CN108290899B - Substituted pyrrolopyrimidine compound and application thereof - Google Patents

Abstract

Provided are a substituted pyrrolopyrimidine compound, a composition containing the same, and applications thereof. Specifically, disclosed is a pharmaceutical composition of a pyrrolopyrimidine compound represented by formula (I), or a crystal form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvate thereof. The compound can be used as a Cyclin Dependent Kinase (CDK) inhibitor, and further can be suitable for preparing medicaments for treating CDK related diseases (such as breast cancer and the like).

Description

Substituted pyrrolopyrimidine compound and application thereof

Technical Field

The invention belongs to the field of medicine. In particular, the present invention relates to substituted pyrrolopyrimidine compounds and uses thereof, and more particularly, to pyrrolopyrimidine compounds and pharmaceutical compositions thereof useful as CDK inhibitors for the treatment and prevention of diseases associated with CDK inhibitors.

Background

Cyclin-dependent kinase (CDK) is a serine-threonine protein kinase that plays a key role in regulating the transitions between different phases of the cell-cycle, such as progression from the resting phase (the interval between mitosis and the onset of DNA replication for a new round of cell division) in G1 to S (the active DNA synthesis phase), or progression from G2 to the M phase, where active mitosis and cell division occur.

CDK complexes are formed by modulating the association of cyclin subunits (e.g., cyclin a, B1, B2, D1, D2, D3 and E) and catalytic kinase subunits (e.g., CDK1, CDK2, CDK4, CDK5 and CDK 6). As the name implies, CDKs show an absolute dependence on cyclin subunits in order to phosphorylate their target substrates, with different kinase/cyclin pairs acting to regulate progression through specific stages 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. The conformational change modulates the activity of the substrate or its ability to interact with other binding ligands. The enzymatic activity of a protein kinase refers to the rate at which the kinase adds phosphate groups to a substrate. It can be measured, for example, by determining 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 CDKs is regulated in a post-translational manner, by transient association with other proteins and by altering their intracellular localization. Tumor development is closely related to genetic changes and dysregulation of CDKs and their regulators, suggesting that CDK inhibitors may be useful anticancer therapeutics. Early results show that: transformed cells and normal cells differ in their requirements for, for example, cyclin a/CDK2, and it is possible to develop novel antineoplastic agents that lack the general host toxicity observed with conventional cytotoxic and cytostatic drugs. While inhibition of cell cycle-associated CDKs is clearly relevant, for example, in oncology applications, inhibition of RNA polymerase-regulated CDKs may also be highly relevant in cancer indications.

CDK has been shown to be involved in cell cycle progression and cell transcription, and loss of growth control has been associated with abnormal cell proliferation of the disease (see e.g. Malumbres and Barbacid, nat. rev. cancer 2001, 1: 222). Increased activity or transient aberrant activation of cyclin-dependent kinases has been shown to lead to the development of human tumors (Sherr C.J., Science 1996, 274: 1672-1677).

Many diseases are associated with abnormal cellular responses triggered by the aforementioned 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, allergies and asthma, alzheimer's disease and hormone-related diseases. Accordingly, there have been a great deal of efforts in medicinal chemistry to find protein kinase inhibitors that are effective as therapeutic agents.

The beginning, progression and end of the mammalian cell cycle are regulated by various cyclin-dependent kinase (CDK) complexes that are critical for cell growth. These complexes comprise at least catalytic (the 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), cyclin E (CDK 2). Each of these complexes is involved in a particular phase of the cell cycle. However, not all CDK family members are only involved in cell cycle control. Thus, CDK7, CDK8 and CDK9 are involved in the regulation of transcription, CDK5 plays a role in neuronal and secretory cell function.

