WO2022161263A1

WO2022161263A1 - New-type hedgehog signaling pathway inhibitor

Info

Publication number: WO2022161263A1
Application number: PCT/CN2022/073087
Authority: WO
Inventors: 陈伟; 范俊婉; 王拥军; 李昊文
Original assignee: 首都医科大学附属北京天坛医院
Priority date: 2021-01-26
Filing date: 2022-01-21
Publication date: 2022-08-04
Also published as: CN114790177B; CN114790177A

Abstract

Disclosed in the present application are a new-type Hedgehog signaling pathway inhibitor and a preparation method therefor. The Hedgehog signaling pathway inhibitor of the present application can effectively inhibit Smo receptor activity and block an SHH signaling pathway, has a good effect on blocking tumor cell proliferation, and has a good application prospect as a candidate drug for tumor treatment.

Description

Novel Hedgehog signaling pathway inhibitor

technical field

The present disclosure relates to the field of biomedicine, in particular to a novel Hedgehog signaling pathway inhibitor.

Background technique

Hedgehog (HH) is an evolutionarily highly conserved gene family that contains 3 homologous genes in mammals: Sonic Hedgehog (SHH), Desert Hedgehog (DHH) and Indian Hedgehog (IHH). Among them, SHH signaling pathway plays a very important role in embryonic development and tissue and organ formation, mainly including: SHH ligand, transmembrane protein Patched (PTCH1), transmembrane protein Smoothened (Smo), Suppressor of Fused (SUFU), G protein-coupled receptor kinases (GRKs), β-arrestins and downstream GLI transcription factors [Gorojankina, T., Cell Mol Life Sci, 2016.73(7): p.1317-32; Ruch, J.M. and E.J. Kim, Drugs , 2013.73(7): p.613-23; Chen, W., et al., Science, 2004.306(5705): p.2257-60]. The transduction of the Hedgehog signaling pathway is mainly mediated by the transmembrane proteins PTCH1 and Smo. In the absence of SHH ligands, PTCH1 binds to Smo and inhibits Smo activity, resulting in GLI forming a complex with SUFU in the cytoplasm, which is hydrolyzed by the proteasome and inhibits the transcription of target genes. Conversely, in the presence of SHH ligands, SHH ligands bind to PTCH1 to relieve its inhibitory effect on Smo; the activated Smo dissociates the SUFU-GLI complex, thereby allowing GLI transcription factors to enter the nucleus and promote the transcription of target genes [Arensdorf , Trends Pharmacol Sci, 2016.37(1): p.62-72; Robbins, D.J., D.L. Fei, and N.A. Riobo, Science Signaling, 2012.5(246): p.re6-re6; Kieran, M.W., Neuro Oncol, 2014.16 ( 8): p. 1037-47].

The Hedgehog (HH) signaling pathway controls many aspects of cellular embryonic development, growth and tissue repair in multicellular organisms. This pathway exists in many vertebrate structural units, such as neurons, bones, skin, and hair. In human tissues, abnormal activation of the HH signaling pathway is associated with a variety of cancers. Although many Smo inhibitors have been used in clinical trials of tumor treatment, most of them show drug resistance and various adverse reactions such as muscle tremors and dysgeusia. For example, GDC-0449/vismodegib was approved by the FDA in 2012 for the treatment of basal cell carcinoma with abnormal activation of the SHH signaling pathway, but due to the drug resistance caused by the molecular target Smo D473H mutation, it has not been used in the treatment of medulloblastoma. success. The research and development of high-efficiency new HH signaling pathway drugs is still a difficult problem that needs to be solved urgently.

SUMMARY OF THE INVENTION

The purpose of the present disclosure is to provide a novel Hedgehog signaling pathway inhibitor and a preparation method thereof, which can effectively inhibit the activity of Smo receptors, block the SHH signaling pathway, thereby reduce the GLI transcription level, and have a good inhibitory effect on tumor cell proliferation. effect. Another object of the present disclosure is to provide a pharmaceutical composition containing the above-mentioned Hedgehog signaling pathway inhibitor. The Hedgehog signaling pathway inhibitor disclosed in the present disclosure can be used as a candidate drug for tumor treatment and has a good application prospect.

Specifically, the present disclosure provides:

A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate:

wherein X ₁ and X ₂ are independently selected from any one of H, Cl, F, and Br, and X ₁ and X ₂ are not H at the same time;

wherein R ₁ is selected from any one of linear or branched C ₁ -C ₄ alkyl groups;

wherein R ₂ is —C(O)(CR ₃ R ₄ ) _m R ₅ , wherein R ₅ is selected from H, hydroxy, or substituted or unsubstituted amino, linear or branched or cyclic C ₁ - C ₆ alkyl, alkoxy, or —S(O) ₂ R ₆ ;

wherein R ₃ , R ₄ are independently selected from H, substituted or unsubstituted C ₁ -C ₆ alkyl, m is selected from an integer of 0-6; R ₆ is selected from linear or branched or cyclic C ₁ -C ₆ alkyl.

