CN118324746A

CN118324746A - Small molecule compounds targeting BCL 9/beta-catenin interactions

Info

Publication number: CN118324746A
Application number: CN202310015931.8A
Authority: CN
Inventors: 王英才; 陈一鸣
Original assignee: Jiangsu Mingsheng Jutai Biotechnology Co ltd
Current assignee: Jiangsu Mingsheng Jutai Biotechnology Co ltd
Priority date: 2023-01-05
Filing date: 2023-01-05
Publication date: 2024-07-12

Abstract

The present invention provides small molecule compounds that target BCL9/β -catenin interactions. In particular, the present invention provides compounds of formula I or pharmaceutically acceptable salts thereof. The compound of the formula I has excellent capability of inhibiting BCL 9/beta-catenin interaction.

Description

Small molecule compounds targeting BCL 9/beta-catenin interactions

Technical Field

The invention belongs to the field of medicines, and particularly relates to a small molecular compound for targeting BCL9 (B-cell lymphoma 9))/beta-catenin to react with each other.

Background

Wnt/β -catenin (catenin) signaling is critical in normal embryonic development and throughout life. Furthermore, aberrant Wnt signaling is associated with a variety of diseases, particularly cancer. Recent studies have shown that direct targeting of β -catenin/B-cell lymphoma 9 (BCL 9) protein-protein interactions (PPI) is a very promising strategy to block the Wnt pathway. With advances in understanding the eutectic complex and mechanism of action of the β -catenin/BCL 9 interaction, the discovery process of inhibitors thereof has been facilitated, but only a few inhibitors have been reported.

Canonical Wnt signaling is a highly conserved developmental signaling pathway that regulates cell proliferation, differentiation, and survival. Beta-catenin is generally considered a key effector of Wnt signaling. In the absence of Wnt single selection (Wntoff), the cytoplasmic pool of β -catenin binds to glycogen synthase kinase 3 β (gsk3β), casein kinase 1 α (ck1α), scaffolding protein AXIN, and tumor suppressor adenomatous polyposis escherichia coli (APC) to regulate phosphorylation, followed by degradation of β -catenin by proteasome. Beta-catenin recruits coactivators, including BCL9 or B cell lymphoma 9-like (B9L), pygo, CREB Binding Protein (CBP), and the like, to promote cell proliferation, migration, and transcription of survivin genes, such as cyclin D1, c-myc, survivin, and LEF1. The occurrence and progression of many types of cancers are closely related to these Wnt target genes, including colorectal cancer, breast cancer, lung cancer, hepatocellular carcinoma, leukemia, and multiple myeloma.

Advances in the use of β -catenin/BCL 9 complexes followed reliable biochemical assays and drug discovery strategies provided further insight into interactions, which could lead to the discovery of new anticancer drugs. To date, several different types of β -catenin/BCL 9PPI inhibitors have been reported. The peptide inhibitors and the non-peptide small molecule inhibitors can be mainly classified into two major classes. However, the search for β -catenin/BCL 9PPI inhibitors, especially non-peptide small molecule inhibitors, is still in the primary research stage.

In view of the foregoing, there is a strong need in the art to develop a small molecule compound that targets BCL9 (B-cell lymphoma 9))/β -catenin interactions.

Disclosure of Invention

The invention aims to provide a novel small molecule compound targeting the mutual reaction of BCL 9/beta-catenin.

In a first aspect of the present invention there is provided a compound, or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystalline form or prodrug thereof, said compound being as shown in formula I

Wherein,

Q is 0,1, 2 or 3;

R ³ is selected from the group consisting of: H. -OR ³¹、-C_1-4 alkylene-OR ³¹、-N(R³²)R³³、-C_1-4 alkylene-N (R ³¹)R³²;

m4 is 0, 1,2, 3, 4, 5, 6 or 7;

R ³¹ is selected from the group consisting of: H. optionally substituted C _1-4 alkyl, R ³⁴、-C_1-4 alkylene-R ³⁴;

R ³² is selected from the group consisting of: H. optionally substituted C _1-4 alkyl;

R ³³ is selected from the group consisting of: H. optionally substituted C _1-4 alkyl, R ³⁴、-C_1-4 alkylene-R ³⁴;

R ³⁴ is selected from the group consisting of: c _3-10 cycloalkyl, 4 to 8 membered heterocycloalkyl, C _6-10 aryl, 5 to 10 membered heteroaryl, C _3-10 cycloalkenyl, 4 to 10 membered heterocycloalkenyl; wherein the cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkenyl, and heterocycloalkenyl are optionally substituted with one or more groups selected from the group consisting of: -NH ₂, R;

W ³ is selected from the group ：-C(O)-、-S(O)-、-S(O)₂-、-C(R^F)₂-C(O)-、-C(R^F)₂-S(O)-、-C(R^F)₂-S(O)₂-(, preferably-C (R ^F)₂ -linked to N (R ^E)) in W ³;

R ^F are each independently selected from the group consisting of: H. optionally substituted C _1-4 alkyl, optionally substituted C _3-10 cycloalkyl, optionally substituted 4 to 10 membered heterocycloalkyl; or two R ^F taken together with the carbon atom to which they are attached form an optionally substituted C _3-6 cycloalkyl or an optionally substituted 4-to 6-membered heterocyclyl;

R ^E is selected from the group consisting of: H. optionally substituted C _1-4 alkyl, optionally substituted C _3-10 cycloalkyl, optionally substituted 4 to 10 membered heterocycloalkyl;

R ^D are each independently selected from the group consisting of: H. optionally substituted C _1-4 alkyl; or two R ^D together with the carbon atoms to which they are attached form a group selected from the group consisting of: optionally substituted C _3-10 cycloalkyl, optionally substituted 4 to 10 membered heterocycloalkyl;

W ² is selected from the group consisting of: -O-, -S-, -N (R ^s1) -;

Ring C is optionally substituted with a ring selected from the group consisting of: c _6-10 aryl, and 5 to 10 membered heteroaryl;

m3=0, 1,2, 3 or 4;

Each R ^C is independently R ^C1 or R ^s1;

Each R ^C1 is independently selected from the group consisting of: halogen, optionally substituted C _1-6 alkyl, optionally substituted C _1-6 haloalkyl, hydroxy and optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 haloalkoxy;

ring B is an optionally substituted ring selected from the group consisting of: c _3-12 cycloalkyl, 4 to 12 membered heterocycloalkyl, C _3-10 cycloalkenyl, 4 to 10 membered heterocycloalkyl;

Each R ^B is independently R ^B1、R^s1 or R ^s2;

Each R ^B1 is independently selected from the group consisting of: halogen, hydroxy, cyano, optionally substituted C _1-6 alkyl, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 alkylthio, optionally substituted C _3-10 cycloalkyl, optionally substituted 4 to 10 membered heterocycloalkyl, optionally substituted C _3-10 cycloalkenyl, optionally substituted 4 to 10 membered heterocycloalkenyl, optionally substituted C _6-10 aryl, and optionally substituted 5 to 10 membered heteroaryl;

m2=0, 1,2, 3 or 4;

L ¹ is-X- (W ¹)_n1 -;

X is selected from the group consisting of: -C (O) -, -S (O) -and-S (O) ₂ -;

Each W ¹ is independently selected from the group consisting of: none, -O-, -S-, -C (O) -, -S (O) ₂-、-N(R^s1)-、-C(R^s2)₂ -;

Subscript n1=0, 1,2, or 3;

Ring a is an optionally substituted ring selected from the group consisting of: c _6-10 aryl; 5 to 10 membered heteroaryl; c _6-10 aryl substituted with C _3-10 cycloalkyl, 4 to 10 membered heterocycloalkyl, C _3-10 cycloalkenyl, 4 to 10 membered heterocycloalkenyl, C _6-10 aryl, or 5 to 10 membered heteroaryl; a 5 to 10 membered heteroaryl substituted with a C _3-10 cycloalkyl, a 4 to 10 membered heterocycloalkyl, a C _3-10 cycloalkenyl, a 4 to 10 membered heterocycloalkenyl, a C _6-10 aryl, or a 5 to 10 membered heteroaryl; c _6-10 aryl fused to C _3-10 cycloalkyl, 4 to 10 membered heterocycloalkyl, C _3-10 cycloalkenyl, 4 to 10 membered heterocycloalkenyl, C _6-10 aryl, or 5 to 10 membered heteroaryl; a 5 to 10 membered heteroaryl group fused to a C _3-10 cycloalkyl, 4 to 10 membered heterocycloalkyl, C _3-10 cycloalkenyl, 4 to 10 membered heterocycloalkenyl, C _6-10 aryl, or 5 to 10 membered heteroaryl group;

m1=0, 1,2, 3 or 4;

each R ^A is independently R ^A1、R^s1 or R ^s2;

each R ^A1 is independently selected from the group consisting of: halogen, optionally substituted C _1-6 alkyl, optionally substituted C _1-6 haloalkyl, optionally substituted C _1-6 alkoxy, optionally substituted C _1-6 alkylthio, optionally substituted C _3-10 cycloalkyl, optionally substituted 4 to 10 membered heterocycloalkyl, optionally substituted C _3-10 cycloalkenyl, optionally substituted 4 to 10 membered heterocycloalkenyl, optionally substituted C _6-10 aryl, optionally substituted 5 to 10 membered heteroaryl; or when two R ^A1 are located on adjacent ring atoms, the two R ^A1 and the ring atoms adjacent to them together form a ring selected from the group consisting of: optionally substituted C _3-10 cycloalkyl, optionally substituted 4 to 10 membered heterocycloalkyl, optionally substituted C _3-10 cycloalkenyl, optionally substituted 4 to 10 membered heterocycloalkenyl, optionally substituted C _6-10 aryl, optionally substituted 5 to 10 membered heteroaryl;

R ⁷ is an optionally substituted group selected from the group consisting of: none, C _1-6 alkyl, C _3-10 cycloalkyl, 4 to 10 membered heterocycloalkyl, C _3-10 cycloalkenyl, 4 to 10 membered heterocycloalkenyl, C _6-10 aryl, and 5 to 10 membered heteroaryl;

R ^s1 are each independently selected from the group consisting of: H. optionally substituted C _1-4 alkyl, optionally substituted C _3-6 cycloalkyl, optionally substituted 4 to 6 membered heterocyclyl;

R ^s2 are each independently selected from the group consisting of: H. optionally substituted C _1-4 alkyl, optionally substituted C _3-6 cycloalkyl, optionally substituted 4 to 6 membered heterocyclyl; or two R ^s2 taken together with the carbon atom to which they are attached form an optionally substituted C _3-6 cycloalkyl or an optionally substituted 4-to 6-membered heterocyclyl;

unless specifically defined, the optional substitution means that one or more (e.g., 1,2, 3, or 4) hydrogens in the unsubstituted or group are replaced with a selected R substituent;