Palbociclib is the first CDK4/6 inhibitor approved by the U.S. FDA for breast cancer treatment. Because all living cells are undergoing cell division, Palbociclib has the ability to arrest the cell division process (also known as the "cell cycle"), and thus has potential broad applicability. Palbociclib in combination with other anti-cancer therapies such as endocrine therapy, chemotherapy, targeted therapy may be effective against a variety of cancers. Clinical trials of either breast or other cancers have shown that Palbociclib is safe when administered once daily, with the major side effect being reversible neutropenia, and that administration should be suspended and resumed at lower doses when count-down side effects occur. Other side effects include fatigue (33%), nausea (30%), diarrhea (18%), constipation (12%), rash (12%). In addition, the CDK inhibitor Ribociclib (LEE011) of noval is still in phase III clinical trials.

Thus, there remains a need to develop inhibitors of protein kinases such as CDK1, CDK2, CDK4, CDK5, CDK6, CDK7, CDK8 and CDK 9; there remains a need for new treatments and therapies for protein kinase-associated disorders. 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 disease.

Disclosure of Invention

In view of the above technical problems, the present invention discloses a pyrrolopyrimidine compound and a composition comprising the same, which is effective as a Cyclin Dependent Kinase (CDK) inhibitor and/or has better pharmacodynamic/pharmacokinetic properties.

In contrast, the technical scheme adopted by the invention is as follows:

it is an object of the present invention to provide a new class of potent Cyclin Dependent Kinase (CDK) inhibitors and/or compounds with better pharmacodynamic/pharmacokinetic properties.

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

in the formula:

R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷independently of each other, selected from the group consisting of "hydrogen (H), deuterium (D)";

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

with the proviso that said pyrrolopyrimidine compound contains at least one deuterium atom.

In another preferred embodiment, the deuterium isotope content of deuterium at the deuterated position is at least greater than the natural deuterium isotope content (0.015%), preferably greater than 30%, more preferably greater than 50%, more preferably greater than 75%, more preferably greater than 95%, more preferably greater than 99%.

Specifically, in the present invention R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶And R²⁷The deuterium isotope content in each deuterated position is at least 5%, 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%, more preferably greater than 40%, more preferably greater than 45%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 99%.

In another preferred embodiment, R of the compound of formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶、R²⁷Wherein at least one R contains deuterium, more preferably two R contain deuterium, more preferably three R contain deuterium, more preferably four R contain deuterium, more preferably five R contain deuterium, more preferably six R contain deuterium,more preferably seven R contain deuterium, more preferably eight R contain deuterium, more preferably nine R contain deuterium, more preferably ten R contain deuterium, more preferably eleven R contain deuterium, more preferably twelve R contain deuterium, more preferably thirteen R contain deuterium, more preferably fourteen R contain deuterium, more preferably fifteen R contain deuterium, more preferably sixteen R contain deuterium, more preferably seventeen R contain deuterium, more preferably eighteen R contain deuterium, more preferably nineteen R contain deuterium, more preferably twenty-one R contain deuterium, more preferably twenty-two R contain deuterium, more preferably twenty-three R contain deuterium, more preferably twenty-four R contain deuterium, more preferably twenty-five R contain deuterium, more preferably twenty-six R contain deuterium, more preferably twenty-seven R contain deuterium.

As a further improvement of the invention, R¹、R²、R³、R⁴、R⁵、R⁷And R⁸Each independently is deuterium or hydrogen.

As a further improvement of the invention, R⁹、R¹⁰And R¹¹Each independently is deuterium or hydrogen.

As a further improvement of the invention, R¹²And R¹³Each independently is deuterium or hydrogen.

As a further improvement of the invention, R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰And R²¹Each independently is deuterium or hydrogen.

As a further improvement of the invention, R²²、R²³、R²⁴、R²⁵、R²⁶And R²⁷Each independently is deuterium or hydrogen.

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

in another preferred embodiment, the compound does not include non-deuterated compounds and only R²²、R²³、R²⁴、R²⁵、R²⁶And R²⁷A compound which is fully deuterated.

