Cxr7 inhibitors for cancer therapy

Abstract

The present invention provides methods of treating cancer in an individual in need thereof, comprising administering a CXCR7 inhibitor to the individual. In some embodiments, an additional therapeutic agent is used. The present invention also provides methods of preventing the development of a pre-cancerous cell that expresses FRS2β into a cancer, comprising administering a CXCR7 inhibitor to an individual having a pre-cancerous cell that expresses FRS2β. In some embodiments, an additional therapeutic agent is used.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 778,605, filed December 12, 2018, pursuant to 35 USC §119(e), the contents of which are incorporated herein by reference in their entirety for all purposes.

[0003] Declaration of rights to inventions made under federally funded research and development

[0004] not applicable

[0005] Refer to the "Sequence List," table, or computer program list appendix submitted on the CD.

[0006] not applicable Background of the Invention

[0008] Tumor tissue is composed of a variety of heterogeneous cell types; not only tumor cells, but also other cell types, including tumor-associated fibroblasts (CAFs), which are a major component of the tumor stroma. In recent years, the tumor microenvironment has received considerable attention as a novel therapeutic target, as these cells appear to support the survival and growth of tumor cells. There is increasing evidence that tumor cells themselves are heterogeneous, including a small number of cancer cells with stem cell characteristics—cancer stem cells (CSCs)—and a large number of rapidly growing, differentiated tumor cells. CSCs are thought to control the CSC niche, the microenvironment surrounding them, to maintain their own survival and growth. An environment rich in inflammatory cytokines is believed to be involved in the microenvironment of both CSCs and the tumor. Some reports indicate that nuclear factor-κB (NFκB) transcription factors play a key role in cytokine production, including insulin-like growth factor (IGF) family cytokines and CXC chemokine ligand (CXCL)12. IGF family cytokines maintain the undifferentiated state of CSCs, and CXCL12 is known to participate in the chemotaxis of CAFs, activating NFκB itself. NF-κB is known to be a major inflammatory transcription factor and a heterodimeric complex (RelA and p50 or RelB and p52) that binds to IκB in its inactive state. Ligand stimulation leads to phosphorylation of IKKα / β and IκB. The phosphorylated IκB then undergoes ubiquitination / degradation, releasing the NF-κB heterodimer, which is transported to the nucleus for transcriptional activation. However, how this occurs in the early stages of tumor development remains unclear, given that only a small number of tumor cells exist within seemingly normal tumor tissue.

[0009] Breast cancer is the most common cancer among women. In recent years, cancer prevention has received widespread attention in order to reduce the number of cancer patients. Increasing evidence suggests a link between inflammation and the development of breast cancer, but the underlying molecular mechanisms remain unclear. Despite advancements in treatment strategies, disease-related mortality remains high due to frequent recurrence. Growing evidence indicates that core cell sacs (CSCs) are a major cause of poor prognosis. They are resistant to various stress conditions and are considered a cause of tumor development, recurrence, and treatment resistance. In many cases, breast cancer tissue contains a large amount of matrix, suggesting that the tumor microenvironment containing cancer cell foci (CCF) plays a crucial role in breast cancer. Therefore, targeting the tumor microenvironment, or CSC niches, to clear CSCs holds promise as an effective strategy for treating breast cancer. Despite this goal, reliable and effective treatment methods are still needed.

[0010] Some breast cancers belong to the human epidermal growth factor receptor 2 (HER2) / ErbB2 positive subtype, in which HER2 gene amplification and / or HER2 protein overexpression can be observed in tumor cells. Herceptin is a humanized antibody against HER2 and is effective in HER2-positive cases; however, Herceptin resistance or relapse still poses serious problems. Overexpression of ErbB2 in the mammary tissue of mouse mammary tumor virus (MMTV)-ErbB2 transgenic mice leads to tumorigenesis. Breast tissue consists of numerous branching tubules, with alveoli at their terminals, both of which expand during pregnancy. The epithelial cells consist of two main cell layers: luminal cells surrounding the luminal tubules and highly elongated myoepithelial cells on the other side. Lumen progenitor cells are thought to be present in the luminal cell layer. There is evidence that luminal progenitor cells are the source of breast tumorigenesis in this model and in human breast cancer. However, effective therapies to prevent or treat the tumorigenesis of these cells remain a hot research area.

[0011] ErbB2 homodimers or heterodimers, along with other members of the ErbB family, activate extracellular signal-regulated kinase (ERK) and phosphoinositol 3-kinase (PI3K) signaling pathways, leading to many aspects of tumor biology. Depending on the cellular environment, ErbB-ERK signaling enhances cell proliferation and differentiation. ErbB-PI3K signaling activates NFκB.

[0012] One adaptor protein, FRS2β, also known as SNT-2 or FRS3, is highly expressed in the brain but only in a few regions of other tissues, while another member of the FRS2 family, FRS2α, is highly expressed in most tissues. More information on FRS2β expression is discussed in Gotoh et al., FEBS Lett. 2004, 564(1-2):14-8. FRS2β, rather than FRS2α, structurally binds to members of the ErbB family, including ErbB2, activating ERK to generate feedback inhibition and fine-tuning ErbB-ERK signaling. FRS2β can also induce ubiquitination and degradation of ErbB1 / 2. However, particularly in tumor development, the role of FRS2β in vivo remains unclear.

[0013] In summary, there remains a need in the art to identify methods that lead to the development of CSC niche environments and drugs that can target appropriate agents to regulate, reduce, or prevent tumor development. This invention addresses this need and also provides related advantages. Summary of the Invention

[0014] In one aspect of the invention, a method for treating cancer in an individual in need is provided, the method comprising administering a CXCR7 inhibitor to the individual, wherein the individual has abnormal FRS2β expression.

[0015] In another aspect of the invention, a method for preventing precancerous cells expressing FRS2β from developing into tumors is provided, the method comprising administering a CXCR7 inhibitor to an individual having precancerous cells expressing FRS2β.

[0016] In some embodiments, CXCR7 inhibitors have the structure of formula I and / or II:

[0017]

[0018] The definitions of each variable group will be explained in more detail below.

[0019] In some embodiments, the CXCR7 inhibitor has the structure of compound 1.

[0020]

[0021] Or its pharmaceutically acceptable salt.

[0022] In some embodiments, the CXCR7 inhibitor has the structure of compound 2.

[0023]

[0024] Or its pharmaceutically acceptable salt.

[0025] In some implementations, the CXCR7 inhibitor has the structure of compound 3.

[0026]

[0027] Or its pharmaceutically acceptable salt.

[0028] In some implementations, the CXCR7 inhibitor has the structure of compound 4.

[0029]

[0030] Or its pharmaceutically acceptable salt.

[0031] In some implementations, the CXCR7 inhibitor has the structure of compound 5.

[0032]

[0033] Or its pharmaceutically acceptable salt.

[0034] In some implementations, the CXCR7 inhibitor has the structure of compound 6.

[0035]

[0036] Or its pharmaceutically acceptable salt.

[0037] In some implementations, the CXCR7 inhibitor has the structure of compound 7.

[0038]

[0039] Or its pharmaceutically acceptable salt.

[0040] In some implementations, the CXCR7 inhibitor has the structure of compound 8.

[0041]

[0042] Or its pharmaceutically acceptable salt.

[0043] In some implementations, the CXCR7 inhibitor has the structure of compound 9.

[0044]

[0045] Or its pharmaceutically acceptable salt.

[0046] In some embodiments, the method provided by the present invention uses one or more therapeutic agents. In some embodiments, the one or more therapeutic agents are IGF1 inhibitors and / or CXCR4 inhibitors. In some embodiments, the IGF1 inhibitor is an anti-IGF1 antibody.

[0047] Other objects, features, and advantages of the present invention will be apparent to those skilled in the art from the following detailed description and accompanying drawings.

[0048] Brief description of the attached figures

[0049] Figure 1A -H: Loss of FRS2β expression in luminal cells significantly delays the development of breast tumors. (A) Representative image of β-galactosidase staining in the mammary gland of a heterozygous mature female with the Frs2β mutant allele. Red arrows indicate FRS2β-positive cells. (B) Schematic diagram of the mammary gland. Numerous branching ducts are surrounded by an inner luminal epithelial cell layer and an outer myoepithelial cell layer. (C) Immunohistochemical staining of female mammary glands with anti-FRS2β antibody and phosphohistone H3 antibody (top) or DAPI (bottom). (D) Immunohistochemical staining of female mammary glands with anti-FRS2β antibody and cytokeratin 18 (top) or cytokeratin 14 (bottom). (E) Representative NMR imaging of the mammary tumor on the front of the mouse, 14 weeks after the start of observation. Left side: head; right side: abdomen. (F) Tumor growth in MMTV-neu(+) / Frs2β(+ / +) and MMTV-neu(+) / Frs2β(- / -) mice. Tumor size was measured weekly for 14 weeks (mean ± SEM, n = 15). FRS2β expression levels in virgin, pregnant, and lactating mice were compared by qRT-PCR (mean ± SEM, n = 4, **P < 0.005, *P < 0.01). (G) Representative images of hematoxylin and eosin stained sections of breast tumors. (H) Immunohistochemical staining of Frs2β(+ / +) and Frs2β(- / -) breast tumors with anti-αSMA antibody. Scale bar: 100 μm.

