Tetrahydroquinoline derivatives for the treatment of metastatic and chemotherapy-resistant cancers

Abstract

Disclosed herein are compounds of structural formula I. Compounds of structural formula I inhibit aldehyde dehydrogenase isoform Ia3 (ALDHIa3) and can be used to treat cancer, for example, metastatic or chemotherapy-resistant cancer, such as chemotherapy-resistant metastatic cancer. Also disclosed herein are compositions comprising compounds of structural formula I and uses of compounds of structural formula I for treating cancer, for example, metastatic or chemotherapy-resistant cancer.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 712,434, filed on July 31, 2018. The entire teachings of the above patent application are incorporated herein by reference. Background Art

[0003] Current therapies for treating aggressive and / or chemotherapy-resistant metastatic cancers have limited efficacy. Thus, there remains a need for therapeutic agents with improved efficacy for the treatment of these cancers. Summary of the invention

[0004] Provided herein are compounds, compositions and methods for treating cancer in a subject in need thereof, e.g., a metastatic cancer (e.g., metastatic breast cancer, such as basal cell-like breast cancer, HER-2 positive breast cancer), a chemotherapy-resistant cancer (e.g., a cancer resistant to paclitaxel and / or doxorubicin).

[0005] One embodiment is a compound of formula II:

[0006]

[0007] or a pharmaceutically acceptable salt thereof, wherein the variable (e.g., R 1 , R 2 , R 3 , R 4 , R 5 , R 9 The values ​​of L, n) are as described herein.

[0008] Another embodiment is a method of treating metastatic or chemotherapy-resistant cancer in a subject in need thereof, comprising administering to the subject a compound of structural formula I:

[0009]

[0010] or a pharmaceutically acceptable salt thereof, wherein the variable (e.g., R 1 , R 2 , R 3 , R 4 , R 5 , R 9 , L, X 1 , X 2 , X 3 , X 4 , X 5) is as described herein. Also provided is a compound for use in the treatment of metastatic or chemotherapy-resistant cancer, wherein the compound is represented by structural formula I, or a pharmaceutically acceptable salt thereof. Also provided is the use of a compound of structural formula I or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of metastatic or chemotherapy-resistant cancer.

[0011] Another embodiment is a method of inhibiting the proliferation of metastatic or chemotherapy-resistant cancer cells, comprising administering to the cells an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. Also provided is a compound for inhibiting the proliferation of metastatic or chemotherapy-resistant cancer cells, wherein the compound is represented by formula I, or a pharmaceutically acceptable salt thereof. Also provided is the use of a compound of formula I or a pharmaceutically acceptable salt thereof for preparing a medicament for inhibiting the proliferation of metastatic or chemotherapy-resistant cancer cells.

[0012] Another embodiment is a method of treating type 2 diabetes in a subject in need thereof, comprising administering to the subject a compound of formula I or a pharmaceutically acceptable salt thereof. Also provided is a compound for use in the treatment of type 2 diabetes, wherein the compound is represented by formula I, or a pharmaceutically acceptable salt thereof. Also provided is the use of a compound of formula I or a pharmaceutically acceptable salt thereof for preparing a medicament for treating type 2 diabetes.

[0013] The compounds, compositions and methods described herein can be used to inhibit an enzyme catalytic target (ALDH1a3) that is important for metastasis and / or chemotherapy resistance and widely expressed in multiple tumor types. Various compounds described herein have low nanomolar affinity for ALDH1a3, and compound MBE1 has been shown in mice to shrink metastatic lesions without toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] This patent or patent application publication contains at least one drawing drawn in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0015] The foregoing will be apparent from the following more particular description of example embodiments.

[0016] Figure 1A is in the presence of various compounds described herein, ALDEFLUOR TM - A bar graph showing the percentage of positive cells using the standard ALDEFLUOR TM Protocol, percentage of SUM159-M1a-Aldh1a3 cells above background fluorescence levels as detected by flow cytometry after incubation with 100 nanomolar compound concentration. Background fluorescence was gated using 1 mM N,N-diethylaminobenzaldehyde (DEAB) as a negative control.

[0017] Figure 1B ALDEFLUOR was detected in the presence of different concentrations of MBE1 or MBE1.5. TM - Line graph of the percentage of positive cells and showing the ALDEFLUOR in combination with dose titration of MBE1 or MBE1-5 according to the standard described herein TM The percentage of SUM159-M1a-Aldh1a3 cells with fluorescence above background level as detected by flow cytometry after incubation with the protocol. The [inh-min] threshold was set to the lower limit of two standard deviations of the control samples, while the IC 50 The threshold was set at 50% of the mean of the control samples.

[0018] Figure 1C is a line graph of the percentage of high ALDH in the presence of different concentrations of MBE1 or MBE1.5, and it shows the standard ALDEFLUOR described herein in combination with dose titrations of MBE1 or MBE1.5. TM Percentage of SUM159-M1a-Aldh1a3 cells at high fluorescence levels as detected by flow cytometry after protocol incubation. High activity was gated at the 99th percentile of cells at 10 nanomolar MBE1.5 concentration. The [inh-min] threshold was set to the lower limit of two standard deviations of the control samples.

[0019] Figure 2A Cells were stably transduced with human Aldh1a1, Aldh1a3, or Aldh3a1 and analyzed by flow cytometry using ALDEFLUOR in combination with DMSO or MBE1 at the listed concentrations. TM The bar graphs of MCF7 cells analyzed by the protocol and showing the effect of DMSO or MBE1 on the ALDEFLUOR induced by each enzyme. TM The influence of activity.

[0020] Figure 2B Cells were transiently transfected with human Aldh1a2 or Aldh1a3 and analyzed by flow cytometry using ALDEFLUOR in combination with DMSO or MBE1.5 at the listed concentrations. TM The bar graphs of 293T cells analyzed by the protocol show the effect of DMSO or MBE1.5 on the ALDEFLUOR induced by each enzyme. TM The influence of activity.

[0021] Figure 3A ALDEFLUOR in multiple cancer types in the presence of DEAB or MBE1 TM- A histogram of positive cells and shows that in addition to M1a (breast) and MDA-468 (breast), ALDH1a3 activity is also observed in multiple cancer types and is inhibited by MBE1 in multiple cancer types.

[0022] Figure 3B ALDEFLUOR in multiple cancer types in the presence of 1 mM DEAB (a pan-ALDH inhibitor) or 100 nM MBE1.5 (a specific ALDH1a3 inhibitor) TM A bar graph of activity is shown and it shows the majority of human cancer cell lines showing Aldh1a3 activity.

[0023] Figure 4A is a flow cytometric profile and it shows that CD45-positive bone marrow cells are ALDEFLUOR TM -positive, but not affected by MBE1.5.

[0024] Figure 4B is a flow cytometric profile and it shows that CD11c-positive bone marrow cells are ALDEFLUOR TM -positive, but not affected by MBE1.5.

[0025] Figure 5A is a diagram of the dosing strategy for administering MBE1 and paclitaxel to mice injected with M1a-Aldh1a3 cells by intravenous tail vein injection, and it shows the design of an in vivo experiment designed to test the efficacy of MBE1 in treating metastatic cancer.

[0026] Figure 5B is a line graph of lung metastasis versus time (days), as measured using bioluminescence imaging (BLI), and in Figure 5A Lung metastasis was compared in the presence and absence of MBE1 in mice from the experiments listed in Student's t-test, two-tailed, assuming unequal variances.

[0027] Fig. 6A is a diagram of the dosing strategy for administering MBE1 and paclitaxel to mice injected with M1a-Aldh1a3 cells by intracardiac injection, and it shows the design of an in vivo experiment designed to test the efficacy of MBE1 in treating metastatic cancer.

[0028] Figure 6B is a line graph of bone metastasis versus time (days), as measured using BLI, and Fig. 6ABone metastasis was compared in the presence and absence of MBE1 in mice from the experiments listed in Student's t-test, one-sided, assuming unequal variances.

[0029] Fig. 7A Figure 2 is a flow cytometric plot showing that genetic knockout of ALDH1a3 in MDA-MB-468 breast cancer cells (middle and rightmost plots) significantly reduced ALDEFLUOR compared to control MDA-MB-468 cells (leftmost plot). TM active.

[0030] Figure 7B is the tumor volume (mm 3 ) is a line graph 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.

[0031] Figure 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 slowed primary tumor growth and sensitized tumors to paclitaxel.

[0032] Fig. 8A is a line graph of bioluminescence (ph / s) versus time (days) and shows the development of lung metastasis in mice injected with SUM159-M1b cells transduced with vectors encoding the three ALDH enzymes ALDH1a1, ALDH1a3 and ALDH3a1.

[0033] Figure 8B is Fig. 8A Figure 3 Plot of ex vivo counts of lung nodules at the experimental endpoint as described in. Student's t-test, two-sided, assuming unequal variances.

[0034] Fig.9A is a flow cytometric profile and it shows that genetic knockout of ALDH1a3 in Sum159-M1a breast cancer cells nearly eliminated ALDEFLUOR in this cell TM The ALDEFLUOR gene can be rescued by transducing the cells with a rescue vector. TM active.

[0035] Fig. 9B is a line graph of bone metastasis versus time (days), as measured by bioluminescence (ph / s), and shows that knockdown of ALDH1a3 in Sum159-M1a breast cancer cells slows bone metastasis growth.

[0036] Fig. 9Cis a Kaplan-Meier plot of bone metastasis-free survival over time, and it shows that knockdown of ALDH1a3 in Sum159-M1a breast cancer cells significantly improved the survival time.

[0037] Fig. 10A Survival curves of patients divided by high (red) and low (black) Aldh1a3 expression are based on a data analysis tool hosted by kmplot.com and show that distant metastasis-free survival of patients with renal clear cell carcinoma is associated with ALDH1a3 expression levels.

[0038] Fig. 10B Survival curves of patients divided by high (red) and low (black) Aldh1a3 expression are based on a data analysis tool hosted by kmplot.com and show that the overall survival of triple-negative breast cancer patients is associated with ALDH1a3 expression levels.

[0039] Fig. 10C Survival curves of patients divided by high (red) and low (black) Aldh1a3 expression are based on a data analysis tool hosted by kmplot.com and show that the overall survival of gastric cancer patients is associated with ALDH1a3 expression levels.

[0040] Fig. 10D Survival curves of patients divided by high (red) and low (black) Aldh1a3 expression are based on a data analysis tool hosted by kmplot.com and show that the overall survival of bladder cancer patients is associated with ALDH1a3 expression levels.

[0041] Fig.10E Survival curves of patients divided by high (red) and low (black) Aldh1a3 expression are based on a data analysis tool hosted by kmplot.com and show that the overall survival of ovarian cancer patients is associated with ALDH1a3 expression levels.

[0042] Fig.10F Survival curves of patients divided by high (red) and low (black) Aldh1a3 expression are based on a data analysis tool hosted by kmplot.com, and show that the overall survival of lung squamous cell carcinoma patients is associated with ALDH1a3 expression levels.

[0043] Figure 10G Patient survival curves divided by high (red) and low (blue) Aldh1a3 expression are based on survival time series data and patient-level RNA expression data from the Cancer Genome Atlas, and show that the overall survival of colorectal cancer patients is associated with ALDH1a3 expression levels.

[0044] Fig. 10H Patient survival curves divided by high (red) and low (blue) Aldh1a3 expression are based on survival time series data and patient-level RNA expression data from the Cancer Genome Atlas, and they show that the overall survival of low-grade glioma patients is associated with ALDH1a3 expression levels.

[0045] Fig.11A is an mRNA expression plot of Aldh1a3 from the METABRIC clinical breast cancer dataset and it shows Aldh1a3 expression and chemotherapy history of breast cancer subtypes.

[0046] Fig. 11B is a set of survival curves based on the Erasmus Medical Center-Memorial Sloan-Kettering (EMC-MSK) data set and shows the survival time of breast cancer patients by subtype and divided by median ALDH1a3 expression level.

[0047] Fig.12 ALDEFLUOR TM Activity versus concentration is plotted and shows the ALDEFLUOR activity of several compounds described herein at concentrations of 10 nM and 100 nM. TM active.

[0048] Fig.13A is a Western blot and it shows the expression of various ALDH isoforms including 1a1, 1a2, 1a3 and 3a1 in MCF7 and SUM 159 cells.

[0049] Fig. 13B ALDEFLUOR expressing the indicated ALDH isoforms TM - Plot of the percentage of positive MCF7 cells versus the logarithm of the MBE 1.5 concentration and it shows that at concentrations below 10 μM, MBE 1.5 specifically inhibits ALDH1a3.

[0050] Fig. 13C ALDEFLUOR expressing the indicated ALDH isoforms TM - A plot of the percentage of positive SUM159 cells versus the logarithm of the MBE 1.5 concentration and shows that at concentrations below 10 μM, MBE 1.5 specifically inhibits ALDH1a3.

