CN112645955B

CN112645955B - [1,2,4] triazolo [4,3-b ] pyridazine compound and preparation method and application thereof

Info

Publication number: CN112645955B
Application number: CN202011556236.5A
Authority: CN
Inventors: 温万东; 石万棋; 陈齐; 史焱; 代明星; 吴瑕; 陈科
Original assignee: Sichuan Guokang Pharmaceutical Co Ltd
Current assignee: Sichuan Guokang Pharmaceutical Co Ltd
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2022-11-04
Anticipated expiration: 2040-12-24
Also published as: CN112645955A

Abstract

The present invention relates to a [1,2,4]]Triazolo [4,3-b ]]Pyridazine compounds and preparation method and application thereof. Specifically provided are compounds represented by formula I, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or optical isomers thereof, or isotopic substitution forms thereof. Experimental results show that the compound provided by the invention has a good inhibition effect on TRK kinase, and has a good inhibition effect on tumor (such as colon cancer) cells related to TRK overexpression. The compound provided by the invention has wide application prospects in preparation of TRK kinase inhibitors and medicines for preventing and/or treating tumors related to TRK overexpression.

Description

[1,2,4] triazolo [4,3-b ] pyridazine compound and preparation method and application thereof

Technical Field

The invention belongs to the field of medicinal chemistry, and particularly relates to a [1,2,4] triazolo [4,3-b ] pyridazine compound and a preparation method and application thereof.

Background

Tropomyosin-related kinase (Trk) is a class of nerve growth factor receptors, and its family consists of highly homologous tropomyosin-related kinase a (TrkA), tropomyosin-related kinase B (TrkB), tropomyosin-related kinase C (TrkC), encoded by NTRK1, NTRK2, and NTRK3, respectively. Trk kinase influences cell proliferation, differentiation, metabolism and apoptosis through Ras/MAPK, PI3K/AKT, PLC gamma and other downstream channels. When NTRK gene fusion occurs in chromosome variation, the regulation of a Trk kinase downstream signal channel can be abnormally caused, and the excessive activation of the channel can cause the generation of cancer.

The research results of recent decades show that Trk kinase has a close relationship with the occurrence, metastasis and exacerbation of various tumors, for example, trk kinase is over-expressed in melanoma, non-small cell lung cancer (NSCLC), thyroid cancer, malignant myeloid leukemia (AML), malignant glioma, astrocytoma, colon cancer and the like, and the over-expression is closely related with the migration of tumor cells.

Trk kinases are activated in malignant tumors by a variety of mechanisms, mainly structural rearrangements and alterations in expression. The gene encoding Trk kinase NTRK rearrangement generates chimeric oncogene, leads to Trk kinase constitutive activation, is no longer regulated and controlled by nerve growth factor ligand, and has carcinogenic risk, such as in colon cancer KM12 cells, tropomyosin-3 gene TPM3 positioned on the long arm of chromosome 1 and NTRK1 generate gene fragment rearrangement, TPM3 is broken between No. 7 and No. 8 exons, NTRK1 is broken inside No. 8 exons, and then TPM3 is directly connected with NTRK1 expressing intracellular structural region, and TPM3-TRKA chimeric protein is abnormally expressed. The change of the gene disrupts the interaction of ligand NGF and TrkA in cells, intracellular TRKA is in an over-expression and continuous activation state, and the downstream PI3K/AKT, ras/MAPK and PLC gamma three signal paths are also in an abnormal activity state.

More and more researches show that the selective inhibitor of the Trk kinase has obvious inhibition effect on the proliferation of human tumor cells, but has lower inhibition effect on normal cells, and the Trk kinase can be used as an effective target point for tumor treatment. Larotrectinib (LOXO-101), an oral selective tropomyosin-related kinase inhibitor developed cooperatively by Bayer and Loxo Oncology in the late 12 th month of 2017, was filed in the United states for new drug marketing applications for treating solid tumors in adults or children carrying NTRK fusion genes. Larotrectinib has previously been qualified by the FDA for orphan medication and for breakthrough therapy. This event is an important step in the accurate medical advance practice and is also another great practice of the innovative clinical Trial-Basket Trial (Basket Trial) based on accurate medical design. Therapeutic efficacy data for Larotrectinib was published as early as 2017 at 6 months in the ASCO conference: in phase i and ii clinical trials, 55 patients with NTRK fusions involving 13 different tumor types were enrolled, including salivary gland cancer (12), sarcoma cancer (10), infantile fibrosarcoma (7), lung cancer (5), thyroid cancer (5), colorectal cancer (4), melanoma (4), cholangiocarcinoma (2), gastrointestinal stromal tumor (2), other cancers (4), with 46 patients having an Overall Response Rate (ORR) of 78%, 2 patients having complete tumor disappearance, and a maximum response time of 23 months.