Each R is independently selected from the group consisting of: D. halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 hydroxyalkyl, C _2-6 alkenyl, C _2-6 alkynyl 、-CN、-OR'、-NO₂、-NR'R"、-SR'、-OC(O)R'、-C(O)R'、-CO₂R'、-CONR'、-OC(O)NR'R"、-NR"C(O)R'、-NR"-C(O)NR'R"、-NR"C(O)₂R'、-S(O)R'、-S(O)₂R'、-S(O)₂NR'R"、-NR"S(O)₂R'、 C _3-10 cycloalkyl optionally substituted with one or more R '", 4 to 10 membered heterocycloalkyl optionally substituted with one or more R'", C _6-10 aryl optionally substituted with one or more R '", 5 to 10 membered heteroaryl optionally substituted with one or more R'", C _1-4 alkylene-C _3-10 cycloalkyl optionally substituted with one or more R '", C _1-4 alkylene-4 to 10 membered heterocycloalkyl optionally substituted with one or more R'", C _1-4 alkylene-C _6-10 aryl optionally substituted with one or more R '", C _1-4 alkylene-5 to 10 membered heteroaryl optionally substituted with one or more R'";

Each R' is independently selected from the group consisting of: H. d, C _1-6 alkyl, C _1-6 haloalkyl, C _3-10 cycloalkyl optionally substituted with one or more R '", 4 to 10 membered heterocycloalkyl optionally substituted with one or more R'", C _6-10 aryl optionally substituted with one or more R '", 5 to 10 membered heteroaryl optionally substituted with one or more R'", C _1-4 alkylene-C _3-10 cycloalkyl optionally substituted with one or more R '", C _1-4 alkylene-4 to 10 membered heterocycloalkyl optionally substituted with one or more R'", C _1-4 alkylene-C _6-10 aryl optionally substituted with one or more R '", C _1-4 alkylene-5 to 10 membered heteroaryl optionally substituted with one or more R'";

Each R "is selected from the group consisting of: H. d, C _1-4 alkyl, C _1-4 haloalkyl, and C _3-4 cycloalkyl;

Each R' "is independently selected from the group consisting of: D. halogen, hydroxy, nitro, CN, C _1-6 alkyl, C _1-6 haloalkyl.

In another preferred embodiment, ring B is an optionally substituted ring selected from the group consisting of: c _3-12 cycloalkyl, 4 to 12 membered heterocycloalkyl.

In another preferred embodiment, ring B isWherein,Is a single bond or a double bond, X ⁷ is N or CH, o1 is 1 or 2, o2 is 0, 1,2 or 3, o3 is 0 or 1, and o4 is 0, 1,2 or 3.

In another preferred embodiment, X ⁷ is N.

In another preferred embodiment, o1 is 1.

In another preferred embodiment, when X ⁷ is N, N in ring B is attached to L ¹.

In another preferred embodiment, ring B isPreferably, N in ring B is connected to L ¹.

In another preferred embodiment, R ^B is R ^s1 or R ^s2.

In another preferred example, m2=0.

In a further preferred embodiment of the present invention,Is that

In a further preferred embodiment of the present invention,Is thatWherein, refer to the connection to L ¹.

In another preferred example, n1=0.

In another preferred embodiment, L ¹ is-X-. In another preferred embodiment, L ¹ is-SO ₂ -.

In another preferred embodiment, ring C isWherein each of X ¹、X²、X³ and X ⁴ is independently selected from the group consisting of: CH. N.

In another preferred embodiment, in ring C, up to 1 or 2 of X ¹、X²、X³ and X ⁴ are N.

In another preferred embodiment, ring C is

In another preferred example, m3=0. In another preferred example, m3=1, 2, 3 or 4.

In another preferred embodiment, R ^C1 is selected from the group consisting of: halogen (preferably F, cl), C _1-6 haloalkyl (preferably trifluoromethyl), and C _1-6 alkoxy (preferably methoxy).

In another preferred embodiment, ring C isM3=0, 1 or 2; r ^C is H or R ^C1; and R ^C1 is selected from the group consisting of: halogen (preferably F, cl), C _1-6 haloalkyl (preferably trifluoromethyl), and C _1-6 alkoxy (preferably methoxy); preferably, R ^C1 is halogen.

In another preferred embodiment, the compound is represented by formula I-1

Wherein,

Is a single bond or a double bond; x ⁷ is N or CH; o1 is 1 or 2, o2 is 0,1, 2 or 3, and o3 is 0 or 1;

X ¹、X²、X³ and X ⁴ are each independently selected from the group consisting of: CH. N.

In another preferred embodiment, the compound is represented by formula I-2

Wherein,

Is a single bond or a double bond; x ⁷ is N or CH; o1 is 1 or 2, o4 is 0, 1, 2 or 3;

In another preferred embodiment, the compound is of formula I-3 or I-4

In another preferred embodiment q is 1 or 2.

In a further preferred embodiment of the present invention,Selected from the group consisting of:

in a further preferred embodiment of the present invention, Selected from the group consisting of:

in another preferred embodiment, the compound is represented by formula A

In a further preferred embodiment of the present invention,Is thatIn another preferred example, m4=0.

In a further preferred embodiment of the present invention,Is thatIn a further preferred embodiment of the present invention,Selected from the group consisting of:

in a further preferred embodiment of the present invention, Is that

In another preferred embodiment, R ³¹ is selected from the group consisting of: H. optionally substituted C _1-4 alkyl. In another preferred embodiment, R ³¹ is selected from the group consisting of: H. c _1-4 alkyl. In another preferred embodiment, R ³¹ is selected from the group consisting of: H. methyl group.

In another preferred embodiment, R ³² and R ³³ are each independently selected from the group consisting of: H. c _1-4 alkyl.

In another preferred embodiment, R ³² and R ³³ are both H.

In another preferred embodiment, R ³ is-N (R ³²)R³³ or-C _1-4 alkylene-N (R ³¹)R³²).

In another preferred embodiment, R ³ is-C _1-4 alkylene-N (R ³¹)R³²).

In another preferred embodiment, R ³ is selected from the group consisting of ：-NH₂、-CH₂-NH₂、-CH₂-N(CH₃)₂、-CH₂-CH₂-NH₂、-CH₂-CH₂-N(CH₃)₂.

In another preferred embodiment, R ⁷ is an optionally substituted group selected from the group consisting of: c _1-6 alkyl, C _3-10 cycloalkyl, 4 to 10 membered heterocycloalkyl, C _3-10 cycloalkenyl, 4 to 10 membered heterocycloalkenyl, C _6-10 aryl, and 5 to 10 membered heteroaryl.

In another preferred embodiment, R ⁷ is an optionally substituted group selected from the group consisting of: c _6-10 aryl, and 5 to 10 membered heteroaryl.

In another preferred embodiment, R ⁷, the heteroaryl group includes 1, 2, or 3 nitrogen heteroatoms as ring atoms, and the remaining ring atoms in the heteroaryl group are all carbon atoms.

In another preferred embodiment, R ⁷ is an optionally substituted group selected from the group consisting of:

In another preferred embodiment, R ⁷ is optionally substituted phenyl or 5 membered heteroaryl.

In another preferred embodiment, in R ⁷, the optional substitution means that 1 or 2 hydrogens in the unsubstituted or group are replaced with a selected R substituent.

In another preferred embodiment, R ⁷ is selected from the group consisting of:

In another preferred embodiment, R ⁷, each R is independently selected from the group consisting of: c _1-6 alkyl, -NR 'R'; wherein each R' is independently selected from the group consisting of: H. c _1-6 alkyl; each R "is selected from the group consisting of: H. c _1-4 alkyl.

In another preferred embodiment, ring a is a ring selected from the group consisting of:

Wherein each of X ¹、X²、X³ and X ⁴ is independently selected from the group consisting of: CH. N.

In another preferred embodiment, ring a is:

In another preferred embodiment, at most 1 or 2 of X ¹、X²、X³ and X ⁴ are N.

In another preferred embodiment, X ¹、X²、X³ and X ⁴ are both CH.

In another preferred example, m1=0, 1 or 2; preferably, m1=0 or 1; more preferably, m=0.

In another preferred embodiment, each R ^A is independently R ^s1 or R ^A1; and R ^A1 is selected from the group consisting of: halogen, optionally substituted C _1-6 alkyl, optionally substituted C _1-6 haloalkyl, and optionally substituted C _1-6 alkoxy (preferably, R ^A1 is halogen).

In another preferred embodiment, each R ^A is independently H, C _1-4 alkyl or R ^A1; and R ^A1 is selected from the group consisting of: halogen, optionally substituted C _1-6 alkyl, optionally substituted C _1-6 haloalkyl, and optionally substituted C _1-6 alkoxy (preferably, R ^A1 is halogen).

In another preferred embodiment, W ² is-O-or-N (R ^s) -; preferably, W ² is-O-or-NH-.

In another preferred embodiment, W ² is-O-.

In another preferred embodiment, each R ^D is independently selected from the group consisting of: H. optionally substituted C _1-4 alkyl; or two R ^D together with the carbon atom to which they are attached form an optionally substituted C _3-10 cycloalkyl group.

In another preferred embodiment, each R ^D is independently selected from the group consisting of: H. optionally substituted C _1-4 alkyl.

In another preferred embodiment, each R ^D is independently selected from the group consisting of: H. methyl, ethyl.

In another preferred embodiment, R ^D are each methyl.

In a further preferred embodiment of the present invention,Is-O-C (R ^D)₂ -C (O) -.Is-O-C (CH ₃)₂ -C (O) -.

In another preferred embodiment, R ^E is selected from the group consisting of: H. methyl, ethyl.

In another preferred embodiment, R ^E is H.

In another preferred embodiment, each R ^F is independently selected from the group consisting of: H. methyl, ethyl.

In another preferred embodiment, R ^F are each H.

In a further preferred embodiment of the present invention,Is-C (O) -CH ₂ -NH-or-S (O) ₂ -NH-.

In another preferred embodiment, in formula I, ring a, ring B, ring C、L¹、W²、W³、R^A、R^B、R^C、R^D、R^E、R³、R⁷、R、 subscript m1, subscript m2, subscript m3, and subscript m4 are each independently a corresponding group in a particular compound of example compounds or tables A, B and C.

In another preferred embodiment, in formulas I-1 and I-2, ring A, X, X ¹、X²、X³、X⁴、X⁷, subscript o1, subscript o2, subscript o3, subscript o4、W¹、W²、W³、R^A、R^B、R^C、R^D、R^E、R³、R⁷、R、 subscript n1, subscript m2, subscript m3, and subscript m4 are each independently a corresponding group in a particular compound of example compounds or tables A, B and C.

In another preferred embodiment, in formulas I-3 and I-4, ring A, X, X ¹、X²、X³、X⁴, subscript o1, subscript o2, subscript o3, subscript o4、W²、W³、R^A、R^B、R^C、R^D、R^E、R³、R⁷、R、 subscript m1, subscript m2, subscript m3, and subscript m4 are each independently a corresponding group in a particular compound of example compound or tables A, B and C.