The present disclosure provides a compound of the above formula (I) or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate,

Preferably, wherein X ₁ , X ₂ are independently selected from Cl or F.

Preferably, wherein R ₂ is —C(O)(CR ₃ R ₄ ) _m S(O) ₂ R ₆ , wherein R ₃ , R ₄ are independently selected from H, substituted or unsubstituted C ₁ -C The alkyl group of ₆ , m is selected from the integer of 0-6; R ₆ is selected from the straight chain or branched chain or cyclic C ₁ -C ₆ alkyl group.

More preferably, the present disclosure provides a compound as follows, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate:

Another aspect of the present disclosure provides a method for preparing any of the compounds described above, comprising the steps of:

1) obtain the compound of formula III through condensation and cyclization reaction with o-phenylenediamine by formula II;

2) formula III compound obtains formula IV through alkylation;

3) formula IV reacts with 1-Boc-piperazine to obtain the compound of formula V;

4) the compound of formula V obtains the compound of formula (I) through reduction and addition reaction;

Wherein, X ₁ , X ₂ , R ₁ , R ₂ are defined as described above.

In another aspect of the present disclosure, there is provided any compound as described above, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, in the preparation for inhibiting Hedgehog Drug application of signaling pathways.

A preferred technical solution of the present disclosure provides any of the aforementioned compounds or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, which is used in the preparation of inhibiting Application of SHH signaling pathway drugs.

In another aspect of the present disclosure, there is provided any compound as described above, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, used in the preparation of an SMO-targeted application in medicine.

A preferred technical solution of the present disclosure provides any of the aforementioned compounds or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, which is used in the preparation of targeting D473H Medicinal applications of mutant SMOs.

Another aspect of the present disclosure provides any of the aforementioned compounds or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, which is used in the preparation of an antitumor drug Applications.

In another aspect of the present disclosure, the tumor as previously described is selected from a tumor associated with the proliferation of cerebellar granule precursor cells.

In another aspect of the present disclosure, the tumor is a tumor with a SMOD473H mutation.

In another aspect of the present disclosure, any of the aforementioned compounds, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, is used in the preparation of antitumor drugs. Application, the tumor is selected from medulloblastoma, basal cell carcinoma, glioma, liver cancer, lung cancer, digestive tract tumor, colorectal cancer, kidney cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, gastric cancer, One or more of oral cancer, nose cancer, throat cancer, bile duct cancer, cervical cancer, uterine cancer, testicular cancer, meningioma, skin cancer, melanoma, lymphoma, leukemia or sarcoma; wherein the tumor is preferably myeloid One or more of blastoma, basal cell carcinoma, glioma, liver cancer, lung cancer, gastrointestinal tumor, pancreatic cancer, and breast cancer.

In another aspect of the present disclosure, there is provided any compound as previously described, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, which is used in the preparation of modulating hair follicle morphology application in medicines.

In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising any of the compounds as previously described, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate substance, and a pharmaceutically acceptable carrier or excipient.

In another aspect of the present disclosure, any one of the aforementioned pharmaceutical compositions, the formulation of the pharmaceutical composition is selected from one or more of oral formulations, intravenous injections, subcutaneous injections, and transdermal patches.

Another aspect of the present disclosure is the use of any of the aforementioned pharmaceutical compositions in the preparation of anti-tumor drugs; the pharmaceutical composition may also include another or more anti-tumor drugs.

The compounds of the present disclosure have the characteristics of low clearance rate and high metabolic stability, and inhibit the expression of target gene Gli1 and SHH signaling pathway in a dose-dependent manner. The compounds of the present disclosure can compete with cyclopamine for binding to wild-type Smo and Smo-D473H receptors, and are not prone to drug resistance. In addition, the compounds of the present disclosure are more effective in preventing the proliferation of cerebellar granule precursor cells than GDC-0449. The Hedgehog signaling pathway inhibitor disclosed in the present disclosure has good application prospects and great clinical significance.

Description of drawings

Figure 1 shows the structure and ¹ HNMR spectrum of compound 0025A of the present disclosure;

Figure 2 shows that 0025A can inhibit the aggregation of βarr2-GFP in cells;

Figure 3 shows the comparison of GDC-0449 and 0025A inhibiting βarr2-GFP aggregation in cells;

Figure 4A shows the effect of 0025A and GDC-0449 on the transcription level of GLI;

Figure 4B shows the effect of 0025A and GDC-0449 on the proliferation of cerebellar granule precursor cells;

Figure 5 shows that 0025A dose-dependently reduced Gli1 mRNA and protein levels;

Figure 6 shows that 0025A inhibits Gli1 expression induced by SAG;

Figure 7A shows that different concentrations of 0025A, GDC-0449 (GDC), cyclopamine (Cyc) compete with bodipy-cyclopamine for binding to wild-type Smo;

Figure 7B shows that 10-6M (1 μM) 0025A, GDC, Cyc competes with bodipy-cyclopamine for binding to mutant Smo;

Figure 8 shows a mouse experiment in which 0025A inhibits SHH-dependent hair growth and hair follicle morphogenesis after depilation.

