Combination therapy using C-C chemokine receptor 4(CCR4) antagonists and one or more checkpoint inhibitors

文档序号：751305 发布日期：2021-04-02 浏览：26次中文

阅读说明：本技术 使用c-c趋化因子受体4(ccr4)拮抗剂和一种或多种检查点抑制剂的联合治疗 (Combination therapy using C-C chemokine receptor 4(CCR4) antagonists and one or more checkpoint inhibitors ) 是由李世杰 V·R·马里 R·辛格杨菊张朋烈于 2019-08-28 设计创作，主要内容包括：本发明涉及在癌症治疗中C-C趋化因子受体4(CCR4)拮抗剂和一种或多种免疫检查点抑制剂的联合疗法。(The present invention relates to combination therapies of C-C chemokine receptor 4(CCR4) antagonists and one or more immune checkpoint inhibitors in the treatment of cancer.)

1. A method of treating cancer in a mammal, said method comprising: administering an effective amount of a C-C chemokine receptor 4(CCR4) antagonist and one or more immune checkpoint inhibitors.

2. The method of claim 1, wherein said CCR4 antagonist is a selective CCR4 antagonist.

3. The method of claim 1 or claim 2, wherein the CCR4 antagonist has the formula:

and pharmaceutically acceptable salts thereof, wherein,

R¹selected from: hydrogen, C_1-8Alkyl radical, C_1-8Haloalkyl, C_1-8Hydroxyalkyl radical, C_3-8Cycloalkyl, halogen, -CN, -SO₂Me and-C (O) NH₂；

Each R is²Selected from: c_1-8Alkyl radical, C_1-8Haloalkyl, halogen, -CN and C_1-8An alkoxy group; or two R attached to adjacent carbon atoms²The groups are optionally joined to form a 5 or 6 membered ring (aliphatic or aromatic, cycloalkyl or heterocycloalkyl);

R³selected from: hydrogen, methyl and C_1-4A haloalkyl group;

R⁴selected from: hydrogen, C_1-8Alkyl radical, C_1-8Haloalkyl and C_1-8A hydroxyalkyl group;

each subscript n is independently an integer of 0 to 3;

b is a bond or C (O);

q is selected from: C. CH, N, O, S (O) and SO₂：

W, X, Y and Z are independently selected from: C. CH and N, but Q and W are not both N;

R⁵and R⁶Absent or independently selected from: H. -OH, C_1-8Alkyl radical, C_1-8Hydroxyalkyl radical, C_1-4alkoxy-C_1-4Alkyl, -C (O) NR^aR^b、C_1-8alkylene-C (O) NR^aR^b、-NH-C_1-4alkylene-C (O) NR^aR^b、-C(O)-C_1-4alkylene-NR^aR^b、-CO₂H and acid isostere, C_1-8alkylene-CO₂H and acid isostere, -N (R)^a)C(O)NR^aR^b、C_1-8alkylene-N (R)^a)C(O)NR^aR^b、-NR^aR^b、C_1-8alkylene-NR^aR^b、C_1-8Alkoxy, -C (O) OR^a、C_1-8alkylene-C (O) OR^a、-CN、-C(O)R^a、-SO₂R^aand-N (R)^a)C(O)R^b；

Wherein the content of the first and second substances,

each R is_aAnd R_bIndependently selected from: hydrogen, C_1-8Alkyl radical, C_1-8Hydroxyalkyl radical, C_1-8Haloalkyl and C_1-8An alkoxy group; and

R⁷absent or selected from: H. c_1-8Alkyl and C_1-8A haloalkyl group.

4. The method of claim 1 or claim 2, wherein the inhibitor of CCR4 has the formula:

or a pharmaceutically acceptable salt thereof.

5. The method of claim 1 or claim 2, wherein the inhibitor of CCR4 has the formula:

or a pharmaceutically acceptable salt thereof.

6. The method of claim 1 or claim 2, wherein the inhibitor of CCR4 has the formula:

or a pharmaceutically acceptable salt thereof.

7. The method of claim 1 or claim 2, wherein the inhibitor of CCR4 has the formula:

or a pharmaceutically acceptable salt thereof.

8. The method of any one of claims 1 to 7, wherein the one or more immune checkpoint inhibitors are cytotoxic T lymphocyte-associated protein 4(CTLA-4) inhibitors or programmed cell death protein 1(PD-1) inhibitors.

9. The method of claim 8, wherein the PD-1 inhibitor is a small molecule PD-1/PD-L1 inhibitor.

10. The method of claim 9, wherein the small molecule PD-1/PD-L1 inhibitor is selected from the group consisting of:

11. the method of claim 9, wherein the PD-1 inhibitor is a compound having formula (II)

Or a pharmaceutically acceptable salt thereof; wherein:

R¹selected from the group consisting of: halogen, C_5-8Cycloalkyl radical, C_6-10Aryl and thienyl, wherein, said C_6-10Aryl and thienyl are optionally substituted with 1 to 5R^xSubstituted by a substituent;

each R is^xIndependently selected from the group consisting of: halogen, -CN, -R^c、-CO₂R^a、-CONR^aR^b、-C(O)R^a、-OC(O)NR^aR^b、-NR^bC(O)R^a、-NR^bC(O)₂R^c、-NR^a-C(O)NR^aR^b、-NR^aR^b、-OR^a、-O-X¹-OR^a、-O-X¹-CO₂R^a、-O-X¹-CONR^aR^b、-X¹-OR^a、-X¹-NR^aR^b、-X¹-CO₂R^a、-X¹-CONR^aR^b、-SF₅and-S (O)₂NR^aR^bWherein each X is¹Is C_1-4An alkylene group; wherein each R is^aAnd R^bIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl groups, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a five-or six-membered ring having 0(ii) to 2 additional heteroatoms selected from N, O or S as ring members; wherein the five or six membered ring is optionally substituted by oxo; each R is^cIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl radical, C_2-8Alkynyl and C_1-8A haloalkyl group; and optionally when two R are^xWhen the substituents are on adjacent atoms, they combine to form a fused five-, six-or seven-membered carbocyclic or heterocyclic ring, which is optionally substituted with 1 to 3 substituents independently selected from: halogen, oxo, C_1-8Haloalkyl and C_1-8An alkyl group;

each R is^2a、R^2bAnd R^2cIndependently selected from the group consisting of: H. halogen, -CN, -R^d、-CO₂R^e、-CONR^eR^f、-C(O)R^e、-OC(O)NR^eR^f、-NR^fC(O)R^e、-NR^fC(O)₂R^d、-NR^e-C(O)NR^eR^f、-NR^eR^f、-OR^e、-O-X²-OR^e、-O-X²-NR^eR^f、-O-X²-CO₂R^e、-O-X²-CONR^eR^f、-X²-OR^e、-X²-NR^eR^f、-X²-CO₂R^e、-X²-CONR^eR^f、-SF₅、-S(O)₂NR^eR^f、C_6-10Aryl and C_5-10Heteroaryl, wherein each X²Is C_1-4An alkylene group; wherein each R is^eAnd R^fIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members; and optionally substituted with oxo; each R is^dIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl and C_1-8A haloalkyl group;

R³selected from the group consisting of: -NR^gR^hAnd C_4-12Heterocyclic group, wherein, said C_4-12Heterocyclyl is optionally substituted by 1 to 6R^ySubstituted;

each R is^yIndependently selected from the group consisting of: halogen, -CN, -Rⁱ、-CO₂R^j、-CONR^jR^k、-CONHC_1-6alkyl-OH, -C (O) R^j、-OC(O)NR^jR^k、-NR^jC(O)R^k、-NR^jC(O)₂R^k、CONOH、PO₃H₂、-NR^j-C_1-6alkyl-C (O)₂R^k、-NR^jC(O)NR^jR^k、-NR^jR^k、-OR^j、-S(O)₂NR^jR^k、-O-C_1-6alkyl-OR^j、-O-C_1-6alkyl-NR^jR^k、-O-C_1-6alkyl-CO₂R^j、-O-C_1-6alkyl-CONR^jR^k、-C_1-6alkyl-OR^j、-C_1-6alkyl-NR^jR^k、-C_1-6alkyl-CO₂R^j、-C_1-6alkyl-CONR^jR^kAnd SF₅，

Wherein R is^yC of (A)_1-6The alkyl moiety being optionally further substituted by OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂H is substituted, wherein each R is^jAnd R^kIndependently selected from: hydrogen, optionally substituted by 1 to 2 groups selected from OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂C substituted by a substituent of H_1-8Alkyl, and optionally substituted by 1 to 2 groups selected from OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂C substituted by a substituent of H_1-8Haloalkyl, or R when attached to the same nitrogen atom^jAnd R^kCan be combined with a nitrogen atom to form a five-or six-membered ring having 0 to2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted by oxo; each R isⁱIndependently selected from the group consisting of: -OH, C_1-8Alkyl radical, C_2-8Alkenyl and C_1-8Haloalkyl, wherein each is optionally substituted with OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂H is substituted;

R^gselected from the group consisting of: H. c_1-8Haloalkyl and C_1-8An alkyl group;