[0051] Fig.14is a line graph of lung metastasis versus time (days) as measured by bioluminescent imaging (BLI) and shows that in a mouse xenograft model, 3 doses of 50 mg / kg MBE1.5 combined with 25 mg / kg paclitaxel administered on days 17, 19 and 21 caused regression of established metastatic disease. Student's t-test, two-sided, assuming unequal variances.

[0052] Fig.15A is a line graph of body weight (g) versus time (days) and shows that there was no overall toxicity associated with MBE1.5 treatment in this experiment.

[0053] Fig. 15B is the tumor volume (mm 3 ) is a line graph against time (days) and shows that 12-day treatment with MBE1.5 causes regression of MDA-MB-468 primary breast tumors. Statistics were performed by Student's t-test. *p<0.05.

[0054] Fig.16A is a line graph of lung metastasis bioluminescence versus time (days), and it shows the development of lung metastasis before and after treatment with MBE1.5 or vehicle. Statistics were performed by Student's t-test. *p<0.05.

[0055] Fig. 16B is a Kaplan-Meier plot of mouse survival over time as a function of treatment group and shows that 12-day treatment with MBE1.5 prolonged survival in mice with late-stage established breast cancer lung metastases. Statistics were calculated by Cox proportional hazards model. DETAILED DESCRIPTION

[0056] The compounds, compositions and methods are based in part on the discovery that aldehyde dehydrogenase (Aldh, ALDH) and specifically ALDH isoform 1a3 (ALDH1a3) are involved in a variety of cancer types and other ALDH1a3-mediated diseases and conditions, such as type 2 diabetes. Therefore, the compounds, compositions and methods provided herein are directed to modulating (e.g., inhibiting) ALDH, e.g., ALDH1a3.

[0057] Example implementations are described below.

[0058] definition

[0059] The compounds described herein include those conventionally described and are further described by the classes, subclasses, and species disclosed herein. Unless otherwise indicated, as used herein, the following definitions shall apply. For purposes of the present invention, the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. represents a chemical element. In addition, the general principles of organic chemistry are described in the following literature: "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and "March's Advanced Organic Chemistry", 5 th Ed., Ed.: Smith, MB and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are incorporated herein by reference.

[0060] Unless otherwise specified in the specification, the nomenclature used in the specification generally follows the examples and rules described 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 chemical structure nomenclature rules. Optionally, a chemical nomenclature program (e.g., Compound names were generated using the PCR product, version 17.0.0.206, PerkinElmer Informatics, Inc.

[0061] The compounds of the present invention may have asymmetric centers, chiral axes and chiral planes (for example, as described in EL Eliel and SH Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pp. 1119-1190), and exist as racemates, racemic mixtures, and as individual diastereomers or enantiomers, and all possible isomers and mixtures thereof, including optical isomers, are included in the present invention.

[0062] "Alkyl" refers to a saturated aliphatic branched or straight chain monovalent hydrocarbon radical having a specified number of carbon atoms. Thus, "(C1-C3)alkyl" means a radical having 1 to 3 carbon atoms in a straight or branched chain arrangement. Alkyl includes methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, and the like. "(C1-C3)alkyl" includes methyl, ethyl, propyl, and isopropyl.

[0063] "Amino" means -NH2.

[0064] "Alkylamino" means (alkyl)(H)-N-, wherein the alkyl is as described herein. In one aspect, the alkylamino is (C1-C3)alkylamino. Specific alkylamino groups include methylamino, ethylamino, propylamino and isopropylamino.

[0065] "Dialkylamino" means (alkyl)2-N-, wherein the alkyl groups are as described herein and may be the same or different. A specific dialkylamino group is ((C1-C3)alkyl)2-N-, wherein the alkyl groups may be the same or different. Dialkylamino groups include dimethylamino, diethylamino and methylethylamino.

[0066] "Halogen" and "halo" are used interchangeably herein and refer to fluorine, chlorine, bromine or iodine, respectively. In some embodiments, the halogen is selected from fluorine (fluoro) or chlorine (chloro).

[0067] "Chloro" means -Cl.

[0068] "Fluoro" means -F.

[0069] "Cyano" means -CN.

[0070] "Nitro" means -NO2.

[0071] "Hydroxyl" means -OH.

[0072] "Mercapto" means -SH.

[0073] "Alkoxy" refers to an alkyl group attached through an oxygen linking atom, wherein "alkyl" is as described herein. "(C1-C6)alkoxy" includes methoxy, ethoxy, propoxy, butoxy, pentyloxy and hexyloxy.

[0074] "Thioalkoxy" refers to an alkyl group attached through a sulfur linking atom wherein "alkyl" is as described herein.

[0075] "Haloalkyl" includes mono-, poly- and perhaloalkyl, wherein each halogen is independently selected from fluorine, chlorine and bromine, and alkyl is as described herein. In one aspect, haloalkyl is perhaloalkyl (e.g., perfluoroalkyl). Haloalkyl includes trifluoromethyl and pentafluoroethyl.

[0076] "Haloalkoxy" refers to a haloalkyl group attached through an oxygen linking atom, wherein "haloalkyl" is as described herein.

[0077] It will be appreciated that one skilled in the art can select substituents on the compounds of the invention to provide compounds that are chemically stable and can be readily synthesized by techniques known in the art and those described below. The substituents contemplated by the invention (e.g., R 1 -R9 , m, n, L, X 1 -X 5 ) are preferably those that result in the formation of stable or chemically feasible compounds. As used herein, the term "stable" refers to a compound that is not significantly altered when placed under conditions that allow its production, detection, and in certain embodiments, its recovery, purification, and use for one or more purposes disclosed herein.

[0078] As used herein, the term "pharmaceutically acceptable salt" means salts that are suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, etc., and commensurate with a reasonable benefit / risk ratio within the scope of reasonable medical judgment. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, and the relevant teachings of the document are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids and bases compatible with the treatment of a subject.

[0079] Examples of pharmaceutically acceptable non-toxic acid addition salts are amino salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid, or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid, or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable acid addition salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentylpropionate, digluconate, dodecylsulfonate, ethanesulfonate, formate, fumarate, gluconate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, 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, valerate, and the like.

[0080] In some embodiments, the exemplary inorganic acid forming a suitable salt includes but is not limited to hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid and acidic metal salts, such as disodium hydrogen orthophosphate and potassium hydrogen sulfate. The exemplary organic acid forming a suitable salt includes mono-, di- and tricarboxylic acids. For example, these acids are exemplified by acetic acid, glycolic acid, lactic acid, pyroglucic 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-hydroxyethylsulfonic acid. Mono- or di-acid salts can be formed, and these salts can exist as hydration, solvation or substantially anhydrous forms. Generally, the acid addition salts of these compounds are more soluble in water and a variety of hydrophilic organic solvents, and generally show a higher melting point than their free alkali forms.

[0081] In some embodiments, acid addition salts are most suitably formed from pharmaceutically acceptable acids and include, for example, those formed with inorganic acids such as hydrochloric acid, sulfuric acid, or phosphoric acid and organic acids such as succinic acid, maleic acid, acetic acid, or fumaric acid.

[0082] 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, alicyclic, or aromatic organic amines, such as methylamine, trimethylamine, and picoline or ammonia. The selection criteria for appropriate salts will be known to those skilled in the art.

[0083] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N + ((C1-C4) alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, lower alkyl sulfonates, and aryl sulfonates, when appropriate.

[0084] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, by replacing a hydrogen with a deuterium or tritium or by replacing a 13 C- or 14 Compounds resulting from the replacement of carbon with a C-enriched carbon are within the scope of the invention. For example, these compounds are useful as analytical tools, as probes in biological assays, or as therapeutic agents according to the invention.

[0085] When introducing elements disclosed herein, the articles "a," "an," and "said" are intended to mean that there are one or more of the 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.

[0086] Compound

[0087] The first embodiment is a compound shown in structural formula I:

[0088]

[0089] or a pharmaceutically acceptable salt thereof, wherein:

[0090] R 1 and R 5 are each independently hydrogen or (C1-C6) alkyl;

[0091] R 2 , R 3 and R 4 Each is independently hydrogen, halogen, halo(C1-C3)alkyl, (C1-C3)alkyl, (C1-C3)alkoxy, (C1-C3)thioalkoxy, halo(C1-C3)alkoxy, amino, (C1-C3)alkylamino, (C1-C3)dialkylamino, cyano, nitro, hydroxyl or thiol;

[0092] L is -C(O)-, -C(R 6 )(R 7 )-, -C(CH2CH2)-, -C(OCH2)- or -C(N(H)CH2)-;

[0093] R 6 and R 7 Each is independently hydrogen, hydroxyl, mercapto, halogen, (C1-C3) alkyl, (C1-C3) alkoxy, (C1-C3) thioalkoxy or halo (C1-C3) alkoxy;

[0094] X 1 , X 2 , X 3 , X 4 and X 5 Each is independently -C(R 8 )-or-N-; and

[0095] Each R 8 are independently hydrogen, halogen, halo(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)thioalkoxy, halo(C1-C6)alkoxy, amino, (C1-C6)alkylamino, (C1-C6)dialkylamino, cyano, nitro, hydroxyl or thiol.

[0096] 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 pharmaceutically acceptable salt of any of the foregoing compounds. The values ​​of the variables are as described in the first embodiment.

[0097] In a second aspect of the first embodiment, R 2 and R 4 The remaining variable values ​​are as described in the first embodiment or the first aspect thereof.

[0098] In a third aspect of the first embodiment, R 1 The values ​​for the remaining variables are as described in the first embodiment, or the first or second aspect thereof.

[0099] In a fourth aspect of the first embodiment, R 5 The remaining variable values ​​are as described in the first embodiment, or first through third aspects thereof.

[0100] In a fifth aspect of the first embodiment, R 3 is hydrogen or a halogen (eg, halogen). The values ​​for the remaining variables are as described in the first embodiment, or first through fourth aspects thereof.

[0101] In a sixth aspect of the first embodiment, R 3 is a halogen (eg, fluorine). The values ​​for the remaining variables are as described in the first embodiment, or first through fifth aspects thereof.

[0102] In a seventh aspect of the first embodiment, L is -C(O)- or -C(R 6 )(R 7 The remaining variable values ​​are as described in the first embodiment or the first to sixth aspects thereof.

[0103] In an eighth aspect of the first embodiment, L is -C(O)-. The remaining variable values ​​are as described in the first embodiment, or first through seventh aspects thereof.

[0104] In a ninth aspect of the first embodiment, L is -CH2-. The values ​​for the remaining variables are as described in the first embodiment, or first through eighth aspects thereof.

[0105] In a tenth aspect of the first embodiment, X 1 , X 2 , X 3 , X 4 and X 5 The remaining variables have values ​​as described in the first embodiment, or first to ninth aspects thereof.

[0106] In the eleventh aspect of the first embodiment, X 3is -N-; and X 1 , X 2 , X 4 and X 5 Each is independently -C(R 8 The remaining variable values ​​are as described in the first embodiment or first to tenth aspects thereof.

[0107] In the twelfth aspect of the first embodiment, each R 8 is independently hydrogen, halogen, halo(C1-C3)alkyl, (C1-C3)alkyl, (C1-C3)alkoxy, (C1-C3)thioalkoxy, halo(C1-C3)alkoxy, amino, (C1-C3)alkylamino, (C1-C3)dialkylamino, cyano, nitro, hydroxyl or thiol. The remaining variable values ​​are as described in the first embodiment or the first to eleventh aspects thereof.

[0108] In the thirteenth aspect of the first embodiment, each R 8 are independently hydrogen, halogen, or (C1-C6)alkyl (eg, hydrogen or (C1-C6)alkyl). The remaining variable values ​​are as described in the first embodiment, or first to twelfth aspects thereof.

[0109] In the fourteenth aspect of the first embodiment, R 6 and R 7 are each independently hydrogen, hydroxy, halogen, (C1-C3) alkyl or (C1-C3) alkoxy. For example, R 6 and R 7 are independently hydrogen, hydroxy or halogen, for example, R 6 and R 7 The remaining variable values ​​are as described in the first embodiment, or first to thirteenth aspects thereof.

[0110] The compounds shown in 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 any pharmaceutically acceptable salts of the above compounds. The compounds shown in formula I also include MBE1.5A, MBE1.5B, MBE1.5C and MBE1.5D, or pharmaceutically acceptable salts of the above compounds.