In a second aspect of the present invention, there is provided a method of preparing a pharmaceutical composition comprising the steps of: mixing a pharmaceutically acceptable carrier with a compound described in the first aspect of the invention, or a crystalline form, a pharmaceutically acceptable salt, a hydrate, or a solvate thereof, 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, a pharmaceutically acceptable salt, a hydrate or a solvate thereof.

Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, any glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersant, disintegrant, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier.

The pharmaceutical composition can be prepared into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powder, granules, paste, emulsion, suspension, solution, suppositories, injection, inhalant, gel, microspheres, aerosol and the like.

Typical routes of administration of the pharmaceutical compositions of the present 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 compositions of the present invention may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

The compounds of the present invention are useful as inhibitors of cyclin dependent kinases. For example, the compounds of the invention are inhibitors of cyclin dependent kinases, with particular preference for cyclin dependent kinases selected from CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK9, with more particular preference for CDK1, CDK2, CDK3, CDK4, CDK5 and CDK 9.

CDKs play a role in cell cycle regulation, apoptosis, transcription, differentiation and CNS function. Thus, CDK inhibitors may be useful in the treatment of diseases having a disorder of cell proliferation, apoptosis or differentiation, such as cancer. In particular, RB + ve tumors may be particularly sensitive to CDK inhibitors. These include ras, Raf, tumor harboring mutations in growth factor receptors or overexpression of growth factor receptors. Tumors with highly methylated promoter regions of CDK inhibitors and cyclin-dependent kinases overexpressing cyclin chaperones 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 (e.g., colorectal cancers such as colon adenocarcinoma and colon adenoma), kidney cancer, epidermal carcinoma, 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, stomach cancer, cervical cancer, thyroid cancer, nasal cancer, head and neck cancer, prostate cancer, or skin cancer such as squamous cell carcinoma; hematopoietic tumors of lymphoid lineage, such as leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, B-cell lymphoma (e.g., diffuse large B-cell lymphoma), T-cell lymphoma, multiple myeloma, hodgkin's lymphoma, non-hodgkin's lymphoma, hairy cell lymphoma, or burkitt's lymphoma; hematopoietic tumors of myeloid lineage, such as acute and chronic myeloid leukemia, myelodysplastic syndrome or promyelocytic leukemia; thyroid follicular cancer; tumors derived from mesenchymal cells, such as fibrosarcoma or rhabdomyosarcoma; tumors of the central or peripheral nervous system, such as astrocytomas, neuroblastoma, glioma or schwannoma; melanoma; seminoma; teratoma; osteosarcoma; xeroderma pigmentosum; keratoacanthoma cell tumor; thyroid follicular carcinoma or kaposi's sarcoma.

The cyclin-dependent kinase inhibitor can be combined with other anticancer drugs for use. For example, cyclin-dependent kinase inhibitors have been used in combination therapy with other anti-cancer drugs.

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

One class of cancers includes human breast cancer (e.g., primary breast tumor, lymph 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 subclass of cancers includes hematopoietic tumors of lymphoid lineage, such as leukemia, chronic lymphocytic leukemia, mantle cell lymphoma, and B-cell lymphoma (e.g., diffuse large B-cell lymphoma).

Another subclass of cancers for which the compounds of the invention may be useful in therapy includes sarcomas, leukemias, gliomas, familial melanomas and melanomas.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

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

Herein, "deuterated", unless otherwise specified, means that one or more hydrogens of a compound or group are replaced with deuterium; deuterium can be mono-, di-, poly-, or fully substituted. The terms "deuterated one or more" and "deuterated one or more" are used interchangeably.

Herein, unless otherwise specified, "non-deuterated compound" means a compound containing deuterium at an atomic ratio of deuterium not higher than the natural deuterium isotope content (0.015%).