[0050] Figure 2A-F: FRS2β expression in coronary progenitor cells supports tumorigenesis in xenografted tumor cells. (A) As shown in the representative image, Frs2β(+ / +) tumor spherocytes were cultured for 14 days and then inoculated into the mammary fat pads of 8-week-old Frs2β(+ / +) or Frs2β(- / -) virgin female mice. (B) Representative tumors were photographed 30 days after transplantation, and (C) the volume of the excised tumors was measured. (D) Tumorigenesis caused by Frs2β(+ / +) tumor spherocytes was observed in Frs2β(+ / +) mice, but not in Frs2β(- / -) mice (n=4). Numerical values ​​represent the ratio of the number of tumors to the number of inoculation sites. (E) Immunohistochemical staining of the mammary glands of MMTV-neu(-) or MMTV-neu(+) female mice with anti-FRS2β and anti-ErbB2 antibodies. Arrows indicate FRS2β-positive coronary cells. (F) Classification of mammary epithelial cells using markers. Further sorting of P1(CD49f) cells using CD61. 低 / CD24 高 ) cavitary cell subsets. Further sorting of P2 (CD49f) cells using FRS2β. 低 / CD24 高 / CD61 + ) cavity progenitor cell subsets to obtain P3 (CD49f 低 / CD24 高 / CD61 + / FRS2β + (subgroup)

[0051] Figure 3A-F: FRS2β-deficient luminal progenitor cells produce a small amount of cytokines. (A) Representative images of breast cancer globules from Frs2β(+ / +) and Frs2β(- / -) breast epithelial cells cultured in SCM. (B) Quantification of breast globule formation efficiency. NT refers to cells not treated with the cytokine combination in SCM. All results are mean ± SEM, n = 4. **P < 0.01, *P < 0.05. (C) Comparison of gene expression profiles between Frs2β(+ / +) and Frs2β(- / -) breast globules using Gene Set Enrichment Analysis (GSEA). Showing two sets of genes highly upregulated in Frs2β(+ / +) breast globules. (D) Comparison of gene expression profiles between Frs2β(+ / +) and Frs2β(- / -) breast cancer preepithelial cells using Gene Set Enrichment Analysis (GSEA). The expression of gene sets in Frs2β(+ / +) or Frs2β(- / -) cells was highly upregulated. ES, enrichment value; NES, normalized enrichment value; FDR, false discovery rate. (E) Expression levels of indicator gene transcripts in Frs2β(+ / +) and Frs2β(- / -) mammary glomeruli were compared using real-time quantitative PCR (qPCR). Results are shown as mean ± SEM, n = 4. **P < 0.01. (F) Immunohistochemical staining was performed on FRS2β(+ / +) and Frs2β(- / -) breast tumors using antibodies against αSMA, CXCL12, and IGF1.

[0052] Figure 4A-I: CXCL12-induced migration of tumor spheres and CAFs by precancerous Frs2β(+ / +) breast cells. (A) Schematic diagram of co-culture of sphere-forming Frs2β(+ / +) tumor cells in the lower layer with Frs2β(+ / +) breast preepithelial cells in the upper layer. (B) Representative images of tumor sphere formation under these conditions, treated with IgG (400 nM) and IGF1 neutralizing antibody (Nab) as controls. NT, untreated (not co-cultured with breast epithelial cells). Scale bar: 100∝μ. (C) Quantification of tumor sphere formation efficiency. Results are shown as mean ± SEM. n = 4. ***P < 0.001, **P < 0.01. (D) Schematic diagram of co-culture of Frs2β(+ / +) or Frs2β(- / -) mammary cells in the lower layer with Frs2β(+ / +) or Frs2β(- / -) CAFs in the upper layer. (E) Comparison of Cxcl12 expression levels in Frs2β(+ / +) and Frs2β(- / -) mammary cells in the upper layer using qPCR. Results are shown as mean ± SEM, n = 6. ***P < 0.001. (F) Representative images of migrating Frs2β(+ / +) CAFs after 24 h of co-culture with Frs2β(+ / +) and Frs2β(- / -) mammary cells in the upper layer. (G) Quantification of migrating Frs2β(+ / +) CAFs. Results are shown as mean ± SEM, n = 4. **P < 0.01. (H) Representative images of migrating CAFs co-cultured with Frs2β(+ / +) cancer cells for 24 h. Cells were treated with specified concentrations of compound 1 and / or +0.1 mg / ml AMD3100 or a control. (I) Quantification of migrating CAFs co-cultured with Frs2β(+ / +) cancer cells for 24 h. Results are shown as mean ± SEM, n = 4. ***P < 0.001 and **P < 0.01.

[0053] Figure 5A-K: Increased FRS2β-dependent AKT-NFκB activation enhances the secretion of IGF1 and CXCL12, promoting tumorigenesis. (A) Schematic diagram of Frs2β(+ / +) breast cancer preepithelial cells cultured in vitro treated with DHMEQ. (B) Comparison of Igf1, Cxcl12, and IκBα expression levels in Frs2β(+ / +) breast cancer preepithelial cells treated with the indicator concentration of DHMEQ by qPCR. Results are shown as mean ± SEM. n = 4. **P < 0.01. (C) Immunoblotting analysis of indicator proteins in lysates of Frs2β(+ / +) or Frs2β(- / -) breast cancer preepithelial tissues. Actin was used as a loading control. (D) Immunoblotting analysis of cytoplasmic and nuclear expression levels of indicator proteins (as nuclear protein controls) in lysates of Frs2β(+ / +) or Frs2β(- / -) breast tissues. PARP1 was used as a representative protein in the nucleus. (E) Immunoblot analysis of indicator proteins in lysates of Frs2β(+ / +) or Frs2β(- / -) mammary tissue. Actin was used as a loading control. (F) Schematic diagram of Frs2β(+ / +) mice treated with DHMEQ. Mice were intraperitoneally injected with 10 μg / g DHMEQ once daily for 3 weeks. (G) Immunohistochemical staining of Frs2β(+ / +) mammary tissue treated with and without 10 μg / g DHMEQ for three weeks, or Frs2β(- / -) mammary tissue treated with anti-RelA antibody. Scale bar: 50 μm. (H) Expression levels of Igf1 and Cxcl12 in Frs2β(+ / +) mammary tissue after three weeks of treatment with and without 10 μg / g DHMEQ. NT, untreated. Results are shown as mean ± SEM. n = 4. ***P < 0.001 and **P < 0.01. (I) Treatment of mice with CXCR7 inhibitors and / or IGF1 antibodies to reduce tumor volume. Frs2β(+ / +) tumor spherocytes were inoculated into the mammary fat pads of 8-week-old female virgin MMTV-neu(+) / Frs2β(+ / +) mice. Seven days later, mice were intraperitoneally injected weekly with 0.1 μg / g IGF1 antibody (R&D Biosystems) and / or intraperitoneally injected daily with 1 μg / g AMD3100 (Sigma) and 1.5 μg / g compound 1. Representative tumors were photographed on day 35 after transplantation of the combination of CXCL12 Inh, AMD3100 and CCX771. Tumor volume (J) and weight (K) of mice treated according to (I) were measured. Results mean ± SEM, n K = 4, *P < 0.05.

[0054] Figure 6. FRS2β-expressing tumor cells in AG cells produce IGF1 and CXCL12, which are associated with abundant stromal mass and poor prognosis. (A) Immunohistochemical staining of Frs2β(+ / +) breast tumors with anti-FRS2b and anti-ErbB2 antibodies. Scale bar, 25∝μ. (B) Comparison of Cxcl12 and Igf1 expression levels in Frs2β(+ / +) and Frs2β(- / -) tumor cells by qPCR. Results are shown as mean ± SEM. n = 4. ***P < 0.001. (C) Immunohistochemical staining with anti-IGF1 and anti-CXCL12 antibodies. Scale bar: 200∝μ. (D) Immunohistochemical staining of tissue arrays with anti-FRS2b antibody or detection of collagen in the stromal using Massen's trichrome staining. Arrows indicate stromal areas. Scale bar: 50∝μ. (E) Tumor specimens were divided into three groups according to the ratio of tumor stromal area to total tumor area (+: 0-10%, ++; 10-20%, +++; >20%). The median FRS2b staining level was used as the cutoff value. n = 30. (F) Kaplan-Meier survival curves were generated using Uppsala cohort (GSE3494). The median value was used as the cutoff value. The p-value was obtained using the log-rank test. (G) FRS2b can trigger the production of cytokines in the luminal cell subset, leading to the creation of a cytokine-rich precancerous microenvironment (top left). Once CSCs are present in the precancerous microenvironment, they can self-renew in the presence of IGF1 and generate tumor cells with the help of CXCL12-mobilized stromal cells, which subsequently become CAFs. CSCs and tumor cells may spontaneously generate IGF1 and CXCL12, leading to rapid growth and tumorigenesis (bottom left). In the absence of FRS2β, cytokines remain at low levels and a suitable precancerous microenvironment is not generated (top right figure); even when CSCs are present, they cannot grow effectively (bottom right figure). Detailed Implementation

[0055] I.General

[0056] This invention demonstrates that aberrant expression of FRS2β maintains suitable microenvironmental conditions for tumor growth and plays a crucial role in creating cytokine-rich CSC niches. Surprisingly, the detrimental effects of this expression can be effectively modulated by administration of CXCR7 inhibitors or in combination with other therapeutic agents.