[0111] In a second embodiment, the compound is a compound represented by structural formula II:

[0112]

[0113] or a pharmaceutically acceptable salt thereof, wherein:

[0114] R 1 and R5 are each independently hydrogen or (C1-C6) alkyl;

[0115] R 2 and R 4 Each is independently hydrogen, halogen, halo(C1-C3)alkyl, (C1-C3)alkyl, (C1-C3)alkoxy, (C1-C3)thioalkoxy, halo(C1-C3)alkoxy, amino, (C1-C3)alkylamino, (C1-C3)dialkylamino, cyano, nitro, hydroxyl or thiol;

[0116] R 3 is halogen, halo(C1-C3)alkyl, (C1-C3)alkyl, (C1-C3)alkoxy, (C1-C3)thioalkoxy, halo(C1-C3)alkoxy, amino, (C1-C3)alkylamino, (C1-C3)dialkylamino, cyano, nitro, hydroxyl or mercapto;

[0117] L is -C(O)-, -C(R 6 )(R 7 )-, -C(CH2CH2)-, -C(OCH2)- or -C(N(H)CH2)-;

[0118] R 6 and R 7 Each is independently hydrogen, hydroxyl, mercapto, halogen, (C1-C3) alkyl, (C1-C3) alkoxy, (C1-C3) thioalkoxy or halo (C1-C3) alkoxy;

[0119] Each R 9 are independently halogen, halo(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)thioalkoxy, halo(C1-C6)alkoxy, amino, (C1-C6)alkylamino, (C1-C6)dialkylamino, cyano, nitro, hydroxyl or mercapto; and

[0120] 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.

[0121] In a first aspect of the second embodiment, each R 9 and (C1-C3)alkyl, (C1-C3)alkyl, (C1-C3)alkoxy, (C1-C3)thioalkoxy, halo(C1-C3)alkoxy, amino, (C1-C3)alkylamino, (C1-C3)dialkylamino, cyano, nitro, hydroxyl or thiol. The remaining variable values ​​are as described in the first embodiment or any aspect thereof or the second embodiment.

[0122] In a second aspect of the second embodiment, each R 9 is independently halogen or (C1-C6)alkyl (eg, halogen or (C1-C3)alkyl). The remaining variable values ​​are as described in the first embodiment, or any aspect thereof, or the second embodiment, or first aspect thereof.

[0123] In a third aspect of the second embodiment, n is 0, 1 or 2 (eg, n is 0, n is 1 or n is 2). The remaining variable values ​​are as described in the first embodiment or any aspect thereof or the second embodiment or first or second aspect thereof.

[0124] In a fourth aspect of the second embodiment, each R 9 The 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.

[0125] In a fifth aspect of the second embodiment, n is 1 or 2 (eg, 1). The remaining variable values ​​are as described in the first embodiment, or any aspect thereof, or the second embodiment, or first to fourth aspects thereof.

[0126] The compound represented by the structural formula II includes MBE1.5 or its pharmaceutically acceptable salts. The compound represented by the structural formula II also includes MBE1.5A, MBE1.5B, MBE1.5C and MBE1.5D or their pharmaceutically acceptable salts.

[0127] The third embodiment is a compound shown in structural formula III:

[0128]

[0129] or a pharmaceutically acceptable salt thereof, wherein the variable values ​​are as described in the first or second embodiment or any aspect above.

[0130] The fourth embodiment is a compound shown in structural formula IV:

[0131]

[0132] or a pharmaceutically acceptable salt thereof, wherein:

[0133] Each R 9 are independently halogen, halo(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)thioalkoxy, halo(C1-C6)alkoxy, amino, (C1-C6)alkylamino, (C1-C6)dialkylamino, cyano, nitro, hydroxyl or mercapto; and

[0134] m is 0, 1, 2, 3, or 4. The variable R 9 Alternative values ​​or values ​​of the remaining variables are as described in the first or second embodiment or any aspect above.

[0135] In a first aspect of the fourth embodiment, X 3 -C(R 8 The values ​​of the remaining variables are as described in the first or second embodiment or any aspect or the fourth embodiment above.

[0136] In a second aspect of the fourth embodiment, X 3 The values ​​of the remaining variables are as described in the first or second embodiment or any aspect above or the fourth embodiment or its first aspect.

[0137] 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 aspect above, or the fourth embodiment, or its first or second aspect.

[0138] In a fourth aspect of the fourth embodiment, R 3 is halogen, halo(C1-C3)alkyl, (C1-C3)alkyl, (C1-C3)alkoxy, (C1-C3)thioalkoxy, halo(C1-C3)alkoxy, amino, (C1-C3)alkylamino, (C1-C3)dialkylamino, cyano, nitro, hydroxyl or thiol. The values ​​of the remaining variables are as described in the first or second embodiment or any aspect above or the fourth embodiment or the first to third aspects thereof.

[0139] In a fifth aspect of the fourth embodiment, the compound is not MBE1 or MBE3.6 or a pharmaceutically acceptable salt thereof. The values ​​of the remaining variables are as described in the first or second embodiment or any aspect above or the fourth embodiment or first to fourth aspects thereof.

[0140] The fifth embodiment is a compound represented by structural formula V:

[0141]

[0142] or a pharmaceutically acceptable salt thereof. The variable values ​​are as described in the first, second or fourth embodiment, or any aspect thereof.

[0143] The compounds represented by structural formula V include MBE1.2, MBE1.3 and MBE1.6, or pharmaceutically acceptable salts of the above compounds.

[0144] The sixth embodiment is a compound shown in structural formula VI:

[0145]

[0146] or a pharmaceutically acceptable salt thereof, wherein R 8' is hydrogen, halogen, halo(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)thioalkoxy, halo(C1-C6)alkoxy, amino, (C1-C6)alkylamino, (C1-C6)dialkylamino, cyano, nitro, hydroxyl or thiol (e.g., hydrogen or (C1-C6)alkyl). The remaining variable values ​​are as described in the first, second or fourth embodiment, or any aspect thereof.

[0147] In a first aspect of the sixth embodiment, R 8 The remaining variable values ​​are as described in the first, second or fourth embodiment or any aspect above or the sixth embodiment.

[0148] The seventh embodiment is a compound shown in structural formula VII:

[0149]

[0150] or a pharmaceutically acceptable salt thereof, wherein R 8 ' is hydrogen, halogen, halo(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, (C1-C6)thioalkoxy, halo(C1-C6)alkoxy, amino, (C1-C6)alkylamino, (C1-C6)dialkylamino, cyano, nitro, hydroxyl or thiol (e.g., hydrogen or (C1-C6)alkyl). The remaining variable values ​​are as described in the first, second, fourth or sixth embodiment, or any aspect thereof.

[0151] Compositions and kits

[0152] Provided herein are compositions (e.g., pharmaceutical compositions) comprising an agent that inhibits ALDH1a3 (e.g., a compound shown in any one of Structural Formula IV or a pharmaceutically acceptable salt of the compound) and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the composition of the present invention is formulated for administration to a subject (e.g., a patient) in need of the composition. In some embodiments, the composition of the present invention is formulated for oral, intravenous, subcutaneous, intraperitoneal, or dermal administration to a subject in need thereof.

[0153] The phrase "pharmaceutically acceptable carrier or excipient" refers to a non-toxic carrier or excipient that does not destroy the pharmacological activity of the agent formulated therewith and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the agent. Pharmaceutically acceptable carriers or excipients that can be used in the compositions of the present invention include, but are 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, dipotassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic (cellulose-based) materials, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin.

[0154] The compositions described herein can be administered orally, parenterally (including subcutaneous, intramuscular, intravenous and intradermal), by inhalation spray, topically, rectally, nasally, buccally, vaginally or by implanted reservoir. In some embodiments, provided compounds or compositions are intravenously and / or intraperitoneally administrable.

[0155] 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 composition is administered orally, subcutaneously, intraperitoneally or intravenously.

[0156] The compositions provided herein can be orally administered in any oral acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, dispersants and solutions. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also usually added. For oral administration in the form of capsules, useful diluents include lactose and dried corn starch. When aqueous suspensions and / or emulsions are needed for oral use, the active ingredient can be suspended or dissolved in an oil phase and mixed with an emulsifying and / or suspending agent. If desired, certain sweeteners, flavorings or coloring agents can also be added.

[0157] In some embodiments, oral formulations are formulated for immediate release or sustained / delayed release.

[0158] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or the following: (a) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as carboxymethyl cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and acacia; (c) diluents such as glycerol; (d) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (e) solution retarders. (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof. For capsules, tablets and pills, the dosage form may also contain a buffer.

[0159] Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.

[0160] Solid compositions of similar types can also be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols, etc. Solid dosage forms of tablets, dragees, capsules, pills and granules can be prepared with coatings and shells such as enteric coatings and other coatings known in the field of pharmaceutical formulations. They can optionally contain opacifiers, and can also be compositions that release active ingredients only or preferentially in certain parts of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0161] The agent may also be in the form of microencapsulation with one or more excipients as described above. In these solid dosage forms, the agent may be mixed with at least one inert diluent such as sucrose, lactose or starch. As in normal practice, these dosage forms may also contain other substances in addition to the inert diluent, for example, tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose.

[0162] Compositions for oral administration can be designed to protect the active ingredient against degradation during passage through the digestive tract, for example, by formulation of an outer coating on a tablet or capsule.

[0163] In another embodiment, the agent can be provided with an extended (or "delayed" or "slow release") release composition. This delayed release composition comprises an agent combined with a delayed release component. This composition enables the provided agent to be targeted and released to the lower gastrointestinal tract, for example, the small intestine, large intestine, colon and / or rectum. In certain embodiments, the delayed release composition also comprises an intestinal or pH-dependent coating, such as cellulose acetate phthalate and other phthalates (e.g., polyvinyl acetate phthalate, methacrylate (Eudragits)). Alternatively, the delayed release composition provides controlled release to the small intestine and / or colon by providing a pH-sensitive methacrylate coating, a pH-sensitive polymer microsphere, or a polymer that undergoes degradation by hydrolysis. The delayed release composition can be formulated with a hydrophobic or gelling excipient or coating. Colonic delivery can also be provided by coatings hydrolyzed 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 linked to azoaromatic bond coatings.

[0164] The compositions described herein can also be administered in suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycols.

[0165] 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.

[0166] Topical application for the lower intestinal tract may be effected in a rectal suppository formulation (see above) or in a suitable enema formulation.Topical transdermal patches may also be used.

[0167] For other topical applications, the composition can be formulated in a suitable ointment, which contains an active component suspended or dissolved in one or more carriers. The carrier for the topical application of the reagent includes but is not limited to mineral oil, liquid petrolatum, white vaseline, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water and penetration enhancer. Alternatively, the composition can be formulated in a suitable lotion or cream, which contains an active component suspended or dissolved in one or more pharmaceutical carriers. Alternatively, the composition can be formulated together with a suitable lotion or cream, which contains an 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, hexadecyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. In other embodiments, suitable carriers are 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.

[0168] For ophthalmic use, the composition can be formulated as a micronized suspension in isotonic, pH-adjusted sterile saline, or preferably, as a solution in isotonic, pH-adjusted sterile saline, with or without a preservative such as benzalkonium chloride. Alternatively, for ophthalmic use, the composition can be formulated in an ointment such as petrolatum.

[0169] The compositions may also be administered by nasal aerosol or inhalation. These compositions are prepared according to techniques well known in the pharmaceutical formulation art and may be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters (to enhance bioavailability), fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0170] The amount of the agent that can be combined with the carrier material to produce a composition in a single dosage form will vary with the host being treated, the particular form of administration, and the activity of the agent 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 the agent can be administered to a subject receiving the composition.

[0171] It will also be understood that the specific dosage 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 specific disease being treated. The amount of the agent in the composition will also depend on the specific agent in the composition.

[0172] Other pharmaceutically acceptable carriers, adjuvants and vehicles that can 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 d-α-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tween or other similar polymer 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 silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic materials, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and lanolin. Cyclodextrins, such as α-, β-, and γ-cyclodextrin, or chemically modified derivatives, such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl-β-cyclodextrin, or other solubilized derivatives may also be advantageously used to enhance delivery of the agents described herein.

[0173] The composition can be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oily suspension. Such suspensions can be prepared using suitable dispersants or wetting agents (e.g., Tween 80) and suspending agents according to techniques known in the art. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally available diluent or solvent, for example, as a solution in 1,3-butanediol. Available carriers and solvents that can be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are generally used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids (such as oleic acid and its glyceride derivatives) are useful in preparing injections, as are natural medicinal oils (such as olive oil or castor oil), particularly in their polyoxyethylated forms. These oily solutions or suspensions may also contain a long-chain alcohol diluent or dispersant or carboxymethylcellulose or similar dispersants commonly used in the formulation of pharmaceutical dosage forms, such as emulsions and / or suspensions. Other commonly used surfactants such as Tweens or Span and / or other similar emulsifiers or bioavailability enhancers commonly used in the production of pharmaceutical solid, liquid or other dosage forms may also be used for formulation purposes.

[0174] In some embodiments, a composition comprising an agent that inhibits ALDH1a3 (e.g., a compound shown in any one of Formulas I-VII or a pharmaceutically acceptable salt thereof) may also comprise one or more other therapeutic agents (e.g., a chemotherapeutic agent, e.g., paclitaxel, doxorubicin, 5-fluorouracil, tamoxifen, octreotide). When the composition of the present invention comprises a combination of an agent that inhibits ALDH1a3 and one or more other therapeutic agents, the agent should be present at a dosage level of about 1 to 100%, and more preferably about 5% to about 95%, of the dose typically administered in a monotherapy regimen. The other agent may be administered separately from the agent that inhibits ALDH1a3 as part of a multiple-dose regimen. Alternatively, the other agent may be part of a single dosage form mixed with the agent that inhibits ALDH1a3 in a single composition.