Detailed ways

According to the above-mentioned content of the present disclosure, and in accordance with common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present disclosure, various other modifications, substitutions or changes can also be made.

I. Definitions

Unless expressly stated otherwise, throughout the specification and claims, the term "comprising" or its conjugations such as "comprising" or "comprising" and the like will be understood to include the stated elements or components, and Other elements or other components are not excluded.

The term "pharmaceutically acceptable" means, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without, commensurate with a reasonable benefit/risk ratio, excessive toxicity, irritation, allergic reaction or other problems or complications of those compounds, materials, compositions and/or dosage forms.

The term "treating" includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.

Use of the terms "reduce", "inhibit", "reduce" or "reduce" is relative to a control. Those skilled in the art will readily determine appropriate controls for each experiment. For example, the reduced response in a subject or cell treated with the compound is compared to the response in a subject or cell not treated with the compound.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount sufficient to treat, inhibit or alleviate one or more symptoms of the disease state being treated or otherwise provide a desired pharmacological and/or physiological effect dose. The precise dosage will vary depending on a variety of factors, such as subject-dependent variables (eg, age, immune system health, etc.), the disease or disease, and the treatment administered. The effect of an effective amount can be relative to a control. These controls are known in the art and discussed herein, and can be, for example, the condition of the subject before or without administration of a drug or drug combination, or in the case of a drug combination, the effect of the combination can be combined Compared to the effect of administering only one drug.

The terms "carrier" and "excipient" are used herein to include any other compound that is not a therapeutic or biologically active compound that may be contained in or on the microparticles. Thus, excipients should be pharmaceutically or biologically acceptable or relevant, eg, excipients are generally not toxic to the subject. "Excipient" includes a single such compound, and is also intended to include multiple compounds.

The term "pharmaceutical composition" means a composition comprising a compound of the present disclosure and, depending on the mode of administration and the nature of the dosage form, at least one pharmaceutically acceptable ingredient selected from the group consisting of, but not limited to, carriers, diluents, adjuvants Agents, excipients, preservatives, fillers, disintegrating agents, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, flavoring agents, antibacterial agents, antifungal agents, lubricants, dispersing agents, Temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

The term "SHH" refers to Sonic Hedgehog. Hedgehog (HH) is an evolutionarily highly conserved gene family that contains 3 homologous genes in mammals: Sonic Hedgehog (SHH), Desert Hedgehog (DHH) and Indian Hedgehog (IHH) ). Among them, SHH signaling pathway plays a very important role in embryonic development and tissue and organ formation.

The term "Gli" refers to the transcription factor Ci (Cubitus interruptus, Gli in vertebrates) of the Hedgehog signaling pathway; Gli1 is a downstream target gene of SHH signaling, which is a marker of whether the SHH signaling pathway is activated.

The term "SMO" refers to Smoothened; Hedgehog (HH) signaling is controlled by two receptors, Patched (Ptc) and Smoothened (Smo), on the target cell membrane. The receptor Ptc is encoded by the tumor suppressor gene Patched and is composed of a single peptide chain with 12 transmembrane regions, which can directly bind to ligands and negatively regulate HH signaling. The receptor Smo is encoded by the proto-oncogene Smoothened and is homologous to G protein-coupled receptors. It is composed of a single peptide chain with seven transmembrane regions. The N-terminus is located outside the cell, and the C-terminus is located in the cell. Conservative, the C-terminal serine and threonine residues are phosphorylation sites, which bind to phosphate groups when catalyzed by protein kinases.

As used herein, the term "tumor" refers to or describes the physiological condition in mammals, especially humans, which is typically characterized by the unregulated growth of cells. Examples of tumors include, but are not limited to, neural tumors, brain tumors, solid tumors, carcinomas, lymphomas, blastomas, sarcomas, leukemias, and the like.

As used herein, the term "modulating follicle morphology" refers to modulating SHH-dependent hair growth and follicle morphogenesis after depilation.

The above-mentioned content of the present disclosure will be further described in detail below through the specific implementation in the form of examples. However, it should not be understood that the scope of the above-described subject matter of the present disclosure is limited to the following examples. All technologies implemented based on the above contents of the present disclosure belong to the scope of the present disclosure.

II. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present disclosure relates to a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate:

In one embodiment, X ₁ and X ₂ are independently selected from any one of H, Cl, F, and Br, and X ₁ and X ₂ are not H at the same time.

In one embodiment, X ₁ and X ₂ are independently selected from any one of Cl and F.

In a specific embodiment, X ₁ is F and X ₂ is Cl.

In another specific embodiment, X ₁ is Cl and X ₂ is F.

In one embodiment, R ₁ is selected from any of linear or branched C1-C4 alkyl groups.

In a specific embodiment, R ₁ can be any one of methyl, ethyl, propyl, isopropyl, butyl, and isobutyl.