R^hselected from optionally CO₂H substituted: -C_1-8Alkyl radical, C_1-8Haloalkyl, C_1-8alkyl-COOH, C_1-8alkyl-OH, C_1-8alkyl-CONH₂、C_1-8alkyl-SO₂NH₂、C_1-8alkyl-PO₃H₂、C_1-8alkyl-CONOH, C_1-8alkyl-NR^h1R^h2、-C(O)-C_1-8Alkyl, -C (O) -C_1-8alkyl-OH, -C (O) -C_1-8alkyl-COOH, C_3-10Cycloalkyl, -C_3-10cycloalkyl-COOH, -C_3-10cycloalkyl-OH, C_4-8Heterocyclyl radical, -C_4-8heterocyclyl-COOH, -C_4-8heterocyclyl-OH, -C_1-8alkyl-C_4-8Heterocyclyl radical, -C_1-8alkyl-C_3-10Cycloalkyl radical, C_5-10Heteroaryl, -C_1-8alkyl-C_5-10Heteroaryl group, C₁₀Carbocyclyl, -C_1-8alkyl-C_6-10Aryl radical, -C_1-8Alkyl- (C ═ O) -C_6-10Aryl radical, -C_1-8alkyl-NH (C ═ O) -C_1-8Alkenyl, -C_1-8alkyl-NH (C ═ O) -C_1-8Alkyl, -C_1-8alkyl-NH (C ═ O) -C_1-8Alkynyl, -C_1-8Alkyl- (C ═ O) -NH-C_1-8Alkyl groups-COOH and-C_1-8Alkyl- (C ═ O) -NH-C_1-8alkyl-OH;

R^hn linked thereto is a mono-, di-or tripeptide comprising 1-3 natural amino acids and 0-2 unnatural amino acids, wherein,

the non-naturalThe amino acid has an alpha carbon substituent selected from the group consisting of: c_2-4Hydroxyalkyl radical, C_1-3Alkyl-guanidino and C_1-4An alkyl-heteroaryl group, which is a cyclic alkyl group,

the alpha carbon of each natural or unnatural amino acid is optionally further substituted with methyl, and

the terminal part of the mono-, di-or tripeptide is selected from the group consisting of: c (O) OH, C (O) O-C_1-6Alkyl and PO₃H₂Wherein, in the step (A),

R^h1and R^h2Each independently selected from the group consisting of: H. c_1-6Alkyl and C_1-4A hydroxyalkyl group;

R^hc of (A)_1-8The alkyl moiety is optionally further substituted with 1 to 3 substituents independently selected from the group consisting of: OH, COOH, SO₂NH₂、CONH₂、CONOH、COO-C_1-8Alkyl, PO₃H₂And optionally substituted by 1 to 2C_1-3C substituted by alkyl substituents_5-6(ii) a heteroaryl group, wherein,

R^hc of (A)₁₀Carbocyclyl, C_5-10Heteroaryl and C_6-10The aryl moiety is optionally substituted with 1 to 3 substituents independently selected from the group consisting of: OH, B (OH)₂、COOH、SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl radical, C_1-4Alkyl radical, C_1-4alkyl-OH, C_1-4alkyl-SO₂NH₂、C_1-4Alkyl CONH₂、C_1-4alkyl-CONOH, C_1-4alkyl-PO₃H₂、C_1-4alkyl-COOH and phenyl and

R^hc of (A)_4-8Heterocyclyl and C_3-10Cycloalkyl moieties optionally substituted with 1 to 4R^wSubstituent group substitution;

each R is^wThe substituents are independently selected from: c_1-4Alkyl radical, C_1-4alkyl-OH, C_1-4alkyl-COOH, C_1-4alkyl-SO₂NH₂、C_1-4Alkyl CONH₂、C_1-4alkyl-CONOH, C_1-4alkyl-PO₃H、OH、COO-C_1-8Alkyl, COOH, SO₂NH₂、CONH₂、CONOH、PO₃H₂And an oxo group;

R⁴selected from the group consisting of: O-C_1-8Alkyl, O-C_1-8Haloalkyl, O-C_1-8alkyl-R^z、C_6-10Aryl radical, C_5-10Heteroaryl, -O-C_1-4alkyl-C_6-10Aryl and-O-C_1-4alkyl-C_5-10Heteroaryl, wherein, said C_6-10Aryl and C_5-10Heteroaryl is optionally substituted with 1 to 5R^zSubstitution;

each R is^zIndependently selected from the group consisting of: halogen, -CN, -R^m、-CO₂Rⁿ、-CONRⁿR^p、-C(O)Rⁿ、-OC(O)NRⁿR^p、-NRⁿC(O)R^p、-NRⁿC(O)₂R^m、-NRⁿ-C(O)NRⁿR^p、-NRⁿR^p、-ORⁿ、-O-X³-ORⁿ、-O-X³-NRⁿR^p、-O-X³-CO₂Rⁿ、-O-X³-CONRⁿR^p、-X³-ORⁿ、-X³-NRⁿR^p、-X³-CO₂Rⁿ、-X³-CONRⁿR^p、-SF₅、-S(O)₂RⁿR^p、-S(O)₂NRⁿR^pAnd a three-to seven-membered carbocyclic or four-to seven-membered heterocyclic ring, wherein the three-to seven-membered carbocyclic or four-to seven-membered heterocyclic ring is optionally substituted with 1 to 5R^tWherein each R is^tIndependently selected from the group consisting of: c_1-8Alkyl radical, C_1-8Haloalkyl, -CO₂Rⁿ、-CONRⁿR^p、-C(O)Rⁿ、-OC(O)NRⁿR^p、-NRⁿC(O)R^p、-NRⁿC(O)₂R^m、-NRⁿ-C(O)NRⁿR^p、-NRⁿR^p、-ORⁿ、-O-X³-ORⁿ、-O-X³-NRⁿR^p、-O-X³-CO₂Rⁿ、-O-X³-CONRⁿR^p、-X³-ORⁿ、-X³-NRⁿR^p、-X³-CO₂Rⁿ、-X³-CONRⁿR^p、-SF₅and-S (O)₂NRⁿR^p；

Wherein each X is³Is C_1-4An alkylene group; each R isⁿAnd R^pIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted with oxo; each R is^mIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl and C_1-8A haloalkyl group; and optionally when two R are^zWhen the substituents are on adjacent atoms, they combine to form a fused five or six membered carbocyclic or heterocyclic ring optionally substituted with oxo;

n is 0, 1,2 or 3;

each R is⁵Independently selected from the group consisting of: halogen, -CN, -R^q、-CO₂R^r、-CONR^rR^s、-C(O)R^r、-OC(O)NR^rR^s、-NR^rC(O)R^s、-NR^rC(O)₂R^q、-NR^r-C(O)NR^rR^s、-NR^rR^s、-OR^r、-O-X⁴-OR^r、-O-X⁴-NR^rR^s、-O-X⁴-CO₂R^r、-O-X⁴-CONR^rR^s、-X⁴-OR^r、-X⁴-NR^rR^s、-X⁴-CO₂R^r、-X⁴-CONR^rR^s、-SF₅、-S(O)₂NR^rR^sWherein each X is⁴Is C_1-4An alkylene group; each R is^rAnd R^sIs independently selected from: hydrogen, C_1-8Alkyl and C_1-8A halogenated alkyl group,or, when attached to the same nitrogen atom, may combine with the nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted by oxo; each R is^qIndependently selected from the group consisting of: c_1-8Alkyl and C_1-8A haloalkyl group;

R^6aselected from the group consisting of: H. c_1-4Alkyl and C_1-4A haloalkyl group;

each R is^6bIndependently selected from the group consisting of: F. c_1-4Alkyl, O-R^u、C_1-4Haloalkyl, NR^uR^vWherein each R is^uAnd R^vIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted with oxo; and

m is 0, 1,2, 3 or 4.

12. The method of claim 8, wherein the PD-1 inhibitor is an anti-PD-1 antibody.

13. The method of claim 12, wherein the anti-PD-1 antibody is selected from the group consisting of: nivolumab, pembrolizumab, and pidilizumab.

14. The method of claim 8, wherein the CTLA-4 inhibitor is an anti-CTLA-4 antibody.

15. The method of claim 14, wherein the anti-CTLA-4 antibody is selected from the group consisting of: ipilimumab, tremelimumab, AGEN1884, and AGEN 2041.

16. The method of any one of claims 1 to 7, wherein the one or more immune checkpoint inhibitors are a cytotoxic T lymphocyte-associated protein 4(CTLA-4) inhibitor and a programmed cell death protein 1(PD-1) inhibitor.

17. The method of claim 16, wherein the PD-1 inhibitor is an anti-PD-1 antibody and the CTLA-4 inhibitor is an anti-CTLA-4 inhibitor.

18. The method of claim 17, wherein the anti-PD-1 inhibitor is selected from the group consisting of: nivolumab, pembrolizumab and pidilizumab and the CTLA-4 inhibitor are selected from the group consisting of: ipilimumab, tremelimumab, AGEN1884, and AGEN 2041.

19. The method of any one of claims 1 to 15, wherein the cancer is a solid cancer.

20. The method of claim 19, wherein the cancer is colon cancer.

21. The method of claim 19, wherein the cancer is pancreatic cancer.

Background

Cancer is a class of diseases in which one class of cells exhibits dysregulation of replication and growth. Recent cancer models have implicated the immune system, including cell homing (cellular homing) and immune checkpoints, in the development and progression of cancer. Although great progress has been made in understanding the biological basis of cancer, this disease remains a leading cause of death.