[0175] For example, the compositions herein can be administered intravenously, intraarterially, intraocularly, intravitreally, subdermally, orally, orally, nasally, transmucosally, topically, in ophthalmic preparations, or by inhalation in a dosage range of about 0.5 mg / kg to about 100 mg / kg body weight, or alternatively, in a dosage range of about 1 mg / dose to about 1000 mg / dose every 4 to 120 hours, or according to the requirements of a particular drug. Typically, the compositions will be administered about 1 to about 6 times a day, or alternatively, the compositions will be administered as a continuous infusion. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary based on the host being treated and the specific form of administration. A typical preparation will contain about 5% to about 95% of the active compound (w / w). Alternatively, the preparation can contain about 20% to about 80% of the active compound (w / w).

[0176] Doses lower or higher than those recited above may be required. The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific agent used, age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, severity and course of the disease, condition or symptom, the patient's susceptibility to the disease, condition or symptom, and the judgment of the treating physician.

[0177] Once the subject's condition improves, if necessary, a maintenance dose of an agent of the present invention (e.g., a compound shown in any one of formulas I-VII or a pharmaceutically acceptable salt of the above compound), a composition, or a combination thereof may be administered. Subsequently, when the symptoms have been alleviated to the desired level, the dosage or frequency of administration or both may be reduced to a level that maintains the improved condition, depending on the symptoms. However, the subject may require intermittent treatment in a long-term manner when symptoms of the disease recur.

[0178] Also provided herein are kits comprising reagents as described herein (e.g., compounds shown in any one of formulas I-VII or pharmaceutically acceptable salts of the above compounds) and other reagents (additional agents). In one embodiment, the kit comprises an effective amount of reagents as described herein to treat a disease, disorder or condition as described herein (e.g., cancer, type 2 diabetes) and an effective amount of other reagents to treat the disease, disorder or condition. In some embodiments, the kit further comprises written instructions for administering the reagents and other reagents to a subject to treat a disease, disorder or condition as described herein (e.g., cancer, type 2 diabetes).

[0179] method

[0180] Aldehyde dehydrogenase (ALDH) belongs to the superfamily of NAD(P+)-dependent enzymes that play a role in aldehyde metabolism by irreversibly catalyzing the oxidation of endogenously and exogenously generated aldehydes to their respective carboxylic acids. ALDH has a broad spectrum of biological activities, including retinoic acid (RA) biosynthesis and alcohol metabolism.

[0181] The ALDH enzyme family contains 19 members with different 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 present in several cellular compartments, however, most are localized to the cytosol or mitochondria. Some ALDH enzymes participate in global metabolism through expression in the liver, where they act to detoxify acetylaldehyde formed by alcohol dehydrogenases, 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 modulate cellular biochemistry, often through unknown mechanisms of action.

[0182] Aldehyde dehydrogenase isoform 1a3 (ALDH1a3) is an isoform / isozyme of the ALDH1a subfamily that is important in the biosynthesis of RA and the regulation of RA signaling and is cell- and disease-specific. Before 2000, ALDH1a3 was called ALDH6 and in 2000-2007 was called Raldh3. Under standard circumstances, ALDH1a3 is required only during embryonic development and is dispensable for healthy adult mice. In contrast to its minor role in normal physiology, ALDH1a3 has recently been shown to be a key regulator of ALDEFLUOR in most cancer types. TM ALDEFLUOR is a major determinant of reactivity. TM Activity has long been used as a marker to distinguish invasive cancer cells from the bulk of the tumor, although the TM Whether / how activity affects tumor development remains significantly unknown.

[0183] ALDEFLUOR has been found to be driven by ALDH1a3 TM Activity is a functional driver of cancer aggressiveness and is critical for tumor development, metastasis, and chemotherapy resistance. Thus, human ALDH1a3 (UniProtKB Accession No.: P47895) is a functional driver of chemotherapy resistance and metastatic phenotypes in cancer, including breast cancer. Therefore, ALDH1a3 represents a potential therapeutic target in a variety of lesions, and targeting ALDH1a3 may overcome current barriers to treating stage 3 / 4 patients whose tumors are resistant to conventional therapy forms.

[0184] Therefore, 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 shown in any one of Structural Formulas I-VII or a pharmaceutically acceptable salt of the above-mentioned compound, or a composition comprising a compound shown in any one of Structural Formulas I-VII or a pharmaceutically acceptable salt of the above-mentioned compound). Therefore, 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 shown in any one of Structural Formulas I-VII or a pharmaceutically acceptable salt of the above-mentioned compound, or a composition comprising a compound shown in any one of Structural Formulas I-VII or a pharmaceutically acceptable salt of the above-mentioned compound. In other embodiments, a method for treating cancer (e.g., metastatic cancer, chemotherapy-resistant cancer) in a subject in need thereof is provided, comprising administering to the subject an effective amount of a compound shown in Table 1 (e.g., MBE1, MBE1.5, MBE1.5A, MBE1.5B, MBE1.5C, MBE1.5D) or a pharmaceutically acceptable salt thereof, or a composition comprising a compound shown in Table 1 (e.g., MBE1, MBE1.5, MBE1.5A, MBE1.5B, MBE1.5C, MBE1.5D) or a pharmaceutically acceptable salt thereof.

[0185] Also provided herein is a method 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 shown in any one of Structural Formulas I-VII or a pharmaceutically acceptable salt of the compound, or a composition comprising a compound shown in any one of Structural Formulas I-VII or a pharmaceutically acceptable salt of the compound). Therefore, in some embodiments, a method for treating type 2 diabetes in a subject in need thereof is provided, comprising administering to the subject an effective amount of a compound shown in any one of Structural Formulas I-VII or a pharmaceutically acceptable salt of the compound, or a composition comprising a compound shown in any one of Structural Formulas I-VII or a pharmaceutically acceptable salt of the compound. In other embodiments, a method for treating type 2 diabetes in a subject in need thereof is provided, comprising administering to the subject an effective amount of a compound shown in Table 1 (e.g., MBE1, MBE1.5, MBE1.5A, MBE1.5B, MBE1.5C, MBE1.5D) or a pharmaceutically acceptable salt thereof, or a composition comprising a compound shown in Table 1 (e.g., MBE1, MBE1.5, MBE1.5A, MBE1.5B, MBE1.5C, MBE1.5D) or a pharmaceutically acceptable salt thereof.

[0186] As used herein, the term "treating" means combating a medical condition (eg, a condition associated with cancer) to the extent that the medical condition is improved (eg, the number of cancer metastases in a subject is reduced) according to clinically acceptable standards.

[0187] In one embodiment, the subject (e.g., 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, sheep, horse, dog, cat or rodent). In a specific embodiment, the subject is a human. "Subject in need thereof" refers to a subject who has or is at risk of developing a disease or condition described herein (e.g., cancer, metastatic cancer). A medical professional (e.g., physician) can easily determine whether a subject has or is at risk of developing a disease or condition described herein (e.g., cancer).

[0188] In one embodiment, the subject in need thereof suffers from cancer (e.g., metastatic cancer, chemotherapy-resistant cancer). The cancer can be a solid tumor, leukemia, lymphoma, or myeloma. In a specific embodiment, the subject in need thereof suffers from 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), clear cell renal cell carcinoma, gastric cancer, bladder cancer, ovarian cancer, squamous cell lung cancer, colorectal cancer, or glioma (e.g., low-grade glioma).

[0189] Cancers include: acute lymphoblastic leukemia, adults; acute lymphoblastic leukemia, children; acute myeloid leukemia, adults; adrenocortical carcinoma; adrenocortical carcinoma, children; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; cerebellar astrocytoma, children; cerebral astrocytoma, children; extrahepatic bile duct cancer; bladder cancer; bladder cancer, children; bone cancer, osteosarcoma / malignant fibrous histiocytoma; brain stem glioma, children; brain tumors, adults; brain tumors, brain stem glioma, children; brain tumors, cerebellar astrocytoma; brain tumors, cerebral astrocytoma / malignant glioma, children; brain tumors, ependymoma, children; brain tumors, medulloblastoma, children; brain tumors, supratentorial primitive neuroectodermal tumor, children; Brain tumors, optic pathway and hypothalamic gliomas, childhood; brain tumors, childhood (other); breast cancer; breast cancer and pregnancy; breast cancer, childhood; breast cancer in males; bronchial adenoma / carcinoid tumors, childhood; carcinoid tumors, childhood; gastrointestinal carcinoid tumors; adrenocortical carcinoma; islet cell carcinoma; carcinoma of unknown primary; primary central nervous system lymphoma; cerebellar astrocytoma, childhood; cerebral astrocytoma / malignant glioma, childhood; cervical cancer; cancer in children; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloid hyperplasia; clear cell sarcoma of the tendon sheath; colon cancer; colorectal cancer, childhood; cutaneous T-cell lymphoma; endometrial cancer; ependymoma, childhood; epithelial ovarian cancer; esophageal cancer; esophageal cancer, childhood; Ewing's sarcoma family; extracranial genital cells, childhood extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; stomach (gastric) cancer; stomach (gastric) cancer in children; gastrointestinal carcinoid tumor; extracranial germ cell tumor in children; extragonadal germ cell tumor; ovarian germ cell tumor; gestational trophoblastic tumor; brainstem glioma in children; optic pathway and hypothalamic glioma in children; hairy cell leukemia; head and neck cancer; adult (primary) hepatocellular (liver) cancer; child (primary) hepatocellular (liver) cancer; adult Hodgkin lymphoma; child Hodgkin lymphoma; Hodgkin lymphoma during pregnancy; tongue cancer; child hypothalamic and optic pathway glioma; intraocular melanoma; islet cell carcinoma (endocrine pancreas); Kaposi's sarcoma; kidney cancer; laryngeal cancer; child Laryngeal cancer; acute lymphoblastic leukemia in adults; acute lymphoblastic leukemia in children; acute myeloid leukemia in adults; acute myeloid leukemia in children; chronic lymphocytic leukemia; chronic myeloid leukemia; hairy cell leukemia; cancer of the lip and oral cavity; liver cancer (primary) in adults; liver cancer (primary) in children; non-small cell lung cancer; small cell lung cancer; acute lymphocytic leukemia in adults; acute lymphocytic leukemia in children; chronic lymphocytic leukemia; AIDS-related lymphoma; lymphoma of the central nervous system (primary); cutaneous T-cell lymphoma; Hodgkin lymphoma in adults; Hodgkin lymphoma in children; Hodgkin lymphoma during pregnancy; non-Hodgkin lymphoma in adults; non-Hodgkin lymphoma in children; non-Hodgkin lymphoma during pregnancy;Primary central nervous system lymphoma; Waldenstrom's macroglobulinemia; Breast cancer in men; Malignant mesothelioma in adults; Malignant mesothelioma in children; Malignant thymoma; Mantle cell lymphoma; Medulloblastoma in children; Melanoma; Intraocular melanoma; Merkel cell carcinoma; Malignant mesothelioma; Metastatic squamous cell carcinoma of the neck with occult primary; Multiple endocrine neoplasia syndrome in children; Multiple myeloma / plasmacytoma; Aliber's disease; Myelodysplastic syndrome; Chronic myeloid leukemia; Acute myeloid leukemia in children; Multiple myeloma; Chronic myelodysplastic disease; Nasal and paranasal sinus cancer; Nasopharyngeal carcinoma; Nasopharyngeal carcinoma in children; Neuroblastoma tumors; non-Hodgkin's lymphoma in adults; non-Hodgkin's lymphoma in children; non-Hodgkin's lymphoma during pregnancy; non-small cell lung cancer; oral cancer in children; oral and lip cancer; oropharyngeal cancer; osteosarcoma / malignant fibrous histiocytoma of bone; ovarian cancer in children; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low-grade malignant potential tumor; pancreatic cancer; pancreatic cancer in children; islet cell pancreatic cancer; sinus and nasal cancer; parathyroid cancer; penile cancer; pheochromocytoma; pineal and supratentorial primitive neuroectodermal tumors in children; 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 in adults; primary liver cancer in children; prostate cancer; colorectal cancer; renal cell (kidney) cancer; renal cell cancer in children; transitional cell cancer of the renal pelvis and ureter; retinoblastoma; rhabdomyosarcoma in children; salivary gland cancer; salivary gland cancer in children; Ewing's sarcoma family of sarcomas; Kaposi's sarcoma; sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; rhabdomyosarcoma in children; soft tissue sarcoma in adults; soft tissue sarcoma in children; Sezary syndrome; skin cancer; skin cancer in children; skin cancer (melanoma); Merkel cell skin cancer; small cell Lung cancer; small intestinal cancer; soft tissue sarcoma in adults; soft tissue sarcoma in children; cervical squamous cell carcinoma with occult metastatic primary; gastric (stomach) cancer; gastric (stomach) cancer in children; supratentorial primitive neuroectodermal tumor in children; cutaneous T-cell lymphoma; testicular cancer; thymoma in children; malignant thymoma; thyroid cancer; thyroid cancer in children; transitional cell carcinoma of the renal pelvis and ureter; gestational trophoblastic tumor; cancer of unknown primary site in children; rare cancers in children; transitional cell carcinoma of the ureter and renal pelvis; urethral cancer; uterine sarcoma; vaginal cancer; optic pathway and hypothalamic glioma in children; vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor.