In a specific embodiment, R ₁ can be methyl.

In one embodiment, R ₂ is -C(O)(CR ₃ R ₄ ) _m R ₅ , wherein R ₅ is selected from H, hydroxy, or substituted or unsubstituted amino, straight or branched chain, or cyclic C ₁ -C ₆ alkyl, alkoxy, or -S(O) ₂ R ₆ ; wherein R ₃ and R ₄ are independently selected from H, substituted or unsubstituted C ₁ -C ₆ Alkyl, m is selected from an integer of 0-6; R ₆ is selected from linear or branched or cyclic C ₁ -C ₆ alkyl.

In a specific embodiment R ₂ is -C(O)(CR ₃ R ₄ ) _m S(O) ₂ R ₆ , wherein R ₃ , R ₄ are independently selected from H, substituted or unsubstituted C ₁ -C ₆ alkyl group, m is selected from an integer of 0-6; R ₆ is selected from straight chain or branched chain or cyclic C ₁ -C ₆ alkyl group.

In a specific embodiment, R ₂ is -C(O)(CR ₃ R ₄ ) _m S(O) ₂ CH ₃ , wherein R ₃ , R ₄ are independently selected from H, substituted or unsubstituted C ₁ -C ₆ alkyl group, m is selected from an integer of 1-3; preferably, R ₃ , R ₄ are independently selected from H, and m is selected from 1 or 2.

In another specific embodiment, R ₂ is -C(O)(CR ₃ R ₄ ) _m R ₅ , wherein R ₃ , R ₄ are independently selected from H, m is selected from an integer of 0-2, and R ₅ is selected from From hydroxyl, or substituted or unsubstituted amino, linear or branched or cyclic C ₁ -C ₆ alkyl, alkoxy; preferably, R ₅ is selected from -OH, -NH-CH ₃ , -N(CH ₃ ) ₂ , -O-CH ₃ , -C(CH ₃ ) ₂ OH,

any of the.

Another aspect of the present disclosure relates to the preparation method of any of the compounds of formula (I) as described above, which specifically comprises the following steps:

a) the compound of formula III is obtained from formula II through condensation and cyclization reaction with o-phenylenediamine;

b) the compound of formula III is subjected to alkylation to obtain formula IV;

c) formula IV reacts with 1-Boc-piperazine to obtain the compound of formula V;

d) compound of formula V obtains the compound of formula (I) through reduction and addition reaction;

Wherein, X ₁ , X ₂ , R ₁ , R ₂ are defined as described above; specifically, it can be carried out with reference to the following reaction formula:

In a specific embodiment, more preferably, the present disclosure provides a compound of the following formula 0025A or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate:

In a specific embodiment, it relates to the preparation method of the compound of formula 0025A as described above; it specifically includes the following steps:

a) by formula 1 through o-phenylenediamine condensation, cyclization reaction to obtain the compound of formula 4;

b) the compound of formula 4 is methylated to obtain the compound of formula 5;

c) the compound of formula 5 is reacted with 1-Boc-piperazine to obtain the compound of formula 7;

d) the compound of formula 7 is reduced and reacted with 3-(methylsulfonyl) propionic acid to obtain the compound of formula 0025A;

Specifically, it can be carried out with reference to the following reaction formula:

It should be noted that the specific method for synthesizing the compound of formula (I) is not particularly limited, and those skilled in the art can select appropriate reaction substrates, intermediates, reaction conditions and other methods according to the specific structure of the compound of formula (I) to synthesize formula ( I) Compounds.

Another aspect of the present disclosure relates to any of the foregoing compounds or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, in the manufacture of a medicament for inhibiting Hedgehog signaling pathway Applications.

In one embodiment, the compound as described above, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, is capable of inhibiting the SHH signaling pathway.

In one embodiment, the compound as described above, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, can inhibit the intracellular aggregation of βarr2-GFP, It inhibits SMO.

In one embodiment, a compound as described above, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, can compete with cyclopamine to compete with wild-type SMO and SMO -D473H mutant receptor binding.

In one embodiment, the aforementioned compound or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate can effectively reduce the transcription level of GLI, and can Reduces Gli1 mRNA and protein levels in a dose-dependent manner.

In one embodiment, the aforementioned compound or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate can be used to prepare a SHH signaling pathway inhibitor.

In one embodiment, the aforementioned compound or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate can be used to prepare a drug targeting SMO, preferably ground, for the preparation of drugs targeting SMO D473H mutants.

Another aspect of the present disclosure relates to any of the aforementioned compounds or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, which are used in the preparation of antitumor drugs applications in .

In one embodiment, the use in preparing an anti-tumor drug as described above, wherein the tumor is selected from tumors related to Hedgehog signaling pathway; preferably, the tumor is selected from tumors related to SHH signaling pathway; preferably, the tumor is selected from tumors related to the SHH signaling pathway The tumor is selected from an SMO-related tumor, more preferably the tumor is selected from a tumor with the SMO D473H mutation.