The invention also relates toIncluding isotopically labeled compounds, equivalent to the original compounds disclosed herein. Examples of isotopes that can be listed as compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, respectively²H，³H，¹³C，¹⁴C，¹⁵N，¹⁷O，¹⁸O，³¹P，³²P，³⁵S，¹⁸F and³⁶and (4) Cl. The compounds of the present invention, or enantiomers, diastereomers, isomers, or pharmaceutically acceptable salts or solvates thereof, wherein isotopes or other isotopic atoms containing such compounds are within the scope of the present invention. Certain isotopically-labelled compounds of the invention, e.g.³H and¹⁴among these, the radioactive isotope of C is useful in tissue distribution experiments of drugs and substrates. Tritium, i.e.³H and carbon-14, i.e.¹⁴C, their preparation and detection are relatively easy, and are the first choice among isotopes. Isotopically labeled compounds can be prepared by conventional methods by substituting readily available isotopically labeled reagents for non-isotopically labeled reagents using the protocols set forth in the examples.

Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Suitable acids for forming the salts include, but are not limited to: inorganic acids such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, nitric acid, phosphoric acid, and the like; organic acids such as formic acid, acetic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, 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 and the like; and amino acids such as proline, phenylalanine, aspartic acid, glutamic acid, etc. Another preferred class of salts are those of the compounds of the invention with bases, for example alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., magnesium or calcium salts), ammonium salts (e.g., lower alkanolammonium salts and other pharmaceutically acceptable amine salts), for example methylamine salts, ethylamine salts, propylamine salts, dimethylamine salts, trimethylamine salts, diethylamine salts, triethylamine salts, tert-butylamine salts, ethylenediamine salts, hydroxyethylamine salts, dihydroxyethylamine salts, triethanolamine salts, and amine salts formed from morpholine, piperazine, lysine, respectively.

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present 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, not being deleterious to the recipient thereof in the amounts employed in the medicament.

The compounds of formula (I) and compositions comprising said compounds are CDK inhibitors and may be used in the treatment, prevention or elimination of various CDK related disorders. Pharmaceutical compositions comprising these compounds are useful for treating, preventing or slowing the progression of diseases or disorders in various therapeutic areas, such as cancer.

Compared with the prior art, the invention has the beneficial effects that: the substituted pyrrolopyrimidine compounds and compositions containing the compounds disclosed herein have excellent CDK inhibitory properties while having better pharmacokinetic profile characteristics. Can change dosage and form long-acting preparation, and improve applicability. Deuterium is used for replacing hydrogen atoms in the compound, and due to the deuterium isotope effect, the medicine concentration of the compound in an animal body can be improved, so that the medicine curative effect is improved. Replacement of hydrogen atoms in compounds with deuterium may increase the safety of the compounds as certain metabolites are inhibited.

Detailed Description

The following describes more specifically the processes for the preparation of the compounds of formula (I) according to the invention, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

In general, in the preparative schemes, each reaction is usually carried out in an inert solvent at a temperature ranging from room temperature to reflux temperature (e.g., from 0 ℃ to 100 ℃, preferably from 0 ℃ to 80 ℃). The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 24 hours.

EXAMPLE 1 preparation of intermediate 2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2, 3-d]Pyrimidine-6-carboxylic acid methyl ester Amide (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 (2.0g, 7.3mmol) and propynal diethyl diacetal (1.1g, 8.6mmol), Pd (dppf) Cl₂(510mg, 0.73mmol), CuI (140mg, 0.73mmol) and 5mL of triethylamine were added to 20mL of Dimethylformamide (DMF), and the mixture was replaced with nitrogen three times, warmed to 100 ℃ and reacted overnight. After completion of the reaction, the reaction mixture was cooled to room temperature, 40mL of water was added, extraction was performed with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to obtain 1.01g of a pale yellow oily substance with a yield of 43%.¹H NMR(300MHz，DMSO-d₆)8.18(s，1H)，7.20(d，J＝7.6Hz，1H)，5.56(s，1H)，4.33(t，J＝7.5Hz，1H)，3.63(ddq，J＝35.4，9.6，7.1Hz，4H)，1.93(s，2H)，1.76-1.47(m，6H)，1.16(t，J＝7.1Hz，6H)；ESI-MS：324[M⁺+1]。

Step 2.2-chloro-7-cyclopentyl-6-diethoxymethyl-7H-pyrrolo [2, 3-d ] pyrimidine (compound 4) synthesis.

