuum. The residue was purified by silica gel flash eluted with PE / EtOAc (10 / 1) to give 12.0 g (73%) of 3-ethyl methyl isonicotinate as a pale yellow liquid. LC-MS (ES) [M+1] + m / z: 166.0.

[0142] (3-Ethylpyridin-4-yl)methanol: A 100-mL round-bottom flask was charged with a mixture of 3-ethylmethyl isonicotinate (3.75 g, 22.7 mmol, 1.00 equiv.), methanol (50 mL), and calcium chloride (2.52 g, 22.7 mmol, 1.00 equiv.). Sodium borohydride (1.72 g, 45.5 mol, 2.00 equiv.) was added portionwise at 0° C. The reaction mixture was stirred at room temperature for 2 h until the starting material disappeared as shown by TLC analysis. The reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (100 mL×3). The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. This gave 1.80 g of (3-ethylpyridin-4-yl)methanol as a white solid. LC-MS (ES) [M+1] + m / z: 138.1.

[0143] 3-Ethylisonicotinaldehyde: A 100-mL round-bottom flask was charged with a mixture of (3-ethylpyridin-4-yl)methanol (823 mg, 6.00 mol, 1.00 equiv.), dichloromethane (25 ml) and DMP (510 mg, 7.20 mol, 1.20 equiv.). The reaction mixture was stirred at room temperature for 4 h until the starting material disappeared as shown by TLC analysis. The reaction mixture was diluted with dichloromethane (25 ml) and washed with saturated sodium bicarbonate solution (25 ml×3). The organic layer was dried over aqueous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography on silica gel (PE / EtOAc=3 / 1). This afforded 350 mg (43%) of 3-ethylisonicotinaldehyde as an orange solid. LC-MS (ES)[M+1] + m / z: 136.1.

[0144] 6-((3-Ethylpyridin-4-yl)methylamino)-7-fluoro-3,4-dihydroquinolin-2(1H)-one: A 40-mL vial was charged with a mixture of 6-amino-7-fluoro-3,4-dihydroquinolin-2(1H)-one (252 mg, 1.40 mmol, 1.00 equiv), methanol (5 ml), 3-ethylisonicotinaldehyde (189 mg, 1.00 mmol, 1.00 equiv), calcium chloride (776 mg, 6.99 mmol, 5.00 equiv), and sodium cyanoborohydride (132 mg, 1.50 mmol, 2.10 equiv). The reaction mixture was stirred at 70° C. for 2 h. The reaction mixture was filtered, and the filtrate was purified by prep-HPLC. This gave 69.9 mg (17%) of 6-((3-ethylpyridin-4-yl)methylamino)-7-fluoro-3,4-dihydroquinolin-2(1H)-one as an off-white solid. LC-MS (ES)[M+1] + m / z: 300.1. 1 1 H NMR (DMSO-r3 / 4, 300 MHz) was consistent with a compound having the structure 6-((3-ethylpyridin-4-yl)methylamino)-7-fluoro-3,4-dihydroquinolin-2(1H)-one.

[0145] Synthesis of 2-ethyl-N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)benzamide (MBE1.5) [ka]

[0146] 2-Ethyl-N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)benzamide: A 40-mL vial was charged with 6-amino-7-fluoro-3,4-dihydroquinolin-2(1H)-one (180 mg, 1.00 mmol, 1.00 equiv.), N,N-dimethylformamide (5 mL), 2-ethylbenzoic acid (135 mg, 1.10 mmol, 1.10 equiv.), N-ethyl-N-isopropylpropan-2-amine (387 mg, 3.00 mmol, 3.00 equiv.), and 1-((dimethylamino)(dimethyliminio)methyl)-1H-[1,2,3]triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (V) (570 mg, 1.00 mmol, 1.00 equiv.). mg, 1.50 mmol, 1.50 equiv). The reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered and the filtrate was purified by prep-HPLC. This gave 181 mg (58%) of 2-ethyl-N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)benzamide as an off-white solid. LC-MS (ES)[M+1] + m / r. 313.1. 1 1 H NMR (DMSO-PBS 3 / 4, 300 MHz) was consistent with a compound having the structure 2-ethyl-N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)benzamide.

[0147] Synthesis of 3-ethyl-N-(2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide (MBE1.6) [ka]

[0148] Ethyl 3-isonicotinate: A 100-mL round-bottom flask was charged with a mixture of methyl 3-ethyl isonicotinate (3.30 g, 20.0 mmol, 1.00 equiv), methanol (20 mL), water (4 mL), and sodium hydroxide (1.60 g, 40.0 mmol, 2.00 equiv). The reaction mixture was stirred at room temperature for 6 h. The pH was adjusted to about pH 4 with 2 M hydrochloric acid solution. The mixture was filtered, and the filter cake was collected. This afforded 2.60 g (86%) of 3-ethyl isonicotinate as a white solid. LC-MS (ES) [M+1] + m / z: 152.0.

[0149] 3-Ethyl-N-(2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide: A 40-mL vial was charged with a mixture of 6-amino-7-fluoro-3,4-dihydroquinolin-2(1H)-one (180 mg, 1.00 mmol, 1.00 equiv.), N,N-dimethylformamide (5 mL), ethyl 3-isonicotinate (166 mg, 1.10 mmol, 1.10 equiv.), N-ethyl-N-isopropylpropan-2-amine (387 mg, 3.00 mmol, 3.00 equiv.), and 1-((dimethylamino)(dimethyliminio)methyl)-1H-[1,2,3]triazolo[4,5-b]pyridine 3-oxide hexafluorophosphate (V) (570 mg, 1.50 mmol, 1.50 equiv.). The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was filtered and the filtrate was purified by prep-HPLC. This gave 260 mg (88%) of 3-ethyl-N-(2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide. LC-MS (ES) [M+1] + m / z:296.1. 1 HNMR (DMSO-i3 / 4, 300 MHz) was consistent with a compound having the structure 3-ethyl-N-(2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide.

