WO2017211245A1 - Substituted pyrrolopyrimidine compound and application thereof - Google Patents

Abstract

Provided are a substituted pyrrolopyrimidine compound and composition containing said compound, and an application thereof. Specifically disclosed is the pyrrolopyrimidine compound represented by formula (I), or a pharmaceutical composition of its crystalline form, pharmaceutically acceptable salt, prodrug, stereoisomer, hydrate, or solvate. The compound may be used as a cyclin-dependent kinase (CDK) inhibitor, and thus is suitable for use as a drug to treat CDK-related diseases (such as breast cancer).

Description

Substituted pyrrolopyrimidine compound and application thereof Technical field

The invention belongs to the field of medicine. In particular, the present invention relates to a substituted pyrrolopyrimidine compound and use thereof, and more particularly to a pyrrolopyrimidine compound and a pharmaceutical composition thereof for use as a CDK inhibitor for the treatment and prevention of diseases associated with CDK inhibitors .

Background technique

Cyclin-dependent kinase (CDK) is a serine-threonine protein kinase that plays a key role in regulating the transition between different phases of the cell-cycle, such as from the quiescent phase of G1 (mitosis and A new round of cell division begins with a pause in DNA replication to the progression of S (active DNA synthesis), or progression from G2 to M phase, in which active mitosis and cell division occur.

CDK complexes regulate the cyclin subunits (eg, cyclin A, B1, B2, D1, D2, D3, and E) and catalytic kinase subunits (eg, CDK1, CDK2, CDK4, CDK5, and CDK6) Formed together. As implied by the name, CDKs show absolute dependence on cyclin subunits in order to phosphorylate their target substrates, and different kinase/cyclin pairs act to regulate progression through specific phases of the cell-cycle. These protein kinases are a class of proteins (enzymes) that regulate a variety of cellular functions. This is accompanied by phosphorylation of specific amino acids on the protein substrate, resulting in a conformational change in the substrate protein. A conformational change modulates the activity of a substrate or its ability to interact with other binding ligands. The enzymatic activity of a protein kinase refers to the rate at which a kinase adds a phosphate group to a substrate. It can be measured, for example, by measuring the amount of substrate converted to product as a function of time. Phosphorylation of the substrate occurs at the active site of the protein kinase.

The activity of CDK is regulated in a post-translational manner by transient association with other proteins by altering their intracellular localization. Tumor development is closely related to genetic changes and dysregulation of CDK and its regulatory factors, suggesting that CDK inhibitors may be useful anticancer therapeutics. Early results indicate that transformed cells and normal cells differ in their demand for, for example, cyclin A/CDK2, and it is possible to develop novel antibiotics that lack the general host toxicity observed with conventional cytotoxic drugs and cytostatic drugs. Tumor agent. Although inhibition of cell cycle-associated CDK is clearly associated with, for example, oncology applications, inhibition of RNA polymerase-regulated CDK can also be highly correlated with cancer indications.

CDK has been shown to be involved in cell cycle progression and cellular transcription, and loss of growth control is associated with abnormal cell proliferation of the disease (see, eg, Malumbres and Barbacid, Nat. Rev. Cancer 2001, 1: 222). Increased or transient abnormal activation of cyclin-dependent kinases has been shown to result in the development of human tumors (Sherr C. J., Science 1996, 274: 1672-1677).

Many diseases are associated with abnormal cellular responses triggered by the above protein kinase-mediated events. These diseases include, but are not limited to, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cancer, cardiovascular diseases, metamorphosis Response to asthma, Alzheimer's disease and hormone-related diseases. Therefore, a great deal of efforts have been made in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents.

The onset, progression, and end of the mammalian cell cycle are regulated by a variety of cyclin-dependent kinase (CDK) complexes that are critical for cell growth. These complexes contain at least the catalytic (CDK itself) and regulatory (cyclin) subunits. Some of the more important complexes for cell cycle regulation include cyclin A (CDK1 and CDK2), cyclin B1-B3 (CDK1), and cyclin D1-D3 (CDK2, CDK4, CDK5, CDK6), cells Cyclin E (CDK2). Each of these complexes is involved in a particular phase of the cell cycle. However, not all members of the CDK family are involved in cell cycle control alone. Thus, CDK7, CDK8 and CDK9 are involved in the regulation of transcription, and CDK5 plays a role in neuronal and secretory cell function.

Palbociclib is the first CDK4/6 inhibitor approved by the US FDA for breast cancer treatment. Because all living cells are undergoing cell division, and Palbociclib has the ability to block the cell division process (also known as the "cell cycle"), it has the potential for broad applicability. Palbociclib in combination with other anti-cancer therapies such as endocrine therapy, chemotherapy, and targeted therapies may be effective against a variety of cancers. Clinical trials, whether breast cancer or other cancers, have shown that it is safe to administer Palbociclib once a day. The main side effect is reversible neutropenia, when the count is reduced, side effects should be suspended, and lower doses should be restarted. Medication. Other side effects include fatigue (33%), nausea (30%), diarrhea (18%), constipation (12%), and rash (12%). In addition, Novartis's CDK inhibitor Ribociclib (LEE011) is still in Phase III clinical trials.

Thus, there is still a need to develop inhibitors of protein kinases such as CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK9; new treatments and therapies for disorders associated with protein kinases are still needed. There remains a need for compounds that can be used to treat or prevent or ameliorate one or more symptoms of cancer, graft rejection, and autoimmune diseases.

Summary of the invention

In view of the above technical problems, the present invention discloses a pyrrolopyrimidine compound and a composition comprising the same as an effective cyclin-dependent kinase (CDK) inhibitor and/or with better pharmacodynamics/ Pharmacokinetic properties.

In this regard, the technical solution adopted by the present invention is:

It is an object of the present invention to provide a novel class of potent cyclin dependent kinase (CDK) inhibitors and/or compounds having better pharmacodynamic/pharmacokinetic properties.

In a first aspect of the invention, there is provided a pyrrolopyrimidine compound of the formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate or a solvent compound:

In the formula:

R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ And R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ are each independently selected from the group consisting of “hydrogen (H), 氘 (D)”;

And physiologically acceptable salts, prodrugs, hydrates, solvates, tautomers and stereoisomers thereof, including mixtures of these compounds in all ratios;

Additionally, the pyrrolopyrimidine compound contains at least one ruthenium atom.

In another preferred embodiment, the cerium isotope content of the cerium in the deuterated position is at least greater than the natural strontium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, and even more preferably greater than 75%. Preferably, the ground is greater than 95%, more preferably greater than 99%.

Specifically, in the present invention, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ and R ¹⁴ , The osmium isotope content of each of the R ⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ is at least 5%. More preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, still more preferably greater than 40%, More preferably more than 45%, more preferably more than 50%, more preferably more than 55%, more preferably more than 60%, more preferably more than 65%, more preferably more than 70%, more preferably more than 75%, more Preferably, it is greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, and even more preferably greater than 99%.

In another preferred embodiment, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² of the compound of formula (I), R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , at least one of R氘, preferably, two R 氘, more preferably three R 氘, more preferably four R 氘, more preferably five R 氘, more preferably six R 氘, more preferably Seven R contain ruthenium, more preferably eight R 氘, more preferably nine R 氘, more preferably ten R 氘, more preferably eleven R 氘, more preferably twelve R氘, preferably thirteen R 氘, more preferably fourteen R 氘, more preferably fifteen R 氘, more preferably sixteen R 氘, more preferably seventeen R氘, preferably 18 R, 更, preferably 19, R, 更, preferably 20, R, preferably 21, R, 二十, 22 R contains 氘, more preferably Twenty-three R contains 氘, more preferably twenty-four R contains 氘, more preferably twenty-five R contains 氘, more preferably twenty-six R contains 氘, more Good land twenty-seven R containing 氘.

