阅读说明：本技术 一种[1,2,4]三唑并[4,3-b]哒嗪类化合物及其制备方法和用途 ([1,2,4] triazolo [4,3-b ] pyridazine compound and preparation method and application thereof ) 是由 温万东 石万棋 陈齐 史焱 代明星 吴瑕 陈科 于 2020-12-24 设计创作，主要内容包括：本发明涉及一种[1,2,4]三唑并[4,3-b]哒嗪类化合物及其制备方法和用途。具体提供了式I所示化合物、或其药学上可接受的盐、或其立体异构体、或其旋光异构体、或其同位素替换形式。实验结果表明,本发明提供的化合物对TRK激酶具有良好的抑制作用,对与TRK过表达相关的肿瘤(例如结肠癌)细胞具有良好的抑制效果。本发明提供的化合物在制备TRK激酶抑制剂,以及制备预防和/或治疗与TRK过表达相关的肿瘤的药物中具有广阔的应用前景。(The 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 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.)

1. A compound of formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof:

wherein X is selected from N or CR₄；

R₁～R₅Each independently selected from hydrogen, halogen, C_1～4Alkyl or C_1～4An alkoxy group;

R₆～R₇each independently selected from hydrogen and C_1～4Alkyl or C_1～4An alkoxy group;

R₈selected from hydroxyl or carboxyl;

n is an integer of 0 to 5.

2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, wherein:

x is selected from N or CR₄；

R₁～R₅Each independently selected from hydrogen, halogen, and at least one of which is halogen;

R₆～R₇each independently selected from hydrogen and C_1～2An alkyl group;

R₈is a hydroxyl group;

n is an integer of 0 to 3.

3. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, wherein: the structure of the compound is shown as a formula II-1 or a formula II-2:

wherein R is₁、R₂、R₃、R₅Each independently selected from hydrogen, fluorine, chlorine or bromine, and at least one of which is fluorine and one is chlorine;

R₆～R₇each independently selected from hydrogen and C_1～2An alkyl group;

n is 1 or 2;

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

wherein R is₁、R₂、R₃、R₄、R₅Each independently selected from hydrogen, fluorine, chlorine or bromine, and at least two of which are fluorine;

R₆～R₇each independently selected from hydrogen and C_1～2An alkyl group;

n is 1 or 2.

4. A compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, wherein: the compound is one of the following compounds:

5. a process for preparing a compound according to any one of claims 1 to 4, wherein: 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 according to any one of claims 1 to 4;

wherein Y is halogen; x, R₁～R₈N is as defined in any one of claims 1 to 4.

6. The method of claim 5, wherein: 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; 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.

7. The method of claim 6, 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 with the volume ratio of 2: 1;

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: mixed solution of methanol in volume ratio of 10: 1.

8. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, for the preparation of a TRK kinase inhibitor.

9. Use of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, for the manufacture of a medicament for the prevention and/or treatment of a tumor associated with TRK overexpression, preferably a salivary gland carcinoma, a lung cancer, a bile duct cancer, a gastrointestinal stromal tumor, a melanoma, a non-small cell lung cancer, a thyroid cancer, a malignant myeloid leukemia, a glioblastoma, an astrocytoma, or a colon cancer, more preferably a colon cancer.

10. A medicament for treating tumors associated with TRK overexpression, comprising: the compound of any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopically substituted form thereof, as an active ingredient, together with a pharmaceutically acceptable excipient.

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, a family of which 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 constitutive activation of Trk kinase, is no longer regulated and controlled by nerve growth factor ligand, and has carcinogenic risk, such as in colon cancer KM12 cells, gene fragment rearrangement of TPM3 and NTRK1 of tropomyosin-3 gene located on the long arm of chromosome 1, breakage of TPM3 between exon 7 and exon 8, breakage of NTRK1 inside exon 8, and then direct connection of TPM3 with NTRK1 expressing intracellular structural region, thereby abnormally expressing TPM3-TRKA chimeric protein. This gene alteration disrupts the interaction of the ligand NGF with TrkA in cells, intracellular TRKA is in an over-expressed and sustained activation state, and the downstream three signaling pathways of PI3K/AKT, Ras/MAPK and PLC gamma are also in an abnormal active 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 phase 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 with an Overall Response Rate (ORR) of 78%, with 2 patients with 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 has great significance for developing drugs for treating TRK over-expressed 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 CR₄；

R₁～R₅Each independently selected from hydrogen, halogen, C_1～4Alkyl or C_1～4An alkoxy group;

R₆～R₇each independently selected from hydrogen and C_1～4Alkyl or C_1～4An alkoxy group;