Therefore, research on more compounds with novel structures and excellent inhibitory effect on Trk kinase is of great importance in developing drugs for treating Trk overexpressed tumors (such as salivary gland cancer, lung cancer, bile duct cancer, gastrointestinal stromal tumor, melanoma, non-small cell lung cancer, thyroid cancer, malignant myeloid leukemia, malignant glioma, astrocytoma, or colon cancer).

Disclosure of Invention

The invention aims to provide a [1,2,4] triazolo [4,3-b ] pyridazine compound which has a novel structure and an excellent inhibitory effect on Trk kinase, and a preparation method and application thereof.

The invention provides a compound shown as a formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substitution form thereof:

wherein X is selected from N or CR ₄ ；

R ₁ ～R ₅ Each independently selected from hydrogen, halogen, C _1～4 Alkyl or C _1～4 An alkoxy group;

R ₆ ～R ₇ each independently selected from hydrogen and C _1～4 Alkyl or C _1～4 An alkoxy group;

R ₈ selected from hydroxyl or carboxyl;

n is an integer of 0 to 5.

Further, the air conditioner is provided with a fan,

x is selected from N or CR ₄ ；

R ₁ ～R ₅ Each independently selected from hydrogen, halogen, and at least one of which is halogen;

R ₆ ～R ₇ each independently selected from hydrogen and C _1～2 An alkyl group;

R ₈ is a hydroxyl group;

n is an integer of 0 to 3.

Further, the structure of the compound is shown as a formula II-1 or a formula II-2:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₅ Each independently selected from hydrogen, fluorine, chlorine or bromine, and at least one of which is fluorine and one is chlorine;

n is 1 or 2;

or, the structure of the compound is shown as formula II-2:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Each independently selected from hydrogen, fluorine, chlorine or bromine, and at least two of which are fluorine;

n is 1 or 2.

Further, the compound is one of the following compounds:

the present invention also provides a process for preparing the above compound, comprising the steps of:

(1) Reacting the compound A ' with the compound B ' to obtain a compound C ';

(2) Reacting the compound C 'with a compound D', namely the compound shown in the formula I;

wherein Y is halogen; x, R ₁ ～R ₈ And n is as described above.

Further, in the step (1), the molar ratio of the compound A 'to the compound B' is 1: (0.8 to 1.2), preferably 1:1; the reaction is carried out under catalysis of an organic base, wherein the organic base is preferably N, N' -diisopropylethylamine or pyridine; the solvent of the reaction is an alcohol solvent, preferably n-butanol; the reaction temperature is 105-115 ℃, preferably 110 ℃, and the reaction time is 2-5 hours, preferably 3 hours; the Y is chlorine;

and/or in the step (2), the molar ratio of the compound C 'to the compound D' is 1 (3-10), preferably 1:4; the solvent of the reaction is an alcohol solvent, preferably n-butanol; the reaction temperature is 105-115 ℃, preferably 110 ℃, and the reaction time is 0.5-3 hours, preferably 1 hour.

Further, in the step (1), after the reaction is finished, the method further comprises the following purification steps: concentrating the system obtained by the reaction, removing the solvent, and then carrying out silica gel column chromatography separation, wherein the mobile phase adopted by the chromatography separation is petroleum ether: a mixed solution of ethyl acetate in a volume ratio = 2;

and/or in the step (2), after the reaction is finished, the method further comprises the following purification steps: concentrating the system obtained by the reaction, removing the solvent, and then carrying out silica gel column chromatography separation, wherein the mobile phase adopted by the chromatography separation is dichloromethane: methanol volume ratio = 10.

The invention also provides application of the compound or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the optical isomer thereof, or the isotopic substitution form thereof in preparing TRK kinase inhibitors.

The invention also provides application of the compound or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the optical isomer thereof, or the isotopic substitution form thereof in preparing a medicament for preventing and/or treating tumors related to TRK overexpression, wherein the tumors related to TRK overexpression are preferably salivary gland cancer, lung cancer, bile duct cancer, gastrointestinal stromal tumor, melanoma, non-small cell lung cancer, thyroid cancer, malignant myeloid leukemia, malignant glioma, astrocytoma or colon cancer, and more preferably colon cancer.