Use of any one of the aforementioned compounds of formula (I), or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, wherein the tumor is selected from medulloblastoma, Basal cell carcinoma, glioma, liver cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nose cancer, laryngeal cancer, bile duct cancer One or more of cancer, cervical cancer, uterine cancer, testicular cancer, meningioma, skin cancer, melanoma, lymphoma, glioma, leukemia or sarcoma.

In one embodiment, the use of any of the aforementioned compounds in the preparation of anti-tumor drugs, wherein the tumor is preferably medulloblastoma, basal cell carcinoma, glioma, liver cancer, lung cancer, digestive tract tumor, One or more of pancreatic cancer and breast cancer.

In a specific embodiment of the present disclosure, any of the aforementioned compounds can inhibit a tumor associated with the proliferation of cerebellar granule precursor cells; preferably, the tumor is a medulloblastoma.

Another aspect of the present disclosure relates to the use of any of the aforementioned compounds or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, in the preparation of a medicament for regulating hair follicle morphology .

In a specific embodiment of the present disclosure, any of the aforementioned compounds inhibits skin pigmentation and hair regrowth.

Another aspect of the present disclosure relates to a pharmaceutical composition comprising the aforementioned compound or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, and a pharmaceutically acceptable Accepted carrier or excipient.

In one embodiment, according to any one of the aforementioned pharmaceutical compositions, the formulation of the pharmaceutical composition is selected from one or more of oral formulations, intravenous injections, subcutaneous injections, and transdermal patches.

In one embodiment, the formulations of the aforementioned pharmaceutical compositions include, but are not limited to, tablets, capsules, lyophilized powders, injection solutions, injections, granules, emulsions, suspensions, oils, nanomaterials one or more of the formulations.

Another aspect of the present disclosure relates to the use of any one of the aforementioned pharmaceutical compositions in the preparation of antitumor drugs.

Another aspect of the present disclosure relates to the use of any one of the aforementioned pharmaceutical compositions in the preparation of a medicament for regulating hair follicle morphology.

In one embodiment, the pharmaceutical composition further includes another one or more anti-tumor drugs, or another drug that modulates hair follicle morphology.

III. Examples

The present disclosure is further explained below with reference to examples. The descriptions of specific exemplary embodiments of the present disclosure have been presented for purposes of illustration and illustration. These descriptions are not intended to limit the disclosure to the precise forms disclosed, and obviously many changes and variations are possible in light of the teachings of the present disclosure. The exemplary embodiments were chosen and described for the purpose of explaining certain principles of the disclosure and its practical application, to thereby enable one skilled in the art to make and utilize various exemplary embodiments and various different aspects of the disclosure. Choose and change.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1: Compound 1-(4-(6-Chloro-2-fluoro-3-(1-methyl-1H-benzo[d]imidazol-2yl]phenyl)piperazin-1-yl)- Synthesis of 3-(methylsulfonyl)propan-1-one (0025A)

resolve resolution:

Step 1): Synthesis of N-(2-aminophenyl)-3-bromo-4-chloro-2 fluorobenzamide

Compound 1 (6.5g, 25.9mmol), compound 2 (o-phenylenediamine, 5.6g, 51.8mmol) were dissolved in 50ml DMF, EDCI (5.9g, 31.07mmol,), HOBT (4.19g, 31.07mmol) were added. DMAP (316 mg, 2.59 mmol), stirred at room temperature, after the reaction was complete, 150 ml of water was added, extracted with ethyl acetate three times (150 mL × 3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography 5.2 g of the product compound 3 (N-(2-aminophenyl)-3-bromo-4-chloro-2fluorobenzamide) was obtained as a yellow solid with a yield of 58.8%.

Structural characterization of compound 1:

LCMS (ESI-MS): [M+H] ⁺ =343.0

₁ HNMR (400MHz, CDCl3): δ7.53-7.48 (m, 1H), 7.39-7.22 (m, 3H), 7.12-7.05 (m, 2H), 6.82-6.78 (m, 3H).

Structural characterization of compound 3:

LCMS (ESI-MS): [M+H] ⁺ = 324.9

₁ HNMR (400MHz, MeOD): δ 7.88-7.84 (m, 1H), 7.75-7.60 (m, 2H), 7.44 (d, J=8.8Hz, 1H), 7.36-7.32 (m, 3H).

Step 2): Synthesis of 2-(3-Bromo-4-chloro-2-fluorophenyl)-1H-benzo[d]imidazole

Compound 3 (4.7 g, 13.8 mmol) was dissolved in 100 ml of acetic acid, heated to 100 degrees and reacted overnight. HPLC showed that the reaction was complete, cooled to room temperature, concentrated, added 50 ml of water, extracted three times with dichloromethane (50 mL*3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column chromatography (PE :EA=1:1) to obtain 2.4 g of product compound 4 (2-(3-bromo-4-chloro-2-fluorophenyl)-1H-benzo[d]imidazole), red solid, yield 51.9%.