As a further improvement of the present invention, R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁷ and R ⁸ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ⁹ , R ¹⁰ and R ¹¹ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ¹² and R ¹³ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ and R ²¹ are each independently hydrazine or hydrogen.

As a further improvement of the present invention, R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ are each independently hydrazine or hydrogen.

In another preferred embodiment, the compound is selected from the group consisting of the compounds or pharmaceutically acceptable salts thereof, but is not limited to the following compounds:

In another preferred embodiment, the compound does not include a non-deuterated compound and only compounds in which R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ^{27 are} all deuterated.

In a second aspect of the invention, there is provided a method of preparing a pharmaceutical composition comprising the steps of: pharmaceutically acceptable carrier and a compound of the first aspect of the invention, or a crystalline form thereof, pharmaceutically acceptable The accepted salt, hydrate or solvate is mixed to form a pharmaceutical composition.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound of the first aspect of the invention, or a crystalline form thereof, a pharmaceutically acceptable salt, hydrated Or a solvate.

Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, any glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents A dispersing agent, a disintegrating agent, a suspending agent, a stabilizer, an isotonic agent, a solvent or an emulsifier.

The pharmaceutical composition of the present invention can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, solutions, suppositories, injections, inhalants, coagulation Glues, microspheres and aerosols.

Typical routes of administration of the pharmaceutical compositions of the invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal , intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.

The pharmaceutical composition of the present invention can be produced by a method known in the art, such as a conventional mixing method, a dissolution method, a granulation method, a sugar-coating method, a pulverization method, an emulsification method, a freeze-drying method, and the like.

The compounds of the invention are useful as inhibitors of cyclin dependent kinases. For example, the compounds of the invention are inhibitors of cyclin-dependent kinases, particularly preferred cyclin-dependent kinases are selected from the group consisting of CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK9, more particularly preferably selected from the group consisting of CDK1, CDK2 CDK3, CDK4, CDK5 and CDK9.

CDK plays a role in cell cycle regulation, apoptosis, transcription, differentiation and CNS function. Thus, CDK inhibitors are useful in the treatment of diseases with cell proliferation, apoptosis or differentiation disorders such as cancer. In particular, RB+ve tumors may be particularly sensitive to CDK inhibitors. These include tumor harboring mutations in ras, Raf, growth factor receptors or overexpression of growth factor receptors. Hypermethylated promoter regions with CDK inhibitors and cyclin-dependent kinase overexpressing cyclin partners additional tumors may also show sensitivity. RB-ve tumors can also be sensitive to CDK inhibitors.

Examples of cancers that can be inhibited include, but are not limited to, cancers such as bladder cancer, breast cancer, colon cancer (eg, colorectal cancer such as colon adenocarcinoma and colon adenoma), kidney cancer, epidermal cancer, liver cancer, lung cancer such as adenocarcinoma , small cell lung cancer and non-small cell lung cancer, esophageal cancer, gallbladder cancer, ovarian cancer, pancreatic cancer such as exocrine pancreatic cancer, gastric cancer, cervical cancer, thyroid cancer, nasal cancer, head and neck cancer, prostate cancer or skin cancer such as squamous cell carcinoma Lymphoid hematopoietic tumors, such as leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma (eg, diffuse large B-cell lymphoma), T-cell lymphoma, multiple myeloma, Hodge Gold lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, or Burkitt's lymphoma; myeloid hematopoietic tumors, such as acute and Chronic myeloid leukemia, myelodysplastic syndrome or promyelocytic leukemia; thyroid follicular carcinoma; derived from stromal cell tumors, such as fibrosarcoma or rhabdomyosarcoma; central or peripheral nervous system tumors, such as astrocytoma, nerve Maternal tumor, glioma or schwannomas; melanoma; seminoma; teratoma; osteosarcoma; xeroderma pigmentosum; keratoacanthoma; thyroid follicular carcinoma or Kaposi's sarcoma .

The cyclin dependent kinase inhibitors of the invention can be used in combination with other anticancer agents. For example, cyclin dependent kinase inhibitors have been used in combination therapy with other anticancer drugs.

Thus, in a pharmaceutical composition, the use or method of the invention for treating a disease or condition comprising abnormal cell growth, in one embodiment the disease or condition comprising abnormal cell growth is cancer.

One type of cancer includes human breast cancer (eg, primary breast tumor, node-negative breast cancer, breast invasive ductal adenocarcinoma, non-endometrioid breast cancer); and mantle cell lymphoma. In addition, other cancers are colorectal cancer and endometrial cancer.

Another subtype of cancer includes lymphoid hematopoietic tumors such as leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, and B-cell lymphoma (eg, diffuse large B-cell lymphoma).

Another subtype of cancer that may be useful in the treatment of a compound of the invention includes sarcoma, leukemia, glioma, familial melanoma, and melanoma.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

Herein, "halogen" means F, Cl, Br, and I unless otherwise specified. More preferably, the halogen atom is selected from the group consisting of F, Cl and Br.

As used herein, unless otherwise specified, "deuterated" means that one or more hydrogens in the compound or group are replaced by deuterium; deuteration may be monosubstituted, disubstituted, polysubstituted or fully substituted. The terms "one or more deuterated" are used interchangeably with "one or more deuterated".

As used herein, unless otherwise specified, "non-deuterated compound" means a compound containing a proportion of germanium atoms not higher than the natural helium isotope content (0.015%).

The invention also includes isotopically labeled compounds, equivalent to the original compounds disclosed herein. Examples of isotopes which may be listed as compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine isotopes such as ² H, ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, respectively. , ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl. a compound, or an enantiomer, a diastereomer, an isomer, or a pharmaceutically acceptable salt or solvate of the present invention, wherein an isotope or other isotopic atom containing the above compound is within the scope of the present invention . Certain isotopically-labeled compounds of the present invention, such as the radioisotopes of ³ H and ¹⁴ C, are also among them, useful in tissue distribution experiments of drugs and substrates.氚, ie ³ H and carbon-14, ie ¹⁴ C, are easier to prepare and detect and are preferred in isotopes. Isotopically labeled compounds can be prepared in a conventional manner by substituting a readily available isotopically labeled reagent with a non-isotopic reagent using the protocol of the examples.

Pharmaceutically acceptable salts include inorganic and organic salts. A preferred class of salts are the salts of the compounds of the invention with acids. Suitable acids for forming salts include, but are not limited to, mineral acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid; formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, Organic acids such as fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, benzoic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid; Amino acids such as amino acid, phenylalanine, aspartic acid, and glutamic acid. Another preferred class of salts are the salts of the compounds of the invention with bases, such as alkali metal salts (for example sodium or potassium salts), alkaline earth metal salts (for example magnesium or calcium salts), ammonium salts (for example lower alkanolammonium). Salts and other pharmaceutically acceptable amine salts), such as methylamine, ethylamine, propylamine, dimethylamine, trimethylamine, diethylamine, triethylamine, tert-butyl A base amine salt, an ethylenediamine salt, a hydroxyethylamine salt, a dihydroxyethylamine salt, a trihydroxyethylamine salt, and an amine salt formed of morpholine, piperazine, and lysine, respectively.

The term "solvate" refers to a complex of a compound of the invention that is coordinated to a solvent molecule to form a specific ratio. "Hydrate" means a complex formed by the coordination of a compound of the invention with water.

The invention also provides a pharmaceutical composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt of said compound, and a pharmaceutically acceptable carrier. The carrier is "acceptable" in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, in a quantity which is not deleterious to the recipient thereof.

The compounds of formula (I) and compositions comprising the compounds are CDK inhibitors and can be used to treat, prevent or ameliorate various CDK related disorders. Pharmaceutical compositions comprising these compounds are useful for treating, preventing, or slowing the progression of the disease or disorder in different therapeutic areas, such as cancer.