[0150] Cell lines and cell culture MDA-MB-468, MCF7, 293T, B16F10, HCT116, HepG2, LN229, HTB140 and SW480 cells were cultured in DMEM + 10% FBS, DET145 cells and their derivatives, PC3, AsPC-1, NCI-H358 were cultured in RPMI-1640 + 10% FBS, and SUM159 cells and their derivatives were cultured in F12 medium supplemented with 10% FBS, 10 pg / mL insulin and 20 ng / mL EGF. Cell lines were labeled with retroviral vectors carrying bicistronic expression of GFP / firefly luciferase to facilitate imaging and flow cytometry experiments. All cell lines were confirmed to be negative for mycoplasma contamination by monthly PCR analysis. The cell lines used here do not appear in the database of commonly misidentified cell lines (ICLAC). All cell lines were verified by STR analysis and compared with NCBI repository data.

[0151] Cloning, virus production, and transduction The coding sequences of Aldh1a1, Aldh1a2, Aldh1a3 and Aldh3a1 were cloned from cDNA generated from a pooled human reference RNA sample. The cloned sequences, flanked by AgeI and XhoI restriction enzyme sites, were inserted into the pLex lentiviral plasmid. Clones were sequenced and compared for accuracy to NCBI expressed sequence tags (ESTs). Viral production of each enzyme was performed by transfecting the 293T packaging cell line with PEI along with PsPax2 and VSVG packaging vectors. Virus was harvested and 0.45 μm filtered, then cells were transduced with polybrene (8 pg / mL) for 12 hours, followed by culturing with 1 pg / mL puromycin for the duration of the experiment. All viral transductions and selections were performed on a whole cell population basis.

[0152] ALDH1a3 in developmental and adult physiology; mechanism of action Although certain enzymes within the ALDH family have well-characterized substrate selectivity, regulation, and function, most members of this family are not well studied or have a primary mechanism of action that is not understood. For example, it has been shown how ALDH1L1 and ALDH1L2 function in folate metabolism by oxidizing 10-formyltetrahydrofolate (10-formyl-THF). Another major example of a well-characterized Aldh enzyme is ALDH3a1, which constitutes 50% of soluble corneal proteins and functions to protect against UV-induced oxidative damage in the retina and cornea by oxidizing 4-hydroxynonenal. Perhaps the most studied ALDH enzyme is ALDH2, the major catalyst of the oxidation of acetylaldehyde to acetate in liver mitochondria. ALDH2 is inhibited by ANTABUSE® (disulfiram), a treatment given to alcoholics to prevent substance abuse. On the other hand, the ALDH1a subfamily has shown widespread significance across developmental biology and various pathologies, although its mechanism of action and key regulators remain to be elucidated.

[0153] The ALDH1a subfamily is the most biologically important group of the ALDH family and has recently been the focus of considerable research, given its importance to developmental biology and the remarkable ability of the ALDEFLUOR™ assay (Stem Cell Technology) to identify stem cells, particularly in cancer. As described herein, the ALDEFLUOR™ assay primarily measures activity from ALDH1a3.

[0154] Total knockout of ALDH1a3 results in postnatal mouse lethality due to nasal congestion defects. Importantly, this phenotype can be rescued by supplementation with all-trans retinoic acid during a short window of gestation, resulting in normal adulthood. In humans, homozygous mutations in ALDH1a3 are associated with microphthalmia, but this phenotype is not fully penetrant, and no additional pathology has been noted. Further supporting the idea that ALDH1a3 is developmentally restricted, recent studies show that ALDH1a3 is specifically repressed in certain developmental tissues to prevent vitamin A signaling. Additional analyses show that ALDH1a3 is dispensable for the developing ovary, and is expressed only in the adult prostate and salivary glands. Of the colon, liver, lung, bladder, prostate, and ovary, only the ovary has a significant ALDEFLUOR™ positive population, and this small population is only partially inhibited by ALDH1a3 inhibitors.

[0155] In metabolic diseases, ALDH1a3 is a marker for incompetent islet cells. Further studies show that ALDH1a3 expression directly reduces insulin secretion by islet cell clones while increasing glucagon secretion, suggesting that ALDH1a3 may also drive the pathology of type 2 diabetes.

[0156] Taken together, these results suggest that ALDH1a3 can potentially be ablated without significant on-target toxicity. The data indicate that ALDH1a3 is dispensable in adult mammals. Inhibition of ALDH1a3 during pregnancy may be contraindicated.

[0157] Published studies claim two main mechanisms for the cancer-promoting action of ALDH1a3. These are divided between detoxification of reactive oxygen species or oxidation of retinal to bioactive retinoic acid. There are multiple reasons for the discrepancy between the number of papers detailing the functional consequences of ALDH1a3 expression compared to its mechanism of action. First, reactive oxygen species are transient and difficult to detect. Current methods use fluorescent reporters such as DCFDA and DHE, both of which are insensitive. Furthermore, oxidative stress in in vitro conditions does not reflect in vivo conditions. Attempts have been made to induce oxidative stress in vitro with paclitaxel and detect it via DCFDA, but the detection range of this assay is smaller than the deviations inherent in the data. However, additional literature evidence supports a ROS-related mechanism: ALDH1a3 was found to detoxify 4-HNE in stallion sperm samples, which led to improved motility. ALDH1a3 is also induced by radiation therapy in head and neck squamous cell carcinoma (SCC), indicating a possible response to cytotoxicity.

[0158] Interestingly, a noteworthy study in melanoma has demonstrated that oxidative stress is a major determinant of metastatic dissemination. In this study, it was shown that oxidative stress is not present in primary tumors, but is dramatically induced and affects the fitness of metastatic cells. Systemic antioxidant delivery was then able to promote lung metastasis in normally non-metastatic cells. Manipulation of ALDH1a3 expression does not strongly affect primary tumor growth until challenged with an oxidative stressor such as chemotherapy. On the other hand, the greatest impact of ALDH1a3 inhibition has been observed on lung or bone metastases, sites with high levels of oxidative stress (Figures 5A-6B).

[0159] The study of ALDH1a3-dependent retinoic acid (RA) signaling is similarly challenging, as it requires exogenous supplementation with retinal in tissue culture conditions and is far from the tumor microenvironment conditions. Data have been developed showing genetic correlations between each ALDH1a enzyme and each component of the RA signaling pathway in tumors from breast cancer patients, and have demonstrated that of ALDH1a1, ALDH1a2, and ALDH1a3, ALDH1a3 shows the lowest correlation with components of the RA signaling pathway in breast cancer. Furthermore, if ALDH1a3 had an effect on the RA signaling pathway, one would expect tissue culture growth or primary tumor growth to be affected. Studies on breast cancer cells have not observed differences in primary tumors or in vitro growth.