[0190] Metastasis of the above cancers can also be treated according to the methods described herein. Thus, in some embodiments, the cancer is a metastatic cancer.

[0191] As used herein, "metastatic cancer" refers to a cancer that spreads to other areas of a subject's body from its original primary site or its initial formation site. 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 suffers from bone metastasis (e.g., spontaneous bone metastasis) of breast cancer. In another embodiment, the subject suffers from lung metastasis of breast cancer. In another embodiment, the subject suffers from bone metastasis.

[0192] In a specific embodiment, the subject in need thereof has breast cancer (eg, metastatic breast cancer). Breast cancer subtypes particularly suitable for treatment using the methods described herein include, but are not limited to, basal cell-like breast cancer and HER2-positive breast cancer.

[0193] As used herein, a "resistant cancer" is a cancer that is unresponsive to treatment. "Resistant cancers" include those that are unresponsive to treatment at the start of treatment and those that become unresponsive to treatment during treatment. In one embodiment, the cancer is a resistant cancer.

[0194] In one embodiment, the subject in need thereof suffers from a cancer that is resistant to treatment with one or more chemotherapeutic agents or a chemotherapy-resistant cancer. As used herein, "chemotherapy-resistant cancer" refers to a cancer that is unresponsive to treatment with one or more chemotherapeutic agents. "Chemotherapy-resistant cancer" includes those that are unresponsive to treatment with one or more therapeutic agents at the beginning of treatment and those that become unresponsive to treatment with one or more therapeutic agents during treatment. Chemotherapy-resistant cancers that are particularly suitable for treatment using the methods described herein include, but are not limited to, cancers that are resistant to treatment with paclitaxel and / or doxorubicin.

[0195] In one embodiment, the subject in need thereof suffers from invasive cancer.As used herein, "invasive cancer" refers to a cancer that develops rapidly (e.g., forms, grows and / or metastasizes), or a cancer that requires more stringent and intense treatment than the typical cancer treatment for the same type of cancer.Invasive cancer includes but is not limited to conventional treatment, for example, chemotherapy (including taxanes, anthracyclines, alkylating agents, etc.), radiotherapy (e.g., proton beam radiation) or targeted biological therapy (e.g., Herceptin, Keytruda, etc.) are unresponsive or develop during the treatment using any accepted method or diagnose (III / IV phase cancer) of lung cancer, breast cancer, pancreatic cancer, kidney cancer, colon cancer, rectal cancer, brain cancer, liver cancer and prostate cancer or hematological cancer.As a specific example, invasive prostate cancer loses the response for anti-androgen therapy or radiation and is usually bone metastasis, and invasive melanoma is any tumor that spreads beyond the dermis.The diagnosis of all glioblastoma multiforme is considered to be invasive. Aggressive cancers often spread to distant sites and cause significant morbidity due to the lack of targeted therapies.

[0196] In some embodiments, the subject in need thereof has advanced (eg, stage III / IV) cancer.

[0197] In some embodiments, the cancer is a primary cancer (eg, a primary solid tumor). As used herein, "primary cancer" refers to the original site of a cancer (eg, a tumor) in a subject's body.

[0198] As used herein, "effective amount" refers to an amount of an agent that is sufficient to achieve the desired therapeutic effect in a subject under the conditions of administration when administered to a subject, such as an amount sufficient to inhibit (e.g., reduce, decrease, prevent) tumor formation, tumor growth (e.g., proliferation, size), tumor vascularization, tumor development (e.g., invasion, metastasis) and / or chemoresistance in a subject (e.g., patient) with cancer. The amount can be determined by any suitable method known to those skilled in the art (e.g., in situ immunohistochemistry, imaging (e.g., ultrasound, CT scan, MRI, NMR), 3 H-thymidine incorporation) determines the effectiveness of therapy (e.g., reduction and / or prevention of tumor metastasis).

[0199] Clinicians with routine techniques can use the guidelines provided herein and other methods known in the art to determine the effective amount of the reagent to be used, and the effective amount depends on several factors, for example, including the specific reagent selected, the age of the experimenter, the sensitivity and tolerance to the drug and the overall health status. For example, a suitable dosage can be about 0.001mg / kg to about 100mg / kg for each treatment, about 0.01mg / kg to about 100mg / kg, about 0.01mg / kg to about 10mg / kg, about 0.01mg / kg to about 1mg / kg body weight. The determination of the dosage of specific reagents, experimenters and cancer is completely within the capabilities of those skilled in the art. Preferably, the dosage does not cause or produce minimum adverse side effects (for example, immunogenic reactions, nausea, dizziness, gastric disturbances, high viscosity syndrome, congestive heart failure, stroke, pulmonary edema).

[0200] Agents that inhibit ALDH1a3 can be administered in a single dose or as multiple doses, for example, in an order and schedule suitable to achieve the desired therapeutic effect (e.g., inhibition of cancer metastasis). Suitable administration doses and schedules can be determined by a clinician with routine skills.

[0201] The agent that inhibits ALDH1a3 can also be administered in combination with one or more other therapies. With respect to the administration of the agent in combination with one or more other therapies (adjuvant, targeted, cancer treatment-related therapies, etc.), the agent is typically administered as a single dose (e.g., by injection, infusion, oral administration), followed by repeated doses at specific intervals (e.g., one or more hours) if necessary or indicated.

[0202] In one embodiment, the agent can be administered in a metronomic dosing regimen, wherein a low dose is administered more often relative to the maximum tolerated dose. Some preclinical studies have demonstrated superior antitumor efficacy, effective antiangiogenic effects, and reduced toxicity and side effects (e.g., bone marrow suppression) of metronomic regimens compared to the corresponding maximum tolerated dose (MTD) (Bocci, et al., Cancer Res, 62: 6938-6943, (2002); Bocci, et al., Proc. Natl. Acad. Sci., 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 shortcomings associated with chemotherapy.

[0203] It can be used as a primary therapy (e.g., as the main therapeutic agent in a therapy or treatment regimen); as an adjunctive therapy (e.g., as a therapeutic agent used 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 adjunctive therapy; as an adjuvant therapy (e.g., as a therapeutic agent administered to a subject in need thereof after the main therapeutic agent in a therapy or treatment regimen has been provided); or in combination with adjuvant therapy, an agent that inhibits ALDH1a3 is administered to a subject in need thereof. For example, adjuvant therapy includes 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 therapy), The invention relates to a method for treating cancer. The invention relates to a method for treating cancer. The invention relates to a method for treating cancer. The invention relates to a method for treating cancer. The invention relates to a method for treating cancer. The invention relates to a method for treating cancer. The invention relates to a method for treating cancer. The invention relates to a method for treating cancer. The invention relates to a method for treating cancer.

[0204] In some embodiments, the method includes administering an effective amount of an agent that inhibits ALDH1a3 in combination with one or more other therapies (e.g., chemotherapy, radiation, and / or surgical removal of a tumor). When administered in a combination therapy, the agent may be administered before, after, or simultaneously with another therapy (e.g., administration of a chemotherapeutic agent such as paclitaxel or doxorubicin). When co-administered simultaneously (e.g., jointly), the agent and the other therapeutic agent may be in separate preparations or in the same preparation. Alternatively, the agent and the other therapeutic agent may be administered sequentially as separate compositions within an appropriate time frame (e.g., within a cancer treatment period / interval, such as about 1.5 to about 5 hours) as determined by a skilled clinician (e.g., a time sufficient to overlap the drug effects of the therapy).

[0205] Depending on the agent and the specific cancer to be treated, the agent that inhibits ALDH1a3 can be administered by a variety of routes of administration, including, for example, oral, dietary, topical, transdermal, rectal, parenteral (e.g., intraarterial, intravenous, intramuscular, subcutaneous, intradermal injection), intravenous infusion, and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal drops) routes of administration. As indicated, administration can be local or systemic. The preferred form of administration can vary based on the specific agent selected.

[0206] For example, a physician can determine the actual dosage of therapeutic agent and treatment regimen by considering the nature of the cancer (primary or metastatic), the number and size of tumors, other therapies being provided, and the characteristics of the subject.

[0207] Also provided herein is a method for inhibiting the proliferation of cancer cells (e.g., metastatic cancer cells, chemotherapy-resistant cancer cells). The method includes administering (e.g., an effective amount of) an agent that inhibits ALDH1a3 to the cell (e.g., a compound shown in any one of formulas I-VII or a pharmaceutically acceptable salt of the above compound, or a composition comprising a compound shown in any one of formulas I-VII or a pharmaceutically acceptable salt of the above compound). In a specific embodiment, the cancer cell is a breast cancer cell (e.g., a basal cell-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 a specific embodiment, the cell is present in a human subject (e.g., a human subject with cancer).

[0208] Also provided herein is a method for inhibiting ALDH1a3 in a subject in need thereof. The method comprises administering to the subject (e.g., an effective amount) an agent that inhibits ALDH1a3 (e.g., a compound shown in any one of Structural Formulas I-VII or a pharmaceutically acceptable salt of the compound, or a composition comprising a compound shown in any one of Structural Formulas I-VII or a pharmaceutically acceptable salt of the compound).

[0209] Also provided herein is a method for treating a disease or condition mediated by ALDH1a3 in a subject in need thereof, such as a cancer type described herein. The method comprises administering to the subject an effective amount of an agent that inhibits ALDH1a3 (e.g., a compound shown in any one of formulas I-VII or a pharmaceutically acceptable salt of the compound, or a composition comprising a compound shown in any one of formulas I-VII or a pharmaceutically acceptable salt of the compound). ALDH1a3 is also involved in type 2 diabetes. Therefore, type 2 diabetes is another example of an ALDH1a3-mediated disease or condition.

[0210] illustration

[0211] Synthesis of N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide (MBE1.2)

[0212] 3-Chloro-N-(3-fluorophenyl)acrylamide: A mixture of 3-fluoroaniline (48.0 g, 0.432 mol, 1.00 equiv) and dichloromethane (440 mL) was placed in a 2-L round-bottom flask. 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 hours until the starting material disappeared as shown by TLC analysis. The reaction mixture was then diluted with water (440 mL) and extracted with ethyl acetate (2.2 L×3). The combined organic layers were washed with brine (1.0 L×2), dried over anhydrous sodium sulfate and concentrated in vacuo to provide 64.0 g (75%) of 3-chloro-N-(3-fluorophenyl)propionamide as a light brown solid. LC-MS (ES) [M+1] + m / z:202.1.

[0213] 7-Fluoro-3,4-dihydroquinolin-2(1H)-one: A mixture of 3-chloro-N-(3-fluorophenyl)propionamide (64.0 g, 0.317 mol, 1.00 equiv) and aluminum chloride (148 g, 1.11 mol, 3.50 equiv) was placed in a 1-L round-bottom flask. The reaction mixture was stirred at 120°C-125°C for 2 hours 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×3) and methyl tert-butyl ether (60 mL×3). This resulted in 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.

[0214] 7-Fluoro-6-nitro-3,4-dihydroquinoline-2(1H)-one: A mixture of 7-fluoro-3,4-dihydroquinoline-2(1H)-one (47.8 g, 0.289 mol, 1.00 equiv) and concentrated sulfuric acid (290 mL) was placed in a 2-L round-bottom flask. Nitric acid (20.8 mL, 0.304 mol, 1.05 equiv) was added dropwise. The reaction mixture was stirred at 0 ° C for 2 hours until the starting material disappeared as shown by TLC analysis. The reaction mixture was poured into ice / water (600 mL) and filtered. The filter residue was collected and washed with water (60 mL×3) and methyl tert-butyl ether (60 mL×3). This resulted in the production of 37.0 g (crude) of 7-fluoro-6-nitro-3,4-dihydroquinoline-2(1H)-one as a light brown solid. LC-MS(ES)[M+1] + m / z:211.1. 1 H NMR (DMSO-d6, 300 MHz) was consistent with a compound having the structure of 7-fluoro-6-nitro-3,4-dihydroquinolin-2(1H)-one.

[0215] 6-Amino-7-fluoro-3,4-dihydroquinolin-2(1H)-one: Into a 3-L round-bottom flask was placed 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 a hydrogen atmosphere for 4 hours 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 on silica gel (DCM / MeOH=200 / 1). This resulted in 12.1 g (two steps, 24%) of 6-amino-7-fluoro-3,4-dihydroquinolin-2(1H)-one as an orange solid. LC-MS(ES)[M+1] + m / z:211.1.

[0216] N-(7-Fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide (MBE1.2): Into a 40-mL vial was placed 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), 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)(dimethylimino)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 preparative HPLC. This resulted in 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 HNMR (DMSO-d6, 300 MHz) was consistent with a compound having the structure of N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide.