[0057] II. Definition

[0058] Unless otherwise stated, the term "alkyl", either on its own or as part of another substituent, refers to an alkyl group having a specified number of carbon atoms (i.e., C40, C50, C60, C70, C6 ... 1-8Alkyl groups are straight-chain or branched hydrocarbon groups (1 to 8 carbon atoms). Examples of alkyl groups include: methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. The term "alkenyl" refers to an unsaturated hydrocarbon group having one or more double bonds. Similarly, the term "alkynyl" refers to an unsaturated hydrocarbon group having one or more triple bonds. Examples of such unsaturated alkyl groups include vinyl, 2-propenyl, crotonyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologues and isomers. The term "cycloalkyl" refers to a cycloalkyl group having a specified number of ring atoms (e.g., C1, C2, C3, C4, C5, C6, C6, C7 ... 3-6 A cycloalkyl group is a hydrocarbon ring that is fully saturated or has no more than one double bond between its ring vertices. "Cycloalkyl" can also refer to bicyclic and polycyclic hydrocarbons, such as bicyclic [2.2.1]heptane, bicyclic [2.2.2]octane, etc. The term "cycloalkenyl" refers to a cycloalkyl group having at least one double bond between its ring vertices. Examples of cycloalkenyl groups are cyclopentenyl and cyclohexenyl. The term "spirocycloalkyl" refers to a cycloalkyl group in which one monocyclic vertices are attached to two other non-hydrogen portions of the molecule. A spirocycloalkyl substituent is a substituent in which two carbon atoms of an alkylene chain (usually the end of the alkylene chain) are attached to the same carbon atom of the remaining portion of the molecule. The term "heterocyclic alkyl" 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 atom is optionally quaternized. The heterocyclic alkyl group can be a monocyclic, bicyclic, or polycyclic system. Non-limiting examples of heterocyclic alkyl groups include: pyrrolidine, imidazoline, pyrazolidine, butyrolactam, valproic acid, imidazolinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrrolidine, thiaran, pyranone, tetrahydrofuran, tetrahydrothiophene, quinine ring, and similar groups. Heterocyclic alkyl groups can be linked to other parts of the molecule via a cyclic carbon or heteroatom.

[0059] The term "alkylene" itself, or as part of another substituent, refers to a divalent group derived from an alkane, such as -CH2CH2CH2CH2-. Typically, alkyl (or alkylene) groups have 1 to 24 carbon atoms, with those having 10 or fewer carbon atoms being preferred in this invention. "Lower alkyl" or "lower alkylene" refers to a short-chain alkyl or alkylene group typically having four or fewer carbon atoms. Similarly, "alkenyl" and "alkynyl" refer to the unsaturated form of an alkylene group having a double or triple bond, respectively.

[0060] As used herein, wavy lines intersecting with single, double, or triple bonds in any chemical structure described herein. This indicates the connection point between a single, double, or triple bond and the rest of the molecule.

[0061] The terms "alkoxy," "alkamino," and "alkathio" (or thioalkoxy) are used in their conventional sense to refer to alkyl groups that are attached to other parts of a molecule via an oxygen, amino, or sulfur atom, respectively. Additionally, for dialkylamino groups, the alkyl moiety can be the same or different and can be attached to 3-7 membered rings via the nitrogen atom to which it is attached. Therefore, groups represented by NRaRb include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl, and similar groups.

[0062] Unless otherwise stated, the terms "halogenated" or "halogen," either on their own or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "halogenated alkyl" are intended to include both monohalogenated and polyhalogenated alkyl groups. For example, the term "C"... 1-4 "Halogenated alkyl" refers to compounds including trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, and 3-bromopropyl.

[0063] Unless otherwise stated, the term "aryl" refers to a polyunsaturated, typically aromatic, hydrocarbon group, which can be monocyclic or fused together or covalently linked polycyclic (up to three rings). The term "heteroaryl" refers to an aryl group (or ring) containing one to five heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom is optionally quaternized. Heteroaryl groups can be linked to other parts of the molecule via heteroatoms. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl; and non-limiting examples of heteroaryl groups include pyridyl, pyrazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purine, benzimidazolyl, benzopyrazolyl, and benzotriazolyl. (lyl), benzoisoxazole, isobenzofuran, isoindolyl, indolazidyl, benzotriazinyl, thienopyridyl, thienopyrimidyl, pyrazolopyrimidyl, imidazoline, benzothiophene, benzothiophene, indolyl, quinolinyl, isoquinolinyl, isothiazolyl, pyrazolyl, indolayl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrroleyl, thiazolyl, furfuryl, thiophene, etc. The substituents used in each of the above-described aryl and heteroaryl ring systems are selected from the acceptable substituents described below.

[0064] The term "aralkyl" refers to those groups in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, etc.). Similarly, the term "heteroarylalkyl" refers to those groups in which a heteroaryl group is attached to an alkyl group (e.g., pyridylmethyl, thiazolylethyl, etc.).

[0065] In some embodiments, the terms above (e.g., alkyl, aryl, and heteroaryl) will include substituted and unsubstituted forms of the indicated groups. Preferred substituents for each group are provided below.

[0066] Alkyl groups (including those commonly referred to as alkylene, alkenyl, ynyl, and cycloalkyl) can be zero to (2m'+1) groups selected from the group consisting of: -halogen, -OR', -NR'R”, -SR', -SiR'R”R”', -C(O)R', -C(O)R', -CO2R', -CONR'R”, -OC(O)NR'R”, -NR”C(O)R', -NR'-C(O) The following are examples of carbon groups: -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 -NO2, where m’ is the total number of carbon atoms in the group. R’, R”, and R”’ each independently refer to hydrogen, unsubstituted carbon, and unsubstituted carbon. 1-8 Alkyl, 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 groups. When R' and R” are attached to the same nitrogen atom, they can combine with the nitrogen atom to form 3, 4, 5, 6, or 7-membered rings. For example, -NR'R” means that it includes 1-pyrrolidinyl and 4-morpholinyl.

[0067] Similarly, aryl and heteroaryl substituents are variable and generally 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”, -N3, perfluoro(C1-C4)alkoxy and perfluoro(C1-C4)alkyl, 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 1-8 Alkyl, C 1-8 Halogenated alkanes, C 3-6 cycloalkyl, C 2-8 alkenyl, C 2-8Alkynyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-C 1-4 Alkyl groups and unsubstituted aryl groups -C 1-4 Alkyl groups. Other suitable substituents include the aforementioned aryl substituents connected to the ring atom via an alkylene group of 1-4 carbon atoms.

[0068] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CH2). q The -U- substituent replaces T and U, where T and U are independently -NH-, -O-, -CH2-, or a single bond, and q is an integer from 0 to 2. Alternatively, the two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by the formula -A-(CH2). r The substituents of -B- are substituted, wherein A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR' or single bonds, and r is an integer from 1 to 3. One of the single bonds in the newly formed ring may optionally be substituted with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be substituted with the formula -(CH2). s -X-(CH2) t The substituent is replaced by a substituent, where s and t are independent integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituent R' in NR'- and -S(O)2NR'- is selected from hydrogen or unsubstituted C. 1-6 alkyl.

[0069] As used in this application, the term "heteroatoms" is intended to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

[0070] As used herein, the terms “progenitor cell” and “stem cell” are used interchangeably. “Progenitor cell” and “stem cell” refer to cells that respond to certain stimuli and are capable of forming differentiated cell lines, including but not limited to hematopoietic cells, mesenchymal cells, epithelial cells, neurons, kidney cells, or myeloid cells. The presence of progenitor cells / stem cells can be assessed by the ability of cells in a sample to form various types of colony-forming units, including, for example, CFU-GM (colony-forming units, granulocyte-macrophage); CFU-GEMM (colony-forming units, pluripotent); BFU-E (burst colony-forming units, bone marrow erythroid); HPP-CFC (highly proliferative colony-forming cells); or other types of differentiated colonies that can be obtained in culture using known methods. Hematopoietic progenitor cells / stem cells are often CD34 positive. However, some stem cells do not contain this marker. These CD34+ cells can be identified using fluorescence activated cell sorting (FACS), and thus their presence can be assessed in a sample using this technique. Alternatively, these cells can be assessed using FACS to determine the presence of the c-kit receptor (CD117) and the absence of lineage-specific markers (e.g., CD2, CD3, CD4, CD5, CD8, NK1.1, B220, TER-119, and Gr-1 in mice, and CD3, CD14, CD16, CD19, CD20, and CD56 in humans).