Compared with the prior art, the beneficial effects of the present invention are that the substituted pyrrolopyrimidine compound disclosed in the present invention and the composition comprising the same have excellent inhibition to CDK and have better pharmacokinetic parameter characteristics. . The dosage can be varied and a long acting formulation can be formed to improve suitability. Replacing a hydrogen atom in a compound with hydrazine can increase the drug concentration of the compound in an animal to improve the efficacy of the drug due to its strontium isotope effect. Substitution of a hydrogen atom in a compound with hydrazine may increase the safety of the compound due to inhibition of certain metabolites.

detailed description

The preparation of the structural compound of the formula (I) of the present invention is more specifically described below, but these specific methods do not constitute any limitation to the present invention. The compounds of the present invention may also be conveniently prepared by combining various synthetic methods described in the specification or known in the art, and such combinations are readily made by those skilled in the art to which the present invention pertains.

Usually, in the preparation scheme, each reaction is usually carried out in an inert solvent at room temperature to reflux temperature (e.g., 0 ° C to 100 ° C, preferably 0 ° C to 80 ° C). The reaction time is usually from 0.1 to 60 hours, preferably from 0.5 to 24 hours.

Example 1 Preparation of intermediate 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound 7)

The specific synthesis steps are as follows:

Step 1. Synthesis of [2-chloro-5-(3,3-diethoxy-prop-1-ynyl)-pyrimidin-4-yl]-cyclopentylamine (Compound 3).

(5-Bromo-2-chloro-pyrimidin-4-yl)-cyclopentylamine (2.0 g, 7.3 mmol) and propargaldehyde diethyl diacetal (1.1 g, 8.6 mmol), Pd (dppf) Cl ₂ (510 mg, 0.73 mmol), CuI (140 mg, 0.73 mmol) and 5 mL of triethylamine were added to 20 mL of dimethylformamide (DMF), and the mixture was replaced with nitrogen three times, and the mixture was warmed to 100 ° C and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature. EtOAc was evaporated. ^{1 H NMR (300MHz, DMSO-} d 6) δ8.18 (s, 1H), 7.20 (d, J = 7.6Hz, 1H), 5.56 (s, 1H), 4.33 (t, J = 7.5Hz, 1H) , 3.63 (ddq, J = 35.4, 9.6, 7.1 Hz, 4H), 1.93 (s, 2H), 1.76 - 1.47 (m, 6H), 1.16 (t, J = 7.1 Hz, 6H); ESI-MS: 324 [M ⁺ +1].

Step 2. Synthesis of 2-chloro-7-cyclopentyl-6-diethoxymethyl-7H-pyrrolo[2,3-d]pyrimidine (Compound 4).

[2-Chloro-5-(3,3-diethoxy-prop-1-ynyl)-pyrimidin-4-yl]-cyclopentylamine (1.01 g, 3.1 mmol) was dissolved in 10 mL of tetrahydrofuran and added Tetrabutylammonium fluoride (2.4 g, 9.3 mmol) was reacted at 65 ° C for 2 hours. The solvent was evaporated, the mixture was evaporated, evaporated, evaporated, evaporated, evaporated. ESI-MS: 324 [M ⁺ +1].

Step 3. Synthesis of 2-chloro-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidin-6-carboxaldehyde (Compound 5).

2-Chloro-7-cyclopentyl-6-diethoxymethyl-7H-pyrrolo[2,3-d]pyrimidine (830 mg, 2.6 mmol) was dissolved in 10 mL of dioxane, and 4 mL was slowly added dropwise. Concentrated hydrochloric acid, stirred at room temperature for 20 minutes, added with 15 mL of water, extracted with EtOAc, EtOAc (EtOAc)EtOAc. . ESI-MS: 250 [M ⁺ +1].

Step 4. Synthesis of 2-chloro-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid (Compound 6).

2-Chloro-7-cyclopentyl-6-diethoxymethyl-7H-pyrrolo[2,3-d]pyrimidin-6-carboxaldehyde (525 mg, 2.1 mmol) was dissolved in 5 mL of DMF and added with peroxy Potassium monosulfonate (1.4 g, 2.3 mmol) was reacted at room temperature for 6 hours. After the completion of the reaction, 10 mL of water was added, and a large amount of solid was precipitated, and dried by filtration to give 475 mg of pale yellow solid. ESI-MS: 266 [M ⁺ +1].

Step 5. Synthesis of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound 7).

2-Chloro-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid (475 mg, 1.8 mmol), O-benzotriazole-tetramethylurea hexafluorophosphate The ester (HBTU, 680 mg, 1.8 mmol) and N,N-diisopropylethylamine (DIEA, 0.9 mL, 5.4 mmol) were dissolved in 10 mL DMF, and 2M dimethylamine in methanol (1.1 mL) After the completion of the dropwise addition, the reaction was carried out for 30 minutes at room temperature, 20 mL of water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with 20 mL of brine, dried over anhydrous sodium sulfate and evaporated. The yield is 80%. ESI-MS: 293 [M ⁺ +1].

Example 2 Preparation of 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl-2,2,3,3,5,5,6,6-d8) Pyridin-2-yl)amino)-7H-pyridyl P-[2,3-d]pyrimidine-6-carboxamide (Compound 14)

The specific synthesis steps are as follows:

Step 1. Synthesis of 1-(6-nitropyridin-3-yl)piperazine-2,2,3,3,5,5,6,6-d8 (Compound 10).

5-Bromo-2-nitropyridine (202 mg, 1 mmol) was dissolved in 10 mL of n-butanol, triethylamine (0.7 mL, 5 mmol) and piperazine-2,2,3,3,5,5,6, 6-d8 hydrochloride (332 mg, 2 mmol), EtOAc (EtOAc: EtOAc. The 175 mg of a yellow solid was obtained by a silica gel column, yield 81%. ESI-MS: 217 [M ⁺ +1].

Step 2. Synthesis of 4-(6-nitropyridin-3-yl)piperazine-2,2,3,3,5,5,6,6-d8-1-carboxylic acid tert-butyl ester (Compound 11) .

1-(6-Nitropyridin-3-yl)piperazine-2,2,3,3,5,5,6,6-d8 (175 mg, 0.8 mmol) was dissolved in 10 mL of dichloromethane. Ethylamine (0.22 mL, 1.6 mmol) and (Boc) ₂ O (210 mg, 0.96 mmol) were stirred at room temperature overnight. After the reaction was completed, 10 mL of water was added, and the layers were separated and the aqueous phase was extracted with dichloromethane. The mixture was washed with saturated brine, dried over anhydrous sodium sulfate and evaporated. _{^{1 H NMR (400MHz, CDCl 3}} ) δ8.18 (d, J = 9.1Hz, 1H), 8.12 (d, J = 3.0Hz, 1H), 7.20 (dd, J = 9.2,3.1Hz, 1H), 1.49 (s, 9H); ESI-MS: 317 [M ⁺ +1].

Step 3. Synthesis of 4-(6-aminopyridin-3-yl)piperazine-2,2,3,3,5,5,6,6-d8-1-carboxylic acid tert-butyl ester (Compound 12).

Dissolve 4-(6-nitropyridin-3-yl)piperazine-2,2,3,3,5,5,6,6-d8-1-carboxylic acid tert-butyl ester (230 mg, 0.73 mmol) 10 mL of isopropanol was added to 30 mg of 10% palladium carbon, and the mixture was replaced with hydrogen three times, and stirred at room temperature under a hydrogen atmosphere of 1 atm. After completion of the reaction, the palladium carbon was filtered off, and the filtrate was concentrated. _{^{1 H NMR (400MHz, CDCl 3}} ) δ7.73 (d, J = 2.9Hz, 1H), 7.19 (dd, J = 8.9,2.9Hz, 1H), 6.51 (dd, J = 8.8,0.7Hz, 1H) , 4.37 (s, 2H), 1.48 (s, 9H); ESI-MS: 287 [M ⁺ +1].

Step 4. 4-(6-((7-Cyclopentyl-6-dimethylamide-7-H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-pyridin-3-yl) Synthesis of piperazine-2,2,3,3,5,5,6,6-d8-1-carboxylic acid tert-butyl ester (Compound 13).