R₈selected 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 CR₄；

R₁～R₅Each independently selected from hydrogen, halogen, and at least one of which is halogen;

R₆～R₇each independently selected from hydrogen and C_1～2An alkyl group;

R₈is 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 is₁、R₂、R₃、R₅Each independently selected from hydrogen, fluorine, chlorine or bromine, and at least one of which is fluorine and one is chlorine;

R₆～R₇each independently selected from hydrogen and C_1～2An alkyl group;

n is 1 or 2;

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

wherein R is₁、R₂、R₃、R₄、R₅Each independently selected from hydrogen, fluorine, chlorine or bromine, and at least two of which are fluorine;

R₆～R₇each independently selected from hydrogen and C_1～2An alkyl group;

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, R₁～R₈And 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; 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 with the volume ratio of 2: 1;

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: mixed solution of methanol in volume ratio of 10: 1.

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 given to them by a person skilled in the art are to be given in light of the disclosure and the context.

The minimum and maximum values of the carbon atom content in the hydrocarbon group are indicated by the prefix C_a～bAlkyl represents any alkyl group containing from "a" to "b" carbon atoms. E.g. C_1～4The alkyl group is a straight-chain or branched alkyl group having 1 to 4 carbon atoms.

C_1～4The alkoxy group means a straight chain or branched chain alkoxy group having 1 to 4 carbon atoms.

Halogen is fluorine, chlorine, bromine or iodine.

"isotopically-substituted forms" refer to compounds wherein one or more than two atoms are replaced by the corresponding isotope. 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 compound¹²Quilt carbon¹¹Or carbon¹³The compound obtained after replacement.

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

"salts" are acid and/or base salts of a compound or a stereoisomer thereof with inorganic and/or organic acids and/or bases, and also include zwitterionic (inner) salts, as well as 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 commercial products.

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 a compound a0.40g (i.e., 2.21mmol), a compound b0.50g (i.e., 2.21mmol), 2.5mL of N, N' -Diisopropylethylamine (DIPEA), and 10mL of N-butanol, and the reaction was stirred at 110 ℃ for 3 hours, TLC showed disappearance of the starting material and completion of the reaction. 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 and ethyl acetate at a volume ratio of 2: 1), to obtain 0.73g of an off-white solid, i.e., compound C, in a yield of 91.7%. LC/MS: M/z 360.1[ M + H ].

(2) Taking 0.50g (namely 1.4mmol) of the compound C, 0.37g (namely the compound D) of ethanolamine (namely 6.0mmol) and 10mL of n-butanol, adding the mixture into a reaction flask, stirring the reaction system at 110 ℃ for 1h, and TLC shows that the raw materials disappear and the reaction is complete. The solvent was removed by concentration under reduced pressure, and the obtained concentrate was subjected to silica gel column chromatography (mobile phase was a mixed solution of dichloromethane and methanol at a volume ratio of 10: 1) to obtain 0.45g of compound 1, with a yield of 83.8%. LC/MS M/z 389.1[ M + H ]]；¹HNMR(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

The compounds A, D in example 1 were replaced with the corresponding compounds in table 1, and the same synthetic routes and methods as those in example 1 were used to prepare the compounds 2 to 4 of the present invention.

The structural characterization of the compounds 2-4 is as follows:

compound 2: LC/MS M/z 417.4[ M + H ]; 1HNMR (dmso-d6):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-d6):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-d6):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).

Preparation raw materials and structures of compounds 2 to 4 in Table 1

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 TKantibodyeuropium cryptate (1:100) diluted with 50mM Hepes/NaOH pH 7.0, 0.1% BSA, 0.8M KF, 20mM EDTA was added to all reaction wells, and after 1h reaction at room temperature, fluorescence signals (320nm stimulation, 665nm, 615nm emission) were detected using a Multilabel Reader (MD, SpectraMax i3x) instrument. 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 IC₅₀。

2. Results of the experiment

TABLE 2 inhibitory Effect of each Compound on TRK kinase Activity

Compound (I)	IC50(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, IC₅₀4.2-35 nM; especially compound 1, compound 2 and compound 3, which have even better inhibitory effects 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 methods

The KM12 cells (human colon cancer cells) were plated on 384-well plates, cultured for 24 hours, and test compounds at different concentrations were added to the culture plates, each test compound was set at 9 concentrations, 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 the half inhibitory concentration IC of each test compound on KM12 cells₅₀. 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 has a good inhibitory effect on KM12 cells, and its IC₅₀0.014-0.102 uM; especially compound 1 and compound 4, IC thereof₅₀As 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 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.