[0071] The term "pharmaceutically acceptable salt" refers to salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific 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 form of these compounds with a sufficient amount of the desired base in a solvent-free or suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, iron, ferrous, lithium, magnesium, trivalent manganese, divalent manganese, 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, and naturally occurring amines, such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucosamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucosamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and similar groups. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of these compounds with a sufficient amount of the desired acid, which may be 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, monohydrophosphoric acid, dihydrophosphoric acid, sulfuric acid, monohydrosulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. Salts of amino acids, such as arginine, and salts of organic acids, such as glucuronic acid or galacturonic acid, are also included (see, for example, Berge, SM, et al., “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specific compounds of the present invention contain both basic and acidic functional groups, such that the compounds can be converted into basic or acid addition salts.

[0072] The neutral form of the compound can be regenerated by contacting the salt with a base or acid and separating the parent compound in a conventional manner. The parent form of the compound differs from its various salt forms in some physical properties, such as solubility in polar solvents, but in other respects the salts are equivalent to the parent form of the compound for the purposes of this invention.

[0073] In addition to the salt form, the present invention provides compounds existing in the form of prodrugs. The prodrugs of the compounds described in this application are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Furthermore, prodrugs can be converted into the compounds of the present invention in an in vitro environment by chemical or biochemical methods. For example, when a prodrug is placed in a transdermal patch reservoir containing suitable enzymes or chemical reagents, the prodrug is slowly converted into the compounds of the present invention.

[0074] Some compounds of the present invention may exist in both solvated and hydrated forms, including hydrated forms. Generally, the hydrated form is equivalent to the hydrated form and is intended to be included within the scope of the present invention. Some compounds of the present invention may exist in polycrystalline or amorphous forms. Generally, all physical forms are equivalent to the intended use of the present invention and are intended to fall within the scope of the present invention.

[0075] Some compounds of the present invention have asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, regioisomers, and monomeric isomers (e.g., single enantiomers) are all intended to be included within the scope of the present invention. In some embodiments, the compounds of the present invention are present in an enantiomer-enriched form, wherein the enantiomer excess of a particular enantiomer is calculated by known methods. The preparation of enantiomer-enriched forms is well known in the art and can be accomplished, for example, by chiral resolution by chromatography or by the formation of chiral salts. Furthermore, the present invention contemplates different conformations and different rotor isomers. A conformation is a conformation that can be distinguished by one or more rotations of σ bonds. A rotor isomer is a conformation that can be distinguished by having only one σ bond. Furthermore, the compounds of the present invention may also contain non-natural proportions of atomic isotopes on one or more atoms constituting these compounds. Thus, in some embodiments, the compounds of the present invention are present in an isotope-enriched form. A non-natural proportion of isotopes can be defined as an amount found in nature up to 100% composed of the atoms in question. For example, the compounds may be incorporated with radioactive isotopes (e.g., tritium (3H), iodine-125 (125I), or carbon-14 (14C)) or non-radioactive isotopes (e.g., deuterium (2H) or carbon-13 (13C)). Such isotopic variations can provide additional utility for those described elsewhere in this application. For example, isotopic variants of the compounds of the present invention may be found to have other uses, including but not limited to, as diagnostic and / or imaging agents, or as cytotoxic / radiotoxic therapeutic agents. Additionally, isotopic variants of the compounds of the present invention may have altered pharmacokinetic and pharmacodynamic characteristics, which may contribute to improved safety, tolerability, or efficacy during treatment. All isotopic variants of the compounds of the present invention, whether radioactive or not, are intended to be included within the scope of the present invention.

[0076] "CXCR7", also known as "RDC1" or "CCXCKR2", refers to a G-protein-coupled receptor (GPCR) with seven transmembrane domains. The canine ortholog of CXCR7 was first identified in 1991. See Libert et al., Science. 244:569-572 (1989). Descriptions of canine sequences include, for example, Libert et al., Nucleic Acids Research. 18(7):1917 (1990). Descriptions of mouse sequences include, for example, Heesen et al., Immunogenetics. 47:364-370 (1998). Descriptions of human sequences include, for example, Sreedharan et al., Proceedings of the National Academy of Sciences of the United States of America. 88:4986-4990 (1991), in which the protein is incorrectly described as a receptor for a vasoactive intestinal peptide.

[0077] The term “therapeutic effective dose” refers to the dose of a subject compound that researchers, veterinarians, physicians, or other treatment providers seek to elicit a biological or medical response in cells, tissues, systems, or animals (such as humans).

[0078] As used herein, the term "composition" is intended to cover products containing specific amounts of specific ingredients, and any product produced directly or indirectly from a combination of specific amounts of specific ingredients. "Pharmaceutically acceptable" means that the carrier, diluent, or excipient must be compatible with the other components of the formulation and harmless to its recipient.

[0079] III. Detailed Implementation

[0080] A: Method

[0081] In one aspect of the invention, a method for treating cancer in an individual in need is provided, the method comprising administering a CXCR7 inhibitor to the individual, wherein the individual has abnormal FRS2β expression.

[0082] In another aspect of the invention, a method for preventing precancerous cells expressing FRS2β from developing into cancer is provided, the method comprising administering a CXCR7 inhibitor to an individual having precancerous cells expressing FRS2β.

[0083] As described in the background section, FRS2β is highly expressed in the brain, but only in a few regions of other tissues. Therefore, many tissues do not naturally express FRS2β. As shown in this article, aberrant expression of FRS2β in cells where this protein should not be expressed can provide a CSC niche, leading to tumorigenesis.

[0084] It should be understood that aberrant expression refers to the expression in the patient's cells, tissues, organs, or body fluids of a protein that is not normally produced in a healthy individual (inappropriate expression), or the expression level of a protein in the subject's cells, tissues, organs, or body fluids being higher than the level detected in the same type of cells, tissues, organs, or body fluids in a healthy individual (differential expression). In some embodiments, the aberrant expression of FRS2β is at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, or higher than the FRS2β expression in healthy individuals. Those skilled in the art will understand that FRS2β expression can be determined using methods known in the art. In some embodiments, FRS2β expression can be detected using the methods described in this invention. In some embodiments, FRS2β expression can be detected using immunohistochemistry. In various embodiments, aberrant expression is detected in ELISA analysis.

[0085] Many CXCR7 inhibitors are known in the art, and further details regarding potentially useful CXCR7 inhibitors are discussed in the following sections.

[0086] A preferred method for treating cancer includes administering to a cancer patient one or more of the above-mentioned compounds (or salts thereof) in a therapeutically effective amount for a period of time to treat the cancer.

[0087] Besides primates, such as humans, a variety of other mammals can be treated using the methods described in this invention. For example, mammals including, but not limited to, cattle, sheep, goats, horses, dogs, cats, guinea pigs, rats, or other cattle, sheep, horses, dogs, cats, rodents, or rats can be treated. However, the methods can also be used on other species, such as bird species (e.g., chickens).

[0088] In some implementations, CXCR7 inhibitors are used to treat cancers such as carcinoma, glioma, mesothelioma, melanoma, lymphoma, leukemia (including acute lymphoblastic leukemia), adenocarcinoma, breast cancer, ovarian cancer, cervical cancer, glioblastoma, leukemia, lymphoma, prostate cancer and Burkitt lymphoma, colon cancer, colorectal cancer, esophageal cancer, gastric cancer, pancreatic cancer, hepatobiliary cancer, gallbladder cancer, small bowel cancer, rectal cancer, kidney cancer, bladder cancer, prostate cancer, penile cancer, urethral cancer, testicular cancer, cervical cancer, vaginal cancer, uterine cancer, ovarian cancer, thyroid cancer, parathyroid cancer, adrenal cancer, pancreatic endocrine carcinoma, carcinoid, bone cancer, skin cancer, retinoblastoma, Hodgkin lymphoma, and non-Hodgkin lymphoma (for other cancers, see Cancer: Principles and Practice (DeVita, VT et al., 1997 ed.)).

[0089] In some implementations, the cancer described in this article is breast cancer.

[0090] In some implementations, individuals have been diagnosed with abnormal FRS2β expression prior to the administration of CXCR7 inhibitors or additional treatments.

[0091] B: CXCR7 inhibitors

[0092] In some implementations, the CXCR7 inhibitor has the structure shown in Formula I.