[0160] The functional importance of ALDH1a3 to therapy resistance, tumor progression and metastasis across most solid tumor types makes it an attractive target for drug discovery. Combined with a low potential for on-target toxicity, systemic pharmacological inhibition of ALDH1a3 alone or in combination with other therapeutics (e.g., approved therapeutics) is expected to be useful in the treatment of primary cancers, as well as indolent and overt metastatic disease.

[0161] ALDEFLUOR™ Assay The ALDEFLUOR™ assay evaluates the ability of cells to oxidize bodipy-aminoacetaldehyde (BAAA) to bodipy-aminoacetic acid (BAA), which spontaneously esterifies to form a non-cell membrane permeable product. This activity can be used to select viable cells and thereby differentiate ALDH activity levels within heterogeneous populations. When it was first discovered in 2007 that ALDEFLUOR™ positive cancer cells were more tumorigenic and predicted worse clinical outcomes, ALDEFLUOR™ activity was presumed to be a marker of a broader transcriptional program that promotes tumor invasiveness. Since these early studies, ALDEFLUOR™ activity has become the most cited method for assessing the "sternness" or invasiveness of tumor cell populations.

[0162] After this promising discovery, ALDEFLUOR™ activity was often assessed with little consideration of the function of the ALDH1 enzyme. Rather, publications showed that the ALDEFLUOR™ assay isolated invasive or metastatic cancer cells regardless of the site of the primary tumor. Since then, hundreds of papers have used the ALDEFLUOR™ assay in assessing cancer cell traits across almost all cancer types. It was not until Marcato et al. in 2011 that ALDH1a3 was shown to be responsible for ALDEFLUOR™ activity in most breast cancer cell lines.

[0163] Since the publication of Marcato et al., an accelerating stream of emerging research has demonstrated that ALDH1a3 is not only responsible for ALDEFLUOR™ activity across most cancer types, but also functionally promotes cancer growth, therapy resistance, and metastasis. Studies of various quality have demonstrated that ALDH1a3 is expressed and is important for proliferation in melanoma patient-derived xenografts or cell lines, metabolism, chemotherapy resistance and radiation resistance in mesenchymal-like gliomas or glioblastomas, tumorigenicity and cisplatin resistance in lung cancer, proliferation and radiation resistance in pancreatic cancer, FAR inhibitor resistance in colon and thyroid cells, cisplatin resistance in mesothelioma, Gleason score and proliferation in prostate cancer, apoptosis resistance and metastasis in breast cancer, and prognosis in cholangiocarcinoma. It is shown herein that ALDH1a3 is the dominant ALDEFLUOR™-inducing enzyme across most solid tumor types (see Figures 3A and 3B).

[0164] Expression and prognostic studies have further shown that ALDH1a3 strongly predicts poor prognosis across cancer types. Hypermethylation of the ALDH1a3 promoter, leading to reduced ALDH1a3 expression, was the strongest predictor of good prognosis in a series of patients with primary glioblastoma. High ALDH1a3 predicted lymph node metastasis in patients with cholangiocarcinoma. ALDH1a3 expression is driven by androgens in prostate cancer, where androgens are the primary mitogens for prostate cancer cells, whereas mirl87 targets ALDH1a3 in prostate cancer, and high mirl87 correlated with good prognosis.

[0165] In the ALDEFLUOR™ assay used herein, cells were grown to 50-80% confluence, harvested with 0.25% trypsin / EDTA (Sigma), and washed once in PBS by centrifugation / resuspension (190 g for 5 min at 4°C). Cells were counted, centrifuged, and resuspended at 1,000,000 cells / mL in ALDEFLUOR™ buffer (Stemcell Technologies). ALDEFLUOR™ substrate (Stemcell Technologies, 1:200) and test compound or 1 mM DEAB were added to the cell suspension and incubated at 37°C for 45 min with vortexing every 15 min. Cells were centrifuged and resuspended in ALDEFLUOR™ buffer containing 5 pg / mL DAPI. Samples were analyzed on a BD LSR2 flow cytometry platform. Gating was performed using DEAB as a negative control.

[0166] Figure 1A is a bar graph of the percentage of ALDEFLUOR™ positive cells in the presence of various compounds described herein, and shows the percentage of SUM159-M1a-Aldh1a3 cells above background fluorescence levels detected by flow cytometry after incubation with 100 nM concentration of compound using the standard ALDEFLUOR™ protocol described herein. Gating for background fluorescence was performed using 1 millimolar DEAB as a negative control. Figure 1A demonstrates that MBE1-5, MBE1 and MBE1-6 are high affinity compounds for inhibiting ALDH1a3 activity.

[0167] FIG. 1B is a line graph of the percentage of ALDEFLUOR™ positive cells in the presence of various concentrations of MBE1 or MBE1.5, and shows the percentage of SErMl59-M1a-Aldh1a3 cells above background fluorescence levels as detected by flow cytometry after incubation according to the standard ALDEFLUOR™ protocol described herein in combination with dose titration of MBE1 or MBE1-5. The [inh-min] threshold was set at the lower limit of two standard deviations of the control sample, while the IC 50 The threshold was set at 50% of the mean value of the control samples. Figure 1B shows that MBE1 and MBE1-5 had IC 50 Values ​​are presented demonstrating that inhibitory activity is detectable at concentrations as low as 2 nanomolar.

[0168] FIG. 1C is a line graph of high ALDH percentage in the presence of various concentrations of MBE1 and MBE1.5, and shows the percentage of SEIM159-M1a-Aldh1a3 cells with high fluorescence levels detected by flow cytometry after incubation according to the ALDEFLUOR™ protocol described herein in combination with dose titration of MBE1 or MBE1.5. High activity was gated on cells at the 99th percentile at a MBE1.5 concentration of 10 nanomolar. The [inh-min] threshold was set at the lower limit of two standard deviations of the control sample. FIG. 1C demonstrates that MBE1.5 is better than MBE1 at inhibiting highly ALDH1a3 active cells.

[0169] Figure 2A is a histogram of MCF7 cells stably transduced with human Aldh1a1, Aldh1a3 or Aldh3a1 and analyzed using the ALDEFLUOR™ protocol by flow cytometry in combination with DMSO or MBE1 at the concentrations listed, and shows the effect of DMSO or MBE1 on ALDEFLUOR™ activity induced by each enzyme. Figure 2A demonstrates that MBE1 does not inhibit Aldh1a1 or Aldh3a1 activity at concentrations below 10 μM, but is effective at inhibiting Aldh1a3 activity in another cancer cell line, MCF7.