2-Chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-6-carboxamide (90 mg, 0.3 mmol) was dissolved in 5 mL of methyl isobutyl ketone Palladium acetate (3 mg, 0.015 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (BINAP, 18 mg, 0.03 mmol), cesium carbonate (146 mg, 0.45 mmol) and 4- (6-Amino-pyridin-3-yl)-piperazine-2,2,3,3,5,5,6,6-d8-1-carboxylic acid tert-butyl ester (97 mg, 3.3 mmol), replaced with nitrogen three times The reaction mixture was reacted at 110 ° C overnight. After the completion of the reaction, the mixture was cooled to room temperature, and the mixture was evaporated. _{^{1 H NMR (300MHz, CDCl 3}} ) δ8.71 (s, 1H), 8.36 (s, 1H), 8.14 (s, 1H), 7.98 (s, 1H), 7.33 (dd, J = 9.1,3.0Hz, 1H), 6.45 (s, 1H), 4.77 (p, J = 8.8 Hz, 1H), 3.15 (s, 6H), 2.08-2.13 (m, 6H), 1.70 (s, 2H), 1.48 (s, 9H) ); ESI-MS: 543 [M ⁺ +1].

Step 5. 7-Cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl-2,2,3,3,5,5,6,6-d8)pyridine Synthesis of 2-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound 14).

4-(6-((7-Cyclopentyl-6-dimethylamide-7-H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-pyridin-3-yl)piperazine -2,2,3,3,5,5,6,6-d8-1-carboxylic acid tert-butyl ester (110 mg, 2 mmol) was dissolved in 5 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added and stirred at room temperature for 2 hours. The solvent was evaporated under reduced pressure. EtOAc (EtOAc m.) The yield is 90%. _{^{1 H NMR (400MHz, CDCl 3}} ) δ8.68 (s, 1H), 8.35 (d, J = 9.1Hz, 1H), 7.99 (d, J = 2.9Hz, 1H), 7.81 (s, 1H), 7.32 (dd, J = 9.1, 3.0 Hz, 1H), 6.43 (s, 1H), 4.82 - 4.75 (m, 1H), 3.15 (s, 4H), 2.58 (s, 2H), 2.04 (m, 4H), 1.71 (m, 4H); ESI-MS: 443 [M ⁺ +1].

Example 3 Preparation of 7-Cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl-3,4,6-d3)amino)-7H -pyrrole [2,3-d]pyrimidine-6-carboxamide (Compound 19)

The specific synthesis steps are as follows:

Step 1. Synthesis of 2-amino-5-(piperazin-1-yl)pyridine-3,4,6-d3 (Compound 16).

4-(6-Aminopyridin-3-yl)piperazine-1-carboxylic acid tert-butyl ester (556 mg, 2 mmol) was added to 10 mL of a 10% aqueous solution of sodium decoxide, and reacted at 200 ° C for 5 hours under nitrogen atmosphere. . After cooling to room temperature, 10 mL of water was added, and the mixture was extracted with methylene chloride. The organic phase was washed with 10 mL of brine, dried over anhydrous sodium sulfate and evaporated. _{^{1 H NMR (300MHz, CDCl 3}} ) δ5.62 (s, 2H), 3.40-3.44 (m, 4H), 2.83-2.89 (m, 4H); ESI-MS: 182 [M + +1].

Step 2. Synthesis of tert-butyl 4-(6-aminopyridin-3-yl-2,4,5-d3)piperazine-1-carboxylate (Compound 17).

2-Amino-5-(piperazin-1-yl)pyridine-3,4,6-d3 (145 mg, 0.8 mmol) was dissolved in dichloromethane (10 mL) and triethylamine (0.22 mL, 1.6 mmol) (Boc) ₂ O (210 mg, 0.96 mmol), stirred at room temperature overnight, after the reaction was completed, 10 mL of water was added, the mixture was stirred and the aqueous phase was extracted with dichloromethane, and the organic phase was combined and washed with 20 mL of brine. The mixture was dried, concentrated, and purified by silica gel column, to yield brown solid (yield: 20%). _{^{1 H NMR (300MHz, CDCl 3}} ) δ5.65 (s, 2H), 3.41-3.45 (m, 4H), 2.82-2.88 (m, 4H), 1.41 (s, 9H); ESI-MS: 282 [M ⁺ +1].

Step 3. 4-(6-((7-Cyclopentyl-6-dimethylamide-7-H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-pyridin-3-yl- Synthesis of 2,4,5-d3) piperazine-1-carboxylic acid tert-butyl ester (Compound 18).

2-Chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-6-carboxamide (90 mg, 0.3 mmol) was dissolved in 5 mL of methyl isobutyl ketone Palladium acetate (3 mg, 0.015 mmol), BINAP (18 mg, 0.03 mmol), cesium carbonate (146 mg, 0.45 mmol) and 4-(6-aminopyridin-3-yl-2,4,5-d3) Tert-butyl piperazine-1-carboxylate (93 mg, 0.33 mmol) was replaced with nitrogen three times and the reaction mixture was reacted at 110 ° C overnight. After the completion of the reaction, 10 mL of water was added, and the mixture was extracted with ethyl acetate. _{^{1 H NMR (300MHz, CDCl 3}} ) δ8.71 (s, 1H), 7.98 (s, 1H), 6.46 (s, 1H), 4.77 (t, J = 8.8Hz, 1H), 3.60 (t, J = 5.1 Hz, 4H), 3.12 (d, J = 17.7 Hz, 10H), 2.54 (s, 2H), 2.04 (s, 4H), 1.73 (s, 2H), 1.49 (s, 9H); ESI-MS: 538 [M ⁺ +1].

Step 4. 7-Cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl-3,4,6-d3)amino)-7H- Synthesis of pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound 19).

4-(6-((7-Cyclopentyl-6-dimethylamide-7-H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)-pyridin-3-yl-2, 4,5-d3) piperazine-1-carboxylic acid tert-butyl ester (116 mg, 2.2 mmol) was dissolved in 5 mL of dichloromethane, 2 mL of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 2 hours, and the solvent was evaporated under reduced pressure. The sodium carbonate solution was stirred for 10 minutes, and the mixture was extracted with methylene chloride. _{^{1 H NMR (300MHz, CDCl 3}} ) δ8.69 (s, 1H), 8.00 (s, 1H), 6.44 (s, 1H), 4.78 (t, J = 9.0Hz, 1H), 3.26 (s, 8H) , 3.15 (s, 6H), 2.57 (s, 2H), 2.12 (s, 4H), 1.67 (s, 2H); ESI-MS: 438 [M ⁺ +1].

Example 4 Preparation of 7-(cyclopentyl-3,4-d2)-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H -pyrrolo[2,3-d] Pyrimidine-6-carboxamide (compound 30)

The specific synthesis steps are as follows:

Step 1. Synthesis of cyclopentylamine-3,4-d2 (Compound 21).

3-Cyclopentenylamine (1.66 g, 20 mmol) was dissolved in 20 mL of CH ₃ OD, 160 mg of 10% palladium carbon was added, and the mixture was replaced by helium gas three times, and stirred at room temperature under a helium atmosphere of 1 atmosphere overnight. After completion of the reaction, palladium carbon was filtered off, and the filtrate was concentrated to give a red-brown solid, 1.57 g, yield 90%. ESI-MS: 88 [M ⁺ +1].

Step 2. Synthesis of 5-bromo-2-chloro-N-(cyclopentyl-3,4-d2)pyrimidine-4-amine (Compound 23).