Structural characterization of compound 4:

LCMS (ESI-MS): [M+H] ⁺ =338.9

₁ HNMR (400MHz, CDCl3): δ7.80 (d, J=7.6Hz, 1H), 7.61 (t, J=7.6Hz, 1H), 7.39-7.34 (m, 1H), 7.32-7.19 (m, 3H) ),3.62(s,3H).

Step 3): Synthesis of 2-(3-Bromo-4-chloro-2-fluorophenyl)-1 methyl-benzo[d]imidazole

Compound 4 (2.4 g, 4.32 mmol) was dissolved in 20 ml of tetrahydrofuran, cooled to zero, 60% sodium hydrogen (345.6 mg, 8.64 mmol) was added, and the mixture was stirred at room temperature for 10 minutes. Then iodomethane (920 mg, 6.48 mmol) was added, and the mixture was stirred at room temperature for 1 hour. HPLC showed that the reaction was complete, 30ml of water was added, extracted three times with ethyl acetate (30mL×3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain 2g of product compound 5 (2-(3-bromo-4). -Chloro-2-fluorophenyl)-1methyl-benzo[d]imidazole), red solid, yield 79.6%.

Structural characterization of compound 5:

LCMS (ESI-MS): [M+H] ⁺ =445.2

₁ HNMR (400MHz, CDCl3): δ7.81 (d, J=6.8Hz, 1H), 7.39-7.19 (m, 4H), 6.97 (t, J=8.8Hz, 1H), 3.62 (s, 3H), 3.60-3.48(m, 4H), 3.15-3.03(m, 2H), 2.97-2.85(m, 2H), 1.43(s, 9H).

Step 4) Synthesis of 1-tert-butoxycarbonyl-4-(6-chloro-2-fluoro-3-(1-methyl-1H-benzo[d]imidazol-2-yl)phenyl)piperazine

Compound 5 (2 g, 5.92 mmol) was dissolved in 20 ml of toluene, compound 6 (1.1 g, 5.92 mmol), Pd ₂ (dba) ₃ (254 mg, 0.296 mmol, ), Cs ₂ CO ₃ (5.77 g, 17.76 mmol, were added) ,) and BINAP (360 mg, 0.592 mmol). The reaction was carried out at 100 degrees overnight under nitrogen protection. HPLC showed that the reaction was complete, cooled to room temperature, added 25ml of water, extracted three times with ethyl acetate (25mL*3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified by silica gel column (PE:EA=1: 1) to obtain 1.7g of product compound 7 (1-tert-butoxycarbonyl-4-(6-chloro-2-fluoro-3-(1-methyl-1H-benzo[d]imidazol-2-yl)benzene) yl)piperazine), yellow solid, yield 64.7%.

Structure characterization of compound 7: LCMS (ESI-MS): [M+H] ⁺ =345.1

Step 5): 1-(4-(6-Chloro-2-fluoro-3-(1-methyl-1H-benzo[d]imidazol-2yl]phenyl)piperazin-1-yl)-3 Synthesis of -(methylsulfonyl)propan-1-one (0025A)

Compound 7 (1.7 g, 3.82 mmol) was dissolved in 10 ml of dichloromethane, and 17 ml of a 4M hydrochloric acid dioxane solution was added. Stir at room temperature. After the reaction is complete, filter, wash the filter cake with petroleum ether, and dry to obtain 1.2 g of compound 8 as a white solid with a yield of 91.1%.

Compound 8 (1.2 g, 3.47 mmol) was dissolved in DMF (15 mL), 3-(methylsulfonyl)propionic acid (529 mg, 3.47 mmol), DIEA (895 mg, 6.94 mmol), HATU (1.98 g, 5.21 mmol) were added mmol). After the reaction at room temperature was completed, 45ml of water was added, extracted three times with ethyl acetate (50mL*3), the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and purified to obtain 380mg of product 0025A, white solid, yield 22.8% .

0025A Structural Characterization:

LCMS (ESI-MS): [M+H] ⁺ =479.1

₁ HNMR (400MHz, DMSO-d6) (Figure 1): δ 7.73-7.66 (m, 2H), 7.48 (d, J=8.8Hz, 1H), 7.37-7.27 (m, 3H), 3.78-3.64 ( m, 7H), 3.36-3.30 (m, 2H), 3.21-3.02 (m, 4H), 3.02 (s, 3H), 2.86 (t, J = 7.6Hz, 2H).

Example 2: In vitro pharmacokinetic detection of 0025A

Using human liver microsomes, the metabolic stability of 0025A was tested in vitro, and compared with the positive control compounds (testosterone, propranolol) and negative control compounds (warfarin), 0025A showed low clearance and high metabolism Stability characteristics (Table 1).

Table 1 shows the in vitro pharmacokinetic analysis of 0025A:

K, clearance coefficient; T _1/2 , half-life; Cl _int , intrinsic elimination rate; Cl _app , apparent half-life rate; Cl _h , hepatic elimination rate; E _h , uptake rate.