[0217] Synthesis of 6-((3-ethylpyridin-4-yl)methylamino)-7-fluoro-3,4-dihydroquinolin-2(1H)-one (MBE1.3)

[0218]

[0219] 6-Amino-3,4-dihydroquinolin-2(1H)-one: Into a 100-mL round-bottom flask was placed 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 hours until the starting material disappeared as shown by TLC analysis. The reaction mixture was filtered and the filtrate was concentrated. This resulted in 1.60 g (99%) of 6-amino-3,4-dihydroquinolin-2(1H)-one as a light green solid. LC-MS (ES) [M+1] + m / z:163.1.

[0220] Methyl 3-ethyl isonicotinate: A mixture of methyl 3-bromoisonicotinate (21.6 g, 100 mmol, 1.00 equiv), dioxane (300 mL) and Pd(dppf)Cl2 (3.66 g, 5.00 mmol, 0.05 equiv) was placed in a 1-L 3-neck round-bottom flask. 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 hours under a 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 in vacuo. The residue was purified by flash silica gel eluting with PE / EtOAc (10 / 1) to provide 12.0 g (73%) of methyl 3-ethyl isonicotinate as a light yellow liquid. LC-MS (ES) [M+1] + m / z:166.0.

[0221] (3-Ethylpyridin-4-yl)methanol: A mixture of methyl 3-ethyl 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) was placed in a 100-mL round-bottom flask. Sodium borohydride (1.72 g, 45.5 mol, 2.00 equiv) was added in portions at 0 ° C. The reaction mixture was stirred at room temperature for 2 hours 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 resulted in the production of 1.80 g of (3-ethylpyridin-4-yl)methanol as a white solid. LC-MS (ES) [M+1] + m / z:138.1.

[0222] 3-Ethylisonicotinaldehyde: 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) was placed in a 100-mL round-bottom flask. The reaction mixture was stirred at room temperature for 4 hours until the starting material disappeared as shown by TLC analysis. The reaction mixture was diluted with dichloromethane (25 mL) and washed with a saturated solution of sodium bicarbonate (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 resulted in 350 mg (43%) of 3-ethylisonicotinaldehyde as an orange solid. LC-MS(ES)[M+1] + m / z:136.1.

[0223]

[0136] 6-((3-ethylpyridin-4-yl)methylamino)-7-fluoro-3,4-dihydroquinolin-2(1H)-one: Into a 40-mL vial was placed 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 preparative-HPLC. These resulted in 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 H NMR (DMSO-d6, 300 MHz) was consistent with a compound having the structure of 6-((3-ethylpyridin-4-yl)methylamino)-7-fluoro-3,4-dihydroquinolin-2(1H)-one.

[0224] Synthesis of 2-ethyl-N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)benzamide (MBE1.5)

[0225]

[0226] 2-Ethyl-N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)benzamide: Into a 40-mL vial was placed 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), 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)(dimethylimino)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 preparative HPLC. This resulted in 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 / z:313.1. 1H NMR (DMSO-d6, 300 MHz) was consistent with a compound having the structure of 2-ethyl-N-(7-fluoro-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)benzamide.

[0227] Synthesis of 3-ethyl-N-(2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide (MBE1.6)

[0228]

[0229] 3-Ethylisonicotinic Acid: Into a 100-mL round bottom flask was placed a mixture of methyl 3-ethylisonicotinate (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 hours. The pH was adjusted to pH about 4 with 2M hydrochloric acid solution. The mixture was filtered and the filter cake was collected. This resulted in 2.60 g (86%) of 3-ethylisonicotinic acid as a white solid. LC-MS (ES) [M+1] + m / z:152.0.

[0230] 3-Ethyl-N-(2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide: Into a 40-mL vial was placed 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), 3-ethylisonicotinic acid (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)(dimethylimino)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 preparative-HPLC. This resulted in 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 H NMR (DMSO-d6, 300 MHz) was consistent with a compound having the structure of 3-ethyl-N-(2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)isonicotinamide.

[0231] Cell lines and cell culture

[0232] MDA-MB-468, MCF7, 293T, B16F10, HCT116, HepG2, LN229, HTB140 and SW480 cells were cultured in DMEM + 10% FBS; DU145 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 μg / mL insulin and 20 ng / mL EGF. Cell lines were labeled with a retroviral vector with bicistronic expression of GFP / firefly luciferase to facilitate imaging and flow cytometry experiments. All cell lines were verified to be negative for mycoplasma contamination by monthly PCR analysis. The cell lines used in this article do not appear in the Commonly Misidentified Cell Lines (ICLAC) database. All cell lines were verified by STR analysis and compared with NCBI database data.

[0233] Cloning, virus production and transduction

[0234] The coding sequences of Aldh1a1, Aldh1a2, Aldh1a3 and Aldh3a1 were cloned from cDNA prepared from mixed human reference RNA samples. The cloned sequences flanked by Age1 and Xho1 restriction sites were inserted into the pLex lentiviral plasmid. The clones were sequenced and compared to NCBI expressed sequence tags (ESTs) for accuracy. Viral production of each enzyme was performed by transfection of 293T packaging cell lines using PEI and PsPax2 and VSVG packaging vectors. The viruses were collected and filtered at 0.45 μm, and then the cells were transduced with polybrene (8 g / mL) for 12 hours, followed by cultivation with 1 g / mL puromycin throughout the experiment. All viral transductions and selections were performed on a population-wide basis.

[0235] ALDH1a3 in development and adult physiology; mechanisms of action

[0236] Although certain enzymes within the ALDH family have well-characterized substrate preferences, regulation, and function, most members of the family are less well-studied or have a primary mechanism of action that is unknown. For example, it has been shown how ALDH1L1 and ALDH1L2 play a role in folate metabolism by oxidizing 10-formyl-THF. Another important example of a well-characterized Aldh enzyme is ALDH3a1, which makes up 50% of soluble corneal proteins and functions to protect against UV-induced oxidative damage to the retina and cornea by oxidizing 4-hydroxynonenal. Perhaps the most studied ALDH enzyme is ALDH2, which is a key catalyst for the oxidation of acetylaldehyde to acetate in liver mitochondria. By (Disulfiram) inhibits ALDH2, which is a therapy provided to alcoholics to prevent substance abuse. On the other hand, the ALDH1a subfamily has shown broad implications in developmental biology and a variety of pathologies, while its mechanisms of action and key regulators remain to be elucidated.

[0237] The ALDH1a subfamily is the most biologically significant group within the ALDH family and given its importance to developmental biology and the ALDEFLUOR TM The remarkable ability to identify stem cells, particularly in cancer, has recently become the focus of considerable research. TM The assay primarily measures activity from ALDH1a3.

[0238] Total knockout of ALDH1a3 results in postnatal mouse death due to nasal closure defects. Importantly, this phenotype can be rescued by addition of all-trans retinoic acid during a brief gestational window, resulting in normal adults. In humans, homozygous mutations in ALDH1a3 are associated with microphthalmia, but the phenotype is not fully penetrant and no other lesions are mentioned. To further support the view that ALDH1a3 is developmentally restricted, recent studies have shown that ALDH1a3 is specifically inhibited in certain developing tissues to prevent vitamin A signaling. Other analyses have shown that ALDH1a3 is not required for the developing ovary and that it is only expressed in the prostate and salivary glands of adults. Among the colon, liver, lungs, bladder, prostate, and ovaries, only the ovary has significant ALDEFLUOR TM -population and this small population was only partially inhibited by ALDH1a3 inhibitor.

[0239] In metabolic diseases, ALDH1a3 is a marker for necrotic islet cells. Other studies have shown that ALDH1a3 expression directly reduces insulin secretion from islet cell clones while increasing glucagon secretion. This suggests that ALDH1a3 can also drive pathology in type 2 diabetes.

[0240] Overall, these results suggest that ALDH1a3 can be potentially eliminated without significant on-target toxicity. The data suggest that ALDH1a3 is dispensable for adult mammals. Inhibition of ALDH1a3 during pregnancy would likely be disabling.

[0241] Published studies have advocated two main mechanisms for the cancer-promoting effects of ALDH1a3. They diverge between the detoxification of reactive oxygen species or the oxidation of retinal to bioactive retinoic acid. There are multiple reasons for the discrepancy between its mechanism of action and the number of papers detailing the functional consequences of ALDH1a3 expression. 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. In addition, oxygen stress under in vitro conditions does not reflect in vivo conditions. We have attempted to induce oxygen stress in vitro with paclitaxel and detect it by DCFDA, however, the detection range of this assay is smaller than the inherent bias in the data. However, other literature evidence does support a ROS-related mechanism: ALDH1a3 has been found to detoxify 4-HNE in stallion semen samples, which leads to improved motility. ALDH1a3 has also been induced in head and neck squamous cell carcinoma (SCC) by radiation therapy, suggesting that it can respond to cellular damage.

[0242] Interestingly, compelling work in melanoma has shown that oxygen stress is a major determinant of metastatic spread. In this study, it has been shown that oxygen stress is not present in the primary tumor, yet it is acutely induced and affects the health of metastatic cells. Systemic antioxidant delivery may then help lung metastasis in cells that normally do not metastasize. Modulation of ALDH1a3 expression does not strongly affect primary tumor growth until stimulated by oxidative stressors, such as chemotherapy. On the other hand, the greatest effects of ALDH1a3 inhibition have been observed in lung or bone metastases, which are sites with high oxygen stress levels ( Figures 5A-6B ).

[0243] Studies of retinoic acid (RA) signaling that are dependent on ALDH1a3 are also difficult because they require exogenous addition of retinal under tissue culture conditions and are far from the conditions of the tumor microenvironment. Data showing genetic correlations between each ALDH1a enzyme and each component of the RA signaling pathway in tumors from breast cancer patients have been developed and indicate that of ALDH1a1, ALDH1a2, and ALDH1a3, ALDH1a3 showed the least correlation with RA signaling pathway components in breast tumors. Furthermore, if ALDH1a3 affects the RA signaling pathway, it is expected that tissue culture growth or primary tumor growth would be affected. No differences in primary tumor or in vitro growth were observed in studies of breast cancer cells.

[0244] The functional importance of ALDH1a3 to treatment resistance, tumor development, and metastasis in most solid tumor types makes it an attractive target for drug discovery. Combined with the low probability of on-target toxicity, it is expected that systemic pharmacological inhibition of ALDH1a3 alone or in combination with other therapeutic agents (e.g., approved therapeutics) will be useful for treating primary cancers as well as slow-growing and overtly metastatic disease.

[0245] ALDEFLUOR TM Determination

[0246] ALDEFLUOR TM The assay evaluates the ability of cells to oxidize bodipy-aminoacetaldehyde (BAAA) to bodipy-aminoacetate (BAA), which spontaneously esterifies to form a non-cell membrane permeable product. This activity can be used to sort live cells and thereby differentiate between ALDH activity levels within heterogeneous populations. When ALDEFLUOR was first discovered in 2007, TM -positive cancer cells are more tumorigenic and predicted to have a worse clinical outcome. TM Activity is a marker of a broader transcriptional program that promotes tumor aggressiveness. TM Activity has become the most cited method for assessing the “stemness” or invasiveness of tumor cell populations.

[0247] After this important discovery, ALDEFLUOR is often evaluated TM activity and little consideration of the function of the ALDH1 enzyme. Then, the publication showed that ALDEFLUOR TM The assay isolates invasive or metastatic cancer cells regardless of the primary tumor site. As a result, hundreds of papers have used ALDEFLUOR TM It was only in 2011 that Marcato and colleagues began to show that in a large proportion of breast cancer cell lines, ALDH1a3 is responsible for ALDEFLUOR TM active.

[0248] Since the publication of Marcato and colleagues, rapidly emerging research has established that ALDH1a3 is not only responsible for ALDEFLUOR in a large proportion of cancer types, but also for TMactivity, and it also functionally promotes cancer growth, therapy resistance, and metastasis. Studies of varying quality have established that ALDH1a3 is expressed and is important for growth in melanoma patient-derived xenografts or cell lines, metabolism in mesenchymal-like gliomas or glioblastomas, chemotherapy resistance and radiation resistance, tumorigenicity and cisplatin resistance in lung cancer, growth and radiation resistance in pancreatic cancer, FAK inhibitor resistance in colon and thyroid cells, cisplatin resistance in mesothelioma, Gleason score and growth in prostate cancer, apoptosis resistance and metastasis in breast cancer, and prognosis in hepatobiliary carcinoma. ALDH1a3 has been shown herein to be the major ALDEFLUOR in most solid tumor types TM -Induced enzyme (see Figure 3A and 3B ).

[0249] Expression and prognostic studies further suggest that ALDH1a3 strongly indicates poor outcomes in cancer types. Hypermethylation of the ALDH1a3 promoter, which results in low ALDH1a3 expression, is the strongest predictor of favorable outcomes in a primary glioblastoma patient group. High ALDH1a3 predicts lymph node metastasis in patients with hepatobiliary carcinoma. ALDH1a3 expression is driven by androgens in prostate cancer, which are the major mitogens for prostate cancer cells, while mir187 targets ALDH1a3 in prostate cancer and high mir187 is associated with a good prognosis.