In another preferred embodiment, the compound is selected from Table A

Table A

In another preferred embodiment, the compound is selected from Table B

Table B

In another preferred embodiment, the compound is selected from Table C

Table C

In a second aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) The compound of the first aspect or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystal form or prodrug thereof; and

(Ii) Pharmaceutically acceptable carriers or excipients.

In a third aspect of the present invention there is provided the use of a compound according to the first or second aspect or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystal form or prodrug thereof, in the manufacture of a medicament for the treatment or prophylaxis of a disease associated with BCL9/β -catenin interactions.

In another preferred embodiment, the disease associated with BCL9/β -catenin interaction comprises: cancer, tumor.

In a fourth aspect of the invention there is provided a method of treating or preventing a disease associated with BCL9/β -catenin interactions comprising the steps of: administering to a subject in need thereof a therapeutically effective amount of a compound according to the first or second aspect or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystalline form or prodrug thereof, or a pharmaceutical composition according to the third aspect.

In a fifth aspect of the invention, there is provided a method of treating or preventing cancer comprising the steps of: administering to a subject in need thereof a therapeutically effective amount of a compound according to the first or second aspect or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystalline form or prodrug thereof, or a pharmaceutical composition according to the third aspect.

In a sixth aspect of the invention there is provided the use of a compound as described in the first aspect or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystal form or prodrug thereof, in the manufacture of a medicament for the treatment or prophylaxis of fibrosis or a disease associated therewith.

In another preferred embodiment, the fibrosis or a disease associated therewith comprises: pulmonary fibrosis, liver fibrosis, non-alcoholic liver steatohepatitis, bone fibrosis, or a combination thereof.

In a seventh aspect of the invention, there is provided a method of treating or preventing a fibrosis-related disease comprising the steps of: administering to a subject in need thereof a therapeutically effective amount of a compound according to the first aspect or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystal form or prodrug thereof, or a pharmaceutical composition according to the third aspect.

In an eighth aspect of the invention, there is provided a method of inhibiting the binding of BCL9 to β -catenin in a subject; and/or modulating Wnt/β -catenin signaling in the subject; and/or reducing regulatory T cell survival in a subject; and/or reducing expression of VEGF in a tumor in a subject; and/or increasing the infiltration of cd4+ T cells and cd8+ T cells into a tumor in a subject; and/or increasing T helper 17 (Th 17) cells into a tumor in a subject; and/or reducing dendritic cells in a tumor in a subject; and/or when administered to a subject, the half-life (T112) is greater than at least 2 hours; and/or inducing a tumor microenvironment conducive to an immune response in the subject; and/or inhibiting tumor growth in a subject; and/or inhibiting proliferation of cancer stem cells in a subject; and/or a method of inhibiting tumor metastasis in a subject, comprising the steps of: administering to a subject a compound according to the first or second aspect or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystal form or prodrug thereof, or a pharmaceutical composition according to the third aspect, or contacting a subject with a compound according to the first aspect or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystal form or prodrug thereof.

In another preferred embodiment, the subject is a mammal, preferably a human.

In another preferred embodiment, the subject is a cell.

In another preferred embodiment, the method is non-therapeutic in vitro.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Without any means for

Detailed Description

The inventors have studied extensively and intensively, and have unexpectedly found that a class of small molecule compounds having a novel structure have an activity of excellently inhibiting the interaction of BCL9 with β -catenin. In addition, the inventor also finds that the compounds have excellent therapeutic and preventive effects in fibrosis and related diseases. Based on this, the inventors completed the present invention.

Terminology

Unless otherwise indicated, each term or abbreviation herein has the conventional meaning as understood by those skilled in the art.

In this context, when a single bond in the structure of a compound is indicated by a broken line, unless otherwise specifiedRepresents the position of attachment to the other part of the molecule.

As used herein, the terms "comprising," "including," or "comprising" mean that the various ingredients can be used together in a mixture or composition of the invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "containing.

Herein, when a subscript is 0, it represents that the group is absent, e.gWhen m4 is 0, only the R ³ substituent is present and the R substituent is absent.

Unless otherwise indicated, the term "alkyl", by itself or as part of another substituent, refers to a straight or branched hydrocarbon radical having the indicated number of carbon atoms (i.e., C _1-6 represents 1-6 carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

The term "alkenyl" refers to an unsaturated alkyl group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated alkyl group having one or more triple bonds. Typically, alkenyl groups have 1-6 carbon atoms (i.e., C _1-6 alkenyl groups) and alkynyl groups have 1-6 carbon atoms (i.e., C _1-6 alkynyl groups). Examples of such unsaturated alkyl groups include: vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl and higher homologs and isomers.

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, amino group or sulfur atom, respectively. In addition, for dialkylamino groups, the alkyl moieties can be the same or different and can be combined with the nitrogen atom to which each alkyl group is attached to form a 3-7 membered ring. Thus, the group represented by-NR ^aR^b is meant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl (azetidinyl), and the like.

The term "alkylene", as used herein, by itself or as part of another substituent, refers to a divalent group derived from an alkane, such as-CH ₂-、-CH₂CH₂ -.

As used herein, the term "aminoalkyl" refers to an alkyl group as defined above having the indicated number of carbon atoms in which 1 or 2 hydrogens are replaced with an amino group. For example, - (CH ₂)₂NH₂).

As used herein, the term "cycloalkyl" refers to a saturated hydrocarbon ring having the specified number of ring atoms (e.g., C _3-10 cycloalkyl, preferably C _3-6 cycloalkyl). "cycloalkyl" may be monocyclic (e.g., cyclopropyl, cyclobutyl, cyclohexyl, etc.), and may also refer to bicyclic and polycyclic hydrocarbon rings (including fused, spiro, bridged, etc.), such as bicyclo [2.2.1] heptane, bicyclo [2.2.2] octane, etc. The term "heterocycloalkyl" refers to cycloalkyl groups containing one to five (preferably 1, 2, 3 or 4) heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Heterocycloalkyl groups can be monocyclic, bicyclic, or polycyclic ring systems (including fused, spiro, bridged, etc.). Typically, heterocyclyl groups typically comprise 4-10 ring atoms (i.e., 4-to 10-membered heterocycloalkyl), preferably 4-7 (e.g., 4, 5, 6) ring atoms (i.e., 4-to 7-membered heterocyclyl, or 4-to 6-membered heterocyclyl) and contain 1, 2, 3, or 4 (preferably 1 or 2) heteroatoms. Non-limiting examples of heterocycloalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidone, hydantoin, dioxolane, phthalimide, piperidine, 1, 4-dioxane, morpholine, thiomorpholine-S-oxide, thiomorpholine-S, S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. Heterocycloalkyl groups can be attached to the remainder of the molecule via a ring carbon or heteroatom (e.g., ring nitrogen).

As used herein, the term "cycloalkenyl", alone or as part of a group, refers to a cyclic hydrocarbon having the specified number of ring atoms (e.g., C3-10 cycloalkenyl, or C _3-6 cycloalkenyl) with 1 or 2 double bonds (preferably, only 1 double bond) between the ring vertices. "cycloalkenyl" may be monocyclic and may also refer to bicyclic and polycyclic hydrocarbon rings (including fused, spiro, bridged, etc.). Examples of cycloalkenyl groups include, for example, cyclopropene, cyclobutene, cyclopentene, cyclopentadiene, and the like. Similarly, the term "heterocycloalkenyl" refers to a cycloalkenyl group containing 1 to 5 (preferably 1, 2, 3 or 4) heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. Heterocycloalkenyl groups can be monocyclic, bicyclic, or polycyclic ring systems (including fused, spiro, bridged, etc.). Generally, heterocycloalkenyl groups typically include 4-10 ring atoms (i.e., 4-to 10-membered heterocycloalkyl), preferably include 4-7 (e.g., 4, 5, 6) ring atoms (i.e., 4-to 7-membered heterocyclyl, or 4-to 6-membered heterocyclyl) and contain 1, 2, 3, or 4 (preferably 1 or 2) heteroatoms.

Terms such as cycloalkylalkyl (alkylene) and heterocyclylalkyl (alkylene) refer to a cyclic alkyl or heterocyclylalkyl group attached to the remainder of the molecule through an alkyl or alkylene linker. For example, cyclobutylmethyl-is a cyclobutyl ring attached to the methylene linker of the rest of the molecule.

Unless otherwise indicated, the term "aryl" refers to a polyunsaturated (usually aromatic) hydrocarbon group, which may be a single ring or multiple rings (up to three rings) fused together or covalently linked. Typically, aryl groups have 6 to 10 ring atoms. The term "heteroaryl" refers to an aryl group (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. Typically, heteroaryl groups have 5-10 ring atoms, i.e., 5-10 membered heteroaryl groups, preferably 5-6 ring atoms, i.e., 5-6 membered heteroaryl groups, and contain 1, 2, 3, or 4 heteroatoms. Heteroaryl groups may be attached to the remainder of the molecule through heteroatoms. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl, while non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl (benzotriazinyl), purinyl, benzimidazolyl, benzopyrazolyl, benzotriazole, benzisoxazolyl, isobenzofuranyl (isobenzofuryl), isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furanyl, thienyl, and the like. The substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

For brevity, when the term "aryl" is used in combination with other terms (e.g., aryloxy, arylthio, aralkyl), aryl and heteroaryl rings as defined above are included. Thus, the term "aralkyl" is meant to include those groups in which an aryl group is attached to an alkyl group attached to the remainder of the molecule (e.g., benzyl, phenethyl, pyridylmethyl, and the like).

In some embodiments, the above terms (e.g., "alkyl", "aryl" and "heteroaryl") are intended to include both substituted and unsubstituted forms of the indicated group. Preferred substituents for each type of group are provided below. For the sake of brevity, the terms aryl and heteroaryl will refer to substituted or unsubstituted forms as provided below, while the term "alkyl" and related aliphatic groups refer to unsubstituted forms unless indicated as substituted.

Substituents for alkyl groups (including those commonly referred to as alkylene, alkenyl, alkynyl and cycloalkyl) may be various groups selected from the group consisting of: halogen 、-OR'、-NR'R"、-SR'、-SiR'R"R"'、-OC(O)R'、-C(O)R'、-CO₂R'、-CONR'R"、-OC(O)NR'R"、-NR"C(O)R'、-NR'-C(O)NR"R"'、-NR"C(O)₂R'、-S(O)R'、-S(O)₂R'、-S(O)₂NR'R"、-NR'S(O)₂R"、-CN and-NO ₂, in amounts from zero to (2 m '+1), where m' is the total number of carbon atoms in such groups. R ', R ' and R ' each independently represent hydrogen, unsubstituted C _1-8 alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C _1-8 alkyl, C _1-8 alkoxy or C _1-8 thioalkoxy, or unsubstituted aryl-C _1-4 alkyl. When R 'and R' are attached to the same nitrogen atom, they may combine with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR' R "is meant to include 1-pyrrolidinyl and 4-morpholinyl. The term "acyl", used alone OR as part of another group, refers to a group in which both substituents are substituted with substituent = O (e.g., -C (O) CH ₃,-C(O)CH₂CH₂ OR', etc.) on the carbon closest to the point of attachment of the group.