5-Bromo-2,4-dichloropyrimidine (3.6 g, 16 mmol) was dissolved in 20 mL of absolute ethanol, triethylamine (4.4 mL, 32 mmol) was added, and cyclopentylamine-3,4- was slowly added dropwise in an ice bath. After the dropwise addition, the mixture was stirred at room temperature for 5 hr. _{^{1 H NMR (400MHz, CDCl 3}} ) δ8.09 (s, 1H), 5.44 (s, 1H), 4.40 (p, J = 6.9Hz, 1H), 2.12 (dt, J = 12.9,6.3Hz, 2H) , 1.75 (d, J = 7.2 Hz, 1H), 1.67 (s, 1H), 1.46 (tt, J = 13.7, 6.3 Hz, 2H); ESI-MS: 278 [M ⁺ +1].

Step 3. 2-Chloro-N-(cyclopentyl-3,4-d2)-5-(3,3-diethoxypropyl-1-yn-1-yl)pyrimidine-4-amine (compound) 24) Synthesis.

5-Bromo-2-chloro-N-(cyclopentyl-3,4-d2)pyrimidin-4-amine (2.0 g, 7.3 mmol) and propargaldehyde diethyl diacetal (1.1 g, 8.6 mmol) ), Pd(dppf)Cl ₂ (510 mg, 0.73 mmol), CuI (140 mg, 0.73 mmol) and 5 mL of triethylamine were added to 20 mL of DMF, replaced with nitrogen three times, and then warmed to 100 ° C, and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, 40 mL of water was added, and the mixture was evaporated. . ESI-MS: 326 [M ⁺ +1].

Step 4. Synthesis of 2-chloro-7-(cyclopentyl-3,4-d2)-6-(diethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (Compound 25) .

2-Chloro-N-(cyclopentyl-3,4-d2)-5-(3,3-diethoxypropyl-1-yn-1-yl)pyrimidine-4-amine (1.01 g, 3.1 mmol) was dissolved in 10 mL of tetrahydrofuran, tetrabutylammonium fluoride (2.4 g, 9.3 mmol) was added, and the mixture was reacted at 65 ° C for 2 hours. The solvent was evaporated, the mixture was evaporated, evaporated, evaporated, evaporated, evaporated. ESI-MS: 326 [M ⁺ +1].

Step 5. Synthesis of 2-chloro-7-(cyclopentyl-3,4-d2)-7H-pyrrolo[2,3-d]pyrimidin-6-carboxaldehyde (Compound 26).

Dissolving 2-chloro-7-(cyclopentyl-3,4-d2)-6-(diethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine (830 mg, 2.6 mmol) 10 mL of dioxane, 4 mL of concentrated hydrochloric acid was added dropwise, and the mixture was stirred at room temperature for 20 minutes, 15 mL of water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with 10 mL of brine, dried over anhydrous sodium sulfate The pale yellow solid was 525 mg in a yield of 82%. ESI-MS: 252 [M ⁺ +1].

Step 6. Synthesis of 2-chloro-7-(cyclopentyl-3,4-d ₂ )-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid (Compound 27).

2-Chloro-7-(cyclopentyl-3,4-d2)-7H-pyrrolo[2,3-d]pyrimidin-6-carboxaldehyde (525 mg, 2.1 mmol) was dissolved in 5 mL of DMF, and a peroxy Potassium sulfonate (1.4 g, 2.3 mmol) was reacted at room temperature for 6 hours. After the completion of the reaction, 10 mL of water was added, and a large amount of solid was precipitated, and dried by filtration to give 475 mg of pale yellow solid. ESI-MS: 268 [M ⁺ +1].

Step 7. 2-Chloro-7-(cyclopentyl-3,4-d2)-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-6-carboxamide (Compound 28) Synthesis.

2-Chloro-7-(cyclopentyl-3,4-d2)-7H-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid (475 mg, 1.8 mmol), HBTU (680 mg, 1.8 mmol) And DIEA (0.9mL, 5.4mmol) dissolved in 10mL DMF, 2M dimethylamine methanol solution (1.1mL, 2.2mmol) was added dropwise on ice bath, the reaction was completed at room temperature for 30 minutes, adding 20mL of water, with acetic acid The organic layer was washed with 20 mL of brine, dried over anhydrous sodium sulfate, and evaporated. ESI-MS: 295 [M ⁺ +1].

Step 8. 4-(6-((7-(Cyclopentyl-3,4-d2)-6-dimethylformamide-7-H-pyrrolo[2,3-d]pyrimidin-2-yl)amino) Synthesis of -pyridin-3-yl)piperazine-1-carboxylic acid tert-butyl ester (Compound 29).

2-Chloro-7-(cyclopentyl-3,4-d2)-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (90 mg, 0.3 mmol) Dissolved in 5 mL of methyl isobutyl ketone, followed by palladium acetate (3 mg, 0.015 mmol), BINAP (18 mg, 0.03 mmol), cesium carbonate (146 mg, 0.45 mmol) and 4-(6-amino-pyridin-3-yl) Tert-butyl piperazine-1-carboxylate (91 mg, 0.33 mmol) was replaced with nitrogen three times and the reaction mixture was reacted at 110 ° C overnight. After the completion of the reaction, the mixture was cooled to room temperature, and then the mixture was evaporated. ESI-MS: 537 [M ⁺ +1].

Step 9. 7-(Cyclopentyl-3,4-d2)-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl)amino)-7H- Synthesis of pyrrolo[2,3-d]pyrimidine-6-carboxamide (Compound 30).

The compound 4-(6-((7-(cyclopentyl-3,4-d2)-6-diformamide-7-H-pyrrolo[2,3-d]pyrimidin-2-yl)amino) -Pyridine-3-yl)piperazine-1-carboxylic acid tert-butyl ester (115 mg, 0.2 mmol) was dissolved in 5 mL of dichloromethane, 2 mL of trifluoroacetic acid was added, and stirred at room temperature for 2 hr. The saturated sodium carbonate solution was stirred for 10 minutes, then extracted with methylene chloride. The organic phase was washed with 10 mL of brine, dried over anhydrous sodium sulfate and evaporated. ^{1 H NMR (300MHz, DMSO-} d 6) δ9.40 (s, 1H), 8.76 (s, 1H), 8.18 (d, J = 9.1Hz, 1H), 8.04 (d, J = 3.0Hz, 1H) , 7.48 (dd, J = 9.1, 3.0 Hz, 1H), 6.60 (s, 1H), 4.73 (t, J = 8.6 Hz, 1H), 3.27 (m, 4H), 3.22 - 3.14 (m, 4H), 3.05 (s, 6H), 2.42 (d, J = 9.6 Hz, 1H), 1.98 (d, J = 6.2 Hz, 4H), 1.63 (s, 1H); ESI-MS: 437 [M ⁺ +1].

Example 5 Preparation of 7-cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl-4-d)amine)-7H-pyrrolo[ 2,3-d]pyrimidine -6-carboxamide-4-d (compound 42)

The specific synthesis steps are as follows:

Step 1. Synthesis of pyrimidine-2,4(1H,3H)-dione-5,6-d2 (compound 32).

Uracil (2.24 g, 20 mmol), 0.2 g of 10% palladium on carbon and 10 mL of heavy water were added to a 20 mL microwave tube, and hydrogen gas was bubbled for 1 minute, and sealed in a microwave reactor. The mixture was subjected to microwave reaction at 130 ° C for 1.5 hours, and then taken out to room temperature, and taken out. 20 mL of heavy water was added thereto, and the temperature was raised to 95 ° C. After heating for 1 hour, it was filtered while hot, and the filtrate was concentrated to give a white solid. ^{1 H NMR (300MHz, DMSO-} d 6) δ10.80 (s, 2H); ESI-MS: 115 [M + +1].

Step 2. Synthesis of 5-bromopyrimidine-2,4(1H,3H)-dione-6-d (Compound 33).

Pyrimidine-2,4(1H,3H)-dione-5,6-d2 (1.82 g, 16 mmol) was added to 20 mL of heavy water, and liquid bromine (0.83 mL, 16 mmol) was slowly added with stirring. The mixture was reacted at 100 ° C for 30 minutes, slowly cooled to room temperature, a large amount of solid was precipitated, filtered, and the filter cake was washed with hot water and dried to give a pale yellow solid, 2.75 g, yield 90%. ESI-MS: 192 [M ⁺ +1].