[0170] Figure 2B is a histogram of 293T cells transiently transfected with human Aldh1a2 or Aldh1a3 and analyzed using the ALDEFLUOR™ protocol by flow cytometry in combination with DMSO or MBE1.5 at the concentrations listed, and shows the effect of either DMSO or MBE1.5 on ALDEFLUOR™ activity induced by each enzyme. Figure 2B demonstrates that MBE1.5 does not inhibit Aldh1a2 activity at concentrations below 10 pM, but is effective at inhibiting Aldh1a3 activity in 293T cells transfected with human Aldh1a3.

[0171] Figure 3A is a bar graph of ALDEFLUOR™ positive cells in various cancer types in the presence of DEAB or MBE1, and shows that ALDH1a3 activity is observed in many cancer types in addition to Suml59-M1a (breast cancer) and MDA-468 (breast cancer), and is inhibited by MBE1 in many cancer types. Figure 3B is a bar graph of ALDEFLUOR™ positive cells in various cancer types in the presence of 1 mM DEAB (pan-ALDH inhibitor) or 100 nM MBE1.5 (specific ALDH1a3 inhibitor), and shows that the majority of human cancer cell lines show ALDH1a3 activity. A notable exception is liver cancer, where a large ALDEFLUOR™ positive population is expected to exist and is driven by ALDH1a1.

[0172] The ALDEFLUOR™ assay was also used to evaluate the relative activity of some of the compounds described herein against SEIM159-M1a-Aldh1a3 cells. The activity of some of the compounds in the assay at a concentration of 100 nM is reported in Table 1, along with the chemical structure and compound name associated with each compound.

[0173] [Table 1-1]

[0174] [Table 1-2]

[0175] [Table 1-3]

[0176] 12 is a graph of ALDEFLUOR™ activity versus concentration and shows the ALDEFLUOR™ activity of several compounds described herein against SUM159-M1a-Aldh1a3 cells at concentrations of 10 nM and 100 nM. Compared to control (DMSO) or DEAB-treated cells, MBE1.5C is nearly twice as potent as MBE1.5, MBE1.5A, or MBE1.5D at a concentration of 10 nM.

[0177] ALDH isoforms 1a1, 1a2, 1a3 and 3a1 were expressed in MCF7 or SEIM159 cells and subsequently used in ALDEFLUOR™ assay. Figure 13A is a Western blot and shows the expression of each ALDH isoform in the indicated cells. Figure 13B is a line graph of the percentage of ALDEFLUOR™ positive MCF7 cells expressing the indicated ALDH isoforms versus the logarithm of MBE1.5 concentration, and shows that MBE1.5 specifically inhibits ALDH1a3 at concentrations less than 10 mM. Figure 13C is a line graph of the percentage of ALDEFLUOR™ positive SETM159 cells expressing the indicated ALDH isoforms versus the logarithm of MBE1.5 concentration, and shows that MBE1.5 specifically inhibits ALDH1a3 at concentrations less than 10 μM.

[0178] Ex vivo analysis of ALDH activity in mouse lymphocytes Bone marrow cells were harvested from 12-week-old C56BL6 mice via PBS-flushed tibias and femurs and passed through a 100-pm cell sieve. Thymocytes and splenocytes were isolated from the corresponding tissues and isolated by physical disruption against a 100 pm cell sieve. Cells were washed by centrifugation / resuspension and then spun for 10 min. 7The cells were then suspended at 1:200 cells / mL. The ALDEFLUOR™ assay was then performed on these cells with or without MBE1 as described, except that either anti-CD3ε (Biolegend #100308), anti-CD11c (BD Pharmigen #561119), anti-CD45 (eBioscience 30-F11), or anti-CD317 (Biolegend 129c1) was added during the incubation period at 1:200.

[0179] Figure 4A is a flow cytometry spectrum and shows that CD45 positive bone marrow cells are ALDEFLUOR™ positive but are not affected by MBE1.5. Figure 4B is a flow cytometry spectrum and shows that CD11c positive bone marrow cells are ALDEFLUOR™ positive but are not affected by MBE1.5. Similar results (not shown) were obtained in other populations tested, including CD45 positive and CD3e positive (CD45+CD3e+) and CD11c positive and CD317 positive (CD11+CD317+). Thus, all populations tested were ALDEFLUOR™ positive but were not affected by MBE1.5, with little potential for toxicity.

[0180] Mouse models and xenografts All mice were initially ordered from Jackson Laboratory (Bar Harbor, ME), and breeding was performed in a specific pathogen-free (SPF) barrier facility. Toxicity experiments were performed on male and female C57B16 mice aged 8–12 weeks. MBE1 was dissolved at 50 mg / mL in DMSO, followed by 1:2 dilution in Kolliphor EL, followed by 1:5 dilution in PBS to obtain a 5 mg / mL MBE1 solution (10% DMSO, 10% Kolliphor EL, 80% PBS). This was administered by IP injection at increasing doses ranging from 12.5 to 200 mg / kg body weight in sets of three mice at 24-h intervals. Body condition scores, food intake, feces / urine production, behavior, and body weight were measured as toxicity readouts. Five mice were then treated with 25 mg / kg MBE1 every 3 days for 18 days. None of the experiments showed any signs of either acute or chronic toxicity.

[0181] All xenograft experiments were performed in 8-week-old female mice (athymic Nu / Nu, or NOD / SCID Gamma). Xenograft experiments were performed with 125,000 SUM159-M1a cells in 100 μL PBS for tail vein or intracardiac injection. Mice were randomized after injection. Bioluminescence imaging (BLI) was performed using an IVIS 200 system and retro-orbital luciferin injection. For drug treatment, MBE1 (prepared as described above) was administered to mice by IP injection in conjunction with paclitaxel (5 mg / mL in 10% ethanol, 10% Kolliphor EL, 80% PBS) at the time intervals and doses shown in Figure 5A (tail vein injection) and Figure 6A (intracardiac injection).

[0182] Figure 5B is a line graph of lung metastases measured using bioluminescence imaging (BLI) versus time (days) and compares lung metastases in the presence and absence of MBE1 in mice from the experiment outlined in Figure 5A. Figure 6B is a line graph of bone metastases measured using BLI versus time (days) and compares bone metastases in the presence and absence of MBE1 in mice from the experiment outlined in Figure 6A. Figures 5B and 6B demonstrate that MBE1 is an effective therapeutic agent for treating established metastatic disease, and demonstrate that MBE1 and compounds disclosed herein can be used to effectively inhibit ALDH1a3 and its downstream effects in vivo.