Similarly, the substituents for aryl and heteroaryl groups are various and are typically selected from: halogen 、-OR'、-OC(O)R'、-NR'R"、-SR'、-R'、-CN、-NO₂、-CO₂R'、-CONR'R"、-C(O)R'、-OC(O)NR'R"、-NR"C(O)R'、-NR"C(O)₂R'、-NR'-C(O)NR"R"'、-S(O)R'、-S(O)₂R'、-S(O)₂NR'R"、-NR'S(O)₂R"、-N₃、 perfluoro (C ₁-C₄) alkoxy and perfluoro (C ₁-C₄) alkyl, in amounts ranging from zero to the total number of open valencies on the aromatic ring system; wherein R ', R ' and R ' are independently selected from the group consisting of hydrogen, C _1-8 alkyl, C _3-6 cycloalkyl, C _2-8 alkenyl, C _2-8 alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl) -C _1-4 alkyl and unsubstituted aryloxy-C _1-4 alkyl. Other suitable substituents include each of the aryl substituents described above attached to a ring atom through an alkylene chain of 1 to 4 carbon atoms.

As used herein, the term "heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si).

As used herein, "halogen" refers to F, cl, br, and I. More preferably, the halogen atom is selected from F, cl and Br.

For the compounds provided herein, a bond from a substituent (typically an R group) to the center of an aromatic ring (e.g., benzene, pyridine, etc.) will be understood to refer to a bond that provides a connection at any available vertex of the aromatic ring. In some embodiments, the description also includes linkages fused to rings of an aromatic ring. For example, a bond drawn to the center of the indole benzene moiety will represent a bond to any available vertex of the six-or five-membered ring portion of the indole.

Unless otherwise indicated, all compounds present in the present invention are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (i.e., racemates). Among all the compounds of the invention, each chiral carbon atom may optionally be in the R configuration or in the S configuration, or in a mixture of R and S configurations. Preferably, in this context, when a single bond in a compound structure is present, unless otherwise specifiedWhen expressed, the compounds include those in which the single bond is in a single configuration, either the S or R configuration, or a mixture of the S and R configurations (e.g., racemates).

Active ingredient

As used herein, the term "compound of the invention" refers to a compound as described in the first aspect of the invention. The term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds according to the first aspect of the invention.

As used herein, the term "pharmaceutically acceptable" ingredient refers to a substance that is suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.

As used herein, the term "therapeutically effective dose" refers to any amount of a drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptoms, or prevention of a disorder or disability caused by the disease. "therapeutically effective dose" of a drug of the present invention also includes "prophylactically effective dose" which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing or suffering from recurrence of the disease, inhibits the occurrence or recurrence of the disease.

The term "pharmaceutically acceptable salts" is intended to include salts of the active compounds prepared with relatively non-toxic acids or bases, depending on the particular substituents on the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base (either solvent-free or in a suitable inert solvent). Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, iron, ferrous iron, lithium, magnesium, manganese, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines and the like, such as arginine, betaine, caffeine, choline, N' -dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine (glucamine), glucosamine (glucosamine), histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid (either solvent-free or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrocarbonic acid, phosphoric acid, monohydrogen phosphoric acid, dihydrogen phosphoric acid, sulfuric acid, monohydrogen sulfuric acid, hydriodic acid, or phosphorous acid, and the like; and salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids, such as arginine salts and the like, and salts of organic acids, such as glucuronic acid (glucuronic acid) or galacturonic acid (galactunoric acid) and the like (see, e.g., berge, s.m. and the like, "pharmaceutically salts (Pharmaceutical Salts)", journal of Pharmaceutical Science,1977,66,1-19). Certain specific compounds of the invention contain both basic and acidic functionalities, thereby enabling the conversion of the compounds into base or acid addition salts.

The neutral form of the compound can be regenerated by contacting the salt with a base or acid and isolating the parent compound in a conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties (e.g., solubility in polar solvents), but in addition, those salts are equivalent to the parent form of the compound for the purposes of the present invention.

In addition to salt forms, the present invention also provides compounds in prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Alternatively, prodrugs can be converted to the compounds of the present invention by chemical or biochemical means in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir containing a suitable enzyme or chemical agent.

Certain compounds of the invention may exist in unsolvated forms as well as solvated forms (i.e., solvates), including hydrated forms (i.e., hydrates). Solvated forms are generally equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the invention may exist in polymorphic or amorphous forms. In general, all physical forms are equivalent for the applications contemplated by the present invention and are intended to be within the scope of the present invention.

Certain compounds of the invention possess an asymmetric carbon atom (optical center) or double bond; racemates, diastereomers, geometric isomers, regioisomers and individual isomers (e.g., isolated enantiomers) are all intended to be included within the scope of the present invention. When compounds provided herein have a defined stereochemistry (denoted R or S, or indicated with dashed or wedge-shaped bonds), those compounds will be understood by those skilled in the art to be substantially free of other isomers (e.g., at least 80%,90%,95%,98%,99% and up to 100% free of other isomers).

The compounds of the present application may also contain non-natural proportions of atomic isotopes at one or more of the isotopic atoms constituting such compounds. The unnatural proportion of an isotope can be defined as from the naturally found amount of the atom in question to 100% of the amount of that atom. For example, the compound may incorporate a radioisotope, such as tritium (³ H), iodine-125 (¹²⁵ I), or carbon-14 (¹⁴ C), or a non-radioisotope, such as deuterium (² H) or carbon-13 (¹³ C). Such isotopic variants may provide additional uses beyond those described herein. For example, isotopic variants of the compounds of the present application can have additional uses including, but not limited to, as diagnostic and/or imaging agents, or as cytotoxic/radiotherapeutic agents. In addition, isotopic variations of the compounds of the present application can have altered pharmacokinetic and pharmacodynamic characteristics to facilitate increased safety, tolerability, or efficacy during treatment. All isotopic variations of the compounds of the present application, whether radioactive or not, are intended to be encompassed within the scope of the present application.

Pharmaceutical compositions and methods of administration

Because the compounds of the present invention have excellent inhibitory activity against the BCL9/β -catenin interaction (BCL 9/β -catenin PPI), the compounds of the present invention and various crystalline forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and pharmaceutical compositions containing the compounds of the present invention as a main active ingredient are useful for treating, preventing and alleviating diseases associated with BCL9/β -catenin interaction. According to the prior art, the compounds of the invention are useful for the treatment of: cancers, tumors, etc., such as Familial Adenomatous Polyposis (FAP), eye cancer, rectal cancer, colon cancer, colorectal cancer, cervical cancer, prostate cancer, breast cancer, bladder cancer, oral cancer, benign tumors and malignant tumors, stomach cancer (stomach cancer), liver cancer, pancreatic cancer, lung cancer, uterine body, ovarian cancer, prostate cancer, testicular cancer, kidney cancer, brain/CNS cancer, laryngeal cancer, multiple myeloma, cutaneous melanoma, acute lymphoblastic leukemia, acute myelogenous leukemia, ewing's sarcoma, kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, vascular endothelial tumor, wilms ' tumor, neuroblastoma, oral/pharyngeal cancer, esophageal cancer, laryngeal cancer, lymphoma, neurofibromatosis, sarcoidosis, hemangioma, gastric cancer (GASTRIC CANCER), ovarian cancer, hepatocellular carcinoma, lymphatic vessels, and the like.

In addition, the compound of the present invention has excellent ability to treat fibrosis, and thus, the compound of the present invention and various crystalline forms thereof, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates thereof, and a pharmaceutical composition containing the compound of the present invention as a main active ingredient are useful for treating, preventing and alleviating fibrosis (fibrosis) and various diseases associated with fibrosis. Fibrosis can occur in a variety of organs, with major pathological changes being increased fibrous connective tissue in organ tissue, reduced parenchymal cells, and continued progression can lead to destruction and hypofunction of organ structure, as well as failure, severely threatening human health and life.

Exemplary diseases of fibrosis and related diseases are as follows:

The pharmaceutical compositions of the present invention comprise a safe and effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of the compound of the invention per dose, more preferably 10-500mg of the compound of the invention per dose. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, and the like), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, and the like), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, and the like), emulsifiers (e.g.) Wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizing agents, antioxidants, preservatives, pyrogen-free water and the like.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-agar or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms of the compounds of the present invention for topical administration include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds.

In some embodiments, pharmaceutical compositions comprising a compound of the invention may further comprise at least one additional agent. In some embodiments, the at least one additional agent is selected from one or more of a checkpoint inhibitor, an EGFR inhibitor, a VEGF inhibitor, a VEGFR inhibitor, and an anti-cancer drug.

In some embodiments, the pharmaceutical compositions described herein may include a checkpoint inhibitor. In one embodiment, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA 4 antibody. In one embodiment, the checkpoint inhibitor targets a stimulus checkpoint molecule, e.g., CD27, CD40, OX40, GITR, or CD138. In yet another embodiment, the checkpoint inhibitor targets a stimulus checkpoint molecule, e.g., A2AR, B7-H3, B7-H4, B and T lymphocyte attenuation factor (BTLA), indoleamine 2, 3-dioxygenase (IDO), killer cell immunoglobulin-like receptor (KIR), lymphocyte activating gene-3 (LAG 3), T cell immunoglobulin and mucin domain protein 3 (TIM-3), VISTA (C10 orf 54), or T cell activating V domain Ig inhibitor.

In some embodiments, the pharmaceutical compositions described herein comprise an EGFR inhibitor. In one embodiment, the EGFR inhibitor is erlotinib, gefitinib, lapatinib, panitumumab, vandetanib, or cetuximab.

In some embodiments, the pharmaceutical compositions described herein may include a VEGF or VEGFR inhibitor. In one embodiment, the VEGF or VEGFR inhibitor is pazopanib, avastin, sorafenib, sunitinib, axitinib, plaitinib, cancerigib, vandetanib, cabotinib, ramucirumab, lenvatinib, or aflibercept.

In some embodiments, the pharmaceutical compositions described herein comprise an anticancer drug. The anticancer agent may be selected from: cyclophosphamide, methotrexate, 5-fluorouracil (5-FU), doxorubicin, nitrogen mustard (mustine), vincristine, methylbenzyl hydrazine, penicortl, dacarbazine, bleomycin, etoposide, cisplatin, epirubicin (epirubicin), capecitabine, leucovorin, actinomycin, all-trans retinoic acid, azacytidine, azathioprine, bortezomib, carboplatin, chlorambucil, cytarabine, daunomycin, european paclitaxel, doxifluridine, fluorouracil, gemcitabine, hydroxyurea, idarubicin (idarubicin), imatinib, elydiginetin, nitrogen mustard (mechlorethamine), mercaptopurine, mitoxantrone, paclitaxel, pemetrexed, teniposide, thioguanine, tolprine, valrubicin, vinblastine, vindesine, wen Nuo and oxaliplatin.