CC chemokine receptor 4(CCR (4)) was first identified by Power et al (1995) journal of biochemistry (J.biol. chem.)270:19495-19500), is a G protein-coupled receptor that binds to encapsulated chemokines, including CCL22, also known as macrophage-derived chemokine (MDC; the CC chemokines are reportedly chemotactic agents for the Th2 subset of peripheral blood T cells, dendritic cells and natural killer cells (NK)), and CCL17, also known as TARC (thymus and activation regulatory chemokine), also produced by monocytes and dendritic cells.

CCR (4) is involved in immunomodulatory processes such as cell homing to specific tissues, including T lymphocyte homing to the skin and lungs (see, e.g., Campbell et al (1999) Nature 400:776-780, Gonzalo et al (1999) J.Immunol 163: 403-5411, Lloyd et al (2000) J.Immunol 191:265-273, Kawasaki et al (2001) J.Immunol 166: 2055-2062). Modulators of CCR4 activity have been described, for example, in WO 2013/082490.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is considered to be a key regulator of the adaptive immune response. In particular, CLTA-4 is understood to play a central role in the maintenance of and the composition used in the maintenance of the T cell burst response. Thus, CTLA-4, as an immune checkpoint inhibitor, is considered a potential therapeutic target for the treatment of cancer and inflammation. CLTA-4 modulators have been described, for example, in WO 2018/035710.

Programmed death-1 (PD-1) is a transmembrane receptor protein that down-regulates T cell function through interaction with its two natural ligands, PD-L1 and PD-L2. Similar to CTLA4, PD-1 is also a major regulator of the immune system and is also considered an immune checkpoint inhibitor. PD-1/PD-L1 modulators have been described, for example, in WO 2018/005374.

Given the role of cell homing and immune checkpoint pathways in cancer development and progression, there is a need to develop combination therapies that can improve cancer treatment.

Disclosure of Invention

The present invention relates to combination therapies of C-C chemokine receptor 4(CCR4) antagonists and one or more checkpoint inhibitors in the treatment of cancer.

In some embodiments, the CCR4 receptor antagonist has the following formula I

Wherein each variable is described later.

In some embodiments, the CCR4 antagonist has the formula

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR4 antagonist has the formula

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR4 antagonist has the formula

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR4 antagonist has the formula

Or a pharmaceutically acceptable salt thereof.

Brief description of the drawings

FIG. 1 illustrates the study design for the KCM orthotopic pancreatic tumor model.

Figures 2A-C show primary tumor + pancreatic weight (group a), total tumor + pancreatic weight (group B), and spleen weight (group C) for each test population. Combination treatment with anti-CTLA-4 antibody and compound 1 reduced tumor weight compared to antibody or compound 1 alone.

FIGS. 3A-F report the relative number of suppressive immune cell populations in each test population. Groups A-C report cells per gram of tissue, while panels D-F report the percentage of CD45+ cells. Groups A and D report T-reg cells; groups B and E report G-MDSC cells; and groups C and F report M-MDSC cells.

Fig. 4 illustrates the study design for the CT26 colon cancer tumor model.

Figure 5 plots survival rates from individual test population mice in the CT26 tumor model over the course of the study.

Figure 6 plots the mean tumor volume of mice from each test population.

Figures 7A-D plot individual tumor volumes of mice from each test population. Group a reports the antibody isotype and vehicle group; group B reports antibody isotypes and compound 1 group; group C reports anti-CTLA-4 antibodies and vehicle groups; group D reports anti-CTLA-4 antibodies and Compound 1.

FIGS. 8A-F plot re-challenge (re-challenge) and blank mice using CT26 colon cancer cells and 4T1 breast cancer cells (bmic) tumor volume. Restimulated mice were those with complete tumor regression previously treated with anti-CTLA-4 (6 mice) or anti-CTLA-4 and compound 1(8 mice). The blank mice refer to mice that had previously been challenged with CT26 colon cancer cells. Group a showed that mice from anti-CTLA-4 treatment group 5/6 were resistant to CT26 tumor reintroduction; group B showed that none of the mice from the anti-CTLA-4 treated group were resistant to 4T1 tumor cells; group C showed that mice from anti-CTLA-4/Compound 1 treatment group 8/8 were resistant to CT26 tumor reintroduction; group D showed that 2 of 8 mice from the anti-CTLA-4/compound 1 treated group were resistant to 4T1 tumor cells; group E showed that all the naive mice had tumor growth on the first challenge with CT26 cells; panel F shows that no blank mice were resistant to 4T1 tumor cells.

FIGS. 9A-C plot the flow cytometry results for blood samples obtained from the mice tested in FIG. 8. Group A showed that prior to re-challenge, mice previously exposed to CT26 cells (and treated with CTLA-4 or anti-CTLA-4 and compound) had cytotoxic T cells against CT26 tumor cells, whereas the blank mice did not. Groups B and C showed the number of cytotoxic T cells responding to CT26 tumor cells before and 1 week after re-challenge.

Detailed Description

I. General purpose

The present invention relates to the surprising and unexpected discovery that combination therapy using a CCR4 antagonist and one or more immune checkpoint inhibitors significantly improves cancer treatment compared to the one or more checkpoint inhibitors themselves.

Abbreviations and Definitions

Unless otherwise indicated, the term "alkyl", by itself or as part of another substituent, means having the indicated number of carbon atoms (i.e., C)_1-8Refers to a straight or branched chain hydrocarbon group of 1 to 8 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. Examples of such unsaturated alkyl groups include: ethenyl, 2-propenyl, crotyl, 2-isopentenyl, 1,2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-and 3-propynyl, 3-butynyl, and higher homologs and isomers. The term "cycloalkyl" refers to a ring having the indicated number of ring atoms (e.g., C)_3-6Cycloalkyl) and fully saturated or having no more than one double bond between the ring vertices. "cycloalkyl" may also refer to bicyclic hydrocarbon rings and polycyclic hydrocarbon rings, e.g., bicyclo [2.2.1]Heptane, bicyclo [2.2.2]Octane, and the like. The term "heterocycloalkyl" refers to a cycloalkyl group containing one to five heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atoms are optionally quaternized. The heterocycloalkyl group may be a monocyclic, bicyclic or polycyclic ring system. Non-limiting examples of heterocycloalkyl groups include: pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1, 4-dioxane, morpholine, thiomorpholine-S-oxide, thiomorpholine-S, S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyranKetones, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. The heterocycloalkyl group can be attached to the rest of the molecule through a ring carbon or a heteroatom. By such as cycloalkylalkyl and heterocycloalkylalkyl, it is meant that the cycloalkyl or heterocycloalkyl group is attached to the rest of the molecule through an alkyl or alkylene linking group. For example, cyclobutylmethyl-is a cyclobutyl ring linked to a methylene linking group in the remainder of the molecule.

The term "alkylene" by itself or as part of another substituent refers to a divalent radical derived from an alkane, such as-CH₂CH₂CH₂CH₂-is shown. Typically, the alkyl (or alkylene) groups will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in this disclosure. "lower alkyl" or "lower alkylene" is a short chain alkyl or alkylene group typically having four or fewer carbon atoms. Similarly, "alkenylene" and "alkynylene" refer to the unsaturated forms of "alkylene" having double or triple bonds, respectively.

Unless otherwise specified, the term "heteroalkyl," by itself or in combination with another term, refers to a stable straight or branched chain or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and one to three heteroatoms selected from O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N and S can be located anywhere within the heteroalkyl group. The heteroatom Si may be located anywhere in the heteroalkyl group, including the position where the alkyl group is attached to the remainder of the molecule. Examples include: -CH₂-CH₂-O-CH₃、-CH₂-CH₂-NH-CH₃、-CH₂-CH₂-N(CH₃)-CH₃、-CH₂-S-CH₂-CH₃、-CH₂-CH₂、-S(O)-CH₃、-CH₂-CH₂-S(O)₂-CH₃、-CH＝CH-O-CH₃、-Si(CH₃)₃、-CH₂-CH＝N-OCH₃and-CH ═ CH-N (CH)₃)-CH₃. Up to two heteroatoms may be consecutive, e.g. -CH₂-NH-OCH₃and-CH₂-O-Si(CH₃)₃. Similarly, unless otherwise specified, the terms "heteroalkenyl" and "heteroalkynyl" by themselves or in combination with another term mean an alkenyl or alkynyl group containing the recited number of carbons and having one to three heteroatoms selected from O, N, Si and S, respectively, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatoms O, N and S can be located anywhere within the heteroalkyl group.

The term "heteroalkylene" by itself or as part of another substituent means a divalent saturated or unsaturated or polyunsaturated radical derived from a heteroalkyl radical, e.g., -CH₂-CH₂-S-CH₂CH₂-and-CH₂-S-CH₂-CH₂-NH-CH₂-、-O-CH₂-CH＝CH-、-CH₂-CH＝C(H)CH₂-O-CH₂-and-S-CH₂-C ≡ C-. For heteroalkylene groups, heteroatoms can also occupy one or both of the chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like).

The terms "alkoxy", "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense to refer to those alkyl groups attached to the rest of the molecule through an oxygen atom, an amino group, or a 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 they are attached to form a 3-7 membered ring. Thus, with-NR^aR^bGroups represented include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl (azetidinyl), and the like.