[0183] In another xenograft experiment, mice were injected with SUM159-Ml-p44 cells by tail vein injection as above and randomized after injection. Metastatic burden was imaged on day 16 via intravital imaging. Mice were then treated with paclitaxel (25 mg / kg) and either vehicle or MBE1.5 (50 mg / kg) on ​​days 17, 19, and 21. Lung metastatic burden was then imaged on day 22. Signal was normalized to day 16.

[0184] FIG. 14 is a line graph of lung metastases measured by bioluminescence imaging (BLI) versus time (days) and shows that three doses of 50 mg / kg MBE1.5 in combination with 25 mg / kg paclitaxel administered on days 17, 19 and 21 caused regression of established metastatic disease in a mouse xenograft model.

[0185] In yet another xenograft experiment, mice were injected with MDA-MB-468 cells via the mammary fat pad and randomized after injection. Mice received MBE1.5 (25 mg / kg daily, n=6) or vehicle (n=12) and paclitaxel (12.5 mg / kg every other day) for 12 days. Primary tumors were measured by caliper between each treatment group.

[0186] Figure 15A is a line graph of body weight (g) versus time (days) and shows that there was no gross toxicity associated with MBE1.5 treatment in this experiment. Figure 15B shows the tumor volume (mm 3 ) versus time (days) and shows that treatment with MBE1.5 for 12 days caused regression of primary mammary tumors. The tumors in two mice in the MBE1.5 treatment group were completely eliminated by the treatment.

[0187] In another xenograft experiment, mice were injected with Sum-l59-M1a-Aldh1a3 cells via tail vein injection and randomized after injection. Mice were treated with MBE1.5 (25 mg / kg daily) or vehicle, and paclitaxel (12.5 mg / kg every other day) for 12 days. The progression of lung metastases was followed by in vivo bioluminescence.

[0188] Figure 16A is a line graph of lung metastasis bioluminescence versus time (days) and shows the progression of lung metastases before and after treatment with MBE1.5 or vehicle. Figure 16B is a Kaplan-Meier plot of mouse survival over time as a function of treatment group and shows that treatment with MBE1.5 for 12 days extended the survival of mice with late-stage established breast cancer lung metastases.

[0189] Pharmacokinetic evaluation Pharmacokinetic experiments were performed on 16-week-old C57BL6 male mice. Mice were administered MBE1.5 (15% DMSO, 5% Kolliphor EL, 80% PBS) by IP injection. Blood was collected by terminal cardiac puncture at 15 min, 30 min, 1 h, 3 h, and 24 h after injection. Serum was obtained by blood separation by centrifugation at 16200 RCF for 1 min. Tissues were then collected after exsanguination. Serum and tissues were snap frozen in liquid nitrogen and subjected to pharmacokinetic evaluation.

[0190] Quantitative determination of plasma concentrations of MBE1.5 by LC / MS / MS for mouse PK studies Plasma MBE1.5 concentrations were measured by LC / MS / MS assay in mouse PK studies.

[0191] Chemicals: MBE1.5 and MBE2.0 were supplied as stocks (1 mg / mL) in MeOH with 1% DMSO. MBE2.0 was used as an internal standard. HPLC grade acetonitrile and water were purchased from JT Baker (Center Valley, PA). Mouse plasma was purchased from Innovative Research (Novi, MI) for preparation of plasma calibration standards.

[0192] LC / MS / MS Assays:

[0193] Preparation of standard solutions: MBE1.5 stock was diluted in control mouse plasma to give a calibration range of 8.2 ng / mL to 18 pg / mL. MBE2.0 at 1 pg / mL in methanol was used as the working internal standard (IS).

[0194] Sample preparation: Plasma standards and mouse plasma samples (20 pL) were spiked with 5 μL IS and deproteinized with 120 μL acetonitrile and methanol (v / v 5:1). After centrifugation at 13000 rpm for 15 min, 25 μL of supernatant was analyzed by LC-MS.

[0195] Liquid chromatography / mass spectrometry: Quantitation of MBE1.5 was performed using a TSQ Quantum triple quadruple mass spectrometer equipped with an API electrospray ionization (ESI) source, a Surveyor MS pump, and a Surveyor autosampler with Xcalibur data acquisition software (ThermoElectron, San Jose, CA, ETSA).

[0196] The LC conditions were as follows: mobile phase A: aqueous solution (0.1% formic acid); mobile phase B: acetonitrile (0.1% formic acid); flow rate 500 pL / min; gradient elution 0-1 min, 60% A; 1-2.5 min, 60%-20% A; 2.5-7.0 min, 0% A, 7.0-9.0 min, 0% A, 9.0-11.0 min, 0%-60% A; and column: Luna® C18, 150X4.6 mm, 5 pm (Phenomenex, Torrance, CA). The retention times of MBE1.5 and IS were 6.3 and 6.6 min, respectively.

[0197] MS / MS conditions were as follows: m / z 313.2 / 178.2 (MBE1.5), m / z 281.2 / 119.1 (MBE2.0, IS) in single reaction monitoring mode; ion spray voltage, 4500v; capillary temperature, 350°C; sheath gas pressure, 20 arbitrary units; Aux gas pressure, 50 arbitrary units; collision pressure, 1.5 arbitrary units. Collision energy and tube lenses were optimized for each compound.

[0198] Calculations: Plasma calibration standards were used for quantification of MBE in plasma samples. Log-linear regression was used on the calibration curve to determine the concentrations in plasma samples.

[0199] result:

[0200] LC / MS / MS Assay: A plasma calibration curve for MBE1.5 was plotted using concentrations ranging from 8.2 ng / mL to 18 pg / mL. The limit of quantification was 8.2 ng / mL (LOD). The LC / MS / MS assay had good linearity in the calibration range (R 2 >0.99). Table 2 summarizes the intraassay accuracy and precision. No matrix effects were observed in the assay.

[0201] [Table 2]

[0202] Plasma PK of MBE1.5 in mice: Plasma concentrations of MBE1.5 in mouse PK studies are shown in Table 3. Preliminary PK data were analyzed using a non-compartmental model with Winnolin 2.0. PK parameters are summarized in Table 4.