Example 3: βarr2-GFP intracellular aggregation assay

1) Identification of Smo inhibitors in U2OS cells

U2OS cells stably overexpressing βarr2-GFP and Smo-633 were added to (A) DMSO, (B) 1 μM cyclopamine (Cyc), (C) 100 nM 0025A or (D) 100

nM

0025A and 1 μM SAG, respectively, and cultured at 37°C 24h, observed by fluorescence microscope. Arrows show intracellular aggregation of βarr2-GFP. (E) The percentage of U2OS cells with βarr2-GFP aggregation was counted.

Using Cyc (Smo inhibitor) and SAG (Smo agonist) as controls, the Smo/βarr2-GFP aggregation experiment verified that 0025A can inhibit the accumulation of βarr2-GFP in cells and inhibit the SHH signaling pathway (Figure 2). .

2) 0025A inhibits the accumulation of βarr2-GFP in cells

(A) Different concentrations of 0025A (10 ^-12 -10 ^-6 M) were added to U2OS cells stably overexpressing βarr2-GFP and Smo-633, incubated at 37°C for 24h, and observed by fluorescence microscopy. (B) The percentage of cells with aggregation of βarr2-GFP after different concentrations of 0025A and GDC-0449 were treated.

The experiment found that the IC50 of 0025A and Roche's marketed drug Smo inhibitor GDC-0449 were 1.7nM and 28nM, respectively (Figure 3), indicating that 0025A can block the SHH signaling pathway more effectively than GDC-0449.

Example 4: GLI-luciferase reporter assay

50ng/mL SHH and different concentrations of 0025A and GDC-0449 were added to NIH3T3 cells stably expressing the reporter gene. After 30 hours of action, GLI-luciferase reporter assay found that both 0025A and GDC-0449 could effectively reduce the transcription level of GLI. There was no difference between the two (Figure 4A).

50ng/mL SHH and different concentrations of 0025A and GDC-0449 were added to the cerebellar granule precursor cells for 48 hours; then, labeled with 3H-thymidine for 16 hours, the amount of incorporation was detected by an isotope detector, and it was found that compared with 0025A The proliferation of cerebellar granule precursor cells was more effectively blocked by GDC-0449 (Fig. 4B).

Conclusion: By GLI-luciferase reporter assay, it was observed that both 0025A and GDC-0449 could effectively reduce the transcription level of GLI, and there was no difference between the two (Fig. 4A). However, the proliferation of cerebellar granule precursor cells was detected by the 3H-thymidine incorporation assay, and it was found that 0025A (IC50=12.3±2.9nM) was more effective than GDC-0449 (IC50=23.1±4.2nM) It inhibited the proliferation of cerebellar granule precursor cells (Figure 4B), confirming that 0025A can effectively inhibit the SHH signaling pathway.

Example 5: Gli1 expression experiment

1) 0025A inhibits Gli1 expression induced by SHH ligand.

(a) Serum-starved NIH3T3 cells were treated with 20% SHH ligand for 24 hours, and the mRNA levels of Gli1 in the cells were analyzed. Serum-starved NIH3T3 cells were treated with 20% SHH-CM and different concentrations of inhibitors (0.01, 0.1, 1, 10

μM

0025A or 1 μM GDC-0449) for 24 h, and the Gli1 mRNA level (b) and protein level (c) in the cells were analyzed. , d). (Figure 5)

2) 0025A inhibits Gli1 expression induced by SAG.

(A) Serum-starved NIH3T3 cells were treated with 100 nM SAG for 24 h, and the mRNA levels of Gli1 in the cells were analyzed. Serum-starved NIH3T3 cells were treated with 100 nMSAG and different concentrations of inhibitors (0.01, 0.1, 1, 10

μM

0025A or 1 μM GDC-0449) for 24 h, and Gli1 mRNA levels (B) and protein levels (C, D) in the cells were analyzed. (Image 6)

Conclusions: We used SHH ligands or SAG, a known Smo agonist, to enhance Gli1 expression. Serum-starved NIH3T3 cells were stimulated with 20% SHH-CM or 100 nM SAG, and Gli1 mRNA levels were found to be increased 4-fold and 3-fold, respectively (Fig. 5A, 6A), indicating that both can activate the SHH signaling pathway. To examine the inhibitory effect of 0025A on the SHH pathway, NIH3T3 cells were treated with 20% SHH-CM and 0025A (0.01, 0.1, 1, 10 μM) or GDC-0449 (1 μM) for 24 h, respectively, and a dose-dependent decrease in 0025A was observed. Gli1 mRNA and protein levels (Fig. 5, b-d). Furthermore, similar to SHH-CM, 0025A inhibited SHH signaling activated by SAG (Fig. 6, B-D). These results suggest that 0025A can act as a potent inhibitor and inhibit the expression of target gene Gli1 and SHH signaling pathway in a dose-dependent manner.