Unless otherwise specified, the term "halo" or "halogen" by itself or as part of another substituent refers to a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are intended to include monohaloalkyl and polyhaloalkyl. For example, the term "C_1-4Haloalkyl "is intended to include: trifluoromethyl, 2,2, 2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

Unless otherwise indicated, the term "aryl" refers to a polyunsaturated, usually aromatic, hydrocarbon group that can be a single ring or multiple rings (up to three rings) that are fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or rings) containing one to five heteroatoms selected from N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atoms are optionally quaternized. Heteroaryl groups may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl groups include: phenyl, naphthyl, and biphenyl; and non-limiting examples of heteroaryl groups include: pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzpyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuranyl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidyl, imidazopyridinyl, 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 aryl and heteroaryl ring systems described above are selected from the group of acceptable substituents described hereinafter.

For the sake of brevity, when the term "aryl" is used in combination with other terms (e.g., aryloxy, arylthio, arylalkyl), aryl includes aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is intended to include those groups in which an aromatic group is attached to an alkyl group that is attached to the rest 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 simplicity, the terms aryl and heteroaryl will refer to the substituted or unsubstituted forms provided below, while the terms "alkyl" and related aliphatic groups refer to the unsubstituted forms, unless indicated to be substituted.

Substituents for alkyl groups (including those groups commonly referred to as alkylene, alkenyl, alkynyl and cycloalkyl) can be various groups selected from: -halogen, -OR ', -NR' R ', -SR', -SiR '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’、-NH-C(NH₂)＝NH、-NR’C(NH₂)＝NH、-NH-C(NH₂)＝NR’、-S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-NR’S(O)₂R ", -CN and-NO₂In a number of from 0 to (2m '+ 1), where m' is the total number of carbon atoms in these groups. R ', R ' and R ' each independently mean hydrogen, unsubstituted C_1-8Alkyl, unsubstituted heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted C_1-8Alkyl radical, C_1-8Alkoxy or C_1-8Thioalkoxy, or unsubstituted aryl-C_1-4An alkyl group. 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 an alkyl group in which the two substituents on the carbon closest to the point of attachment of the group are substituted with a substituent ═ O (e.g., c (O) CH)₃,-C(O)CH₂CH₂OR', etc.).

Similarly, the substituents for aryl and heteroaryl groups are varied 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”’、-NH-C(NH₂)＝NH、-NR’C(NH₂)＝NH、-NH-C(NH₂)＝NR’、-S(O)R’、-S(O)₂R’、-S(O)₂NR’R”、-NR’S(O)₂R”、-N₃Perfluoro (C)₁-C₄) Alkoxy and perfluoro(C₁-C₄) Alkyl groups in a number ranging from zero to the total number of open valences on the aromatic ring system; and R ', R ", and R'" are independently selected from: hydrogen, C_1-8Alkyl radical, C_3-6Cycloalkyl radical, C_2-8Alkenyl radical, C_2-8Alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl) -C_1-4Alkyl and unsubstituted aryloxy-C_1-4An alkyl group. 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.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be substituted by a group of formula-T-C (O) - (CH)₂)_q-U-substituent, wherein T and U are independently-NH-, -O-, -CH₂-or a single bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally substituted by a group of formula-A- (CH)₂)_r-B-substituent, wherein A and B are independently-CH₂-、-O-、-NH-、-S-、-S(O)-、-S(O)₂-、-S(O)₂NR' -or a single bond, and r is an integer of 1 to 3. One of the single bonds of the new ring so formed may be optionally substituted by a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally substituted by a group of formula- (CH)₂)_s-X-(CH₂)_t-substituent (S) wherein S and t are independently an integer from 0 to 3, and X is-O-, -NR' -, -S (O)₂-or-S (O)₂NR' -. At NR' -and-S (O)₂The substituents R 'in NR' are selected from: hydrogen or unsubstituted C_1-6An alkyl group.

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

For the compounds provided herein, a bond drawn 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 attachment at any available vertex of the aromatic ring. In some embodiments, the description will also include a connection on a ring fused to the aromatic ring. For example, a bond drawn to the center of the benzene portion of an indole would represent a bond to any available vertex of the six-or five-membered ring portion of the indole.

The term "pharmaceutically acceptable salt" is intended to include salts of the active compounds prepared with relatively nontoxic acids or bases, depending on the particular substituents present 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 forms of these compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include: aluminum salt, ammonium salt, calcium salt, copper salt, ferric salt, ferrous salt, lithium salt, magnesium salt, trivalent manganese salt, divalent manganese salt, potassium salt, sodium salt, zinc salt 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, 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 forms of these compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as salts derived from relatively nontoxic organic acids such as acetic, propionic, isobutyric, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like. Also included are Salts of amino acids such as arginine and the like, and Salts of organic acids such as glucuronic acid or galacturonic acid and the like (see, e.g., Berge, s.m., et al, "pharmaceutically acceptable Salts", Journal of Pharmaceutical Science (1977, 66, 1-19). Certain specific compounds of the invention contain both basic and acidic functionalities that allow the compounds to be converted into base or acid addition salts.

The neutral form of the compound may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salt is equivalent to the parent form of the compound for purposes of the present invention.

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

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in polycrystalline or amorphous form. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

Certain compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers, and individual isomers (e.g., separated enantiomers) are intended to be included within the scope of the present invention. When the compounds provided herein have a defined stereochemistry (indicated as R or S, or indicated by a dashed line or a wedge bond), those skilled in the art will understand that those compounds are 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 invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. Unnatural proportions of isotopes can be defined from amounts found in nature to 100% of the amount made up of the atoms in question. For example, the compounds may incorporate radioactive isotopes, such as tritium(s) (iii)³H) Iodine-125 (¹²⁵I) Or carbon-14 (¹⁴C) Or non-radioactive isotopes, such as deuterium (I)²H) Or carbon-13 (¹³C) In that respect Such isotopic variations may provide additional utility to those described elsewhere in this application. For example, isotopic variants of the compounds of the present invention may find other uses, including but not limited to, as diagnostic and/or imaging agents, or as cytotoxic/radiotoxic therapeutic agents. In addition, isotopic variations of the compounds of the present invention can have altered pharmacokinetic and pharmacodynamic profiles that can contribute to improved safety, tolerability, or efficacy during treatment. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

As used herein, the term "selective CCR4 antagonist" refers to a highly recognition-competent compound that inhibits CCR4 activity and has little or no cross-reactivity to non-target proteins (such as CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR12, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and/or CXCR 7). In some embodiments, a "selective CCR4 antagonist" has an IC50 value that is at least 10 lower than that of proteins such as CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR12, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6 and/or CXCR7 when tested in the assay used in example 3 of the present application; 100, respectively; 500, a step of; 1,000; 2,000; 5,000; or more than a multiple of the IC50 value. In some embodiments, a "selective CCR4 antagonist" does not inhibit CCR1, CCR2, CCR3, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CCR12, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, and/or CXCR7 at a concentration of 1 μ M or less in the assays used in example 2 herein. Such proteins are considered "uninhibited" when they maintain 100%, 99%, 95%, 90% or 85% of their activity under reference conditions using a selective CCR4 antagonist.

Combination therapy using a CCR4 antagonist and one or more immune checkpoint inhibitors

Provided herein are methods, compositions and kits for treating cancer using the synergistic effect of a CCR4 antagonist and an immune checkpoint inhibitor. Combination therapy comprising a CCR4 antagonist and one or more immune checkpoint inhibitors is more effective in treating cancer than either alone.

Cancer generally comprises any of a variety of malignancies and is characterized by the proliferation of undifferentiated cells that tend to invade surrounding tissues and metastasize to new body sites. Non-limiting examples of different types of cancers that are suitable for treatment using the compositions of the present disclosure include: ovarian cancer, breast cancer, lung cancer (such as non-small cell lung cancer), bladder cancer, thyroid cancer, liver cancer, pleural cancer, pancreatic cancer, cervical cancer, prostate cancer, testicular cancer, colon cancer, anal cancer, colorectal cancer, bile duct cancer, gastrointestinal carcinoid tumor, esophageal cancer, gallbladder cancer, rectal cancer, appendiceal cancer, small intestine cancer, gastric (stomach) cancer, kidney cancer (i.e., renal cell carcinoma), central nervous system cancer, skin cancer, choriocarcinoma, head and neck cancer, bone cancer, osteogenic sarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, leukemia (e.g., acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, or hairy cell leukemia), lymphoma (e.g., non-hodgkin's lymphoma, B-cell lymphoma, or Burkitt's lymphoma), and multiple myeloma.

In some embodiments, the cancer is lung cancer (e.g., non-small cell lung cancer), melanoma, epithelial cancer (e.g., prostate cancer, ovarian cancer, breast cancer), or blood cancer (e.g., leukemia, lymphoma, multiple myeloma).

In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is colon cancer.

A. CCR4 antagonists,

CCR4 antagonists are compounds that reduce or inhibit CCR4 activity. Many such compounds are known in the art. In some embodiments, the CCR4 antagonists of the invention are selective CCR4 antagonists.

In some embodiments, the CCR4 antagonist is a small molecule inhibitor of CCR4 having the following formula (I):

and pharmaceutically acceptable salts thereof, wherein,

R¹selected from: hydrogen, C_1-8Alkyl radical, C_1-8Haloalkyl, C_1-8Hydroxyalkyl radical, C_3-8Cycloalkyl, halogen, -CN, -SO₂Me and-C (O) NH₂；

R³selected from: hydrogen, methyl and C_1-4A haloalkyl group;

R⁴selected from: hydrogen, C_1-8Alkyl radical, C_1-8Haloalkyl and C_1-8A hydroxyalkyl group;

each subscript n is independently an integer of 0 to 3;

b is a bond or C (O);

q is selected from: C. CH, N, O, S (O) and SO₂：

W, X, Y and Z are independently selected from: C. CH and N, but Q and W are not both N;

Wherein the content of the first and second substances,

each R is^aAnd R^bIndependently selected from: hydrogen, C_1-8Alkyl radical, C_1-8Hydroxyalkyl radical, C_1-8Haloalkyl and C_1-8An alkoxy group; and

R⁷absent or selected from: hydrogen, C_1-8Alkyl and C_1-8A haloalkyl group.