Step 3. Synthesis of 5-bromo-2,4-dichloropyrimidine-6-d (Compound 34).

Add 1 mL of N,N-dimethylaniline to 5-bromopyrimidine-2,4(1H,3H)-dione-6-d (2.75 g, 14.4 mmol), stir, and let the reaction mixture Cold phosphorus oxychloride (7 mL, 72 mmol) was slowly added dropwise, and the mixture was heated to reflux under reflux for 2 hours. After cooling to room temperature, the phosphorus oxychloride was distilled off under reduced pressure, and toluene was taken up three times. To the concentrated residue, ice water was slowly added, and the mixture was adjusted to neutral with aqueous ammonia, and extracted with ethyl acetate. The organic phase was washed with 10 mL of brine. The residue was dried over anhydrous sodium sulfate and evaporated. ESI-MS: 228 [M ⁺ +1].

Step 4. Synthesis of 5-bromo-2-chloro-N-cyclopentylpyrimidine-6-d-4-amine (Compound 35).

Dissolve 5-bromo-2,4-dichloropyrimidine-6-d (2.22 g, 9.78 mmol) in 20 mL absolute ethanol, add triethylamine (2.7 mL, 19.6 mmol), and slowly add cyclopentane in an ice bath. The amine (0.92 g, 10.8 mmol) was stirred at room temperature for 5 hr. ^{1 H NMR (300MHz, DMSO-} d 6) δ7.37 (d, J = 7.6Hz, 1H), 4.31 (h, J = 7.3Hz, 1H), 1.97-1.84 (m, 2H), 1.74-1.48 ( m, 6H); ESI-MS: 277 [M ⁺ +1].

Step 5. Synthesis of 2-chloro-N-cyclopentyl-5-(3,3-diethoxypropyl-1-yn-1-yl)pyrimidine-6-d-4-amine (Compound 36) .

5-Bromo-2-chloro-N-cyclopentylpyrimidine-6-d-4-amine (2.0 g, 7.3 mmol) and propargaldehyde diethyl diacetal (1.1 g, 8.6 mmol), Pd ( Dppf)Cl ₂ (510 mg, 0.73 mmo), CuI (140 mg, 0.73 mmol) and 5 mL of triethylamine were added to 20 mL of DMF, replaced with nitrogen three times, warmed to 100 ° C, and allowed to react overnight. After the reaction was completed, the mixture was cooled to room temperature, 40 mL of water was added, and the mixture was evaporated. . ESI-MS: 325 [M ⁺ +1].

Step 6. Synthesis of 2-chloro-7-cyclopentyl-6-(diethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidine-4-d (Compound 37).

Dissolving 2-chloro-N-cyclopentyl-5-(3,3-diethoxypropyl-1-yn-1-yl)pyrimidine-6-d-4-amine (1.01 g, 3.1 mmol) To 10 mL of tetrahydrofuran, tetrabutylammonium fluoride (TBAF, 2.4 g, 9.3 mmol) was added, and the mixture was reacted at 65 ° C for 2 hours. The solvent was evaporated, the mixture was evaporated, evaporated, evaporated, evaporated. ESI-MS: 325 [M ⁺ +1].

Step 7. Synthesis of 2-chloro-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidin-4-d-6-carbaldehyde (Compound 38).

2-Chloro-7-cyclopentyl-6-(diethoxymethyl)-7H-pyrrolo[2,3-d]pyrimidin-4-d (830 mg, 2.6 mmol) was dissolved in 10 mL of dioxane The mixture was slowly added dropwise with 4 mL of concentrated hydrochloric acid. The mixture was stirred at room temperature for 20 minutes, and then 15 mL of water was added, and the mixture was extracted with ethyl acetate. The organic phase was washed with 10 mL of brine, dried over anhydrous sodium sulfate and evaporated. The yield was 82%. ESI-MS: 251 [M ⁺ +1].

Step 8. Synthesis of 2-chloro-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidin-4-d-6-carboxylic acid (Compound 39).

2-Chloro-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidin-4-d-6-carboxaldehyde (525 mg, 2.1 mmol) was dissolved in 5 mL of DMF and potassium peroxymonosulfonate was added ( 1.4 g, 2.3 mmol), reacted at room temperature for 6 hours. After the completion of the reaction, 10 mL of water was added, and a large amount of solid was precipitated, and dried by filtration to give 475 mg of pale yellow solid. ESI-MS: 267 [M ⁺ +1].

Step 9. Synthesis of 2-chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-d-6-carboxamide (Compound 40).

2-Chloro-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidin-4-d-6-carboxylic acid (475 mg, 1.8 mmol), HBTU (680 mg, 1.8 mmol) and DIEA (0.9) mL, 5.4 mmol) was dissolved in 10 mL of DMF, and a 2M solution of dimethylamine in methanol (1.1 mL, 2.2 mmol) was added dropwise in an ice bath. After the dropwise addition, the reaction was carried out at room temperature for 30 minutes, 20 mL of water was added, and extracted with ethyl acetate. The organic phase was washed with 20 mL of brine, dried over anhydrous sodium sulfate, and evaporated. ^{1 H NMR (300MHz, DMSO-} d 6) δ6.80 (s, 1H), 4.80 (p, J = 8.7Hz, 1H), 3.03 (d, J = 12.9Hz, 6H), 2.29-2.16 (m, 2H), 2.08 - 1.88 (m, 4H), 1.64 (dtd, J = 12.1, 7.1, 6.6, 2.6 Hz, 2H); ESI-MS: 294 [M ⁺ +1].

Step 10. 4-(6-((7-Cyclopentyl-6-dimethylamide-7-H-pyrrolo[2,3-d]pyrimidin-2-yl-4-d)amino)-pyridine- Synthesis of tert-butyl 3-phenyl)piperazine-1-carboxylate (Compound 41).

2-Chloro-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-d-6-carboxamide (90 mg, 0.3 mmol) was dissolved in 5 mL of A In the isobutyl ketone, palladium acetate (3 mg, 0.015 mmol), BINAP (18 mg, 0.03 mmol), cesium carbonate (146 mg, 0.45 mmol) and 4-(6-amino-pyridin-3-yl)-piperazine were sequentially added. tert-Butyl 1-carboxylate (91 mg, 0.33 mmol) was replaced with nitrogen three times and the reaction mixture was reacted at 110 ° C overnight. After the reaction was completed, the mixture was cooled to room temperature, and then added with 10 mL of water, and the mixture was washed with ethyl acetate. The organic phase was washed with 10 mL of brine, dried over anhydrous sodium sulfate, and evaporated. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 9.36 (s, 1H), 8.17 (d, J = 9.1 Hz, 1H), 8.1 (d, J = 2.9 Hz, 1H), 7.46 (dd, J = 9.1, 3.0 Hz, 1H), 6.60 (s, 1H), 4.72 (q, J = 8.9 Hz, 1H), 3.48 (t, J = 5.0 Hz, 4H), 3.09 - 3.05 (d, J = 9.6 Hz, 10H), 2.43 (s, 2H), 1.98 (s, 4H), 1.70 - 1.59 (m, 2H), 1.42 (s, 9H); ESI-MS: 536 [M ⁺ +1].

Step 11. 7-Cyclopentyl-N,N-dimethyl-2-((5-(piperazin-1-yl)pyridin-2-yl-4-d)amine)-7H-pyrrolo[2 ,3-d]pyrimidine-6-formyl Synthesis of amine-4-d (compound 42).