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 1 to 2000mg, preferably 20 to 500mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

BCL-9, beta-catenin and Wnt signaling

Aberrant activation of Wnt signaling is implicated in a variety of cancers, as tumors can grow and survive dependent on Wnt signaling. Up to 90% of all sporadic colorectal cancer cases are associated with constitutive activation of Wnt signaling.

Beta-catenin is a protein that can be involved in protein-protein interactions that stimulate Wnt signaling, resulting in changes in transcriptional activation that may lead to tumor growth and development. Beta-catenin is usually phosphorylated and targeted for degradation by the axin complex. If stimulation of the Wnt signaling pathway is present, unphosphorylated β -catenin accumulates and binds to lymphokine/T-cell factor (LEF/TCF) and is transferred into the nucleus to stimulate transcription of the Wnt target gene. The Wnt target gene comprises c-myc and CD44 as upregulation genes in tumor models. BCL9 is a protein required for efficient beta-catenin-mediated transcription in mammalian cells.

"Canonical" Wnt/β -catenin signaling is a pathway activated by binding of Wnt ligands to the Frizzled family of cell surface receptors, which then regulate β -catenin expression and intracellular localization. In the absence of Wnt ligands, β -catenin is phosphorylated and ubiquitinated in the disrupted complex consisting of Adenomatous Polyposis Coli (APC), glycogen synthase kinase-3 (GSK-3), casein kinase-1 (CK 1) and axin, and targeted for degradation in a proteasome-dependent manner. In the presence of Wnt ligand, β -catenin ubiquitination is inhibited in the complex, resulting in saturation of phosphorylated β -catenin, which is then stabilized and transferred to the nucleus. There, phosphorylated β -catenin is involved in nuclear T-cell factor (TCF) transcription factors, such as lymphokine/3 (LEF/TCF), to induce expression of genes that promote cell proliferation, migration and survival, including c-Myc and Cyclin D.

Several molecules comprising BCL9 and its homolog B cell lymphoma 9-like (B9L) have been shown to be coactivators of Wnt/β -catenin transcription. The formation of a complex consisting of TCF, β -catenin and BCL9 (or B9L) enhances β -catenin dependent Wnt transcriptional activity. In normal cells, this transcriptional pathway is shut down when Wnt ligands decouple from their receptors. However, various loss-of-function mutations in APC and axin, as well as activating mutations in β -catenin itself, enable β -catenin to escape the destructive complex and accumulate in the nucleus. Such inappropriate persistence of β -catenin promotes the development of a wide range of common human epithelial cancers, including hepatocellular carcinoma, breast cancer, colorectal cancer, and hematologic malignancies, such as multiple myeloma. In addition, active β -catenin signaling leads to T cell rejection, particularly cd8+ T cell rejection, which leads to therapy resistance and shortens patient survival. Thus, blocking Wnt signaling by targeting β -cat may provide a powerful approach for the treatment of CRC, potentially preventing tumor development and metastasis.

Like other transcription factors, the development of selective nontoxic β -catenin inhibitors and their conversion to the clinic has proven to be a considerable challenge, as β -catenin interacts with most of its protein partners through the same binding surface. Thus, wnt pathway inhibitors targeting this common binding surface show significant adverse effects in animal and clinical trials. Only a few drugs targeting β -catenin exist in clinical trials, including PRI-724 (EisaiPharmaceuticals; stage II), LGK974 (Novartis; stage I), and OMP-54F28 and OMP-18R5 (OncoMed/Bayer; stage I). In addition, disruption of LEF/TCF interactions by small molecule and peptide inhibitors of β -cat may produce serious side effects, including treatment of severe myelodysplasia, anemia, and systemic wasting in mice—which may be the result of disruption of Wnt signaling in homeostasis in normal hematopoietic stem cells and intestinal stem cells. Such therapeutic limitations may be derived from disruption of β -catenin-TCF and β -catenin-E-cadherin interactions, which may affect epithelial tissue integrity. In addition, biological agents targeting Frizzled receptors (OMP-54F 28 and OMP-18R 5) showed significant bone marrow toxicity during clinical trials. Wnt ligands are required for Wnt/β -cat activation, but APC and β -catenin mutations in cancer cells can induce downstream transcription in the absence of Wnt ligand activation, thus blocking Wnt secretion fails to inhibit endogenous oncogenic Wnt activity caused by APC and β -catenin mutations inducing downstream gene transcription. As identified by certain biomarkers, LGK974 targets only a small population of patients. PRI-724, a small molecule inhibitor, is being tested in phase II using daily infusion, but Intravenous (IV) doses more than once a week exhibit undesirable and non-foot-holding properties for clinical development.

Traditionally, the Wnt signaling pathway involves three different types of signaling: canonical Wnt signaling pathways, wherein Wnt modulates various transcription target genes by β -catenin dependent means; non-canonical Wnt signaling pathways that are primarily involved in planar cell polarity, wherein Wnt may function independently of β -catenin; and a non-canonical Wnt/calcium pathway that regulates intracellular calcium levels. In the present application, "canonical Wnt signaling" is interchangeably referred to as "canonical Wnt/β -catenin signaling" or "Wnt signaling. As described herein, canonical Wnt/β -catenin signaling may refer to a component of the pathway that controls the amount of β -catenin in a patient or sample by modulating the stability of β -catenin. In some embodiments, canonical Wnt/β -catenin signaling includes transcriptional transduction of pathway components that transduce one or more genes such as c-myc, ccnd1, cd44, LGR5, VEGFA, AXIN2, and LEF1. In some embodiments, canonical Wnt/β -catenin signaling comprises pathway components that are modulated by interactions between β -catenin and BCL 9. In some embodiments, canonical Wnt/β -catenin signaling comprises one or more genes that are transcriptional controlled by interactions between β -catenin and BCL 9. The one or more genes controlled by the interaction between β -catenin and BCL9 may comprise c-myc, ccnd1, cd44, LGR5, VEGFA, AXIN2, and LEF1. In some embodiments, canonical Wnt/β -catenin signaling comprises one or more proteins whose transcriptional expression is modulated by an interaction between β -catenin and BCL 9. These components may include, for example, c-Myc, cyclin D1, CD44, LGR5, VEGFA, AXIN2, and LEF1.

Application method

In some embodiments, administration of a compound of the invention to a subject inhibits Wnt signaling in the subject. In some embodiments, administration of a compound of the invention inhibits BCL9 binding to β -catenin. In some embodiments, a compound of the invention is administered canonical Wnt/β -catenin signaling. In some embodiments, the compounds of the invention are administered to treat a disease in a subject.

In some embodiments, the compounds of the invention are capable of inhibiting BCL9 binding to β -catenin in vitro and/or in vivo. In some embodiments, the compounds of the invention have one or more improved effects. The one or more effects may be selected from one or more of the following: (1) inhibiting BCL9 binding to β -catenin; (2) inhibiting canonical Wnt signaling; (3) decreasing regulatory T cell survival; (4) reducing VEGF expression in the tumor; (5) Increasing infiltration of cd4+ T cells and cd8+ T cells into the tumor; (6) increasing T helper 17 (Th 17) cells into the tumor; (7) reduction of intratumoral dendritic cells; (8) When administered to a subject, the half-life (T1/2) is greater than at least 2 hours; (9) inducing a tumor microenvironment conducive to an immune response; and (10) inhibiting tumor growth, tumor stem cell proliferation and/or tumor metastasis.

In some embodiments, the compounds of the invention exhibit advantageous biological functions in some or each of the classes listed above, e.g., efficacy in various biochemical and cellular bioassays, including cell-based Wnt and/or β -catenin transcription assays.

BCL9 binding to β -catenin

Pygopus (Pygo) and legress (Lgs) are found in drosophila as novel components of Wnt signaling necessary for armadillo-mediated transcription during normal development. Pygo and BCL9/Legless transduce Wnt signals by promoting the transcriptional activity of β -catenin/Armadillo in normal and malignant cells. The ability of compounds to inhibit BCL9 binding to β -catenin can be assessed in various assays of inhibition in the art. In some embodiments, the ability of the compounds of the invention to inhibit BCL9 binding to β -catenin can be assessed using a Homogeneous Time Resolved Fluorescence (HTRF) binding assay. In this assay, the compound/small molecule is bound to a label that recognizes another label attached to another labeled target protein (i.e., β -catenin). When a compound/small molecule binds to a target protein and thus two labels are adjacent, a signal is generated and can be read quantitatively to calculate the binding affinity of the compound/small molecule. In some embodiments, the binding affinity of the compound/small molecule in this assay is compared to the binding affinity of a control to detect an improved binding affinity compared to the binding affinity of the control.

In some embodiments, the ability of the compounds of the invention to inhibit BCL9 binding to β -catenin can be evaluated in Amplified Luminescent Proximity Homogeneous Assays (ALPHA). In this assay, the compound is conjugated to a donor bead and its target protein (i.e., β -catenin) is attached to an acceptor bead. When two beads are in proximity due to binding of a compound to a target protein, a signal is generated and the binding affinity of the compound can be quantitatively calculated. In some embodiments, the binding affinity of the compound in this assay is compared to the binding affinity of the vehicle or control to detect an improved binding affinity compared to the binding affinity of the vehicle or control.

In various embodiments, the ability of a compound of the invention to inhibit BCL9 binding to β -catenin can be evaluated in a Wnt transcription assay. In some embodiments, the Wnt transcription assay is a cell-based assay. In some embodiments, the cell-based Wnt transcription assay is a beta-lactamase (bla) reporter assay. Various cell lines, transformed cell lines or primary cells derived from healthy subjects or subjects suffering from a disease can be used in this assay. Cell lines known to be dependent on canonical Wnt/β -catenin signaling for their survival can also be used. In some embodiments CellSensor ^TM LEF/TCF-bla HCT-116 cells and CIGNAL WNT reporters are used for this reporter assay. These cells contain a beta-lactamase (BLA) reporter gene under the control of a beta-lactamase/LEF/TCF response element stably integrated into HCT-116 cells. Since the cells constitutively express beta-lactamase, the addition of a compound that inhibits BCL9 binding to beta-catenin in this assay reduces beta-lactamase production. Thus, the efficacy of a compound in inhibiting Wnt transcription can be quantitatively calculated in this assay.

In some embodiments, the ability of a compound of the invention to inhibit the binding of BLC9 to β -catenin may be evaluated in a cell viability assay. In some embodiments, the cell viability assay is a celltiter glo luminescence assay, wherein cell viability is quantitatively measured. Various cell lines, transformed cell lines or primary cells derived from healthy subjects or subjects suffering from a disease can be used in this assay.