In one set of embodiments, the compounds provided herein are those wherein X and Y are not both N. In another set of embodiments, R³Is H, and each R²Independently selected from: c_1-8Alkyl radical, C_1-8Haloalkyl, halogen and-CN.

In another set of embodiments, the compounds provided herein have the following formula (Ia):

wherein each R is¹、R²、R⁴、R⁵、R⁶、R⁷X, Y, Z, W, Q, B and subscript n are as described for formula I. In selected embodiments, X is C or CH.

In another set of embodiments, the compounds provided herein have the following formula (Ib):

wherein each R is²Selected from: c_1-8Alkyl radical, C_1-8Haloalkyl, halogen and-CN, and each R¹、R⁴、R⁵、R⁶、R⁷X, Z, W, Q, B and subscript n are as described for formula I.

In another set of embodiments, the compounds provided herein have the following formula (Ic):

wherein each R is²Selected from: c_1-8Alkyl radical, C_1-8Haloalkyl, halogen, and-CN; subscript n is 0 or 1; and each R¹、R⁴、R⁶、R⁷X, Y, Z and Q are as described for formula I. In selected embodiments, n is 1, and R is⁴Is hydrogen or methyl.

In another set of embodiments, the compounds provided herein have the following formula (Id):

wherein each R is²Is a member selected from: c_1-8Alkyl radical, C_1-8Haloalkyl, halogen, and-CN; subscript n is 0 or 1; and each R¹、R⁴、R⁶、R⁷X, Z and Q are as described for formula I. In selected embodiments, n is 1, and R is⁴Is hydrogen or methyl.

In another set of embodiments, the compounds provided herein have the following formula (Ie):

wherein the content of the first and second substances,each R is²Selected from: c_1-8Alkyl radical, C_1-8Haloalkyl, halogen, and-CN; subscript n is 0 or 1; and each R¹、R⁴、R⁶、R⁷Y, Z and Q are as described for formula I. In selected embodiments, n is 1, and R is⁴Is hydrogen or methyl.

In another set of embodiments, the compounds provided herein have the following formula (If):

wherein each R is²Selected from: c_1-8Alkyl radical, C_1-8Haloalkyl, halogen, and-CN; and each R¹、R⁴、R⁶、R⁷And Q is as described for formula I. In selected embodiments, R⁴Is hydrogen or methyl.

In still other embodiments, the compounds provided herein have the following formulas (I), (Ia) and (Ib), including the specific embodiments provided above, wherein B is c (o). Still further, there is provided a compound wherein the ring having Z as the ring apex is selected from pyrrolidine and piperidine. In selected embodiments, compounds are provided wherein the ring having Z as the ring apex is selected from pyrrolidin-2-yl and piperidin-2-yl, and R is⁵、R⁶And R⁷At least one of which is not hydrogen.

In still other embodiments, the compounds provided herein have the following formulas (I), (Ia) and (Ib), including the specific embodiments provided above, wherein B is a bond. In related embodiments, the ring in which B is a bond and Z is the apex of the ring is selected from pyrrolidine, piperidine, and cyclohexane. In a particular embodiment, the ring in which B is a bond and has Z as a ring apex is selected from pyrrolidin-1-yl, pyrrolidin-2-yl, piperidin-1-yl, piperidin-2-yl, piperidin-3-yl, and cyclohexane. In still other embodiments, B is a bond and the ring having Z as the ring apex is selected from the group consisting of: pyrrolidin-1-yl, pyrrolidin-2-yl, piperidin-1-yl, piperidin-2-yl, piperidin-3-yl and cyclohexaAn alkane; and R⁵、R⁶And R⁷At least one of which is not hydrogen.

In one set of embodiments, Z is CH or N.

In some embodiments, the CCR4 antagonist has the formula

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR4 antagonist has the formula

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR4 antagonist has the formula

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR4 antagonist has the formula

Or a pharmaceutically acceptable salt thereof.

In some embodiments, the CCR4 antagonist is selected from a compound or pharmaceutical composition disclosed in WO2013/082490 filed by chemicentryx. The contents of which are incorporated herein for all purposes.

B. Immunodetection point inhibitors

Immune checkpoints are signaling proteins that stimulate or suppress immune responses. Compositions targeting immune checkpoints can modulate these proteins, thereby altering the innate immune response of an individual. This targeted approach is useful because specific cancer cells can bypass these checkpoints to escape additional innate immune responses. Two specific immune checkpoints are programmed cell death protein 1(PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4).

PD-1 inhibitors

Programmed cell death protein-1 (PD-1) is an immune checkpoint protein, most commonly found on T cells. PD-1 normally binds to its natural ligands PD-L1 and PD-L2 and is expressed on the surface of different cells. When bound to its native ligand, T cells are considered to be in the "off" position. Notably, some cancer cells express abnormally high levels of PD-L1, which means that T cell activity and associated anti-cancer immune responses are abnormally inhibited. Most critically, the use of PD-1 inhibitors that block the interaction with their natural ligands can stimulate an immune response to help fight cancer.

The PD-1 inhibitors of the present invention include small molecules and antibodies.

In some embodiments, the PD-1 inhibitor is a small molecule PD-1/PD-L1 inhibitor.

In some embodiments, the small molecule PD-1/PD-L1 inhibitor has the formula:

in some embodiments, the small molecule PD-1/PD-L1 inhibitor is a compound having formula (II)

Or a pharmaceutically acceptable salt thereof; wherein the content of the first and second substances,

R¹selected from the group consisting of: halogen, C_5-8Cycloalkyl radical, C_6-10Aryl and thienyl, wherein, C_6-10Aryl and thienyl are optionally substituted with 1 to 5R^xSubstituted by a substituent;

each R is^xIndependently selected from the group consisting of: halogen, -CN, -R^c、-CO₂R^a、-CONR^aR^b、-C(O)R^a、-OC(O)NR^aR^b、-NR^bC(O)R^a、-NR^bC(O)₂R^c、-NR^a-C(O)NR^aR^b、-NR^aR^b、-OR^a、-O-X¹-OR^a、-O-X¹-CO₂R^a、-O-X¹-CONR^aR^b、-X¹-OR^a、-X¹-NR^aR^b、-X¹-CO₂R^a、-X¹-CONR^aR^b、-SF₅and-S (O)₂NR^aR^bWherein each X is¹Is C_1-4An alkylene group; wherein each R is^aAnd R^bIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members; wherein the five or six membered ring is optionally both substituted with oxo; each R is^cIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl radical, C_2-8Alkynyl and C_1-8A haloalkyl group; and optionally when two R are^xWhen the substituents are on adjacent atoms, they combine to form a fused five-, six-or seven-membered carbocyclic or heterocyclic ring, which is optionally substituted with 1 to 3 substituents independently selected from: halogen, oxo, C_1-8Haloalkyl and C_1-8An alkyl group;

each R is^2a、R^2bAnd R^2cIndependently selected from the group consisting of: H. halogen, -CN, -R^d、-CO₂R^e、-CONR^eR^f、-C(O)R^e、-OC(O)NR^eR^f、-NR^fC(O)R^e、-NR^fC(O)₂R^d、-NR^e-C(O)NR^eR^f、-NR^eR^f、-OR^e、-O-X²-OR^e、-O-X²-NR^eR^f、-O-X²-CO₂R^e、-O-X²-CONR^eR^f、-X²-OR^e、-X²-NR^eR^f、-X²-CO₂R^e、-X²-CONR^eR^f、-SF₅、-S(O)₂NR^eR^f、C_6-10Aryl and C_5-10Heteroaryl, wherein each X²Is C_1-4An alkylene group; each R is^eAnd R^fIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O and S as ring members, and optionally substituted with oxo; each R is^dIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl and C_1-8A haloalkyl group;

R³selected from the group consisting of: -NR^gR^hAnd C_4-12Heterocyclic group, wherein, said C_4-12Heterocyclyl is optionally substituted by 1 to 6R^ySubstituted;

Wherein R is^yC of (A)_1-6The alkyl moiety being optionally further substituted by OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂H is substituted, wherein each R is^jAnd R^kIndependently selected from: hydrogen, optionally substituted by 1 to 2 groups selected from OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂C substituted by a substituent of H_1-8Alkyl, and optionally substituted by 1 to 2 groups selected from OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂C substituted by a substituent of H_1-8Haloalkyl, or R when attached to the same nitrogen atom^jAnd R^kMay be combined with a nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted by oxo; each R isⁱIndependently selected from the group consisting of: -OH, C_1-8Alkyl radical, C_2-8Alkenyl and C_1-8Haloalkyl, each of which is optionally substituted with OH, SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl or CO₂H is substituted;

R^gselected from the group consisting of: H. c_1-8Haloalkyl and C_1-8An alkyl group;