The compound 4-(6-((7-cyclopentyl-6-dimethylamide-7-H-pyrrolo[2,3-d]pyrimidin-2-yl-4-d)amino)-pyridine-3 Tert-butyl piperazine-1-carboxylate (115 mg, 0.2 mmol) was dissolved in 5 mL of dichloromethane, 2 mL of trifluoroacetic acid was added, stirred at room temperature for 2 hr, solvent was evaporated under reduced pressure, and 10 mL of saturated sodium carbonate solution was added. The mixture was stirred for 10 minutes, extracted with methylene chloride. EtOAc was evaporated. ^{1 H NMR (300MHz, DMSO-} d 6) δ9.39 (s, 1H), 8.19 (s, 1H), 8.03 (s, 1H), 7.49 (s, 1H), 6.60 (s, 1H), 4.74 ( m,1H),3.23-3.28(m,4H),3.14-3.20(m,4H),3.05(s,6H),2.34-2.44(m,2H),1.98(d,4H),1.64(s, 2H); ESI-MS: 436 [M ⁺ +1].

Biological activity test.

(1) Determination of enzyme activity.

Experimental Materials:

CDK2/cyclin A, CDK4/cyclin D1, CDK6/cyclin D1. ULight labeled polypeptide substrates ULight-4E-BP1 and ULight-MBP. The vision-labeled anti-myelin basic protein antibody and the sputum-labeled rabbit-derived antibody were detected by Envision multi-label analyzer.

experimental method:

The compound to be tested was diluted threefold, including 10 concentration gradients. The initial concentration of CDK2/cyclin A test compound was 10 uM, and the initial concentration of CDK4/cyclin D1 and CDK6/cyclin D1 test compounds was 1 uM.

Enzyme reaction system of CDK2/cyclin A:

The standard Lance Ultra method was performed by a 10 microliter enzyme reaction system containing 0.5 nanomolar CDK2/cyclin A protein, 100 nanomolar ULight-MBP polypeptide, and 25 micromolar ATP. They were dissolved in the enzyme buffer. The buffer components were: hydroxyethylpiperazine ethanesulfuric acid solution 50 mM in PH7.5, ethylenediaminetetraacetic acid 1 mM, magnesium chloride 10 mM, 0.01% Brij-35. , dithiothreitol 2 mmol. After starting the reaction, the OptiPlate 384 well plate was sealed with a top heat seal film TopSeal-A and incubated for 60 minutes at room temperature.

Enzyme reaction system of CDK4/cyclin D1:

The standard Lance Ultra method was performed by a 10 microliter enzyme reaction system containing 1 nanomolar CDK4/cyclin D1 protein, 50 nanomolar ULight-4E-BP1 polypeptide, and 350 micromolar ATP. They were dissolved in the enzyme buffer. The buffer components were: hydroxyethylpiperazine ethanesulfuric acid solution 50 mM in PH7.5, ethylenediaminetetraacetic acid 1 mM, magnesium chloride 10 mM, 0.01% Brij-35. , dithiothreitol 2 mmol. After the reaction was started, the OptiPlate 384 well plate was sealed with a top heat seal film TopSeal-A and incubated at room temperature for 90 minutes.

Enzyme reaction system of CDK6/cyclin D1:

The standard Lance Ultra method was performed by a 10 microliter enzyme reaction system containing 0.8 nanograms of CDK6/cyclin D1 protein, 50 nanomolar ULight-4E-BP1 polypeptide, and 250 micromolar ATP. They were dissolved in an enzyme buffer, which consisted of 50 mM hydroxyethylpiperazine ethanesulfuric acid solution at pH 7.5, 1 mM ethylenediaminetetraacetic acid, 10 mmol of magnesium chloride, and 0.01% Brij-35. Dithiothreitol 2 mmol. After the reaction was started, the OptiPlate 384 well plate was sealed with a top heat seal film TopSeal-A and incubated at room temperature for 180 minutes.

The enzyme reaction termination buffer was prepared, and EDTA was dissolved in a 1-fold diluted assay buffer, and the reaction was terminated at room temperature for 5 minutes. Five microliters of the assay mixture (configured with tritiated anti-myelin basic antibody and tritiated rabbit-derived antibody, respectively) was added to the CDK2/cyclin A, CDK4/cyclin D1, and CDK6/cyclin D1 reactions, respectively. After incubating for 60 min at room temperature, the reaction signal was detected using an Envision instrument according to the principle of time-resolved fluorescence resonance energy transfer.

data analysis:

The original data is converted to the inhibition rate using the equation (Max-Ratio)/(Max-Min)*100%, and the value of IC50 can be obtained by curve fitting with four parameters.

The compounds of Examples 2-5 and their non-deuterated compound Ribociclib were tested as described above, and the kinase inhibitory effects are shown in Table 1 below.

Table 1:

As shown in Table 1, the compound of the present invention exhibited excellent inhibitory activity against CDK4/cyclin D1 and CDK6/cyclin D1, and showed low inhibitory activity against CDK2/cyclin A. In particular, the compounds of Examples 4 and 5 have superior inhibitory activity against CDK4/cyclin D1 and CDK6/cyclin D1 than the non-deuterated compound Ribociclib.

(2) Cytotoxicity test

The inhibitory effect of the compounds on the activity of MCF-7 and MDA-MB-436 cells was examined.

Reagents and consumables

1. Cell culture: RPMI-1640 medium, fetal bovine serum, antibiotic (Penicillin-Streptomycin)

2. Cell lines: MCF-7 and MDA-MB-436

3. Detection reagent: live cell detection kit CellTiter-Glo

4. Other major consumables and reagents: compound dilution plate, intermediate plate, test plate, DMSO

Experimental principle

The amount of ATP directly reflects the number of cells and the state of the cells, and the number of viable cells in the culture is detected by quantitatively measuring ATP. The live cell assay kit uses luciferase as a test substance. The kit uses a stable glow-type signal generated by UltraGlow luciferase. During the luminescence process, luciferase requires the participation of ATP, and the respiration of metabolically active cells. And other life activity processes can produce ATP. Was added to the cell culture CellTiter-Glo ^TM Reagent, luminescence is measured, and the optical signal is directly proportional to the amount of ATP system, ATP and the number of viable cells was positively correlated, which can detect cell proliferation. The assay plate was analyzed using PE company's Envision.

experimental method

1. Preparation of cell plates

MCF-7 and MDA-MB-436 cells were separately seeded in 384-well plates, MCF-7 assay contained 200 cells per well, and MDA-MB-436 assay contained 600 cells per well. The cell plates were placed in a carbon dioxide incubator for overnight culture.

2. Prepare the compound

A double-fold experiment was performed by 3-fold dilution with ECHO and 10 compound concentrations.

3. Compound treatment of cells

Compounds were transferred to cell plates at a starting concentration of 10 uM. The cell plates were incubated in a carbon dioxide incubator for 6 days.

4. Detection

The Promega CellTiter-Glo reagent was added to the cell plate and incubated for 10 minutes at room temperature to stabilize the luminescence signal. Readings were performed using a PerkinElmer Envision multi-label analyzer.

The compounds of Examples 2-5 and their non-deuterated compound Ribociclib were tested as described above, and the cytotoxicity experiments are shown in Table 2 below.

Table 2

Numbering MCF-7 MDA-MB-436 Ribociclib 468.69 >10000 Example 2 530.42 >10000 Example 3 1315.43 >10000 Example 4 363.49 >10000 Example 5 456.22 >10000

As shown in Table 1, the compound of the present invention exhibited excellent inhibitory activity against MCF-7 and MDA-MB-436 cells. In particular, the compounds of Examples 4 and 5 have superior inhibitory activity against MCF-7 and MDA-MB-436 cells than the non-deuterated compound Ribociclib. Therefore, the compounds of the present invention are promising as a drug for treating ER-positive, HER2-negative breast cancer.

(3) Evaluation of metabolic stability.

Microsomal experiments: human liver microsomes: 0.5 mg/mL, Xenotech; rat liver microsomes: 0.5 mg/mL, Xenotech; coenzyme (NADPH/NADH): 1 mM, Sigma Life Science; magnesium chloride: 5 mM, 100 mM phosphate buffer Agent (pH 7.4).