[0250] The ALDEFLUOR TM In the assay, cells were grown until they reached 50-80% confluence, harvested by 0.25% trypsin / EDTA (Sigma) and washed once with PBS by centrifugation / resuspension (190 g, 5 min at 4°C). Cells were counted, centrifuged and plated at 1,000,000 cells / mL in ALDEFLUOR TM Resuspend in buffer (Stemcell Technologies). TM Substrate (Stemcell Technologies, 1:200) and test compounds 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 incubated in ALDEFLUOR with 5 μg / mL DAPI. TM The samples were analyzed by BD LSR2 flow cytometry platform. DEAB was used as a negative control for gating.

[0251] Figure 1A is in the presence of various compounds described herein, ALDEFLUORTM - A bar graph showing the percentage of positive cells using the standard ALDEFLUOR TM Protocol, percentage of SUM159-M1a-Aldh1a3 cells above background fluorescence levels as detected by flow cytometry after incubation with compounds at 100 nM concentration. Background fluorescence was gated using 1 mM DEAB as a negative control. Figure 1A It was confirmed that MBE1-5, MBE1 and MBE1-6 are high-affinity compounds for ALDH1a3 activity inhibition.

[0252] Figure 1B ALDEFLUOR was detected in the presence of different concentrations of MBE1 or MBE1.5. TM - Line graph of the percentage of positive cells and showing the ALDEFLUOR in combination with dose titration of MBE1 or MBE1-5 according to the standard described herein TM The percentage of SUM159-M1a-Aldh1a3 cells with fluorescence above background level as detected by flow cytometry after incubation with the protocol. The [inh-min] threshold was set to the lower limit of two standard deviations of the control samples, while the IC 50 The threshold was set at 50% of the mean of the control samples. Figure 1B MBE1 and MBE1-5 were confirmed to show IC values ​​in the 8-10 nanomolar range. 50 values, which detected inhibitory activity at concentrations as low as 2 nanomolar.

[0253] Figure 1C is a line graph of the percentage of high ALDH in the presence of different concentrations of MBE1 or MBE1.5, and it shows the ALDEFLUOR according to the present invention in combination with dose titration of MBE1 or MBE1.5. TM Percentage of SUM159-M1a-Aldh1a3 cells at high fluorescence levels as detected by flow cytometry after protocol incubation. High activity was gated at the 99th percentile of cells at 10 nanomolar MBE1.5 concentration. The [inh-min] threshold was set to the lower limit of two standard deviations of the control samples. Figure 1C MBE1.5 was shown to be better than MBE1 at inhibiting high ALDH1a3-active cells.

[0254] Figure 2A Cells were stably transduced with human Aldh1a1, Aldh1a3, or Aldh3a1 and analyzed by flow cytometry using ALDEFLUOR in combination with DMSO or MBE1 at the listed concentrations. TMThe bar graphs of MCF7 cells analyzed by the protocol and showing the effect of DMSO or MBE1 on the ALDEFLUOR induced by each enzyme. TM The influence of activity. Figure 2A It was demonstrated that MBE1 did not inhibit Aldh1a1 or Aldh3a1 activity at concentrations below 10 μM, although it was effective in inhibiting Aldh1a3 activity in a separate cancer cell line, MCF7.

[0255] Figure 2B Cells were transiently transfected with human Aldh1a2 or Aldh1a3 and analyzed by flow cytometry using ALDEFLUOR in combination with DMSO or MBE1.5 at the listed concentrations. TM The bar graphs of 293T cells analyzed by the protocol show the effect of DMSO or MBE1.5 on the ALDEFLUOR induced by each enzyme. TM The influence of activity. Figure 2B It was demonstrated that MBE1.5 did not inhibit Aldh1a2 activity at concentrations below 10 μM, although it was effective in inhibiting Aldh1a3 activity in 293T transfected with human Aldh1a3.

[0256] Figure 3A ALDEFLUOR in multiple cancer types in the presence of DEAB or MBE1 TM - A histogram of positive cells and shows that ALDH1a3 activity is observed in multiple cancer types, except for Sum159-M1a (breast) and MDA-468 (breast), and is inhibited by MBE1 in multiple cancer types. Figure 3B ALDEFLUOR in multiple cancer types in the presence of 1 mM DEAB (pan-ALDH inhibitor) or 100 nM MBE1.5 (specific ALDH1a3 inhibitor) TM - Histogram of positive cells and it shows the majority of human cancer cell lines showing Aldh1a3 activity. The notable exception is liver cancer, where a large ALDEFLUOR TM -positive population and it is driven by ALDH1a1.

[0257] ALDEFLUOR TM The assay was also used to evaluate the relative activity of several compounds described herein against SUM159-M1a-Aldh1a3 cells. The activity of several 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.

[0258] Table 1.

[0259]

[0260]

[0261]

[0262] Fig.12 is a graph of ALDEFLUORTM activity versus concentration, and it shows the ALDEFLUORTM activity of several compounds described herein against SUM159-M1a-Aldh1a3 cells at concentrations of 10 nM and 100 nM. At 10 nM concentration, MBE 1.5C was nearly twice as potent as MBE 1.5, MBE 1.5A or MBE 1.5D compared to control (DMSO) or DEAB treated cells.

[0263] ALDH isoforms 1a1, 1a2, 1a3, and 3a1 were expressed in MCF7 or SUM159 cells and subsequently used in the ALDEFLUOR™ assay. Fig.13A are Western blots and show the expression of each ALDH isoform in the indicated cells. Fig. 13B is a graph of the percentage of ALDEFLUOR™-positive MCF7 cells expressing the indicated ALDH isoforms versus the logarithm of the MBE 1.5 concentration and shows that at concentrations below 10 μM, MBE 1.5 specifically inhibits ALDH1a3. Fig. 13C is a graph of the percentage of ALDEFLUOR™-positive SUM159 cells expressing the indicated ALDH isoforms versus the logarithm of the MBE 1.5 concentration and shows that at concentrations below 10 μM, MBE 1.5 specifically inhibits ALDH1a3.

[0264] In vitro analysis of ALDH activity in murine lymphocytes

[0265] Bone marrow cells were collected from 12-week-old C56BL6 mice by washing the tibia and femur with PBS and passing through a 100-μm cell sieve. Thymocytes and splenocytes were isolated from the corresponding tissues and separated by physical disruption of the 100 μm cell sieve. Cells were washed by centrifugation / resuspending and then resuspended at 107 cells / mL. Then, except for the addition of antibodies at 1:200 during the incubation period, These cells were subjected to the ALDEFLUOR™ assay with or without MBE1 as described, in addition to anti-CD11c (Biolegend #100308), anti-CD11c (BD Pharmigen #561119), anti-CD45 (eBioscience 30-F11), or anti-CD317 (Biolegend 129c1).

[0266] Figure 4A is a flow cytometric profile and it shows that CD45-positive bone marrow cells are ALDEFLUOR™-positive but are not affected by MBE1.5. Figure 4B Flow cytometry spectra, and it shows that CD11c-positive bone marrow cells are ALDEFLUORTM-positive, but not affected by MBE1.5. Similar results were obtained in other populations tested, including CD45-positive and CD3e-positive (CD45+CD3e+) and CD11c-positive and CD317-positive (CD11+CD317+) (not shown). Therefore, all populations tested are ALDEFLUORTM-positive, but not affected by MBE1.5, thereby showing less toxic potential.

[0267] Mouse models and xenografts

[0268] All mice were originally ordered from Jackson Laboratory (Bar Harbor, ME) and bred in a barrier facility free of specific pathogens (SPF). Toxicity experiments were performed on male and female C57B16 mice aged 8-12 weeks. MBE1 was dissolved in DMSO at 50 mg / mL, then diluted 1:2 to Kolliphor EL, then diluted 1:5 to PBS to obtain a 5 mg / ml MBE1 solution (10% DMSO, 10% Kolliphor EL, 80% PBS). It was administered by IP injection in groups of 3 mice at 24h intervals with increasing doses from 12.5 to 200 mg / kg body weight. Body condition scores, food absorption, feces / urine production, behavior, and body weight were measured as toxicity readings. Then, 5 mice were treated with 25 mg / kg MBE1 every 3 days for 18 days. No indication of acute or chronic toxicity was shown in any experiment.

[0269] All xenograft experiments were performed on 8-week-old female mice (athymic Nu / Nu or NOD / SCIDγ). Xenograft experiments were performed using 125,000 SUM159-M1a cells in 100 μL PBS for tail vein or intracardiac injection. After injection, mice were randomized into groups. Bioluminescence imaging (BLI) was performed using the IVIS200 system and retro-orbital luciferin injection. For drug treatment, Figure 5A (tail vein injection) and Fig. 6A Mice were given MBE1 (prepared as above) by IP injection in combination with paclitaxel (5 mg / mL in 10% ethanol, 10% Kolliphor EL, 80% PBS) at the time intervals and doses indicated in (intracardial injection).

[0270] Figure 5B is a line graph of lung metastasis versus time (days), as measured using bioluminescence imaging (BLI), and in Figure 5A Lung metastasis was compared in mice in the presence and absence of MBE1 in the experiments listed in . Figure 6B is a line graph of bone metastasis versus time (days), as measured using BLI, and Fig. 6A Bone metastasis was compared in mice in the presence and absence of MBE1 in the experiments listed in . Figure 5B and 6B MBE1 was demonstrated to be an effective therapeutic agent for the treatment of established metastatic disease and it was determined that MBE1 and the compounds disclosed herein can be used to effectively inhibit ALDH1a3 and its downstream effects in vivo.

[0271] In another xenograft experiment, mice were injected with SUM159-M1-p44 cells by tail vein injection as described above and randomized after injection. Metastatic burden was imaged by intravital imaging on day 16. Mice were then treated with paclitaxel (25 mg / kg) and vehicle or MBE1.5 (50 mg / kg) on ​​days 17, 19, and 21. Lung metastatic burden was then imaged on day 22. Signals were normalized to day 16.

[0272] Fig.14 is a line graph of lung metastasis versus time (days) as measured by bioluminescence imaging (BLI) and shows that in a mouse xenograft model, 3 doses of 50 mg / kg MBE1.5 combined with 25 mg / kg paclitaxel administered on days 17, 19 and 21 caused regression of established metastatic disease.

[0273] In another xenograft experiment, mice were injected with MDA-MB-468 cells via mammary fat pads and randomly divided into groups after injection. Mice were treated with MBE1.5 (25 mg / kg per day, n=6) or vehicle (n=12) and paclitaxel (12.5 mg / kg, every other day) for 12 days. Primary tumor measurements were performed by calipers between each treatment group.

[0274] Fig.15A is a line graph of body weight (g) versus time (days) and shows that there was no overall toxicity associated with MBE1.5 treatment in this experiment. Fig. 15B is a line graph of tumor volume (mm3) versus time (days) and shows that 12 days of treatment with MBE1.5 caused regression of primary breast tumors. The tumors of two mice in the MBE1.5 treated group were completely eliminated by treatment.

[0275] In another xenograft experiment, mice were injected with SUM-159-M1a-Aldh1a3 cells by tail vein injection and randomized after injection. Mice were treated with MBE1.5 (25 mg / kg per day) or vehicle and paclitaxel (12.5 mg / kg every other day) for 12 days. Lung metastasis development was tracked by in vivo bioluminescence.

[0276] Fig.16A is a line graph of lung metastasis bioluminescence versus time (days) and shows the development of lung metastasis before and after treatment with MBE1.5 or vehicle. Fig. 16B is a Kaplan-Meier plot of mouse survival over time as a function of treatment group and shows that 12-day treatment with MBE1.5 prolonged survival in mice with advanced established breast cancer lung metastases.

[0277] Pharmacokinetic evaluation

[0278] Pharmacokinetic experiments were performed on 16-week-old C57BL6 male mice. MBE1.5 (15% DMSO, 5% Kolliphor EL, 80% PBS) was administered to mice by IP injection. Blood was collected by terminal cardiac puncture at 15 minutes, 30 minutes, 1 hour, 3 hours and 24 hours after injection. Serum was obtained by blood separation by centrifugation at 16200RCF for 1 min. Then, tissues were collected after exsanguination. Serum and tissues were quickly frozen in liquid nitrogen and submitted for pharmacokinetic evaluation.

[0279] For mouse PK studies, plasma concentration quantification of MBE1.5 by LC / MS / MS

[0280] In a mouse PK study, plasma MBE1.5 concentrations were measured by LC / MS / MS assay.

[0281] Chemicals: MBE1.5 and MBE2.0 were provided as stock solutions (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 the preparation of plasma calibration standards.

[0282] LC / MS / MS assay:

[0283] Preparation of standard solutions: The MBE1.5 stock solution was diluted in control mouse plasma to obtain a calibration range of 8.2 ng / ml to 18 μg / ml. A 1 μg / ml solution of MBE2.0 in methanol was used as a working internal standard (IS).