Canonical Wnt/beta-catenin signaling

In certain embodiments, the ability of a compound of the invention to inhibit canonical Wnt/β -catenin signaling can be assessed in various in vitro and/or in vivo assays. In some embodiments, the effect of a compound of the invention on canonical Wnt/β -catenin signaling is evaluated in a cell-based Wnt transcription assay, such as a β -lactamase (bla) reporter assay. The beta-lactamase (bla) reporter assay measures the intensity of canonical Wnt/beta-catenin signaling by its ability to control beta-catenin/LEF/TCF response elements, and thus can be used to evaluate whether an agent can attenuate or increase the intensity of control of canonical Wnt/beta-catenin signaling transcription on its transcriptional targeting.

The ability of the compounds of the invention to inhibit canonical Wnt/β -catenin signaling can also be assessed by measuring gene expression and/or protein expression of a target gene that is transcriptionally controlled by canonical Wnt/β -catenin signaling. Expression of a target gene can be assessed in transcribed cells contacted with a compound of the invention or in subjects administered with such compounds. Target genes include, for example, CMYC, CCND1, CD44, LGR5, VEGFA, AXIN2, and LEF1. The expression level of one or more target genes associated with canonical Wnt/β -catenin signaling may be analyzed using methods known in the art, such as cell staining, flow cytometry, immunoblotting, and/or real-time quantitative PCR (rt-qPCR) analysis.

Regulatory T cell survival

It is known to express various markers, such as CD4, FOXP3 and CD25, on regulatory T cells. The ability of the compounds of the invention to reduce the survival of regulatory T cells can be assessed by counting the total number of regulatory T cells present in the blood and/or in a specific tissue (e.g., tumor). For example, a sample obtained from a subject contacted with a compound of the invention may be stained with an antibody that detects a marker associated with regulatory T cells. Samples can also be processed and labeled with antibodies that detect such markers and analyzed by flow cytometry. Gene and/or protein expression of such markers can be determined in a sample and analyzed by, for example, immunoblotting and/or rt-qPCR.

VEGF expression in tumors

Various assays can be used to measure gene expression and/or protein expression of VEGF in tumor samples. For example, after contacting a subject with a compound, tumor cells can be collected and stained with an anti-VEGF antibody to detect VEGF protein. Cells can also be analyzed by, for example, rt-qPCR to determine gene expression of VEGF. Other assays that indicate changes in VEGF expression may be employed. For example, tumor samples from subjects contacted with a compound of the invention can be analyzed to detect various angiogenic markers controlled by VEGF. In some embodiments, the compounds of the invention reduce VEGF expression more effectively than the vehicle or control.

Infiltration of CD4+ and/or CD8+ T cells into tumors

Infiltration of cd4+ T cells and/or cd8+ T cells into a tumor may be assessed by counting the total number of cd4+ T cells and/or cd8+ T cells present in the tumor or in a sample from the tumor (e.g., biopsy). It is known to express various markers, such as CD4 and CD45, on cd4+ T cells (also known as helper T cells). It is known to express various markers, such as CD8 and CD45, on cd8+ T cells (also known as cytotoxic T cells). The ability of a compound to increase the infiltration of cd4+ and/or cd8+ T cells into a tumor can be assessed in vivo by administering the compound to a subject having a tumor. Tumor samples may be collected from subjects and stained with antibodies that detect markers associated with cd4+/cd8+ T cells. The sample may also be processed and labeled with, for example, antibodies that detect such markers, and analyzed, for example, by flow cytometry. Gene and/or protein expression of such markers can also be determined in a sample and analyzed by, for example, immunoblotting and/or rt-qPCR.

T helper 17 cell infiltration into tumors

In some embodiments, the compounds of the invention are capable of increasing T-helper 17 cell infiltration into a tumor when administered to a subject bearing the tumor. Entry of T-helper 17 cells into a tumor can be assessed by counting the total number of T-helper 17 cells present in the tumor. It is known to express various markers, e.g., IL-17, on T helper 17 cells. The ability of a compound to increase T-helper 17 cell infiltration into a tumor can be assessed in vivo by administering the compound to a subject having a tumor. Tumor samples can be collected from subjects and stained with, for example, antibodies that detect markers associated with T-helper 17 cells. Samples can also be processed and labeled with antibodies that detect such markers and analyzed by flow cytometry. Gene and/or protein expression of such markers can also be determined in a sample and analyzed by, for example, immunoblotting and/or rt-qPCR. The sample can be analyzed to detect the amount of IL-17 present in the sample.

Dendritic cells in tumors

In some embodiments, the compounds of the invention are capable of modulating dendritic cells present in a tumor when administered to a subject bearing the tumor. The number of dendritic cells present in a tumor can be assessed, for example, by staining the tumor with an antibody that recognizes one or more markers associated with the dendritic cells. It is known to express various markers, e.g., CD11c, on dendritic cells. The ability of a compound to reduce dendritic cells in a tumor can be assessed in vivo by administering the compound to a subject. Tumor samples can be collected from subjects and stained with antibodies that detect markers associated with dendritic cells. The sample may also be processed and labeled, for example, with antibodies that detect such markers, and analyzed, for example, by flow cytometry. Gene and/or protein expression of such markers is analyzed by, for example, immunoblotting and/or rt-qPCR.

Biomarkers and their use

The present disclosure also encompasses methods of measuring at least one biomarker for monitoring the therapeutic efficacy of a compound or pharmaceutical composition of the present invention or for selecting a subject for treatment with such a compound or pharmaceutical composition. In some embodiments, the biomarker is one or more of BCL9, CD44, axin2, cMyc, LGR5, VEGFA, sox2, oct4, nanog, and/or active β -catenin. As used herein, active β -catenin refers to the non-phosphorylated form of β -catenin.

Various known methods can be used to measure the gene expression level and/or protein level of such biomarkers. For example, a sample from a subject treated with a compound or pharmaceutical composition, such as a biopsy of a tumor, blood, plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes or spleen may be obtained. In some embodiments, the sample is a tumor biopsy in a subject. Samples obtained from a subject may be stained with one or more antibodies or other detection reagents that detect such biomarkers. The sample may also or alternatively be processed to detect the presence of nucleic acid (e.g., mRNA) encoding the biomarker by, for example, rt-qPCR methods.

In some embodiments, reduced levels of gene expression and/or protein levels of BCL9, CD44, axin2, cMyc, LGR5, VEGFA, sox2, oct4, nanog, and/or active β -catenin are indicative of therapeutic efficacy of a compound or pharmaceutical composition described herein. The level of expression of such biomarkers can be measured, for example, 1 day, 2 days, 3 days, 4 days, 5 days, one week, or two weeks after administration of the compound or pharmaceutical composition, or any period of time therebetween. In some embodiments, a method is disclosed that includes measuring the level of one or more biomarkers after one or more rounds of use of a compound or pharmaceutical composition of the invention. In some embodiments, the method further comprises continuing to administer the compound or pharmaceutical composition if the biomarker level decreases. In some embodiments, the method further comprises administering an increased dose of a compound or pharmaceutical composition of the invention, or increasing the frequency of subsequent administration, if the biomarker level is not reduced. In some embodiments, if biomarker levels do not decrease after initial administration, treatment is stopped. In various embodiments, marker levels are also measured prior to the first use of the compounds or pharmaceutical compositions of the invention and compared to levels after one or more rounds of administration, wherein treatment efficacy and duration of treatment steps are determined based on the change in one biomarker level from one or more levels prior to administration.

In some embodiments, increased levels of gene expression and/or protein levels of BCL9, CD44, axin2, cMyc, LGR5, VEGFA, sox2, oct4, nanog, and/or active β -catenin indicate that a subject would benefit from treatment with a compound or pharmaceutical composition of the invention as compared to a subject without increased levels of gene expression and/or protein levels. In some embodiments, methods of treatment are disclosed that include selecting patients with increased biomarker levels and administering a compound or pharmaceutical composition of the invention.

In certain embodiments, subjects with elevated levels of gene and/or protein expression of BCL9, CD44, axin2, cMyc, LGR5, VEGFA, sox2, oct4, nanog, and/or active β -catenin are selected for treatment with a compound or pharmaceutical composition of the invention. In some embodiments, after obtaining a tumor sample from a subject and identifying BCL9, CD44, axin2, cMyc, LGR5, VEGFA, sox2, oct4, nanog, and/or active β -catenin elevated gene and/or protein expression, a subject with a tumor is selected for treatment. In some embodiments, after obtaining a tumor sample from a subject and identifying elevated gene and/or protein expression of BCL9, the subject with the tumor is selected for treatment. In some embodiments, after obtaining a tumor sample from a subject and identifying elevated gene and/or protein expression of CD44, the subject with the tumor is selected for treatment. In some embodiments, after obtaining a tumor sample from a subject and identifying elevated gene and/or protein expression of active β -catenin, the subject with the tumor is selected for treatment.

Half-life in receptors

In some embodiments, the compounds of the invention have one or more improved pharmacokinetic parameters as compared to the vehicle or control. Such pharmacokinetic parameters may include, for example, maximum observed concentration (Cmax), time to maximum concentration (Tmax), final half-life (T1/2), systemic Clearance (CL), volume of distribution (Vz), area under the curve from time of administration to last measurable concentration (AUC 0-T), area under the curve extrapolated from time of administration to infinity (AUC 0-inf), and bioavailability.

Methods for assessing the pharmacokinetics of a pharmaceutical agent are known in the art. For example, blood samples from subjects to which the compounds described herein were administered may be obtained 5 minutes, 1, 2, 4, 6, 8, 12, and 24 hours after administration. The concentration of compounds in blood samples may be analyzed by various analytical means, e.g. LC/MS. Pharmacokinetic parameters were calculated based on the compound concentration at each time point. As used herein, the term "maximum observed concentration (C _max)" refers to the maximum serum concentration that a compound achieves after administration. In connection with the concept of C _max, the time to maximum concentration (T _max) is the time for the compound to reach the maximum serum concentration. The terms "terminal half-life (T _1/2)" and "half-life (T _1/2)" are used interchangeably and refer to the time at which a compound loses half its serum concentration. Systemic Clearance (CL) refers to the amount of blood that completely eliminates a compound per unit time. The term "distribution volume (V _z)" refers to a theoretically calculated volume that needs to contain the total amount of compound administered to a subject at the same concentration observed in blood. The term "bioavailability" refers to the extent and rate at which a drug is absorbed into a biological system, or the extent and rate available at a physiologically active site. Bioavailability may be a function of several of the properties previously described, including stability, solubility, immunogenicity, and pharmacokinetics, and may be assessed using methods known to those of skill in the art.

The pharmacokinetic parameters of the compounds may be assessed in mammals (including, for example, mice, rats, or humans). Parameters may also be assessed using various routes of administration, such as intravenous, intraperitoneal, subcutaneous, and intramuscular routes of administration. In some embodiments, the pharmacokinetic parameters of the compounds of the present invention are evaluated in mice. In some embodiments, the pharmacokinetic parameters of the compounds described herein are evaluated in mice administered the compounds subcutaneously. In some embodiments, the pharmacokinetic parameters of the compounds of the present invention are evaluated in humans. In some embodiments, the pharmacokinetic parameters of the compounds of the present invention are assessed in humans following subcutaneous administration.