Preparation of the stock solution: A certain amount of the compound Ribociclib and the powder of Example 2-5 were accurately weighed and dissolved in 5 mM with DMSO, respectively.

Preparation of phosphate buffer (100 mM, pH 7.4): Mix 150 mL of pre-formed 0.5 M potassium dihydrogen phosphate and 700 mL of 0.5 M potassium dihydrogen phosphate solution, and adjust the mixture with 0.5 M potassium dihydrogen phosphate solution. The pH was adjusted to 7.4, diluted 5 times with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100 mM) containing 100 mM potassium phosphate, 3.3 mM magnesium chloride, and a pH of 7.4.

A solution of NADPH regeneration system (containing 6.5 mM NADP, 16.5 mM G-6-P, 3 U/mL G-6-P D, 3.3 mM magnesium chloride) was prepared and placed on wet ice before use.

Formulation stop solution: acetonitrile solution containing 50 ng/mL propranolol hydrochloride and 200 ng/mL tolbutamide (internal standard). Take 25057.5 μL of phosphate buffer (pH 7.4) into a 50 mL centrifuge tube, add 812.5 μL of human liver microsomes, and mix to obtain a liver microsome dilution with a protein concentration of 0.625 mg/mL. 25057.5 μL of phosphate buffer (pH 7.4) was taken into a 50 mL centrifuge tube, and 812.5 μL of SD rat liver microsomes were added and mixed to obtain a liver microsome dilution having a protein concentration of 0.625 mg/mL.

Incubation of the sample: The stock solution of the corresponding compound was diluted to 0.25 mM with an aqueous solution containing 70% acetonitrile as a working solution, and was used. 398 μL of human liver microsomes or rat liver microsome dilutions were added to 96-well incubation plates (N=2), and 2 μL of 0.25 mM working solution was added and mixed.

Determination of metabolic stability: 300 μL of pre-cooled stop solution was added to each well of a 96-well deep well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system were placed in a 37 ° C water bath, shaken at 100 rpm, and pre-incubated for 5 min. 80 μL of the incubation solution was taken from each well of the incubation plate, added to the stopper plate, and mixed, and 20 μL of the NADPH regeneration system solution was added as a sample of 0 min. Then, 80 μL of the NADPH regeneration system solution was added to each well of the incubation plate to start the reaction and start timing. The corresponding compound had a reaction concentration of 1 μM and a protein concentration of 0.5 mg/mL. 100 μL of the reaction solution was taken at 10, 30, and 90 min, respectively, and added to the stopper, and the reaction was terminated by vortexing for 3 min. The plate was centrifuged at 5000 x g for 10 min at 4 °C. 100 μL of the supernatant was taken into a 96-well plate to which 100 μL of distilled water was previously added, mixed, and sample analysis was performed by LC-MS/MS.

Data analysis: The peak area of the corresponding compound and the internal standard was detected by LC-MS/MS system, and the ratio of the peak area of the compound to the internal standard was calculated. The slope is measured by the natural logarithm of the percentage of the remaining amount of the compound versus time, and t _1/2 and CL _{int are} calculated according to the following formula, where V/M is equal to 1/protein concentration.

The metabolic stability of human and rat liver microsomes was evaluated by simultaneously testing the compounds of the present invention and their compounds without deuteration. The half-life and liver intrinsic clearance as indicators of metabolic stability are shown in Table 3. The undeuterated compound Ribociclib was used as a control sample in Table 3. As shown in Table 1, the compounds of the present invention can significantly improve metabolic stability by comparison with the undeuterated compound Ribociclib.

table 3

(4) Pharmacokinetic evaluation in rats.

Eight male Sprague-Dawley rats, 7-8 weeks old, weighing 210 g, were divided into 2 groups, 4 in each group, 5 mg/kg in a single oral dose; (a) Control group: Ribociclib; (b) Test group: Example compounds were compared for differences in pharmacokinetics.

Rats were fed a standard diet and given water. Fasting began 16 hours before the test. The drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the eyelids at a time point of 0.083 hours, 0.25 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after administration.

Rats were briefly anesthetized after inhalation of ether, and 300 μL of blood samples were collected from the eyelids in test tubes. There was 30 μL of 1% heparin salt solution in the test tube. The tubes were dried overnight at 60 ° C before use. After the blood sample collection was completed at a later time point, the rats were anesthetized with ether and sacrificed.

Immediately after the blood sample is collected, gently invert the tube at least 5 times to ensure adequate mixing and place on ice. Blood samples were centrifuged at 5000 rpm for 5 minutes at 4 ° C to separate plasma from red blood cells. Pipette 100 μL of plasma into a clean plastic centrifuge tube, indicating the name and time of the compound. Plasma was stored at -80 °C prior to analysis. The concentration of the compound of the invention in plasma was determined by LC-MS/MS. Pharmacokinetic parameters were calculated based on the plasma concentration of each animal at different time points.

The experimental results show that the compound of the present invention has better pharmacokinetics in animals relative to the control compound, and thus has better pharmacodynamics and therapeutic effects.

It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention, and the experimental methods in which the specific conditions are not indicated in the examples, usually in accordance with conventional conditions or in accordance with the conditions suggested by the manufacturer. Parts and percentages are parts by weight and percentage by weight unless otherwise stated.

The above is a further detailed description of the present invention in connection with the specific preferred embodiments, and the specific embodiments of the present invention are not limited to the description. It will be apparent to those skilled in the art that the present invention may be made without departing from the spirit and scope of the invention.

Claims (10)

A substituted pyrrolopyrimidine compound characterized by a pyrrolopyrimidine compound of the formula (I), or a crystalline form thereof, a pharmaceutically acceptable salt, a hydrate or a solvent compound,

In the formula: R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ , R ¹² , R ¹³ , R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ And R ¹⁸ , R ¹⁹ , R ²⁰ , R ²¹ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ are each independently selected from the group consisting of “hydrogen (H), 氘 (D)”; And physiologically acceptable salts, prodrugs, hydrates, solvates, tautomers and stereoisomers thereof, including mixtures of these compounds in all ratios; Additionally, the pyrrolopyrimidine compound contains at least one ruthenium atom. The pyrrolopyrimidine compound according to claim 1, wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁷ and R ⁸ are each independently hydrazine or hydrogen. The pyrrolopyrimidine compound according to claim 1, wherein each of R ⁹ , R ¹⁰ and R ^{11 is} independently hydrazine or hydrogen. The pyrrolopyrimidine compound according to claim 1, wherein each of R ¹² and R ^{13 is} independently hydrazine or hydrogen. The pyrrolopyrimidine compound according to claim 1, wherein R ¹⁴ , R ¹⁵ , R ¹⁶ , R ¹⁷ , R ¹⁸ , R ¹⁹ , R ²⁰ and R ²¹ are each independently hydrazine or hydrogen. The pyrrolopyrimidine compound according to claim 1, wherein R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ and R ²⁷ are each independently hydrazine or hydrogen. The pyrrolopyrimidine compound according to claim 1, wherein the compound is selected from the group consisting of the following compounds or a pharmaceutically acceptable salt thereof, but is not limited to the following compounds:

A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a substituted pyrrolopyrimidine compound according to any one of claims 1 to 6, or a crystalline form thereof, a pharmaceutically acceptable salt, A pharmaceutical composition of a hydrate or a solvate, a stereoisomer, a prodrug or an isotope variant is used as an active ingredient and contains a conventional pharmaceutical carrier. Use of the pyrrolopyrimidine compound according to any one of claims 1 to 7, which is for use in the preparation of a medicament for preventing or treating cancer. The use according to claim 8, wherein said cancer is selected from the group consisting of breast cancer, pancreatic cancer, colorectal cancer, lung cancer and melanoma, sarcoma, leukemia, glioma, familial melanoma and melanoma, and set of Cellular lymphoma and non-small cell lung cancer.