[0093]

[0094] Or its pharmaceutically acceptable salts, hydrates, N-oxides, isotope concentrates, or enantiomers thereof, wherein

[0095] n is an integer from 0 to 2;

[0096] When R 1 When it exists, each R 1 Select independently from the following group: C 1-4 Alkyl, -CO2R a -X-CO2R a -CONR a R b and XCONR a R b ;

[0097] R 2 and R 3 Each is independently selected from the following groups: H, -R a -XR a -XNR a R b -XNHCONR a R b -XNHCOR a -XO-CONR a R b -XNHSO2R a -CO2R a -X-CO2R a -CONR a R b -X-CONR a R b , or together it is oxo;

[0098] C 1 Selected from the group consisting of monocyclic or fused-bicyclic aryl and heteroaryl groups, wherein the heteroaryl group has 1-3 heteroatoms selected from N, O or S as ring members; and wherein the aryl and heteroaryl groups are optionally surrounded by 1-3 R atoms. 4Substituent substitution;

[0099] C 2 It is a quaternary, pentagonal, hexanal, or heptagonal monocyclic ring selected from the group consisting of benzene, heterocyclic aromatics, cycloalkanes, and heterocyclic alkanes, wherein the heterocyclic aromatics and heterocyclic alkanes have 1-3 heteroatoms selected from N, O, or S as ring members; wherein each of the said monocyclic rings has C 2 The ring can be randomly divided into 1-3 Rs 5 Substituent substitution;

[0100] C 3 Selected from the following group: hydrogen, C 1-8 Alkyl, C 3-8 cycloalkyl, aryl, aryl-C 1-4 Alkyl, heteroaryl, heteroaryl-C 1-4 Alkyl groups and 4-6 membered heterocyclic alkyl groups, wherein the heterocyclic alkyl group or part thereof has 1-3 heteroatoms selected from N, O or S, and wherein the heteroaryl group has 1-3 heteroatoms selected from N, O or S as ring members, and each C 3 Optional land is covered by 1-3 R 6 Substituent substitution;

[0101] Each R 4 Each is independently selected from the following groups: halogen, -CN, -NO2, -R c -CO2R a -NR a R b -OR a -X-CO2R a -CONRaR b and -X-CONR a R b ;

[0102] Among them, in each R 1 R 2 and R 3 and R 4 In, each R a and R b Each is independently selected from the following groups: hydrogen, C 1-8 Alkyl, C 3-7 cycloalkyl, C 1-8 Halogenated alkyl groups and 4-6 membered heterocyclic alkyl groups, or when attached to the same nitrogen atom, can combine with a nitrogen atom to form a four-, five-, or six-membered ring having 0-2 additional heteroatoms selected from N, O, or S as ring members; wherein, R 4 and R c Each of them independently selected from the following group: C 1-8 Alkyl, C 1-8 Haloalkyl, C 3-6Cycloalkyl, aryl, and heteroaryl, wherein R a R b and R c The aliphatic and cyclic moieties are optionally further substituted with 1-3 halogens, hydroxyl groups, methyl groups, alkoxy groups, amino groups, alkylamino groups, dialkylamino groups, formamide groups, carboxylalkyl esters, carboxylic acids, heteroaryl groups, and 4-6 membered heterocyclic alkyl groups; and wherein R 2 R 3 and R 4 The heterocyclic alkyl portion is optionally substituted with an oxo group; and optionally, when both R groups are substituted with an oxo group... 4 When the substituents are located on adjacent atoms, they combine to form fused five- or six-membered rings with carbon and oxygen atoms as ring members;

[0103] Each R 5 Each is independently selected from the following groups: halogen, -CN, -NO2, -R f -CO2R d -COR d -NR d R e -OR d -X-CO2R d -CONR d R e and -X-CONR d R e ; where each R d and R e Each is independently selected from the following groups: hydrogen, C 1-8 Alkyl, C 1-8 Halogenated alkanes, C 3-6 cycloalkyl, C 3-6 Cycloalkyl and four- to six-membered heterocyclic alkyl groups, or when attached to the same nitrogen atom, can combine with a nitrogen atom to form a ring member having 0-2 additional heteroatoms selected from N, O, or S; each R f Each of them independently selected from the following group: C 1-8 Alkyl, C 1-8 Halogenated alkyl groups and C 3-6 cycloalkyl, wherein R d R e and R f The aliphatic and cyclic moieties are optionally further substituted with 1-3 halogens, hydroxyl groups, methyl groups, alkoxy groups, amino groups, alkylamino groups, dialkylamino groups, formamide groups, carboxylalkyl esters, carboxylic acids, heteroaryl groups, or 4-6 membered heterocyclic alkyl groups.

[0104] Each R 6 Each is independently selected from the following groups: halogen, -CN, -NO2, -R i -CO2R g -CORg -NR g R h OR g -X-CO2R g -X-COR g -CONR g R h and -X-CONR g R h , where each R g and R h Each is independently selected from the following groups: hydrogen, C 1-8 Alkyl and C 1-8 Halogenated alkyl groups; each R i Select independently from the following group: C 1-8 Alkyl and C 1-8 Halogenated alkyl groups; and

[0105] Each X is a linking group having the following formula: -(CH2) m O(CH2) P , where the subscripts m and p are integers from 0 to 5, and m+p is between 0 and 6, wherein the methylene group is optionally replaced by 1 to 2 methyl groups.

[0106] In some embodiments, the CXCR7 inhibitor has the structure of compound 1.

[0107]

[0108] Or its pharmaceutically acceptable salt.

[0109] In some embodiments, the CXCR7 inhibitor has the structure of compound 2.

[0110]

[0111] Or its pharmaceutically acceptable salt.

[0112] In some embodiments, the CXCR7 inhibitor has the structure of compound 3.

[0113]

[0114] Or its pharmaceutically acceptable salt.

[0115] In some embodiments, the CXCR7 inhibitor has the structure of compound 4.

[0116]

[0117] Or its pharmaceutically acceptable salt.

[0118] In some embodiments, the CXCR7 inhibitor has the structure of compound 5.

[0119]

[0120] Or its pharmaceutically acceptable salt.

[0121] In some embodiments, the CXCR7 inhibitor has the structure of compound 6.

[0122]

[0123] Or its pharmaceutically acceptable salt.

[0124] In some embodiments, the CXCR7 inhibitor is selected from compounds or pharmaceutical compositions disclosed in PCT Publication WO2010 / 054006, which originates from PCT application US2009 / 063298 filed by ChemoCentryx on November 4, 2009, and is incorporated herein by reference for all purposes.

[0125] In some embodiments, the CXCR7 inhibitor has the structure shown in Formula II.

[0126]

[0127] Or its pharmaceutically acceptable salts, hydrates, N-oxides, isotope concentrates or their enantiomers or their rotational isomers, wherein

[0128] Each ring vertex X a X b and X c Each is independently selected from the following groups: N, NH, N(R) 2 ), O, CH and C(R) 2 );

[0129] The subscript n is selected from the following groups: 0, 1, and 2;

[0130] Z is selected from the following group:

[0131] (i) Monocyclic or fused-bicyclic aryl and heteroaryl groups, wherein the heteroaryl group has 1-4 heteroatoms selected from N, O or S as ring members; and wherein the aryl and heteroaryl groups are optionally surrounded by 1-5 R atoms. 5 Substituent substitution;

[0132] (ii) A monocyclic ring selected from the group consisting of tetracyclic, pentacyclic, hexacyclic, or heptagonal members: cycloalkanes and heterocyclic alkanes, wherein the heterocyclic alkanes have 1-3 heteroatoms selected from N, O, or S as ring members; wherein each of the monocyclic Z-rings is optionally surrounded by 1-3 R-rings. 5 Substituent substitution;

[0133] R 1 Selected from the following groups: H and C 1-8 Alkyl group, wherein the alkyl portion is optionally coated with halogen or -NR. a R b -OR a -CO2R a or -CONR a R b replace;

[0134] Each R 2 Each is independently selected from the following groups: H, halogen, CN, C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8 Hydroxyalkyl, -OR a -CO2R a -X-CO2R a -NR a R b -CONR a R b and -X-CONR a R b ;

[0135] R 3 Selected from the following groups: H, C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8 Hydroxyalkyl, -CO2R a -X-CO2R a -CONR a R b and -X-CONR a R b ;

[0136] When R 4 When it exists, each R 4 Each of them independently selected from the following group: C 1-8 Alkyl, C 1-8 Haloalkyl, C 1-8 Hydroxyalkyl, -OR a -CO2R a -X-CO2R a -NR a R b -CONR a R b and -X-CONR a R b ;

[0137] Each R 5 Each is independently selected from the following groups: halogen, CN, -X-CN, C1-8 Alkyl, C 3-8 cycloalkyl, C 3-8 Cycloalkenyl, C 3-5 Spirocycloalkyl, C 2-8 alkenyl, C 2-8 alkynyl group, C 1-8 Haloalkyl, C 1-8 Hydroxyalkyl, -OR a -CO2R a -X-CO2R a -NR a R b -CONR a R b -X-CONR a R b aryl, 5- or 6-membered heteroaryl, and 3-, 4-, 5- or 6-membered heterocycles, wherein the heteroatom present as the ring apex on the heteroaryl and heterocycle is selected from N, O or S, and wherein R 5 The aryl, heteroaryl, and heterocyclic moieties are optionally further reinforced by 1-3 R a replace;

[0138] Each R a and R b Each is independently selected from the following groups: hydrogen, hydroxyl, halogen, cyano, C 1-8 Alkyl, C 1-8 Alkoxy, C 1-8 Haloalkyl, C 3-6 cycloalkyl, C 3-6 Cycloalkyl, alkyl, amino, C 1-8 Alkylamino, bis(C) 1-8 Alkylamino, formamide, carboxyl C 1-4 Alkyl esters, carboxylic acids and -SO2-C 1-8 alkyl;

[0139] Each X is a linking group having the following formula: -(CH2) m O(CH2) P - where the subscripts m and p are integers from 0 to 5, and m+p is between 0 and 6, where any methylene group in X is optionally replaced by 1 to 2 methyl groups.