Example 6: Competitive binding of 0025A to Smo receptors

To further verify the binding of 0025A to the Smo receptor, we performed a bodipy-cyclopamine competition binding experiment. We found that a known Smo antagonist (cyclopamine, GDC-0449) and 0025A were able to displace 5 nM of bodipy-cyclopamine from Smo with similar affinity, thereby reducing green fluorescence in cells (Fig. 7A).

In order to test whether 0025A can bind to Smo with D473H mutation, we overexpressed Smo-D473H in HEK293 cells and performed competition binding experiments. The results showed that 0025A could effectively compete with 5nM bodipy-cyclopamine in Smo-D473H. However, GDC-0449 could not bind to mutant Smo-D473H at low concentration, and showed partial effect at high concentration (Fig. 7B). These results indicate that 0025A can compete with cyclopamine to bind to wild-type Smo and Smo-D473H receptors, and is not prone to drug resistance.

Example 7: 0025A inhibits skin pigmentation and hair regeneration in mice

Studies have shown that the SHH signaling pathway is closely related to the regulation of hair follicle morphogenesis. To verify the inhibitory effect of 0025A on SHH signaling in vivo, we subjected 8-week-old female C57BL/6 mice to back shaving and depilatory cream, which induces hair follicle development into the growth phase. After depilation, we treated the depilated skin area with a control reagent (95% acetone/5% DMSO) and an experimental reagent (95% acetone/5% DMSO + 5 mM 0025A) once a day for 14 days to observe pigmentation and hair formation regeneration situation. We found that control mice had more graying (pigment accumulation) in their skin after 10 days. After 14 days most of the hair grows back. However, the hair growth of mice in the 0025A experimental group was blocked (Fig. 8A). Histological analysis showed that pigment formation and hair follicle morphogenesis were blocked in the 0025A experimental group (Fig. 8B).

Claims

A compound of formula (I), or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate:

wherein X 1 and X 2 are independently selected from any one of H, Cl, F, and Br, and X 1 and X 2 are not H at the same time;

R 1 is selected from any one of linear or branched C 1 -C 4 alkyl groups;

R 2 is -C(O)(CR 3 R 4 ) m R 5 ,

wherein R 3 , R 4 are independently selected from H, substituted or unsubstituted C 1 -C 6 alkyl,

wherein R 5 is selected from H, hydroxy, or substituted or unsubstituted amino, linear or branched or cyclic C 1 -C 6 alkyl, alkoxy, or -S(O) 2 R 6 ;

R 6 is selected from linear or branched or cyclic C 1 -C 6 alkanes;

m is selected from an integer of 0-6.
The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate,

wherein X 1 and X 2 are independently selected from Cl or F.
The compound of formula (I) according to claim 1, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate,

wherein R 2 is -C(O)(CR 3 R 4 ) m S(O) 2 R 6 ,

wherein R 3 , R 4 are independently selected from H, or a substituted or unsubstituted C 1 -C 6 alkyl group, and m is selected from an integer of 0-6;

R 6 is selected from linear or branched or cyclic C 1 -C 6 alkyl groups.
The following compounds or pharmaceutically acceptable salts thereof, or isomers thereof, or pharmaceutically acceptable solvates or hydrates:
The preparation method of the compound described in any one of claim 1-4, described compound preparation method comprises the steps:

1) obtain the compound of formula III through condensation and cyclization reaction with o-phenylenediamine by formula II;

2) formula III compound obtains formula IV through alkylation;

3) formula IV reacts with 1-Boc-piperazine to obtain formula V compound;

4) the compound of formula V obtains the compound of formula (I) through reduction and addition reaction;

Wherein, X 1 , X 2 , R 1 , R 2 are defined as described in claim 1 .
The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, which is used in the preparation of a compound for inhibiting Hedgehog signaling pathway. The application in medicine; preferably in the preparation of medicine for inhibiting SHH signaling pathway.
Use of the compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, in the preparation of an antitumor drug.
The use according to claim 7, wherein the tumor is preferably a tumor with the SMO D473H mutation.
The application according to claim 7, wherein the tumor is selected from the group consisting of medulloblastoma, basal cell carcinoma, glioma, liver cancer, lung cancer, gastrointestinal tumor, colorectal cancer, kidney cancer, pancreatic cancer, breast cancer, and ovarian cancer , prostate cancer, stomach cancer, oral cancer, nose cancer, throat cancer, bile duct cancer, cervical cancer, uterine cancer, testicular cancer, meningioma, skin cancer, melanoma, lymphoma, leukemia or sarcoma one or more.
The use according to claim 7, wherein the tumor is selected from tumors associated with the proliferation of cerebellar granule precursor cells.
The compound according to any one of claims 1-4 or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, which is used in the preparation of a medicament for regulating hair follicle morphology application.
A pharmaceutical composition comprising a compound according to any one of claims 1-4, or a pharmaceutically acceptable salt thereof, or an isomer thereof, or a pharmaceutically acceptable solvate or hydrate, and a pharmaceutically acceptable Carrier.