[0203] [Table 3]

[0204] [Table 4]

[0205] ALDH1a3 gene knockout CRISPR-Cas9 vectors containing both the Cas9 gene and gRNA sequences containing homologous sequences to genomic Aldh1a3 were transduced into MDA-MB-468 cells by lentiviral infection, followed by puromycin selection for viral integration, and the resulting cells were analyzed by ALDEFLUOR™ assay. Two ALDH1a3-targeting gRNA vectors were used to generate two derivative cell lines, and one gRNA with a scrambled sequence was used to generate a control derivative cell line.

[0206] Figure 7A is a flow cytometry spectrum and shows that genetic knockout of ALDH1a3 in MDA-MB-468 breast cancer cells (middle and rightmost spectra) substantially reduces ALDEFLUOR™ activity compared to control MDA-MB-468 cells (leftmost spectrum). Figure 7B shows the relationship between tumor volume (mm 37A is a line graph of tumor mass (g) versus time (days) and shows that gene knockout of ALDH1a3 in MDA-MB-468 breast cancer cells slows primary tumor growth and sensitizes tumors to paclitaxel. FIG. 7C is a bar graph of tumor mass (g) versus gene knockout and shows that gene knockout of ALDH1a3 in MDA-MB-468 breast cancer cells slows primary tumor growth and sensitizes tumors to paclitaxel.

[0207] CRISPR-Cas9 vectors containing both the Cas9 gene and gRNA sequences containing homologous sequences to genomic Aldh1a3 were transfected into Suml59-M1a cells, followed by selection for positively transfected cells by flow cytometry. Positively transfected cells were then virally transduced with lentiviral vectors containing either the vector backbone or full-length human Aldh1a3. Positive transductants were selected by puromycin, and the resulting cells were analyzed by ALDEFLUOR™ assay. In xenograft experiments, knockout vector or knockout Aldh1a3 or wild-type vector or wild-type Aldh1a3 cells were injected intracardially into mice, and bone metastasis growth was tracked by in vivo bioluminescence imaging. Bone metastasis-free survival was tracked by bioluminescence and plotted using the Kaplan-Meier model.

[0208] Figure 9A is a flow cytometry spectrum and shows that gene knockout of ALDH1a3 in Suml59-M1a breast cancer cells nearly abolishes ALDEFLUOR™ activity in the cells, and ALDEFLUOR™ activity can be rescued by transducing the cells with a rescue vector. Figure 9B is a line graph of bone metastasis measured by bioluminescence (ph / s) versus time (days) and shows that knockout of ALDH1a3 in Suml59-M1a breast cancer cells slows bone metastasis growth. Figure 9C is a Kaplan-Meier plot of bone metastasis-free survival over time and shows that knockout of ALDH1a3 in Suml59-M1a breast cancer cells significantly increases survival time.

[0209] Gene expression of ALDH1a3 Lentiviral vectors encoding one of three human ALDH genes, ALDH1a1, ALDH1a3 or ALDH3a1, were introduced into luciferase-labeled Suml59-Mlb cells by viral transduction, followed by positive selection with puromycin, and the transduced cells were injected into mice via tail vein injection. Growth of lung metastases was tracked by weekly in vivo bioluminescence imaging. Lung nodules were counted ex vivo.

[0210] Figure 8A is a line graph of bioluminescence (ph / s) versus time (days) and shows the development of lung metastases in mice injected with SUM159-Mlb cells transfected with vectors encoding the three ALDH enzymes, ALDH1a1, ALDH1a3, and ALDH3a1. Figure 8B is a plot of lung nodules counted ex vivo at the endpoint of the experiment described in Figure 8A.

[0211] Survival prediction TCGA and Kaplan-Meier plotter (kmplot.com) data were used to generate expression data and survival curves for various cancers as a function of ALDH1a3 expression levels. Cancer patients from each dataset were stratified by high or low Aldh1a3 expression according to either the median expression value or the optimal stratification value. Kaplan-Meier analysis was then used to plot patient survival, whether measuring distant metastasis-free survival or overall survival, and to evaluate the relationship between the relative levels of Aldh1a3 among patients and the corresponding survival indicators.

[0212] 10A-10H are prognostic patient survival curves stratified by high (red) and low (black) Aldh1a3 expression based on the data analysis tools provided at kmplot.com, and show distant metastasis-free survival of triple-negative breast cancer patients (FIG. 10A), and overall survival of renal clear cell carcinoma, gastric cancer, bladder cancer, ovarian cancer, lung squamous cell carcinoma, colorectal cancer, and low-grade glioma cancer patients as a function of ALDH1a3 expression level (FIGS. 10B-10H, respectively).

[0213] In another set of predictions, Aldh1a3 mRNA expression from the METABRIC clinical breast cancer dataset was separated by tumor type and chemotherapy pretreatment. Survival curves for the EMC-MSK dataset were generated by dividing patients according to subtype and stratifying by median Aldh1a3 expression.

[0214] Figure 11A is a graph of ALDH1a3 mRNA expression from the METABRIC clinical breast cancer dataset, showing ALDH1a3 expression by breast cancer subtype and chemotherapy history. Figure 11B is a predicted survival curve based on the EMC-MSK dataset, showing predicted survival of breast cancer patients by subtype and median ALDH1a3 expression level.

[0215] Table 5 reports the hazard ratios (p-values) for patients with high expression of ALDH1a1 or ALDH1a3 in estrogen receptor (ER)-negative breast cancer derived from the Kaplan-Meier plotter database, and includes Her2 and triple-negative breast cancer (TNBC) populations at high risk for developing metastases. ALDH1a3 is a poor prognostic predictor in ER-negative breast cancer patients, the population most likely to develop metastases.