Tumor microenvironment conducive to immune response

In various embodiments, the compounds of the invention induce a tumor microenvironment that favors an immune response. In various embodiments, the compounds of the invention induce a tumor microenvironment that favors an immune response over a vehicle or control.

Various parameters can be used to evaluate tumor microenvironment. For example, an increased ratio between cytotoxic T cells and regulatory T cells in and/or around tumor tissue may indicate that the tumor microenvironment is favorable for an immune response. A reduction in the number of dendritic cells and/or regulatory T cells in and/or around the tumor tissue may also indicate that the tumor microenvironment is favorable for the immune response. Other parameters include an increase in circulating T cells in peripheral blood and an increase in the ratio between T helper 17 cells and regulatory T cells in and/or around tumor tissue. These parameters may indicate that the tumor microenvironment is favorable for the immune response.

In some embodiments, the compounds of the invention can increase the ratio of the amount of cytotoxic T cells to the amount of regulatory T cells in the tumor microenvironment. In some embodiments, the change in ratio caused by the compound is greater than the change in ratio caused by the vehicle or control.

Tumor growth, tumor stem cell proliferation and/or tumor metastasis

Since Wnt signaling is a modulator of tumor growth, compounds can be evaluated in animal models for their therapeutic efficacy in affecting BCL9 binding to β -catenin.

The in vivo efficacy of the invention can be assessed in a human cancer model using, for example, BALB/c nude mice, as xenografts of human cancer cells will grow into tumors in these mice. For example, colo320DM tumor cells, a commercially available cell line derived from human colon cancer tissue, can be used to form tumors in BALB/c nude mice. Additional in vivo models may also be utilized to assess in vivo efficacy of the compounds disclosed herein. For example, human DLD-1 colon cancer cells can be implanted into nude mice to assess tumor growth. A CT26 isogenic mouse model of colon cancer can also be used, as the model allows assessment of tumor growth in the context of an intact immune system. Other types of cancer cells, such as B16 melanoma, 4T1 breast cancer, human kidney cancer, and Lewis lung cancer cells, can also be used in these known animal models to assess the in vivo efficacy of the compounds disclosed herein.

By administering a compound of the invention to one or more animal models, the effect of the compound in reducing tumor growth in vivo can be assessed. Based on animal data treated with stabilized BCL9 peptide, the ability of the peptide to inhibit Wnt signaling can be assessed by, for example, staining a tissue sample with a marker of Wnt signaling. These downstream markers of Wnt signaling include Axin2 and CD44, for example.

In situ mouse models can be used to assess the effect of the compounds described herein on tumor metastasis. For example, orthotropic animal models can be injected with cells carrying luciferase constructs and then administered with their prescribed treatment. The presence of injected cells can be detected by administering a luciferin substrate to each treated animal. The intensity of the bioluminescent signal can be quantitatively measured and used as an indicator of cell growth.

In some embodiments, the effect of a compound of the invention on cancer stem cell proliferation can be assessed by measuring biomarkers for various cancer stem cells. For example, the expression level of CD44 and/or LGR5 may be indicative of the amount of cancer stem cells present in the sample. Tumor samples can be collected from subjects and stained with antibodies that detect markers associated with cancer stem cells. The sample may also be processed and labeled, for example, with antibodies that detect such markers, and analyzed, for example, by flow cytometry. Gene and/or protein expression of such markers may be detected and analyzed by, for example, immunoblotting and/or rt-qPCR.

Diseases in which Wnt/beta-catenin signaling is abnormal

Aberrant Wnt/β -catenin signaling is associated with malignant transformation of normal cells into cancer cells. Activation of Wnt signaling and β -catenin nuclear localization is associated with tumor phenotypes in various models.

The present disclosure encompasses compositions for use and methods of using the compounds disclosed herein to inhibit the binding of BCL9 to β -catenin in a subject by administering the compounds or pharmaceutical compositions comprising the compounds to the subject. The present disclosure also encompasses the inhibition of canonical Wnt/β -catenin signaling in a subject by administering a compound or pharmaceutical composition disclosed herein. The present disclosure further encompasses methods of treating a disease in a subject by administering to the subject a compound or pharmaceutical composition of the present invention. The disease may be a cancer or other neoplastic disease associated with aberrant canonical Wnt/β -catenin signaling.

In some embodiments, the disease, disorder, or condition may be a disease that can benefit from inhibiting canonical Wnt/β -catenin signaling. In some embodiments, such a disease, disorder, or condition is cancer. In some embodiments, the cancer is a cancer that is highly expressed in BCL9 and/or β -catenin. In some embodiments, the cancer is a cancer in which BCL9 and β -catenin are co-localized in the nucleus of the cancer cell. In some embodiments, the cancer is selected from: familial Adenomatous Polyposis (FAP), eye cancer, rectal cancer, colon cancer, colorectal cancer, cervical cancer, prostate cancer, breast cancer, bladder cancer, oral cancer, benign tumors and malignancies, gastric cancer (stomach cancer), liver cancer, pancreatic cancer, lung cancer, uterine body, ovarian cancer, prostate cancer, testicular cancer, renal cancer, brain/CNS cancer, laryngeal cancer, multiple myeloma, cutaneous melanoma, acute lymphoblastic leukemia, acute myelogenous leukemia, ewing's sarcoma, kaposi's sarcoma, basal cell carcinoma and squamous cell carcinoma, small cell lung cancer, choriocarcinoma, rhabdomyosarcoma, angiosarcoma, vascular endothelial tumor, wilms ' tumor, neuroblastoma, oral/pharyngeal cancer, esophageal cancer, laryngeal cancer, lymphoma, neurofibromatosis, nodular sclerosis, hemangioma, gastric cancer (GASTRIC CANCER), ovarian cancer, hepatocellular carcinoma and lymphangiogenesis. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is cutaneous melanoma. In some embodiments, the cancer is lung cancer.

In some embodiments, any of the compounds or variants disclosed herein or pharmaceutical compositions comprising such compounds may be used to treat a disease, such as the cancers listed above.

The treatment and measured treatment parameters may be assessed after administration of the compound or pharmaceutical composition alone or in combination with one or more additional therapeutic agents (e.g., as a single bolus or administered separately sequentially). The additional agent may be any additional therapeutic agent mentioned herein or known to those of skill in the art. Depending on the regimen selected, the compound or pharmaceutical composition comprising the compound and/or additional agent may be administered one or more times.

The invention also encompasses compounds or pharmaceutical compositions disclosed herein for treating a disease in a subject. In some embodiments, the disease may benefit from inhibiting canonical Wnt/β -catenin signaling. In some embodiments, the disease is cancer.

The present disclosure further encompasses the use of a compound or pharmaceutical composition disclosed herein in the manufacture of a medicament for treating a disease in a subject. In some embodiments, the disease may benefit from inhibiting canonical Wnt/β -catenin signaling. In some embodiments, the disease is cancer.

In another embodiment, the disease treated is a disease other than cancer. In certain embodiments, the disease is bone density deficiency, ocular vascular deficiency, familial exudative vitreoretinopathy, early coronary heart disease, alzheimer's disease, autosomal dominant oligodendropathy, retinal angiogenesis, osteogenesis imperfecta, tetraamilia's syndrome (Tetra-Amelia syndrome), miaole's tube degeneration and maleation (Mullerian-duct regression andvirilization), SERKAL syndrome, type II diabetes, fullman syndrome (Fuhrmannsyndrome), odontoea dermis dysplasia, obesity, hand and foot crack deformity, tail replication, tooth hypoplasia, skeletal dysplasia, localized dermal hypoplasia, autosomal recessive scleroderma, neural tube defect or sclerosteosis, and Van Buchem disease (Van Buchem disease).

Combination therapy

In certain embodiments, a compound or pharmaceutical composition disclosed herein is administered with at least one additional agent. That is, the compounds of the present disclosure and the additional agents may be administered to a patient sequentially or simultaneously in separate dosage forms as described herein. In some embodiments, the at least one additional agent is selected from the group consisting of checkpoint inhibitors, EGFR inhibitors, VEGF inhibitors, VEGFR inhibitors, anticancer drugs (e.g., any of the additional therapeutic agents described herein) stapling peptides, and additional agents can be administered in a therapeutically effective amount.

In certain embodiments, the subject to whom the compounds or pharmaceutical compositions disclosed herein are administered is also treated with radiation therapy and/or chemotherapy before, after, or simultaneously with administration of the compounds or pharmaceutical compositions.

Kit for detecting a substance in a sample

The invention also encompasses pharmaceutical kits useful, for example, in the treatment of the disorders, diseases and conditions described herein, comprising one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention. Such kits may also comprise, if desired, various conventional pharmaceutical kit components, e.g., one or more of a container with one or more pharmaceutically acceptable carriers, additional containers, etc. Instructions for the administration of the components, instructions for the administration and/or instructions for mixing the components, as inserts or as labels, may also be included in the kit.

Also disclosed herein are kits for performing the methods described herein. In various embodiments, kits for making the compounds of the invention are provided. In some embodiments, the kit includes a compound capable of undergoing a reaction for forming one or more hydrocarbon linkages. In some embodiments, the kit includes a metal catalyst for performing metal-mediated ring-closing metathesis.

In some embodiments, the kit comprises an agent for detecting gene and/or protein expression of BCL9, CD44, axin2, cMyc, LGR5, VEGFA, sox2, oct4, nanog, and/or active β -catenin.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Preparation example

General synthetic method

The compounds of the present invention may be prepared, isolated or obtained by any method apparent to those skilled in the art. The compounds of the present invention may also be prepared according to the exemplary preparation schemes provided below (as in the examples). The reaction conditions, steps and reactants not provided in the exemplary preparation schemes are obvious and known to those skilled in the art. As used herein, the symbols and conventions used in these processes, schemes and examples, whether or not a particular abbreviation is specifically defined, have the meanings well known to those skilled in the art.

In particular, but not limited to, the following abbreviations may be used in the examples and throughout the specification: t. (room temperature); g (g); milligrams (milligrams); mL (milliliters); mu L (microliters); millimeter (millimoles); μM (micromolar); MHz (hertz); MHz (megahertz); mmol (millimoles); hr (hours); min (min); MS) (mass spectrometry); ESI (electrospray ionization); TLC (thin layer chromatography); HPLC (high performance liquid chromatography); BOC (t-butoxycarbonyl); tBu (t-butyl); HATU (2- (7-azabenzotriazol) -N, N' -tetramethylurea hexafluorophosphate); TFA (trifluoroacetic acid); pd ₂(dba)₃ (tris (dibenzylideneacetone) dipalladium); DIPEA (N, N-diisopropylethylamine).