[0140] In some implementations, the CXCR7 inhibitor has the structure of compound 7.

[0141]

[0142] Or its pharmaceutically acceptable salt.

[0143] In some implementations, the CXCR7 inhibitor has the structure of compound 8.

[0144]

[0145] Or its pharmaceutically acceptable salt.

[0146] In some implementations, the CXCR7 inhibitor has the structure of compound 9.

[0147]

[0148] Or its pharmaceutically acceptable salt.

[0149] In some embodiments, the CXCR7 inhibitor is selected from compounds or pharmaceutical compositions disclosed in PCT Publication WO2014 / 085490, which originates from PCT application US2013 / 072067 filed by ChemoCentryx on November 26, 2013, and is incorporated herein by reference for all purposes.

[0150] C. Combined therapy

[0151] The method for treating cancer disclosed in this invention further includes one or more additional therapeutic agents.

[0152] Additional therapeutic agents used in this invention include compounds or compositions with antitumor activity. In some embodiments, the CXCR7 modulator of this invention may be administered in combination with chemotherapeutic agents or radiation.

[0153] Further examples of therapeutic agents that can be used in combination with the compounds or compositions described in this invention, including those administered alone and in the manner of the same pharmaceutical composition, include, but are not limited to: IGF1 inhibitors (e.g., antibodies or small molecules), CXCR4 inhibitors (e.g., AMD3100), immunomodulators, cisplatin, paclitaxel, methotrexate, cyclophosphamide, ifosfamide, chlorambucil, carmustine, carboplatin, vincristine, vinblastine, thiotepa, lomustine, sermustine, 5-fluorouracil, and cytarabine. In some embodiments, the one or more additional therapeutic agents are anti-IGF1 antibodies and / or CXCR4 inhibitors. In some embodiments, the one or more additional therapeutic agents are CXCR4 inhibitors. In some embodiments, the one or more additional therapeutic agents are anti-IGF1 antibodies.

[0154] Numerous CXCR4 inhibitors are known in the art, including small molecules, peptides, and antibodies. Each CXCR4 inhibitor is applicable in this invention. Some exemplary CXCR4 inhibitors include AMD3100, as well as the CXCR4 inhibitors provided in WO2007115232, WO2007115231, US20070275965, US20130289020, US20140286936, and US20170226106, which are incorporated herein by reference for all purposes.

[0155] Similar to CXCR4, many small molecule inhibitors and antibodies are known to target IGF1. Exemplary inhibitors include AG538, AG1024, NVP-AEW541, and figitumumab, as well as the inhibitors provided in US20090068110, US20140045832, US20050281812, US20050244408, US20120005767, US20140044720, and US20080161278, all of which are incorporated herein by reference for all purposes.

[0156] The weight ratio of the compound of the present invention to the second active ingredient varies widely and depends on the effective dose of each ingredient. Generally, the effective dose of each ingredient is used. Therefore, for example, when the compound of the present invention is combined with a second anticancer agent, the weight ratio of the compound of the present invention to the second anticancer agent is typically about 1000:1 to about 1:1000, preferably about 200:1 to about 1:200. Combinations of the compound of the present invention with other active ingredients are generally also within the above range, but in each case, the effective dose of each active ingredient should be used.

[0157] It should be understood that, compared to administering a second therapeutic agent without a CXCR7 modulator, this administration method, which allows for administration before, after, or concurrently with the second therapeutic agent, results in a stronger therapeutic effect. Based on conventional pharmaceutical principles, those skilled in the art can select suitable agents for combination therapy. The combination of therapeutic agents can have a synergistic effect; using this method, therapeutic effects can be achieved with lower doses of each drug, thereby reducing the likelihood of adverse side effects.

[0158] D. Administration method

[0159] Typically, the treatment methods described herein involve administering an effective amount of one or more of the compounds CXCR7 described herein to a patient. In a preferred embodiment, the compounds of the present invention are preferably administered orally to the patient (e.g., a human). Treatment regimens may vary depending on the compound used and the specific condition to be treated; for the treatment of most diseases, administration is preferably four times daily or less. Generally, a twice-daily dosing regimen is more preferred, and a once-daily dosing regimen is particularly preferred. However, it should be understood that the specific dose level and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, weight, general health condition, sex, diet, route of administration, excretion rate, combination of medications (i.e., other medications given to the patient), and the severity of the specific disease being treated, as well as the prescribing physician's judgment. Generally, it is preferred to use the minimum dose sufficient to provide effective treatment. The effectiveness of treatment in patients can generally be monitored using medical or veterinary standards appropriate to the condition being treated or prevented.

[0160] Depending on the cancer to be treated and the patient's condition, the compounds and compositions of the present invention can be administered orally, parenterally (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisional injection or infusion, subcutaneous injection or implantation), by inhalation, nasal, vaginal, rectal-sublingual, or local routes of administration. They can be formulated alone or together in suitable dosage units containing conventionally non-toxic, pharmaceutically acceptable carriers, adjuvants, and excipients suitable for each dose. The present invention also contemplates the administration of the compounds and compositions of the present invention in storage formulations.

[0161] It can be used at dose levels ranging from approximately 0.1 mg to approximately 140 mg per kilogram of body weight per day (approximately 0.5 mg to approximately 7 g per person per day). The amount of active ingredient that can be combined with carrier materials to produce a single dosage form can vary depending on the host being treated and the specific route of administration. A unit dosage form typically contains between approximately 1 mg and approximately 500 mg of the active ingredient. Sufficient amounts of the compound should be administered to achieve serum concentrations of 50 ng / ml to 200 ng / ml.

[0162] The compounds and compositions of the present invention can be combined with other compounds and compositions having related efficacies such as cancer prevention and treatment. Such other medicaments can be administered simultaneously or sequentially with the compounds or compositions of the present invention via their usual route of use and in the usual amounts. When a CXCR7 inhibitor is used concurrently with one or more other medicaments, a pharmaceutical composition containing other medicaments besides the CXCR7 inhibitor is preferred. Therefore, in addition to CXCR7 inhibitors, the pharmaceutical compositions of the present invention also include one or more other active ingredients or therapeutic agents.

[0163] Additional therapeutic agents used in combination therapy—whether compounds or antibodies—can be administered orally, parenterally (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracerebral injection or infusion, subcutaneous injection or implantation), by inhalation, nasal, vaginal, rectal, sublingual, or local routes. Furthermore, compounds and / or antibodies can be formulated alone or together in suitable dosage unit formulations comprising conventional, non-toxic, pharmaceutically acceptable carriers, adjuvants, and delivery systems suitable for each dose. The invention also contemplates administering the compounds and antibodies of the invention in stock formulations.

[0164] However, it should be understood that the specific dose level and frequency for any particular patient may vary and depend on a variety of factors, including the activity of the specific compound used, its metabolic stability and duration of action, age, weight, genetic characteristics, general health status, sex, diet, route and timing of administration, excretion rate, drug combination, severity of the specific disease state, and the host receiving treatment.

[0165] Combination therapy includes co-administration of a CXCR7 inhibitor and one or more additional therapeutic agents, sequential administration of a CXCR7 inhibitor and one or more additional therapeutic agents, or simultaneous administration of a single composition (one of which contains a CXCR7 inhibitor) and one or more containing one or more additional therapeutic agents.

[0166] Combination administration includes administering the CXCR7 inhibitor described herein within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours after administration of one or more additional therapeutic agents. Furthermore, the CXCR7 inhibitor and one or more additional therapeutic agents may be administered once daily, or twice, three times, or more daily, to provide a preferred daily dose level.

[0167] IV. Examples

[0168] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0169] Example 1: FRS2β expression by cavitary progenitor cells creates a microenvironment conducive to breast tumor development.

[0170] To investigate the role of FRS2β in vivo, we mutated the Frs2β gene in mice using gene-targeting technology. The mutant mice exhibited normal growth, fertility, and no obvious abnormalities. Frs2β promoter activity was detected using β-galactosidase staining of mature female mammary tissue. The Frs2β mutant allele was heterozygous. Figure 1AThe number of Frs2β transcripts increased significantly during pregnancy and lactation, then decreased after weaning (3 weeks after birth) during the resolution phase (data not shown). Immunohistochemistry confirmed FRS2β expression in a minority of cells within the mammary lobules. Figure 1C Most FRS2β-positive cells are negative for phosphohistone H3 (a nuclear marker used for cell division), indicating that they proliferate more slowly than other cells, consistent with the negative role of FRS2β in cell proliferation. Figure 1C FRS2β is expressed in a minority of cells that are positive for cytokeratin 18 (a lumen cell marker) but not for cytokeratin 14 (a myoepithelial cell marker). Figure 1D These data indicate that a small number of luminal cells in the mammary gland express FRS2β. On the other hand, whole-embryonic staining of the mammary gland in mutant mice showed no obvious structural abnormalities. This allows us to investigate the pathological role of FRS2β in tumorigenesis.