[0284] Sample preparation: Plasma standards and mouse plasma samples (20 μL) 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 the supernatant was analyzed by LC-MS.

[0285] Liquid chromatography / mass spectrometry: Quantification of MBE1.5 was performed using a TSQ Quantum triple quadrupole mass spectrometer equipped with an API electrospray ionization (ESI) source, a Surveyor MS pump, a Surveyor autosampler, and Xcalibur data acquisition software (Thermo Electron, San Jose, CA, USA).

[0286] 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 μL / min; gradient elution, 0 to 1 minute, 60% A; 1 to 2.5 minutes, 60% to 20% A; 2.5 to 7.0 minutes, 0% A, 7.0 to 9.0 minutes, 0% A, 9.0 to 11.0 minutes, 0% to 60% A; and column: C18, 150×4.6 mm, 5 μm (Phenomenex, Torrance, CA). The retention times of MBE1.5 and IS were 6.3 min and 6.6 min, respectively.

[0287] 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, 4500 V; capillary temperature, 350°C; sheath gas pressure, 20 arbitrary units; auxiliary gas pressure, 50 arbitrary units; collision gas pressure, 1.5 arbitrary units. Collision energy and tube mirror were optimized for each compound.

[0288] Calculation: Plasma calibration standards were used for quantification of MBE in plasma samples. Log linear regression was used on the calibration curve to determine the concentration in plasma samples.

[0289] result:

[0290] LC / MS / MS Assay: A plasma calibration curve for MBE1.5 was plotted using a concentration range of 8.2 ng / mL to 18 μg / mL. The limit of quantification was 8.2 ng / mL (LOD). The LC / MS / MS assay was in good linearity (R2>0.99) within the calibration range. Table 2 summarizes the accuracy and precision within the assay. No matrix effects were observed in this assay.

[0291] Table 2. Intra-assay accuracy and precision of the MBE1.5 LC / MS / MS assay

[0292]

[0293] Plasma PK of MBE1.5 in mice: The plasma concentrations of MBE1.5 in the mouse PK study are listed in Table 3. The basic PK data were analyzed by Winnolin 2.0 using a non-compartmental model. The PK parameters are summarized in Table 4.

[0294] Table 3. Concentrations of MBE1.5 in mouse plasma (μg / ml).

[0295]

[0296] *The integrated peak area is less than the LOD (limit of detection), forcing the calculation.

[0297] Table 4. Pharmacokinetic parameters of MBE1.5 in mice (dose 25 mg / kg, ip)

[0298]

[0299]

[0300] Gene knockout of ALDH1a3

[0301] The CRISPR-Cas9 vector containing both the Cas9 gene and the gRNA sequence containing the homologous sequence of genomic Aldh1a3 was transduced into MDA-MB-468 cells by lentiviral infection, followed by puromycin selection for viral integration and ALDEFLUOR TM The resulting cells were assayed. 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.

[0302] Fig. 7A Figure 2 is a flow cytometric plot showing that genetic knockout of ALDH1a3 in MDA-MB-468 breast cancer cells (middle and rightmost plots) significantly reduced ALDEFLUOR compared to control MDA-MB-468 cells (leftmost plot). TM active. Figure 7B is the tumor volume (mm 3 ) is a line graph 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 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 slowed primary tumor growth and sensitized tumors to paclitaxel.

[0303] The CRISPR-Cas9 vector containing both the Cas9 gene and the gRNA sequence containing the homologous sequence of genomic Aldh1a3 was transfected into Sum159-M1a cells, and the positive transfected cells were subsequently selected by flow cytometry. Then, the positive transfected cells were virally transduced by lentiviral vector containing the vector backbone or full-length human Aldh1a3. Positive transducants were selected by puromycin and expressed by ALDEFLUOR. TM The resulting cells were assayed. In xenograft experiments, knockout-vector or knockout-Aldh1a3 or wild-type vector or wild-type-Aldh1a3 cells were injected into mice by intracardiac injection, 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.

[0304] Fig.9A is a flow cytometric profile and it shows that genetic knockout of ALDH1a3 in Sum159-M1a breast cancer cells nearly eliminated ALDEFLUOR in the cells TM The activity of ALDEFLUOR can be rescued by transducing the cells with a rescue vector TM active. Fig. 9B is a line graph of bone metastasis versus time (days), as measured by bioluminescence (ph / s), and shows that knockdown of ALDH1a3 in Sum159-M1a breast cancer cells slows bone metastasis growth. Fig. 9C is a Kaplan-Meier plot of bone metastasis-free survival over time, and it shows that knockdown of ALDH1a3 in Sum159-M1a breast cancer cells significantly improved the survival time.

[0305] Gene expression of ALDH1a3

[0306] Lentiviral vectors encoding one of the three human ALDH genes ALDH1a1, ALDH1a3, or ALDH3a1 were introduced into luciferase-labeled Sum159-M1b cells by viral transduction, followed by positive selection by puromycin, and transduced cells were injected into mice by tail vein injection. Lung metastatic growth was tracked once a week by in vivo bioluminescence imaging. Lung nodules were counted ex vivo.

[0307] Fig. 8A is a line graph of bioluminescence (ph / s) versus time (days) and shows the development of lung metastasis in mice injected with SUM159-M1b cells transfected with vectors encoding the three ALDH enzymes ALDH1a1, ALDH1a3 and ALDH3a1. Figure 8B is Fig. 8A Figure 3 shows ex vivo counts of lung nodules at the experimental endpoint described in the experiment.

[0308] Survival prediction

[0309] TCGA and Kaplan-Meier plotter (kmplot.com) data were used to generate expression data and survival curves for various cancers in relation to ALDH1a3 expression levels. Cancer patients from each data set were divided by high or low Aldh1a3 expression based on the median expression value or the optimal partition value. Kaplan-Meier analysis was then used to plot patient survival (whether measuring free of distant metastasis or overall survival) to evaluate the relationship between relative levels of Aldh1a3 between patients and the corresponding survival measure.

[0310] Figures 10A-10H The prognostic patient survival curves are based on the data analysis tool hosted by kmplot.com, divided by high (red) and low (black) Aldh1a3 expression, and it shows the distant metastasis-free survival of patients with triple-negative breast cancer ( Fig. 10A ) and overall survival of patients with renal clear cell carcinoma, gastric cancer, bladder cancer, ovarian cancer, lung squamous cell carcinoma, colorectal cancer, and low-grade glioma were associated with ALDH1a3 expression levels (respectively Figures 10B-10H ).

[0311] In another set of predictions, the mRNA expression of Aldh1a3 from the METABRIC clinical breast cancer dataset was segregated by tumor type and prior treatment with chemotherapy. Survival curves in the EMC-MSK dataset were generated by categorizing patients by subtype and partitioning by median Aldh1a3 expression.

[0312] Fig.11A is a graph of ALDH1a3 mRNA expression from the METABRIC clinical breast cancer dataset and shows ALDH1a3 expression and chemotherapy history by breast cancer subtype. Fig. 11B is a prediction survival curve based on the EMC-MSK data set and it shows the predicted survival time of breast cancer patients by subtype and median ALDH1a3 expression level.

[0313] Table 5 reports the hazard ratios (p-values) of patients expressing high ALDH1a1 or ALDH1a3 in estrogen receptor-negative (ER) breast cancer derived from the Kaplan-Meier plotter database, and includes Her2 and triple-negative breast cancer (TNBC) groups at high risk of metastasis. ALDH1a3 is a poor prognostic predictor in ER-negative breast cancer patients (a group that is likely to metastasize).

[0314] Table 5. Hazard ratios (p-values) for patients expressing high ALDH1a1 or ALDH1a3 in the ER-negative breast cancer patient population derived from the Kaplan-Meier plotter database

[0315]

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[0390] The teachings of all patents, published patent applications, and references cited herein are incorporated by reference in their entirety.

[0391] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments as encompassed by the appended claims.

Claims

1. A compound represented by any one of the following structural formulas: or a pharmaceutically acceptable salt of the above compound.

2. Use of an effective amount of a compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating metastatic or chemotherapy-resistant cancer in a subject in need thereof, the treatment comprising administering the medicament to the subject.

3. Use of an effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for inhibiting the proliferation of metastatic or chemotherapy-resistant cancer cells, wherein the inhibition comprises administering the medicament to the cells.

4. Use of an effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating type 2 diabetes in a subject in need thereof, the treatment comprising administering the medicament to the subject.

5. Use of an effective amount of a compound of formula (VII) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating metastatic or chemotherapy-resistant cancer in a subject in need thereof, the treatment comprising administering the medicament to the subject: in: R 1 and R 5 are each independently hydrogen or (C1-C6) alkyl; R 2 , R 3 and R 4 Each is independently hydrogen, halogen, halo(C1-C3)alkyl, (C1-C3)alkyl, (C1-C3)alkoxy, halo(C1-C3)alkoxy, amino, (C1-C3)alkylamino, (C1-C3)dialkylamino, cyano, nitro, hydroxyl or thiol; L is -C(O)-; X 3 -C(R 8 )-or-N-; R 8 is a halo(C1-C6)alkyl group, a (C2-C6)alkyl group, a (C1-C6)alkoxy group, a halo(C1-C6)alkoxy group, an amino group, a (C1-C6)alkylamino group, a (C1-C6)dialkylamino group, a cyano group, a nitro group, a hydroxyl group or a mercapto group; and R 8 ' is hydrogen, halogen, halo(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, amino, (C1-C6)alkylamino, (C1-C6)dialkylamino, cyano, nitro, hydroxyl or mercapto.

6. The use according to claim 5, wherein R 2 and R 4 Each is hydrogen.

7. The use according to claim 5 or 6, wherein R 1 It's hydrogen.

8. The use according to claim 5 or 6, wherein R 5 For hydrogen.

9. The use according to claim 5 or 6, wherein R 3 is hydrogen or a halogen.

10. The use according to claim 9, wherein R 3 It's fluorine.

11. The use according to claim 5 or 6, wherein R 8 ' is hydrogen, halogen or (C2-C6) alkyl.

12. The use according to claim 5 or 6, wherein the compound is represented by the following structural formula: or a pharmaceutically acceptable salt thereof.

13. The use according to claim 5, wherein the compound is represented by MBE1, MBE1.5 or MBE1.6, or the compound is a pharmaceutically acceptable salt of any of the above compounds, 14. The use according to claim 5 or 6, wherein the cancer is a metastatic cancer.

15. The use according to claim 14, 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.

16. The use according to claim 14, wherein the subject has bone metastasis.

17. The use according to claim 5 or 6, wherein the cancer is a chemotherapy-resistant cancer.

18. Use of an effective amount of a compound represented by formula (VII) or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting the proliferation of metastatic or chemotherapy-resistant cancer cells, wherein the inhibition comprises administering the drug to the cells: in: R 1 and R 5 are each independently hydrogen or (C1-C6) alkyl; R 2 , R 3 and R 4 Each is independently hydrogen, halogen, halo(C1-C3)alkyl, (C1-C3)alkyl, (C1-C3)alkoxy, halo(C1-C3)alkoxy, amino, (C1-C3)alkylamino, (C1-C3)dialkylamino, cyano, nitro, hydroxyl or thiol; L is -C(O)-; X 3 -C(R 8 )-or-N-; R 8 is a halo(C1-C6)alkyl group, a (C2-C6)alkyl group, a (C1-C6)alkoxy group, a halo(C1-C6)alkoxy group, an amino group, a (C1-C6)alkylamino group, a (C1-C6)dialkylamino group, a cyano group, a nitro group, a hydroxyl group or a mercapto group; and R 8 ' is hydrogen, halogen, halo(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, amino, (C1-C6)alkylamino, (C1-C6)dialkylamino, cyano, nitro, hydroxyl or mercapto.

19. Use of an effective amount of a compound represented by formula (VII) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating type 2 diabetes in a subject in need thereof, the treatment comprising administering the medicament to the subject: in: R 1 and R 5 are each independently hydrogen or (C1-C6) alkyl; R 2 , R 3 and R 4 Each is independently hydrogen, halogen, halo(C1-C3)alkyl, (C1-C3)alkyl, (C1-C3)alkoxy, halo(C1-C3)alkoxy, amino, (C1-C3)alkylamino, (C1-C3)dialkylamino, cyano, nitro, hydroxyl or thiol; L is -C(O)-; X 3 -C(R 8 )-or-N-; R 8 is a halo(C1-C6)alkyl group, a (C2-C6)alkyl group, a (C1-C6)alkoxy group, a halo(C1-C6)alkoxy group, an amino group, a (C1-C6)alkylamino group, a (C1-C6)dialkylamino group, a cyano group, a nitro group, a hydroxyl group or a mercapto group; and R 8 ' is hydrogen, halogen, halo(C1-C6)alkyl, (C1-C6)alkyl, (C1-C6)alkoxy, halo(C1-C6)alkoxy, amino, (C1-C6)alkylamino, (C1-C6)dialkylamino, cyano, nitro, hydroxyl or mercapto.

Images