The invention also provides a medicine for treating tumors related to TRK overexpression, which is a preparation prepared by taking the compound, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or optical isomers thereof, or isotopic substitution forms thereof as active ingredients and adding pharmaceutically acceptable auxiliary materials.

Definitions of terms used in connection with the present invention: the initial definitions provided herein for a group or term apply to that group or term throughout the specification unless otherwise indicated; for terms not specifically defined herein, the meanings that would be afforded to them by a person skilled in the art, in light of the disclosure and context, should be given.

The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by the prefix C _a～b Alkyl represents any alkyl group containing from "a" to "b" carbon atoms. For example, C _1～4 Alkyl refers to a straight or branched chain alkyl group containing 1 to 4 carbon atoms.

C _1～4 Alkoxy means a straight or branched chain alkoxy group containing 1 to 4 carbon atoms.

Halogen is fluorine, chlorine, bromine or iodine.

"isotopic substitution pattern"Refers to a compound obtained by replacing one or more atoms in the compound with their corresponding isotopes. Such as compounds obtained by replacing one or more hydrogens (H) in the compound with deuterium (D) or tritium (T); such as one or more than two carbons in a compound ¹² Quilt carbon ¹¹ Or carbon ¹³ The compound obtained after replacement.

By "pharmaceutically acceptable" is meant a carrier, cargo, diluent, excipient, and/or salt that is generally chemically or physically compatible with the other ingredients that make up the pharmaceutical dosage form, and with the recipient.

"salts" are acidic and/or basic salts formed from a compound or stereoisomer thereof with inorganic and/or organic acids and/or bases, and also include zwitterionic (inner) salts, and also include quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. The compound, or a stereoisomer thereof, may be obtained by appropriately (e.g., equivalentlymixing) a certain amount of an acid or a base. These salts may form precipitates in the solution which are collected by filtration, or they may be recovered after evaporation of the solvent, or they may be prepared by reaction in an aqueous medium followed by lyophilization.

The "pharmaceutically acceptable salt" in the present invention may be hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate of the compound.

Experimental results show that the compound provided by the invention has a good inhibitory effect on TRK kinase and a good inhibitory effect on tumor (such as colon cancer) cells related to TRK overexpression. The compound provided by the invention has wide application prospects in preparation of TRK kinase inhibitors and medicines for preventing and/or treating tumors related to TRK overexpression.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Detailed Description

The raw materials and equipment used in the invention are known products, and are obtained by purchasing products sold in the market.

EXAMPLE 1 preparation of Compound 1 of the present invention

Compound 1 was prepared according to the following synthetic route:

the preparation method comprises the following steps:

(1) To a reaction flask were added 0.40g (i.e., 2.21 mmol) of compound A0.50g (i.e., 2.21 mmol) of compound B0.50g (i.e., 2.21 mmol), 2.5mL of N, N' -Diisopropylethylamine (DIPEA), and 10mL of n-butanol, and the reaction was heated to 110 ℃ and stirred for 3h, TLC showed disappearance of the starting material, and the reaction was complete. The solvent was removed by concentration under reduced pressure, and the resulting concentrate was separated by silica gel column chromatography (mobile phase was a mixed solution of petroleum ether: ethyl acetate volume ratio = 2). LC/MS: m/z =360.1[ m + H ].

(2) Taking 0.50g (namely 1.4 mmol) of compound C, 0.37g (namely 0.37g,6.0 mmol) of ethanolamine and 10mL of n-butanol, adding the mixture into a reaction flask, stirring the reaction system at 110 ℃ for 1h, and TLC (thin layer chromatography) to show that the raw materials disappear and the reaction is complete. The solvent was removed by concentration under reduced pressure, and the resulting concentrate was separated by silica gel column chromatography (mobile phase is a mixed solution of dichloromethane: methanol volume ratio = 10). LC/MS m/z =389.1[ m ] +H ]]； ¹ HNMR(dmso-d6):9.01(1H)，8.10(d,J＝7.5,1H)，7.35(d,J＝7.51H),7.05-7.19(m，3H)，4.65(s,1H),4.35(m,1H),3.50(t,2H),3.07-3.14(m,4H),1.55-1.78(m,4H)。

EXAMPLE 2 preparation of Compounds 2 to 4 of the invention

Compounds 2 to 4 according to the present invention were obtained by replacing compounds a and D in example 1 with the corresponding compounds in table 1, respectively, and using the same synthetic route and method as in example 1.