[0171] We crossed Frs2β mutant mice with MMTV-neu(+) mice to produce MMTV-neu(+) / Frs2β(+ / +) and MMTV-neu(+) / Frs2β(- / -) mice, hereinafter referred to as Frs2β(+ / +) mice and Frs2β(- / -) mice, respectively. We observed that tumor development in 8-week-old MMTV-neu(+) mice (23.4 ± 1.9 weeks, 83%, n = 8) was earlier than in non-pregnant MMTV-neu(+) mice (32.6 ± 2.6 weeks, 23.4%, n = 8). Therefore, we examined tumor development in the mice immediately after the end of gestation and lactation. We used nuclear magnetic resonance (NMR) imaging to detect tumors, a highly sensitive method that can detect tumors as small as 1 mm in diameter. 20 ( Figure 1E We observed small tumors 5–8 weeks after the start of the measurements and found that the tumor growth rate in Frs2β(- / -) mice was significantly slower than that in Frs2β(+ / +) mice. Figure 1E The tumor incidence rates were similar in both Frs2β (+ / +) and Frs2β (- / -) mice, with an incidence of 83.2% (n=18) and 88.2% (n=17) in Frs2β (+ / +) mice. These results indicate that Frs2β plays an important role in breast tumorigenesis. To explore the underlying molecular mechanisms, we first compared the histology of the tumors. Frs2β (+ / +) tumors contained abundant matrix, reminiscent of human breast cancer tissue. Figure 1GHowever, Frs2β(- / -) tumors contain very little matrix. Considering that the tumor matrix is ​​a major component of the tumor microenvironment, we hypothesize that FRS2β plays a role in creating a tumor-promoting microenvironment in breast tissue.

[0172] Histological studies showed that Frs2β(+ / +) tumors contained a large amount of matrix, reminiscent of human breast cancer tissue. Figure 1G (The arrows in the image indicate this). Conversely, very little stroma was observed in Frs2β(- / -) tumors. High concentrations of smooth muscle actin (SMA)-positive CAFs were observed in the stroma of Frs2β(+ / +) tumors, but in Frs2β(- / -) tumors (…). Figure 1H (The arrow in the image) was not found. These results indicate that FRS2β is required for the formation of the tumor stroma.

[0173] To verify the role of FRS2β in creating the tumorigenesis-required microenvironment in breast tissue prior to tumorigenesis, we conducted xenograft experiments. Frs2β(+ / +) tumor cells were seeded into premammary breast tissue from juvenile virgin Frs2β(+ / +) mice and juvenile virgin Frs2β(- / -) mice. We cultured the Frs2β(+ / +) tumor cells in serum-free suspension to enrich cancer cell stem cells (CSCs) as tumor spheres. 15，21 After a certain dilution, the tumor spheres were inoculated into the mammary tissue of 8-week-old Frs2β(+ / +) mice or the mammary tissue of 8-week-old Frs2β(- / -) mice. Figure 2A Interestingly, tumors formed only in the mammary tissue of Frs2β(+ / +) mice and grew rapidly within one month; however, they did not form in the mammary tissue of Frs2β(- / -) mice. Figure 2B (2C, 2D). This result indicates that CSCs disappear in the microenvironment of Frs2β(- / -) mammary tissue. As expected, when Frs2β(+ / +) tumor cells were inoculated into the mammary pads of Frs2β(+ / +) male mice (data not shown), tumors did not form, confirming the important role of mammary tissue in tumorigenesis. Therefore, FRS2β-expressing precancerous breast cells appear to create a microenvironment that supports CSC growth and allows tumorigenesis.

[0174] Immunohistochemistry revealed a similar number of coelomic cells expressing FRS2β in both MMTV-neu(-) and MMTV-neu(+) mice. Figure 2E In MMTV-neu(-) mice, endogenous ErbB2 expression was slightly reduced in FRS2β-positive cells (yellow arrow), which is consistent with the fact that FRS2β is involved in the ubiquitination and degradation of ErbB219.19 In MMTV-neu(+) mice, ErbB2 was overexpressed in FRS2β-positive cells (white arrow). To further investigate which columellar cells FRS2β is expressed in, we sorted mammary cells using surface markers. It is well known that columellar cells are rich in CD49f. 低 / CD24 高 cell population 22 Furthermore, coronary progenitor cells can be further fractionated with CD61 to obtain CD49f. 低 / CD24 高 / CD61 + cell population 23 CD49f 低 / CD24 + High / CD61 + Cells that significantly expressed FRS2β accounted for 23.6% of the coelomic progenitor cell population. Figure 2F We confirmed that CD49f derived from Frs2β(- / -) mammary cells... 低 / CD24 高 / CD61 + The luminal progenitor cell population lacks FRS2β. These data suggest that a subset of mammary luminal progenitor cells expresses FRS2β.

[0175] Example 2: Precancerous breast cells express cytokines dependent on FRS2β expression

[0176] Next, we investigated the molecular mechanism by which FRS2β expression in lumbar progenitor cells creates a favorable microenvironment for tumorigenesis. We cultured Frs2β(+ / +) or Frs2β(- / -) precancerous breast cells in serum-free suspension, enriched undifferentiated cells or progenitor cells into spheroids, and measured their spheroid-forming ability (…). Figure 3A and 3B We dissociated these initial mammary globules into single-cell suspensions and cultured them to produce secondary mammary globules. Secondary mammary globules are thought to accurately reflect the incidence of globules, undifferentiated cells, or progenitor cells. We found that a deficiency of FRS2β significantly reduced globule-forming ability. Figure 3A (3B). There was no significant difference in the diameter of the mammary globules, indicating that the proliferation rates of Frs2β(+ / +) and Frs2β(- / -) precancerous mammary cells were similar. To determine which progenitor cells' function was impaired due to FRS2β loss, we compared the transcriptomic profiles of Frs2β(+ / +) and Frs2β(- / -) mammary globules using DNA microarrays. Gene set enrichment analysis (GSEA) showed that, compared to Frs2β(- / -) cells, stem cell function-related gene sets and interferon signaling-related gene sets were abundant in Frs2β(+ / +) mammary globule cells. Figure 3CGSEA results of precancerous breast epithelial cells also showed that, compared to Frs2β(- / -) cells, gene sets related to NFκB targets, stem cell function, and matrix were abundant in Frs2β(+ / +) cells. Figure 3D Compared to Frs2β(+ / +) cells, the expression of ERK pathway-related genes was upregulated in Frs2β(- / -) cells, which was expected because Frs2β inhibits ERK signaling. The expression of many genes encoding cytokines was upregulated in Frs2β(+ / +) cells; among them, 18 genes showed a >1.5-fold increase in expression in Frs2β(- / -) cells compared to Frs2β(- / -) cells (data not shown).

[0177] We then focused on two genes with the highest differential expression in Frs2β(+ / +) cells—IGF1, which is included in the stem cell function-related gene set, and CXCL12, which is included in the interferon signaling-related gene set and the matrix-related gene set. Quantitative PCT (qPCT) confirmed that Igf1 and CXCL12 transcripts were strongly expressed in heterozygous Frs2β(+ / -) mammary cells compared to Frs2β(- / -) cells, while the expression of cell differentiation markers (keratin 8, keratin 18, and keratin 14) was upregulated in Frs2β(- / -) cells. Figure 3E Immunohistochemistry confirmed that protein levels of IGF1, CXCL12, and the CAF marker αSMA were higher in Frs2β(+ / +) breast tissue. Figure 3F Strong staining of αSMA confirmed the mobilization of CAFs in wild-type breast tissue.

[0178] Example 3: Increased CXCL12 production in pre-breast cancer cells is associated with FRS2β. Treatment of tumorigenesis with CXCR7 inhibitors or CXCR7 inhibitors in combination with other therapeutic agents regulates tumor growth.