Test examples

I test method

(1) FP (BCL-9 affinity):

200 μl of FP buffer (25mM HEPES,100Mm Nacl,0.01% Triton X-100,0.1% BSA) and 1 μM beta-catenin were added to a black 96-well plate, and the compounds of the present invention were added to the wells at different concentrations, with concentration gradients of 0.625, 1.25, 2.5, 5, 10 μmol/L, respectively, and three sub-wells were made for each concentration condition. Incubation with horizontal shaker for 2H. Then 2nM FAP-Bcl9 tracer was added to each well and absorbance was measured after 2H incubation in a horizontal shaker. The positive control representing 100% inhibition contained only the tracer. The negative control representing 0% inhibition contained the tracer and β -catenin.

(2)CCK8

HCT116 cell plating

Cell digestion, counting, plating 96-well plates (flat bottom transparent), 10000 cells per well per 100ul DMEM (10% fbs);

2. the cell state is observed the next day, and medicine adding is started after the cell wall is intact;

3. Dilution 514 with DMEM (2% fbs), gradient dilution at concentrations of 20 μm, 10 μm, 5 μm, 2.5 μm, 1.25 μm, 0.625 μm, 0.3125 μm, 0.15625 μm, 0 μm (equal volume DMSO);

4. 100 μl of 514 of the above different concentrations was added to each well, 2 wells were made per concentration gradient; 3 blank wells (DMEM 2% fbs medium alone, no cells);

Culturing for 24 hours at the temperature of 5.37 ℃;

6. Mu.l of CCK-8 enhancement solution was added per well: because the CCK-8 is added in a small amount to each hole, errors possibly caused by the reagent being stuck to the hole wall are likely to occur, and the culture plate is suggested to be gently knocked after the reagent is added so as to help uniform mixing;

7. Incubating in the incubator for 0.5-4 hours: the amount of Formazan formed varies depending on the cell type and for most cases incubation for 1 hour is sufficient. If the color development is insufficient, the culture may be continued to confirm the optimal conditions.

8. Measuring the absorbance at 450nm and the absorbance at 600nm (excluding the interference of the absorbance values of the reagent background and the well plate itself);

9. the final absorbance value adopts OD450nm-OD600nm, and the inhibition rate is calculated;

inhibition ratio = [ (Ac-As)/(Ac-Ab) ]. Times.100%

As: absorbance of experimental wells (cell-containing medium, CCK-8, drug to be tested);

ac: absorbance of control wells (medium containing cells, CCK-8, no drug to be tested);

ab: absorbance of blank wells (medium without cells and drug to be tested, CCK-8).

(3)qPCR

HCT116 cell plating

Cell digestion, counting, plating 24 wells (flat bottom transparent), 3x10 x 5 cells/500 ul DMEM (10% FBS) per well

2. The following day is to observe the cell state, and the drug addition is started after the cell wall is intact

Dilution 514 with DMEM (2% FBS), gradient dilution at concentrations of 20. Mu.M, 10. Mu.M, 5. Mu.M, 2.5. Mu.M, 1.25. Mu.M, 0.625. Mu.M, 0.3125. Mu.M, 0.15625. Mu.M, 0. Mu.M (equal volume DMSO)

500 Μl of 514 of the above different concentrations was added to each well, and each concentration gradient was single well (multiple wells were performed in qPCR) except for 3 multiple wells at 0 μM concentration

Culturing at 3.37℃for 24 hours

4. The supernatant was discarded, washed once with P BS and added to 500/well trizol

RNA extraction (step. Omitted)

6. Reverse transcription (step omitted, according to kit)

QPCR (step omitted, according to kit procedure)

Primer human AXIN2

H-AXIN2-F cggaaactgttgacagtggat

H-AXIN2-R ggtgcaaagacatagccagaa

Humanβ-actin

8. Inhibition rate calculation

Calculating 2 (-DeltaDeltaCT) of each concentration gradient

Inhibition = (1-experimental well/control well) ×100%

(4) Conversion of fibroblasts to myofibroblasts (anti-fibrosis test)

1. Main materials

No	Material name	Branding	Goods number
				1	Human cell line HFL1	Kebai (Kebai)
2	TGF-β1	MCE	HY-P70543
				3	a-SMA	Shanghai zhen Ke
4	Collagen type I Assay Kit	Nanjing build	H142-1-2
				5	Collagen type III Assay Kit	Nanjing build	H144-1-2

HFL1 medium: F12K+10% FBS, wall-attached growth

2. Experimental setup

1. Normal group (medium only), model group (TGF-. Beta.1 induction of 20 ng/ml), sample group to be tested (TGF-. Beta.1+ different final concentration compounds of 20 ng/ml)

2.5 Compounds, 3 concentration gradients, 2 multiplex wells per gradient.

3. Experimental procedure

1. Cell digestions were counted, 5e ⁵/well/2 ml, seeded in 6-well plates.

2. After adherence, TGF- β1 was added at a final concentration of 20ng/ml and stimulated for 48h. Different final concentrations (0, 5um,20 um) of compound were added.

3. Collecting the supernatant, and detecting the amount of col1 and col3 in the supernatant by using the kit; cells are lysed with lysate, centrifuged, the supernatant is collected,

QPCR detection of a-SMA isogenic

(5) SRP test

1. Experimental setup

1. The analyte is in the form of a powder.

2. The temperature was 25 ℃.

3. The analytical equipment used a Biacore T200 instrument.

2. Sample dilution

1. Ligand beta-catenin was diluted with HEPES (pH 7.4), 0.5mg/mL

2. The analyte was dissolved in DMSO and diluted with 0.1% DMSO HEPES (pH 7.4)

3. Experimental procedure

1. And starting up according to Biacore T200 instrument standard operation.

2. Buffer HEPES (pH 7.4) and 500ml of deionized water (having been filtered through a 0.22 μm membrane) were prepared to clean the needle.

3. And starting to install the chip, and installing the CM5 chip according to a standard flow.

4. In preparation for starting the formal experiment, the buffer will flush the flow system inside the whole system at a higher flow rate.

5. A suitable procedure is selected according to the sample size.

6. Capture of the chip was started and a sufficient volume of ligand beta-catenin, EDC/NHS, blocking solution was prepared. The coupling procedure was started with a coupling time of 7 minutes and a flow rate of 10. Mu.l/min, and the final ligand coupling amount was about 16000RU.

7. Sample detection was started after the coupling was completed, the analyte binding time was set to 120s,

The flow rate is 30 mu L/min; dissociation time is 200s, flow rate is 30 mu L/min; the regeneration time was 30s and the time required for the regeneration,

The flow rate was 30. Mu.L/min.

8. And preparing a corresponding sample to be detected according to the requirement, and starting an automatic running program to detect.

9. And (3) analyzing results, namely carrying out fitting analysis on the data according to the operation results to obtain a final affinity fitting KD value.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A compound or pharmaceutically acceptable salt thereof, or isomer, solvate, crystal form or prodrug thereof, wherein the compound is shown as formula I

Wherein,

Q is 0,1, 2 or 3;

m4 is 0, 1,2, 3, 4, 5, 6 or 7;

W ³ is selected from the group consisting of ：-C(O)-、-S(O)-、-S(O)₂-、-C(R^F)₂-C(O)-、-C(R^F)₂-S(O)-、-C(R^F)₂-S(O)₂-;

W ² is selected from the group consisting of: -O-, -S-, -N (R ^s1) -;

m3=0, 1,2, 3 or 4;

Each R ^C is independently R ^C1 or R ^s1;

Each R ^B is independently R ^B1、R^s1 or R ^s2;

m2=0, 1,2, 3 or 4;

L ¹ is-X- (W ¹)_n1 -;

X is selected from the group consisting of: -C (O) -, -S (O) -and-S (O) ₂ -;

Subscript n1=0, 1,2, or 3;

m1=0, 1,2, 3 or 4;

each R ^A is independently R ^A1、R^s1 or R ^s2;

Each R is independently selected from the group consisting of: D. halogen, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 hydroxyalkyl, C _2-6 alkenyl, C _2-6 alkynyl, -CN, -OR ', -NO ₂、-NR'R"、-SR'、-OC(O)R'、-C(O)R'、-CO₂ R ', -CONR ',

-OC(O)NR'R"、-NR"C(O)R'、-NR"-C(O)NR'R"、-NR"C(O)₂R'、-S(O)R'、-S(O)₂R'、-S(O)₂NR'R"、-NR"S(O)₂R'、 C _3-10 cycloalkyl optionally substituted with one or more R ' ", 4 to 10 membered heterocycloalkyl optionally substituted with one or more R '", C _6-10 aryl optionally substituted with one or more R ' ", 5 to 10 membered heteroaryl optionally substituted with one or more R '", C _1-4 alkylene optionally substituted with one or more R ' "

-C _3-10 cycloalkyl, -C _1-4 alkylene-4 to 10 membered heterocycloalkyl optionally substituted by one or more R ' ", -C _1-4 alkylene-C _6-10 aryl optionally substituted by one or more R '", and-C _1-4 alkylene-5 to 10 membered heteroaryl optionally substituted by one or more R ' ";

2. A compound according to claim 1 wherein,

The compound is shown as a formula I-1

Wherein,

X ¹、X²、X³ and X ⁴ are each independently selected from the group consisting of: CH. N;

Or alternatively

The compound is shown as a formula I-2

Wherein,

3. The compound according to claim 2, wherein the compound is represented by formula I-3 or I-4

4. A compound according to any one of claim 1 to 3,Is thatWherein R ³ is-N (R ³²)R³³ or-C _1-4 alkylene-N (R ³¹)R³²;R³² and R ³³ are each independently selected from the group consisting of H, C _1-4 alkyl).

5. A compound according to any one of claims 1 to 3, wherein the compound has one or more of the following characteristics:

(a) R ⁷ is selected from the group consisting of:

(b) In R ⁷, each R is independently selected from the group consisting of: c _1-6 alkyl, -NR 'R'; wherein each R' is independently selected from the group consisting of: H. c _1-6 alkyl; each R "is selected from the group consisting of: H. c _1-4 alkyl;

(c) Ring a is a ring selected from the group consisting of:

wherein each of X ¹、X²、X³ and X ⁴ is independently selected from the group consisting of: CH. N;

(d) Each R ^A is independently H, C _1-4 alkyl or R ^A1; and R ^A1 is selected from the group consisting of: halogen, optionally substituted C _1-6 alkyl, optionally substituted C _1-6 haloalkyl, and optionally substituted C _1-6 alkoxy;

(e) W ² is-O-or-N (R ^s) -.

6. The compound of claim 1, wherein said compound is selected from the group consisting of table a, table B and table C.

7. A pharmaceutical composition comprising:

(i) The compound of claim 1, or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystal form or prodrug thereof; and

(Ii) Pharmaceutically acceptable carriers or excipients.

8. Use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystal form or prodrug thereof, in the manufacture of a medicament for the treatment or prophylaxis of a disease associated with BCL9/β -catenin interactions.

9. The use according to claim 9, wherein the disease associated with BCL9/β -catenin interactions comprises: cancer, tumor, or a combination thereof.

10. Use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, or an isomer, solvate, crystal form or prodrug thereof, in the manufacture of a medicament for the treatment or prevention of fibrosis or a related disease thereof.