The structures of compounds 2 to 4 are characterized as follows:

compound 2: LC/MS m/z =417.4, M + H ];1HNMR (dmso-d 6): 8.35 (s, 1H), 8.10 (d, J =7.5, 1h), 7.35 (d, J =7.5, 1h), 7.05-7.19 (m, 3H), 4.41 (s, 1H), 4.30-4.38 (m, 1H), 4.18 (m, 1H), 3.82-3.84 (m, 2H), 3.07-3.14 (m, 2H), 1.55-1.78 (m, 6H), 1.26 (d, 3H).

Compound 3: LC/MS m/z =406.1, [ M ] +H ];1HNMR (dmso-d 6): 9.01 (1H), 8.52 (s, 1H), 8.10 (d, J =7.5, 1H), 7.92 (s, 1H) 7.35 (d, J =7.5, 1H), 4.69 (s, 1H), 4.35 (m, 1H), 3.50 (t, 2H), 3.07-3.14 (m, 4H), 1.55-1.78 (m, 4H).

Compound 4: LC/MS m/z =435.1[ M + H ];1HNMR (dmso-d 6): 8.56 (s, 1H), 8.35 (s, 1H), 8.10 (d, J =7.5, 1H), 7.92 (s, 1H) 7.35 (d, J =7.5, 1H), 4.41 (s, 1H), 4.38 (m, 1H), 4.13 (m, 1H), 3.87 (t, 2H), 3.07-3.14 (m, 2H), 1.55-1.78 (m, 6H), 1.27 (d, 3H).

TABLE 1 preparation of Compounds 2 to 4 starting materials and structures

The beneficial effects of the present invention are demonstrated by the following experimental examples.

Experimental example 1 inhibitory Effect of the Compound of the present invention on TRK kinase

1. Experimental methods

TK-biotin substrate, ATP, enzyme and a certain concentration of compound to be detected are reacted in a corresponding 1 x Kinase Buffer at room temperature. The final concentration of the test compound was 1uM and 0.1uM, and DMSO in the reaction system was controlled to be 1%. Mu.l of a mixed assay solution of Streptavidin-XL665 and TK antibody europeium cryptate (1The fluorescence signal (320 nm stimulation, 665nm,615nm emission) was detected with a Multilabel Reader (MD, spectraMax i3 ×). Adding TRK kinase A, after the activity is stable, adding the compound to be tested and LOXO-101, calculating the concentration of the compound to be tested which needs to be added when the activity inhibition rate of the compound to be tested on the TRK kinase reaches 50%, and marking as IC ₅₀ 。

2. Results of the experiment

TABLE 2 inhibitory Effect of each Compound on TRK kinase Activity

Compound (I)	IC ₅₀ (nM)
		LOXO-101	32
Compound 1	4.2
		Compound 2	18.2
Compound 3	24
		Compound 4	35

The results are shown in Table 2. It can be seen that the compounds of the present invention have good inhibitory effect on TRK kinase, IC ₅₀ 4.2-35 nM; especially compound 1, compound 2 and compound 3, which have even better inhibitory effect on the activity of TRK kinase than the positive control drug LOXO-101.

Experimental example 2 inhibitory Effect of the Compound of the present invention on Trk overexpression tumor cell lines

1. Experimental method

The KM12 cells (human colon cancer cells) were plated on 384-well plates, cultured for 24 hours, and test compounds of different concentrations were added to the culture well plates, 9 concentrations were set for each test compound, with the highest concentration of 10uM, according to a 1:3 proportion gradient dilution. The cells were then cultured at 37 ℃ for 24 hours. CellTiter-Glo reagent was added and detection was performed by envision method. Calculating half inhibition concentration IC of each test compound on KM12 cells ₅₀ . The positive control was the FDA-approved Trk kinase inhibitor LOXO-101, which was reported in China as a clinical trial.

2. Results of the experiment

TABLE 3 inhibitory Effect of each of the compounds on KM12 cells

The results are shown in Table 3. As can be seen, the compound of the present invention had a good inhibitory effect on KM12 cells, and its IC ₅₀ 0.014-0.102 uM; especially compound 1 and compound 4, IC thereof ₅₀ As low as 0.014uM and 0.054uM, respectively, the inhibitory effect on KM12 cells was even better than that of the positive control drug LOXO-101.