[0179] Tumor spheroid formation reflects the characteristics of breast cancer cell-derived stem cells (CSCs), and CSC growth depends on cytokines in culture. To determine whether IGF1 derived from breast cancer preepithelial cells plays a role in tumor spheroid formation, we cultured Frs2β(+ / +) cells in serum-free suspensions without cytokine combinations, both in the presence and absence of Frs2β(+ / +) breast cancer preepithelial cells. Figure 4A In the presence of Frs2β(+ / +) breast cancer preepithelial cells, we observed Frs2β(+ / +) tumor cells forming tumor spheroids; however, in the absence of Frs2β(+ / +) breast cancer preepithelial cells, we did not observe Frs2β(+ / +) tumor cells forming tumor spheroids. Figure 4B and4C The control group IgG and the untreated group [NT] were compared. Treatment with the IGF1 neutralizing antibody (IGF1NAb) significantly reduced the formation of tumor spheres in Frs2β(+ / +) tumor cells co-cultured with Frs2β(+ / +) breast cancer pre-cure cells. Figure 4B and 4C These findings suggest that IGF1 originates from nearby Frs2β(+ / +) breast cancer preepithelial cells and plays an important role in tumor spheroid formation. Therefore, IGF1 derived from Frs2β(+ / +) breast cancer preepithelial cells may support the growth of CSCs.

[0180] To determine whether CXCL12 derived from breast cancer preepithelial cells plays a role in cancer-associated fibroblasts (CAFs), we co-cultured Frs2β(+ / +) CAFs with either Frs2β(+ / +) or Frs2β(- / -) breast cancer preepithelial cells. Figure 4D We confirmed that under these culture conditions, the Cxcl12 expression level in Frs2β(+ / +) pre-mastoma cells was higher than that in Frs2β(- / -) pre-mastoma cells. Figure 4E We observed that, compared to co-culturing with Frs2β(+ / +) pre-mastoma cells, the number of migrating CAFs significantly increased. Figure 4F and 4G CXCL12 binds to CXC receptors (CXCR)4 and CXCR7. We did not observe a significant effect on CAF mobilization by using the optimal concentration (100 μg / mL) of the reported CXCR4 inhibitor AMD3100 or by using compound 1 alone (100 μg / mL) (data not shown); however, when treated with both inhibitors in combination, CAF mobilization was significantly reduced in a dose-dependent manner. Figure 4H These findings suggest that CXCL12 originates from nearby Frs2β(+ / +) breast cancer precancerous cells and plays an important role in the mobilization of CAFs. Therefore, it appears that FRS2β-dependent increased production of cytokines, including IGF1 and CXCL12, maintains CSCs and mobilizes CAFs.

[0181] What are the molecular mechanisms that induce IGF1 and CXCL2 expression in pre-cancerous breast tissue? Because IGF1 and CXCL2 belong to the NFκB target gene set (…). Figure 3DFurthermore, the AKT–NFκB axis is activated by numerous signaling pathways, including ErbB2 and CXCL12. We investigated whether NFκB activation is involved in the production of these cytokines. To this end, we cultured Frs2β(+ / +) breast cancer preepithelial cells and treated them with DHMEQ, a specific inhibitor of NFκB. Figure 5A DHMEQ inhibits the expression of Igf1, Cxcl12, and IkBα in a dose-dependent manner; IkBα is a well-known NFκB-induced gene. Figure 5B This indicates that NFκB activation plays an important role in the expression of IGF1 and CXCL12 in preepithelial cells of breast cancer.

[0182] Next, we examined the activation of the AKT–NFκB axis in pre-breast cancer tissue in vivo. Immunoblotting of pre-breast cancer tissue lysates showed that, compared to Frs2β(- / -) tissue, Frs2β(+ / +) tissue had higher levels of phosphorylated AKT, higher levels of the NFκB components RelA and RelB in the nucleus, higher levels of phosphorylated IKKb, and lower levels of IkBa. Figure 5C-5E As expected, the level of phosphorylated ERK1 / 2 was lower in Frs2β(+ / +) tissues than in Frs2β(- / -) tissues. Figure 5C Furthermore, immunohistochemical results showed that the proportion of RelA in the nuclei of Frs2β(+ / +) precancerous luminal cells was greater than that in Frs2β(- / -) precancerous luminal cells. Figure 5G (Red arrows on the left and in the middle). In vivo DHMEQ treatment significantly reduced the number of Frs2β(+ / +) precancerous luminal cells containing RelA in their nuclei. Figure 5F (and G, right figure) and inhibiting the expression of Igf1 and Cxcl12 transcripts in precancerous breast tissue ( Figure 5H These results indicate that NFκB activation in precancerous cavitary cells plays a crucial role in the expression of IGF1 and CXCL12 in preepithelial breast cancer cells in vivo. It appears that FRS2β induces the AKT-NFκB axis in precancerous cavitary cells, thereby inducing the production of cytokines including IGF1 and CXCL12, which in turn activates NFκB through autocrine or paracrine processes, spreading the NFκB activation effect to surrounding mammary epithelial cells.

[0183] To determine whether IGF1 and CXCL12 expression in the pre-mammary microenvironment of breast cancer leads to tumorigenesis, we treated Frs2β(+ / +) mice with IGF1 neutralizing antibodies and / or CXCR4 and CXCR7 inhibitors (compound 1) (both together, a CXCL12 inhibitor) after inoculating them with Frs2β(+ / +) tumor cells. Treatment with either IGF1 neutralizing antibodies or CXCL12 inhibitors significantly reduced tumorigenesis, and the combined treatment with both IGF1 neutralizing antibodies and CXCL12 inhibitors showed a significant inhibitory effect on tumor volume and weight. Figure 5I-5K No significant changes in body weight (data not shown) indicate no toxic effects. These results suggest that the increased production of FRS2β-dependent IGF1 and CXCL12 in pre-cancerous breast tissue creates a necessary microenvironment for tumorigenesis.

[0184] Next, we examined the expression of FRS2β in breast tumors. Immunohistochemical results showed the presence of cells expressing FRS2β in breast tumors. Figure 6A The expression levels of Igf1 and Cxcl12 were higher in Frs2β(+ / +) tumors than in Frs2β(- / -) tumors. Figure 6B Immunohistochemical results showed that the expression levels of IGF1 and CXCL12 were higher in Frs2β(+ / +) tumors than in Frs2β(- / -) tumors. Figure 6C Therefore, it is reasonable to speculate that FRS2β induces the AKT-NFκB axis to induce the production of IGF1 and CXCL12 in tumor tissue.

[0185] Finally, we used immunohistochemistry to detect the expression of FRS2β in human breast cancer tissue. The expression level of FRS2β varied among different cancer cells. Figure 6D Compared to breast cancer tissues with moderate (++) or low (+) FFRS2β expression levels, breast cancer tissues with high (+++) FFRS2β expression levels had significantly higher levels of cancer stromal tissue (p = 0.0499, Barnard test). Figure 6E Furthermore, analysis of publicly available gene expression profiles indicates that patients with higher FRS2β expression levels in breast cancer tissue have a poorer prognosis. Figure 6F ).

[0186] In this study, we demonstrated that the FRS2β protein is expressed in a subset of luminal cells and induces the production of cytokines, including IGF1 and CXCL12. FRS2β stimulates the AKT-NFκB axis to promote cytokine production while inhibiting ERK signaling. These cytokines appear to, in turn, activate NFκB in peripheral breast luminal cells via autocrine or paracrine signaling, leading to a precancerous microenvironment rich in cytokines and containing a certain amount of matrix before tumorigenesis. Figure 6G (Top left image). Once cancerous stromal cells (CSCs) are present in the precancerous microenvironment, they can self-renew in the presence of IGF1 and generate tumor cells with the help of CXCL12-mobilized stromal cells, subsequently becoming cancerous fibroblasts (CAFs). CSCs and tumor cells may spontaneously generate IGF1 and CXCL12, leading to rapid growth and tumorigenesis. Figure 6G (See bottom left image). Without FRS2β, cytokines remain at low levels, preventing the formation of a suitable precancerous microenvironment. Figure 6G (See top right image); even if CSCs appear, they cannot grow effectively. Figure 6G (See bottom right figure). Based on these findings, we believe FRS2β is a promising target for breast cancer prevention. Furthermore, we demonstrate that combination therapy targeting IGF1 and CXCL12 effectively prevents tumor development in its early stages.

[0187] The tumor microenvironment is composed of multiple cell types: CAFs, mesenchymal stem cells, bone marrow-derived dendritic cells, immune cells, and neovascularization (3). On the other hand, it remains unclear which cell types in the precancerous microenvironment contribute to tumorigenesis. We found that luminal cells and luminal progenitor cells are important cell types in the precancerous microenvironment, and that FRS2β expressed by luminal cells and luminal progenitor cells plays a key role in cytokine production, thereby leading to the formation of a cytokine-rich precancerous microenvironment necessary for tumorigenesis.

[0188] Although the foregoing invention has been described in detail by way of illustration and examples for purposes of clarity, those skilled in the art will understand that certain changes and modifications may be made within the scope of the appended claims. Furthermore, each reference provided herein is incorporated by way of citation as if it were incorporated individually. In the event of any conflict between this application and the references provided herein, this application shall prevail.

Claims

1. Use of a combination of a CXCR7 inhibitor and a CXCR4 inhibitor in the preparation of a medicament for treating cancer in individuals of need, wherein, The individual expresses FRS2β in one or more coelomic progenitor cells, and the CXCR7 inhibitor has the structure shown in compound 1: Compound 1 Or its pharmaceutically acceptable salt. The cancer mentioned is breast cancer.

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