[0216] [Table 5]

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[0218] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

[0219] Although exemplary embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and detail can be made therein without departing from the scope of the embodiments encompassed by the appended claims. [Explanation of symbols]

[0220] Figure 1A Cells cells Lead name Figure 1B Drug Concentration Control Poly. Linear Figure 1C High Drug Concentration Control Poly. Linear Figure 2A Count Specimen Specimen stable expression Figure 2B Specimen Specimen contour transfectants Figure 3A positive positive Pancreatic Mouse Melanoma Prostate Lung Colon Liver Melanoma Glioblastoma Prostate Figure 3B Activity Breast Colon Liver Lung Melanoma Pancreatic Prostate Control Cancer cellline Cancer cell line Figure 4A specimen-boneMarrow contour Figure 4B specimen-BoneMarrow Figure 5A Day 18, 22, 25, 28, 32 intravenous Day 1 Tail-vein injection cells cells Day 16 normalized Day 18.5, 22.5, 25.5, 28.5, 32.5 Paclitaxel intraperitoneal Day 36 Final BLI measurement Final BLI measurement Figure 5B Lung Metastasis Vehicle Solvent Days Figure 6A Day 9, 11, 13, 16 MBE1 or vehicle intraperitoneal Day 1 Intra-cardiac injection cells cells Day 8 normalized Paclitaxel intraperitoneal Day 27 Measurement Figure 6B Bone Metastasis Days Vehicle Solvent One-tailed Figure 7B Tumor Volume Days Figure 7C Tumor mass Figure 8A Bioluminescence Days Vector Vector Figure 8B Lung nodes Figure 9A Wild-typeCell Wild-type cell Vector Vector Knockout Figure 9B Bone Metastasis Days Vector Vector Knockout Rescue Figure 9C Bone metastasis-free survival Days Knockout Rescue Vector Vector Figure 10A Probability Time (months) Period (months) logrank Expression low High Figure 10B Probability Time (months) Period (months) logrank Expression low High Figure 10C Probability Time (months) Period (months) logrank Expression low High Figure 10D Probability logrank Expression low High Figure 10E Probability Time (months) Period (months) logrank Expression low High Figure 10F Probability Time (months) Period (months) logrank Expression low High Figure 10G Survival probability Time in days Expression Level High expression Low / Medium-expression low / medium low / medium High Figure 10H Survival probability Time in days Expression Level High expression Low / Medium-expression low / medium low / medium High Figure 11A Expression Chemotherapy Normal Basal Figure 11B Basal Survival Survival Months Low expression High expression Normal Survival Survival Months High expression Low expression Figure 12 Activity median Inhibitor Control Figure 13A β-actin Figure 13B positive positive Vector Vector Figure 13C positive positive Vector Vector Figure 14 Lung Metastasis 3 Doses vehicle or MBE1.5 Day 17, 19, 21 and Paclitaxel Vehicle Solvent Figure 15A Treatment period Body Mass Days Vehicle Solvent Figure 15B Tumor volume Days Vehicle Solvent Figure 16A Treatment period Lung Metastasis Bioluminescence Days Vehicle Solvent Figure 16B Survival Survival rate Days Hazard ratio p-value P value Untreated Paclitaxel Vehicle Solvent

Claims

1. A compound represented by any one of the following structural formulas (1) to (9): 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof.

2. 10. A composition comprising a compound of claim 1, or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier or excipient.

3. Any compound selected from the following: 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof for treating metastatic or chemotherapy-resistant cancer.

4. Any compound selected from the following: 【Chemistry 3】 or a pharma- ceutically acceptable salt thereof for inhibiting the proliferation of metastatic or chemotherapy-resistant cancer cells.

5. Any compound selected from the following: 【Chemistry 4】 or a pharma- ceutically acceptable salt thereof for treating type 2 diabetes.

6. A compound represented by the following structural formula (54): 【Chemistry 5】 or a pharma- ceutically acceptable salt thereof for treating metastatic or chemotherapy-resistant cancer. (In the formula, R １ and R ５ are each independently hydrogen or (C １ -C ６ ) alkyl; R ２ , R ３ and R ４ are each independently hydrogen, halo, halo(C １ -C ３ ) alkyl, (C １ -C ３ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, amino, (C １ -C ３ ) alkylamino, (C １ -C ３ ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl; L is -C(O)- or -C(R ６ ) (R ７ )—wherein R ６ and R ７ are each independently hydrogen, hydroxy, sulfhydryl, halo, (C １ -C ３ ) alkyl, (C １ -C ３ ) alkoxy or halo(C １ -C ３ ) alkoxy; X １ , X ２ , X ３ , X ４ and X ５ Each independently represents -C(R ８ )- or -N-; and Each R ８ are independently hydrogen, halo, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, halo (C １ -C ６ ) alkoxy, amino, (C １ -C ６ ) alkylamino, (C １ -C ６ ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl).

7. R ２ and R ４ The method of claim 6, wherein each of is hydrogen.

8. R １ The method of claim 6 or 7, wherein is hydrogen.

9. R ５ The agent according to any one of claims 6 to 8, wherein is hydrogen.

10. R ３ The agent according to any one of claims 6 to 9, wherein is hydrogen or halo.

11. R ３ The agent according to claim 10, wherein is fluoro.

12. L is -C(R ６ ) (R ７ 12. The method according to claim 6, wherein said compound is selected from the group consisting of aryl, aryl, aryl and aryl groups.

13. The agent according to claim 12, wherein L is -C(O)-.

14. L is -CH ２ The method of claim 12, wherein

15. Each R ８ are independently hydrogen, halo, halo(C １ -C ３ ) alkyl, (C １ -C ３ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, amino, (C １ -C ３ ) alkylamino, (C １ -C ３ 15. The agent according to any one of claims 6 to 14, which is dialkylamino, cyano, nitro, hydroxy or sulfhydryl.

16. Each R ８ are independently hydrogen, halo or (C １ -C ６ ) alkyl. The method of any one of claims 6 to 14.

17. The compound is a compound represented by the following structural formula (55): 【Chemistry 6】 or a pharma- ceutical agent according to any one of claims 6 to 14, which is ... (In the formula, Each R ９ are independently halo, halo (C １ -C ６ ) alkyl, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, halo (C １ -C ６ ) alkoxy, amino, (C １ -C ６ ) alkylamino, (C １ -C ６ ) dialkylamino, cyano, nitro, hydroxy, or sulfhydryl; and m is 0, 1, 2, 3 or 4).

18. X ３ But -C(R ８ 18. The method of claim 17, wherein

19. X ３ The agent according to claim 17, wherein is -N-.

20. Each R ９ However, independently, Halo, Halo (C １ -C ３ ) alkyl, (C １ -C ３ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, amino, (C １ -C ３ ) alkylamino, (C １ -C ３ 20. The agent according to any one of claims 17 to 19, which is dialkylamino, cyano, nitro, hydroxy or sulfhydryl.

21. Each R ９ are independently halo or (C １ -C ６ 20. The method according to any one of claims 17 to 19, wherein said aryl group is aryl.