In conclusion, the invention provides a [1,2,4] triazolo [4,3-b ] pyridazine compound shown in a formula I. Experimental results show that the compound provided by the invention has a good inhibition effect on TRK kinase, and has a good inhibition effect on tumor (such as colon cancer) cells related to TRK overexpression. The compound provided by the invention has wide application prospects in preparation of TRK kinase inhibitors and medicines for preventing and/or treating tumors related to TRK overexpression.

Claims

1. A compound of formula I, or a pharmaceutically acceptable salt, or stereoisomer thereof:

wherein X is selected from N or CR ₄ ；

R ₁ ~R ₅ Each independently selected from hydrogen, halogen, and at least one of which is halogen;

R ₆ ~R ₇ each independently selected from hydrogen, C _1~2 An alkyl group;

R ₈ is a hydroxyl group;

n is an integer of 0 to 3.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: the structure of the compound is shown as formula II-1 or formula II-2:

R ₆ ~R ₇ each independently selected from hydrogen, C _1~2 An alkyl group;

n is 1 or 2;

or, the structure of the compound is shown as formula II-2:

wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ Each independently selected from hydrogen, fluorine, chlorine or bromine, and at leastTwo are fluorine;

R ₆ ~R ₇ each independently selected from hydrogen and C _1~2 An alkyl group;

n is 1 or 2.

3. The compound according to any one of claims 1 to 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, wherein: the compound is one of the following compounds:

。

4. a process for the preparation of a compound according to any one of claims 1 to 3, characterized in that: the method comprises the following steps:

(1) Reacting the compound A ' with the compound B ' to obtain a compound C ';

(2) Reacting the compound C 'with a compound D' to obtain a compound as defined in any one of claims 1 to 3;

wherein Y is halogen; x, R ₁ ~R ₈ N is as defined in any one of claims 1 to 3.

5. The method of claim 4, wherein: in the step (1), the molar ratio of the compound A 'to the compound B' is 1: (0.8 to 1.2); the reaction is carried out under the catalysis of organic base; the solvent for the reaction is an alcohol solvent; the reaction temperature is 105-115 ℃, and the reaction time is 2-5 hours; the Y is chlorine;

and/or in the step (2), the molar ratio of the compound C 'to the compound D' is 1 (3 to 10); the solvent for the reaction is an alcohol solvent; the reaction temperature is 105-115 ℃, and the reaction time is 0.5-3 hours.

6. The method of claim 5, wherein: in the step (1), the molar ratio of the compound A 'to the compound B' is 1:1; the organic base is N, N' -diisopropylethylamine or pyridine; the solvent of the reaction is n-butyl alcohol; the reaction temperature is 110 ℃, and the reaction time is 3 hours;

and/or, in the step (2), the molar ratio of the compound C 'to the compound D' is 1:4; the solvent of the reaction is n-butyl alcohol; the reaction temperature was 110 ℃ and the reaction time was 1 hour.

7. The method of claim 5, wherein: in the step (1), after the reaction is finished, the method further comprises the following purification steps: concentrating the system obtained by the reaction, removing the solvent, and then carrying out silica gel column chromatography separation, wherein the mobile phase adopted by the chromatography separation is petroleum ether: a mixed solution of ethyl acetate in a volume ratio = 2;

and/or, in the step (2), after the reaction is finished, the method further comprises the following purification steps: concentrating the system obtained by the reaction, removing the solvent, and then carrying out silica gel column chromatography separation, wherein the mobile phase adopted by the chromatography separation is dichloromethane: methanol volume ratio = 10.

8. Use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, for the preparation of a TRK kinase inhibitor.

9. Use of the compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, in the preparation of a medicament for preventing and/or treating tumors associated with TRK overexpression.

10. Use according to claim 9, characterized in that: the tumor related to TRK overexpression is salivary gland cancer, lung cancer, bile duct cancer, gastrointestinal stromal tumor, melanoma, thyroid cancer, malignant myeloid leukemia, malignant glioma, astrocytoma or colon cancer.

11. Use according to claim 10, characterized in that: the lung cancer is non-small cell lung cancer.

12. Use according to claim 10, characterized in that: the tumor related to TRK overexpression is colon cancer.

13. A medicament for treating tumors associated with TRK overexpression, comprising: the compound is a compound as defined in any one of claims 1 to 3, or a pharmaceutically acceptable salt or a stereoisomer thereof as an active ingredient, and is prepared by adding a pharmaceutically acceptable auxiliary material.