R^hthe combination of N linked thereto is a mono-, di-or tripeptide comprising 1-3 natural amino acids and 0-2 unnatural amino acids, wherein,

the unnatural amino acid has an alpha carbon substituent selected from the group consisting of: c_2-4Hydroxyalkyl radical, C_1-3Alkyl-guanidino and C_1-4An alkyl-heteroaryl group, which is a cyclic alkyl group,

the alpha carbon of each natural or unnatural amino acid is optionally further substituted with methyl, and

the terminal part of the mono-, di-or tripeptides is selected from the group consisting of: c (O) OH, C (O) O-C_1-6Alkyl and PO₃H₂Wherein, in the step (A),

R^h1and R^h2Each independently selected from the group consisting of: H. c_1-6Alkyl and C_1-4A hydroxyalkyl group;

R^hc of (A)_4-8Heterocyclyl and C_3-10Cycloalkyl moieties optionally substituted with 1 to 4R^wSubstituent group substitution;

R⁴selected from the group consisting of: O-C_1-8Alkyl, O-C_1-8Haloalkyl, O-C_1-8alkyl-R^z、C_6-10Aryl radical, C_5-10Heteroaryl, -O-C_1-4alkyl-C_6-10Aryl and-O-C_1-4alkyl-C_5-10Heteroaryl of which C is_6-10Aryl and C_5-10Heteroaryl is optionally substituted with 1 to 5R^zSubstitution;

n is 0, 1,2 or 3;

each R is⁵Independently selected from the group consisting of: halogen, -CN, -R^q、-CO₂R^r、-CONR^rR^s、-C(O)R^r、-OC(O)NR^rR^s、-NR^rC(O)R^s、-NR^rC(O)₂R^q、-NR^r-C(O)NR^rR^s、-NR^rR^s、-OR^r、-O-X⁴-OR^r、-O-X⁴-NR^rR^s、-O-X⁴-CO₂R^r、-O-X⁴-CONR^rR^s、-X⁴-OR^r、-X⁴-NR^rR^s、-X⁴-CO₂R^r、-X⁴-CONR^rR^s、-SF₅、-S(O)₂NR^rR^sWherein each X is⁴Is C_1-4An alkylene group; each R is^rAnd R^sIs independently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted with oxo; each R is^qIndependently selected from the group consisting of: c_1-8Alkyl and C_1-8A haloalkyl group;

R^6aselected from the group consisting of: H. c_1-4Alkyl and C_1-4A haloalkyl group;

m is 0, 1,2, 3 or 4.

In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has formula (IIa)

In some embodiments, the compound, or a pharmaceutically acceptable salt thereof, has formula (IIb)

In some embodiments, R¹Selected from the group consisting of: phenyl and thienyl, wherein said phenyl and thienyl are optionally substituted with 1 to 5R^xSubstituted by a substituent. In some embodiments, R¹Is optionally substituted by 1 or 2R^xSubstituted phenyl, wherein each R is^xIndependently selected from: halogen, C_1-8Alkyl, O-C_1-8Alkyl, O-C_1-8Haloalkyl, -NR^aR^bAnd CN, and optionally when two R are^xWhen the substituents are on adjacent atoms, they combine to form a fused six-membered heterocyclic ring optionally substituted with 1 to 3 substituents independently selected from the group consisting of: oxo radical, C_1-8Haloalkyl and C_1-8An alkyl group. In some embodiments, R¹Is phenyl optionally substituted by F. In some embodiments, R¹Selected from the group consisting of:

in some embodiments, each R is^2a、R^2bAnd R^2cIndependently selected from the group consisting of: H. halogen, -CN, -R^d、-NR^eR^f、-OR^e、-X²-OR^e、-X²-NR^eR^fWherein X is²Is C_1-4An alkylene group; each R is^eAnd R^fIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl, or when attached to the same nitrogen atom, can combine with the nitrogen atom to form a five-or six-membered ring having from 0 to 2 additional heteroatoms selected from N, O or S as ring members, and optionally substituted with oxo; each R is^dIndependently selected from the group consisting of: c_1-8Alkyl radical, C_2-8Alkenyl and C_1-8A haloalkyl group; in some embodiments, R^2bAnd R^2cAre all H and R^2aSelected from the group consisting of: halogen, C_1-4Alkyl radical, C_2-4Alkenyl radical, C_1-3Haloalkyl, -CN, -OMe and OEt. In some embodiments, R^2bAnd R^2cAre all H and R^2aIs halogen. In some embodiments, R^2bAnd R^2cAre all H and R^2aIs Cl.

In some embodiments, n is 0, 1 or 2 and each R is⁵Independently selected from the group consisting of: halogen, -CN, -R^q、-NR^rR^sand-OR^rWherein each R is^rAnd R^sIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8Haloalkyl and each R^qIndependently selected from the group consisting of: c_1-8Alkyl and C_1-8A haloalkyl group. In some embodiments, n is 0.

In some embodiments, R^6aIs H. In some embodiments, m is 0. In some embodiments, m is 1 and R^6bSelected from the group consisting of: F. c_1-4Alkyl, O-R^u、C_1-4Haloalkyl and NR^uR^vWherein each R is^uAnd R^vIndependently selected from: hydrogen, C_1-8Alkyl and C_1-8A haloalkyl group. In some embodiments, m is 1 and R^6bIs F.

In some embodiments of the present invention, the substrate is,is composed of

In some embodiments, R⁴Selected from the group consisting of: O-C_1-4Alkyl, O-C_1-6alkyl-R^z、C_6-10Aryl radical, C_5-10Heteroaryl, -O-C_1-4alkyl-C_6-10Aryl and-O-C_1-4alkyl-C_5-10Heteroaryl, wherein, said C_6-10Aryl and said C_5-10Heteroaryl is optionally substituted with 1 to 2R^zWherein each R is^zIndependently selected from the group consisting of: halogen, -CN, -R^m、-CO₂Rⁿ、-CONRⁿR^p、-C(O)Rⁿ、-OC(O)NRⁿR^p、-NRⁿC(O)R^p、-NRⁿC(O)₂R^m、-NRⁿ-C(O)NRⁿR^p、-NRⁿR^p、-ORⁿ、-S(O)₂NRⁿR^pA three-to seven-membered carbocyclic ring and a four-to seven-membered heterocyclic ring, wherein the three-to seven-membered carbocyclic or four-to seven-membered heterocyclic ring is optionally substituted with 1 to 2R^tWherein each R is^tIndependently selected from the group consisting of: c_1-8Alkyl radical, C_1-8Haloalkyl, -CO₂Rⁿ、-CONRⁿR^p、-C(O)Rⁿ、-OC(O)NRⁿR^p、-NRⁿC(O)R^p、-NRⁿC(O)₂R^m、-NRⁿ-C(O)NRⁿR^p、-NRⁿR^p、-ORⁿand-S (O)₂NRⁿR^p. In some embodiments, R⁴Selected from the group consisting of: O-C_1-4Alkyl, O-C_1-6alkyl-CN, phenyl, pyridyl, -O-C_1-2Alkyl-pyridyl, -O-C_1-2Alkyl-pyrimidinyl, -O-C_1-2Alkyl-pyridazinyl and-O-C_1-2Alkyl-phenyl, wherein the pyridyl, phenyl, pyrimidinyl and pyridazinyl are optionally substituted with 1 to 2R^zWherein each R is^zIndependently selected from the group consisting of: halogen, -CN, -CO₂Rⁿ、-NRⁿR^p、-ORⁿAnd piperidinyl optionally substituted with OH.

In some embodiments, R⁴Selected from the group consisting of:

in some embodiments, R⁴Is composed of

In some embodiments, R³Selected from the group consisting of: NR (nitrogen to noise ratio)^gR^hAnd C_4-6Heterocyclic group, wherein, said C_4-6Heterocyclyl is optionally substituted with 1 to 3R^yIn which R is^gSelected from the group consisting of: H. c_1-8Haloalkyl and C_1-8Alkyl, and wherein R^his-C substituted with 1 to 3 substituents independently selected from_1-8Alkyl groups: OH, COOH, SO₂NH₂、CONH₂、CONOH、COO-C_1-8Alkyl radical, C_5-6Heteroaryl group, C_5-6Heterocyclic group and PO₃H₂Wherein, C_5-6Heteroaryl and C_5-6The heterocyclyl is optionally substituted with 1 to 3 substituents independently selected from: OH, B (OH)₂、COOH、SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl radical, C_1-4Alkyl radical, C_1-4alkyl-OH, C_1-4alkyl-SO₂NH₂、C_1-4Alkyl CONH₂、C_1-4alkyl-CONOH, C_1-4alkyl-PO₃H₂And C_1-4alkyl-COOH and wherein, said C_5-6The heterocyclyl is also optionally substituted by oxo. In some embodimentsIn, R³Selected from the group consisting of: azetidinyl, pyrrolidinyl and piperidinyl, wherein said azetidinyl, pyrrolidinyl or piperidinyl is linked through a nitrogen atom, and wherein said azetidinyl, pyrrolidinyl or piperidinyl is optionally substituted with 1 to 3R^yWherein each R is^yIndependently selected from the group consisting of: -CO₂H、CONOH、PO₃H₂、OH、SO₂NH₂、CONH₂And COO-C_1-8An alkyl group. In some embodiments, R³Is NHR^hWherein R is^his-C substituted with 1 to 2 substituents independently selected from_1-8Alkyl groups: OH, COOH, CONH₂、PO₃H₂Tetrazolyl, tetrazolonyl (tetrazolonyl) and pyrazolyl. In some embodiments, R³Selected from the group consisting of:

in some embodiments, R³is-NR^gR^h. In some embodiments, R^hN linked thereto is a mono-, di-or tripeptide comprising 1-3 natural amino acids and 0-2 unnatural amino acids, wherein,

the alpha carbon of each natural or unnatural amino acid is optionally further substituted with methyl, and

the terminal part of the mono-, di-or tripeptide is selected from the group consisting of: c (O) OH, C (O) O-C_1-6Alkyl and PO₃H₂。

In some embodiments, R^hEach natural amino acid of (a) is independently selected from the group consisting of: serine, alanine, glycine, lysine, arginine, threonine, phenylalanine, tyrosine, aspartic acid, asparagine, histidine and leucine.