22. The agent according to any one of claims 17 to 21, wherein m is 0, 1 or 2.

23. The compound is a compound represented by the following structural formula (56): 【Chemistry 7】 The agent according to any one of claims 6 to 19, which is a medicament for treating or preventing osteoarthritis, which is a medicament for treating osteoarthritis, or a medicament for treating osteoarthritis, which is ...

24. The compound is MBE1, MBE1.2, MBE1.3, MBE1.5, MBE1.6, MBE2, MBE3.1, MBE3.2, MBE3.3, MBE3.4, MBE3.5 or MBE3.6 as defined in Table A below: 【Table A】 or a pharma- ceutically acceptable salt thereof.

25. The method according to any one of claims 6 to 24, wherein the cancer is a metastatic cancer.

26. 26. The method of claim 25, wherein the metastatic cancer is selected from the group consisting of metastatic breast cancer, metastatic colon cancer, metastatic lung cancer, metastatic pancreatic cancer, metastatic prostate cancer, metastatic bone cancer, metastatic blood cancer, metastatic brain cancer, and metastatic liver cancer.

27. The method of claim 25 or 26, wherein the subject has bone metastasis.

28. The agent according to any one of claims 6 to 27, wherein the cancer is a chemotherapy-resistant cancer.

29. A compound represented by the following structural formula (57): 【Chemistry 8】 or a pharma- ceutically acceptable salt thereof for inhibiting the proliferation of metastatic or chemotherapy-resistant cancer cells. (In the formula, R １ and R ５ are each independently hydrogen or (C １ -C ６ ) alkyl; R ２ , R ３ and R ４ are each independently hydrogen, halo, halo(C １ -C ３ ) alkyl, (C １ -C ３ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, amino, (C １ -C ３ ) alkylamino, (C １ -C ３ ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl; L is -C(O)- or -C(R ６ ) (R ７ )—wherein R ６ and R ７ are each independently hydrogen, hydroxy, sulfhydryl, halo, (C １ -C ３ ) alkyl, (C １ -C ３ ) alkoxy or halo(C １ -C ３ ) alkoxy; X １ , X ２ , X ３ , X ４ and X ５ Each independently represents -C(R ８ )- or -N-; and Each R ８ are independently hydrogen, halo, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, halo (C １ -C ６ ) alkoxy, amino, (C １ -C ６ ) alkylamino, (C １ -C ６ ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl).

30. A compound represented by the following structural formula (58): 【Chemistry 9】 or a pharma- ceutically acceptable salt thereof for treating type 2 diabetes (In the formula, R １ and R ５ are each independently hydrogen or (C １ -C ６ ) alkyl; R ２ , R ３ and R ４ are each independently hydrogen, halo, halo(C １ -C ３ ) alkyl, (C １ -C ３ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, amino, (C １ -C ３ ) alkylamino, (C １ -C ３ ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl; L is -C(O)- or -C(R ６ ) (R ７ )—wherein R ６ and R ７ are each independently hydrogen, hydroxy, sulfhydryl, halo, (C １ -C ３ ) alkyl, (C １ -C ３ ) alkoxy or halo(C １ -C ３ ) alkoxy; X １ , X ２ , X ３ , X ４ and X ５ Each independently represents -C(R ８ )- or -N-; and Each R ８ are independently hydrogen, halo, halo(C １ -C ６ ) alkyl, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, halo (C １ -C ６ ) alkoxy, amino, (C １ -C ６ ) alkylamino, (C １ -C ６ ) dialkylamino, cyano, nitro, hydroxy or sulfhydryl).

31. R ２ and R ４ The agent according to claim 29 or 30, wherein each is hydrogen.

32. R １ The agent according to any one of claims 29 to 31, wherein is hydrogen.

33. R ５ The agent according to any one of claims 29 to 32, wherein is hydrogen.

34. R ３ The agent according to any one of claims 29 to 33, wherein is hydrogen or halo.

35. R ３ The agent of claim 34, wherein is fluoro.

36. L is -C(R ６ ) (R ７ 36. The method according to any one of claims 29 to 35, wherein

37. The agent according to claim 36, wherein L is -C(O)-.

38. L is -CH ２ The method of claim 36, wherein

39. Each R ８ are independently hydrogen, halo, halo(C １ -C ３ ) alkyl, (C １ -C ３ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, amino, (C １ -C ３ ) alkylamino, (C １ -C ３ 39. The agent according to any one of claims 29 to 38, which is dialkylamino, cyano, nitro, hydroxy or sulfhydryl.

40. Each R ８ are independently hydrogen, halo or (C １ -C ６ ) alkyl. The method of any one of claims 29 to 38,

41. The compound is a compound represented by the following structural formula (59): 【Chemistry 10】 or a pharma- ceutical agent according to any one of claims 29 to 38, which is ... (In the formula, Each R ９ are independently halo, halo (C １ -C ６ ) alkyl, (C １ -C ６ ) alkyl, (C １ -C ６ ) alkoxy, halo (C １ -C ６ ) alkoxy, amino, (C １ -C ６ ) alkylamino, (C １ -C ６ ) dialkylamino, cyano, nitro, hydroxy, or sulfhydryl; and m is 0, 1, 2, 3 or 4).

42. X ３ But -C(R ８ 42. The method of claim 41, wherein

43. X ３ The method of claim 41, wherein is -N-.

44. Each R ９ But independently, Halo, Halo (C １ -C ３ ) alkyl, (C １ -C ３ ) alkyl, (C １ -C ３ ) alkoxy, halo (C １ -C ３ ) alkoxy, amino, (C １ -C ３ ) alkylamino, (C １ -C ３ 44. The agent according to any one of claims 41 to 43, which is dialkylamino, cyano, nitro, hydroxy or sulfhydryl.

45. Each R ９ are independently halo or (C １ -C ６ 44. The agent according to any one of claims 41 to 43, wherein said alkyl is aryl.

46. The agent according to any one of claims 41 to 45, wherein m is 0, 1 or 2.

47. The compound is a compound represented by the following structural formula (60): 【Chemistry 11】 or a pharma- ceutically acceptable salt thereof.

48. The compound is MBE1, MBE1.2, MBE1.3, MBE1.5, MBE1.6, MBE2, MBE3.1, MBE3.2, MBE3.3, MBE3.4, MBE3.5 or MBE3.6 as defined in Table B below: Table B 31. The agent of claim 29 or 30, which is:

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