In some embodimentsIn the formula, R¹Is optionally substituted by 1 to 3R^xSubstituted phenyl radicals, R^6bIs H, R⁴Selected from the group consisting of: O-C_1-4Alkyl, O-C_1-6alkyl-CN, phenyl, pyridyl, -O-C_1-2Alkyl-pyridyl, -O-C_1-2Alkyl-pyrimidinyl, -O-C_1-2Alkyl-pyridazinyl and-O-C_1-2Alkyl-phenyl, wherein the pyridyl, phenyl, pyrimidinyl and pyridazinyl are optionally substituted with 1 to 2R^zWherein each R is^zIndependently selected from the group consisting of: halogen, -CN, -CO₂Rⁿ、-NRⁿR^p、-ORⁿAnd piperidinyl optionally substituted with OH, and R³Selected from the group consisting of: NR (nitrogen to noise ratio)^gR^hAnd C_4-6Heterocyclic group, wherein, said C_4-6Heterocyclyl is optionally substituted with 1 to 3R^yIn which R is^gSelected from the group consisting of: H. c_1-8Haloalkyl and C_1-8Alkyl, and wherein R^his-C substituted with 1 to 3 substituents independently selected from_1-8Alkyl groups: OH, COOH, SO₂NH₂、CONH₂、CONOH、COO-C_1-8Alkyl radical, C_5-6Heteroaryl group, C_5-6Heterocyclic group and PO₃H₂Wherein, C_5-6Heteroaryl and C_5-6The heterocyclyl is optionally substituted with 1 to 3 substituents independently selected from: OH, B (OH)₂、COOH、SO₂NH₂、CONH₂、CONOH、PO₃H₂、COO-C_1-8Alkyl radical, C_1-4Alkyl radical, C_1-4alkyl-OH, C_1-4alkyl-SO₂NH₂、C_1-4Alkyl CONH₂、C_1-4alkyl-CONOH, C_1-4alkyl-PO₃H₂And C_1-4alkyl-COOH, and wherein said C_5-6The heterocyclyl is also optionally substituted by oxo.

In some embodiments, R¹Is optionally substituted by 1 or 2R^xSubstituted phenyl, wherein each R^xIndependently selected from: halogen, C_1-8Alkyl, O-C_1-8Alkyl, O-C_1-8Haloalkyl group、-NR^aR^bAnd CN, wherein R^2bAnd R^2cAre all H, R^2aSelected from the group consisting of: halogen, C_1-4Alkyl radical, C_1-3Haloalkyl, -CN, -OMe and OEt, R^6aIs H, m is 0, n is 0, R⁴Is composed ofAnd R³Selected from the group consisting of: NHR^hAzetidinyl, pyrrolidinyl and piperidinyl, wherein said azetidinyl, pyrrolidinyl or piperidinyl is attached through a nitrogen atom, and wherein said azetidinyl, pyrrolidinyl or piperidinyl is optionally substituted with 1 to 3R^yWherein each R is^yIndependently selected from the group consisting of: CO 2₂H、CONOH、PO₃H₂、OH、SO₂NH₂、CONH₂And COO-C_1-8Alkyl, and wherein R^hIs C substituted with 1 to 2 substituents independently selected from_1-8Alkyl groups: OH, COOH, CONH₂、PO₃H₂Tetrazolyl, tetrazolonyl (tetrazolonyl) and pyrazolyl. In some embodiments, R^2aIs halogen.

In some embodiments, the small molecule PD-1/PD-L1 inhibitor is selected from a compound or pharmaceutical composition disclosed in WO2018/005374 filed by chemicentryx on 26/6/2017. The contents of which are incorporated herein for all purposes.

In some embodiments, the PD-1 inhibitor is an antibody. In some embodiments, the PD-1 inhibitor antibody is selected from the group consisting of: nivolumab (Nivolumab), Pembrolizumab (Pembrolizumab), and Pidilizumab (Pidilizumab). In some embodiments, the PD-1 inhibitor antibody is nivolumab. In some embodiments, the PD-1 inhibitor antibody is pembrolizumab. In some embodiments, the PD-1 inhibitor antibody is pidilizumab.

The PD-1 inhibitors of the present invention can be prepared using methods known in the art. For example, SCID mice can be used to prepare human monoclonal antibodies of the disclosure into which human immune cells have been recombined so that upon immunization a human antibody response can be generated. Such mice are described, for example, in Wilson et al, U.S. patent nos. 5,476,996 and 5,698,767. The PD-1 inhibitors of the present disclosure may be formulated to delay degradation of the compound or antibody or to minimize immunogenicity of the antibody. A variety of techniques are known in the art to achieve this.

CTLA-4 inhibitors

Cytotoxic T lymphocyte-associated protein 4(CTLA-4) is an immune checkpoint protein, most commonly expressed by activated T cells. Like PD-1, CTLA-4 also acts as a negative regulator of T cell immune function. By providing inhibitors of CTLA-4, down-regulation is reduced and the anti-tumor activity of T cells is increased.

In some embodiments, the CTLA-4 inhibitor is an antibody. In some embodiments, the CLTA-4 inhibitor antibody has about 10⁸M^-1Or higher binding affinity. In some embodiments, the CLTA-4 inhibitor antibody has about 10⁹M^-1Or higher binding affinity. In some embodiments, the CTLA-4 inhibitor antibody can inhibit binding of human CTLA-4 to B7-1 or B7-2.

In some embodiments, the CTLA-4 inhibitor antibody is selected from the group consisting of: ipilimumab (Ipilimumab), Tremelimumab (Tremelimumab), AGEN1884, and AGEN 2041. In some embodiments, the CTLA-4 inhibitor antibody is ipilimumab. In some embodiments, the CTLA-4 inhibitor antibody is tremelimumab. In some embodiments, the CTLA-4 inhibitor antibody is age 1884. In some embodiments, the CTLA-4 inhibitor antibody is age 2041.

In some embodiments, the CTLA-4 inhibitor is selected from a compound or pharmaceutical composition disclosed in WO/2009/100140 or WO 2017/194265. The respective contents are incorporated herein for all purposes.

The CTLA-4 inhibitors of the invention can be prepared using methods known in the art. For example, SCID mice can be used to prepare human monoclonal antibodies of the disclosure into which human immune cells have been recombined so that upon immunization a human antibody response can be generated. Such mice are described, for example, in Wilson et al, U.S. patent nos. 5,476,996 and 5,698,767. CTLA-4 inhibitors of the disclosure can also be formulated to delay degradation of the compound or antibody or to minimize immunogenicity of the antibody. A variety of techniques are known in the art to achieve this.

Administration of combination therapy

In another aspect, the present disclosure provides a combination therapy for treating cancer. The combination therapy comprises a therapeutically effective amount of a CCR4 antagonist and a therapeutically effective amount of one or more immune checkpoint inhibitors. In some embodiments, the one or more immune checkpoint inhibitors are PD-1 inhibitors. In some embodiments, the one or more immune checkpoint inhibitors are CTLA-4 inhibitors. In some embodiments, the one or more immune checkpoint inhibitors are a PD-1 inhibitor and a CTLA-4 inhibitor. The combined synergistic effect of the therapeutic agents affects the treatment or prevention of cancer.

The term "therapeutically effective amount" refers to the amount of a compound of interest that will elicit the biological or medical response of a cell, tissue, system or animal (e.g., human) that is being sought by a researcher, veterinarian, medical doctor or other treatment provider.

Depending on the disease state and condition of the subject, the compounds, antibodies and formulations of the present disclosure may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection or implant), inhalation, nasal, vaginal, rectal, sublingual or topical routes of administration. In addition, the compounds and antibodies can be formulated, alone or together, into suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration. The present disclosure also contemplates that the compounds and antibodies of the present disclosure are administered in long acting formulations.

It will be understood that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of the compound, the age, body weight, genetic characteristics, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular disease state, and the host undergoing therapy.

Combination therapy includes co-administration (co-administration) of a CCR4 antagonist and one or more immune checkpoint inhibitors, sequential administration of a CCR4 antagonist and one or more checkpoint inhibitors, administration of a composition comprising a CCR4 antagonist and one or more checkpoint inhibitors, or simultaneous administration of separate compositions (one composition containing a CCR4 antagonist and one or more compositions containing one or more checkpoint inhibitors). In embodiments where two immune checkpoint inhibitors are administered, it will be appreciated that each immune checkpoint inhibitor may be formulated separately or in a single dose unit.

Co-administration includes administering a CCR4 antagonist of the present disclosure within 0.5, 1,2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of administering one or more immune checkpoint inhibitors of the present disclosure. Co-administration also includes simultaneous administration, about simultaneous administration (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequential administration in any order. In addition, the CCR4 antagonist and the one or more checkpoint inhibitors may each be administered once daily, or twice, three times or more daily to provide the preferred dosage level per day.

V. kit

In certain aspects, provided herein are kits containing a CCR4 antagonist and one or more immune checkpoint inhibitors, which may be used to treat cancer. One kit may comprise a pharmaceutical composition comprising a CCR4 antagonist compound (e.g., a small molecule inhibitor of CCR4) and one or more pharmaceutical compositions comprising an immune checkpoint inhibitor (e.g., an anti-PD-1 inhibitor and/or an anti-CTLA-4 inhibitor). In certain instances, the kit includes written material, e.g., instructions for use of the compound, antibody, or pharmaceutical composition thereof. Without limitation, the kit may include buffers, diluents, filters, needles, syringes, and package inserts with instructions to perform any of the methods disclosed herein.

VI. examples

Example 1: in a mouse tumor model, C-C chemokine receptor 4(CCR4) antagonists could potentiate the effects of checkpoint inhibition (summary)

Chemokines and their receptors affect many of the hallmark processes of cancer: they act not only on infiltrating leukocytes, but also on fibroblasts, endothelial cells, and directly on certain types of tumor cells. C-C chemokine receptor 4(CCR4) and its ligands were found to be highly expressed in various types of human tumors and associated with poor prognosis. CCR4 antagonists have been shown to reduce tumor growth in various mouse tumor models. Here, we evaluated small molecule inhibition of CCR4 as a therapeutic agent to enhance the effect of checkpoint inhibitors in CT26 and KCM tumor models.

Method

The effect of the combination of CCR4 inhibitor (compound 1) and anti-CTLA-4 antibody (BioXcell: anti-murine CTLA-4 (Clone: 9H10), Syrian Hamster (Syrian Hamster) culture, purified with protein G) was evaluated using the subcutaneous CT26 colon cancer model and the orthotopic KCM pancreatic cancer model. CT26 cells were implanted into the flanks of 9-week old female Balb/c mice. Mice were randomized to study groups according to tumor size on day 6, and administration of compound 1 and anti-CTLA-4 was started on day 7. For the in situ pancreatic cancer model, KCM cells were directly implanted into the pancreas, and compound 1 and anti-CTLA-4 were also administered beginning on day 7. Compound 1 is administered orally 2 times daily at a dose of 30mg/kg, and anti-CTLA-4 is administered by intraperitoneal Injection (IP) on days 7, 11, and 15 at a dose of 100 μ g/mouse.

Results

Blockade of CCR4 significantly enhanced the therapeutic efficacy of anti-CTLA-4 in both models. The combination of an anti-CTLA-4/CCR 4 inhibitor significantly reduced tumor size and increased the proportion of long-term survivors in the CT26 model. The anti-tumor response was specific for CT 26; long-term survivors were resistant to re-inoculation with CT26 cells (no drug required to be re-administered), but 4T1 breast tumors grew well after challenge with CT26 survivors. Tumor-depleted mice exhibited a high proportion of CD 8T cells that recognized CT 26-specific neoantigen, as shown by AH1 peptide-MHC tetramer staining.

Although long-term survival of the KCM model has not been studied, compound 1 alone can significantly reduce tumor burden in 3 independent studies. anti-CTLA-4 alone provided substantial inhibition of tumor growth in this model, which was further enhanced by compound 1.

Example 2: compound 1 is a highly potent and selective CCR4 inhibitor

By determining the IC of relatively distinct sets of receptors, enzymes and ion channels₅₀Values were tested for selectivity of compound 1. Compound 1 was found to be highly specific for CCR 4. See table 1.

TABLE 1 Compound 1 Selectivity

The binding activity of compound 1 in human and mouse is reported in table 2.

TABLE 2 Cross-species Activity

Example 3: combination therapy using KCM in situ pancreatic tumor model.

As briefly introduced in example 1, a KCM pancreatic cancer model was used to assess the effectiveness of CCR4 inhibitors in combination with anti-CTLA-4 antibodies. The study design is shown in figure 1. Mice were divided into 4 groups (each group n-12): antibody isotype + vehicle; antibody isotype + compound 1; anti-CTLA-4 + vehicle; anti-CTLA-4 + Compound 1. The mice used were C57BL/6 female mice aged 7-8 weeks. anti-CTLA-4 (BioXcell: anti-murine CTLA-4 Clone (Clone): 9H10), Syrian Hamster (Syrian Hamster) culture, purified with protein G) and isotype matched control antibodies were injected intraperitoneally (i.p.) at a dose of 100 μ G/mouse on days 7, 11 and 16. Compound 1(40mg/kg) and vehicle were administered p.i. twice daily (bid). KCM cells in the basal gel (matrigel) were injected into the pancreas.

After the study was completed, tumor/pancreas weight was measured for each group. The results are shown in FIGS. A-C. As the figure shows, compound 1 alone or in combination with anti-CTLA-4 reduced tumor burden in KCM tumor models. In this model, most mice develop secondary tumors on the abdominal wall near the incision site. These secondary tumors were dissected and their weight was included in the total tumor plus pancreatic weight. Since the primary tumor and pancreas could not be surgically isolated, they were weighed together. Spleen weight was also reduced by compound 1 treatment. Adequate compound levels were confirmed by measuring plasma compound concentrations at trough on day 12.

At the completion of this study, changes in the inhibitory immune cell population were also measured. On day 25 after KCM cell inoculation, primary (along with pancreas) and secondary tumors were dissected, minced and digested with collagenase D for immune cell analysis with flow cytometry. Pancreas from sham operated mice was processed and analyzed. The results from looking at the changes in T regulatory cells, G-MDSC and M-MDSC in the processed material are shown in FIGS. 3A-F. These cell populations are shown as the number of cells per gram of tissue (groups A-C) or as the percentage of CD45+ cells (groups D-F). Although no change in T regulatory (CD4+/FoxP3+) cells was observed, monocyte and granulocyte myeloid-derived suppressor cells (mMDSC-CD11b +/Ly6G-/Ly 6C-high, gMDSC-CD11b +/Ly6G +/Ly 6C-low) showed a downward trend with a-CTLA-4/Compound 1 treatment compared to a-CTLA-4 alone treatment.

Example 4: combination therapy using the CT26 colon cancer model.

As briefly introduced in example 1, the CT26 colon cancer model was used to evaluate the effectiveness of CCR4 inhibitors in combination with anti-CTLA-4 antibodies. The study design is shown in figure 4. Mice were divided into 4 groups (each group n-12): antibody isotype + vehicle; antibody isotype + compound 1; anti-CTLA-4 + vehicle; anti-CTLA-4 + Compound 1. The mice used were C57BL/6 female mice aged 7-8 weeks. anti-CTLA-4 (BioXcell: anti-murine CTLA-4 Clone (Clone): 9H10), Syrian Hamster (Syrian Hamster) culture, purified with protein G) and isotype matched control antibodies were injected intraperitoneally (i.p.) at a dose of 100 μ G/mouse on days 7, 11 and 16. Compound 1(40mg/kg) and vehicle were administered p.i. twice daily (bid). At the start of the study, CT25 cells were injected subcutaneously (s.c.).

Tumor volume and percent survival were monitored throughout the study. Survival rates are plotted in fig. 5, and mean tumor volumes are plotted in fig. 6. As can be seen in figure 6, the combination of compound 1 with anti-CTLA-4 reduced tumor growth and improved survival. Individual tumor sizes for each treatment group are shown in fig. 7A-D. Note how in particular, the tumors of the mice in the ctla-4+ vehicle group (group C) did not respond at all to the ctla-4 treatment, while the combination of ctla + compound 1 showed that all tumors responded to the treatment (group D).

After 3 months of the last administration, mice with complete tumor regression, previously treated with a-CTLA-4(6 mice) or a-CTLA-4/compound 1(8 mice), were re-challenged with CT26 tumor cells injected subcutaneously (s.c.) in the left flank, and these mice were also injected subcutaneously (s.c.) in the right flank into 4T1 mouse breast cancer cells. The results are shown in FIGS. 8A-F. As can be seen from this figure, 1 of 6 mice previously treated with a-CTLA-4 developed CT26 tumor (group a) and 8 mice previously treated with a-CTLA-4/compound 1 had no mice and developed CT26 tumor (group B). All 6 mice previously treated with a-CTLA-4 did not develop 4T1 tumor (group D), and 2 of 8 mice previously treated with a-CTLA-4/compound 1 did not develop 4T1 tumor (group E). All 5 naive mice developed CT26 and 4T1 tumors (groups C and F).

The blood cells of the mice in fig. 8 were stained with a fluorescently labeled AH1 peptide before and 7 days after re-challenge with CT 26. AH1 is an immunodominant peptide of cytotoxic T cells in response to CT26 tumor cells, which can label CT26 antigen reactive CD8+ T cells. Flow cytometry analysis of these samples is shown in FIGS. 9A-C. These CT26 antigen-reactive CD8+ T cells were largely absent from the peripheral blood of the blank mice, but were abundant in CD8+ T cells in most of the surviving CT26 mice 3 months after the last administration (groups a and B). Re-challenge with CT26 cells further increased the number of these cells in previously treated surviving mice, but had little effect in the blank mice (group C).

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference were individually incorporated by reference. In the event of a conflict between the present application and a reference provided herein, the present application controls.

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Combination therapy using C-C chemokine receptor 4(CCR4) antagonists and one or more checkpoint inhibitors

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