Reacting [ 2-chloro-5- (3, 3-diethoxy-prop-1-ynyl) -pyrimidin-4-yl]Cyclopentylamine (1.01g, 3.1mmol) was dissolved in 10mL tetrahydrofuran, tetrabutylammonium fluoride (2.4g, 9.3mmol) was added, and the solution was dissolved in 6The reaction was carried out at 5 ℃ for 2 hours. The solvent was removed by rotary evaporation, 20mL of water was added, extraction was performed with ethyl acetate, the organic phase was washed with 20mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to give 830mg of a pale yellow oil with a yield of 82%. ESI-MS: 324[ M ]⁺+1]。

Step 3.2-chloro-7-cyclopentyl-7H-pyrrolo [2, 3-d ] pyrimidine-6-carbaldehyde (compound 5) was synthesized.

2-chloro-7-cyclopentyl-6-diethoxymethyl-7H-pyrrolo [2, 3-d)]Pyrimidine (830mg, 2.6mmol) was dissolved in 10mL dioxane, 4mL concentrated hydrochloric acid was slowly added dropwise, stirred at room temperature for 20 minutes, 15mL water was added, extraction was performed with ethyl acetate, the organic phase was washed with 10mL saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column to give 525mg of pale yellow solid with a yield of 82%. ESI-MS: 250[ M ]⁺+1]。

Step 4.2-chloro-7-cyclopentyl-7H-pyrrolo [2, 3-d ] pyrimidine-6-carboxylic acid (compound 6) synthesis.

2-chloro-7-cyclopentyl-6-diethoxymethyl-7H-pyrrolo [2, 3-d)]Pyrimidine-6-carbaldehyde (525mg, 2.1mmol) was dissolved in 5ml of DMF, and potassium peroxomonosulfonate (1.4g, 2.3mmol) was added thereto to react at room temperature for 6 hours. After the reaction, 10mL of water was added, and a large amount of solid was precipitated, which was filtered and dried to obtain 475mg of pale yellow solid in 85% yield. ESI-MS: 266[ M ]⁺+1]。

Step 5.2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2, 3-d ] pyrimidine-6-carboxamide (compound 7) synthesis.

2-chloro-7-cyclopentyl-7H-pyrrolo [2, 3-d)]Pyrimidine-6-carboxylic acid (475mg, 1.8mmol), O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU, 680mg, 1.8mmol) and N, N-diisopropylethylamine (DIEA, 0.9mL, 5.4mmol) were dissolved in 10mL of dmf, 2M dimethylamine methanol solution (1.1mL, 2.2mmol) was added dropwise under ice bath, reaction was performed at room temperature for 30 minutes after completion of dropwise addition, 20mL of water was added, extraction was performed with ethyl acetate, the organic phase was washed with 20mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column to obtain 420mg of red solid with a yield of 80%. ESI-MS: 293[ M ]⁺+1]。

Example 2 preparation of 7-cyclopentyl-N, N-dimethyl-2- ((5- (piperazine-1-)Radical-2, 2, 3, 3, 5, 5, 6, 6-d8) Pyridin-2-yl) amino) -7H-pyrrolo [2, 3-d]Pyrimidine-6-carboxamide (Compound 14)

The specific synthesis steps are as follows:

step 1.1 synthesis of- (6-nitropyridin-3-yl) piperazine-2, 2, 3, 3, 5, 5, 6, 6-d8 (compound 10).

Dissolving 5-bromo-2-nitropyridine (202mg, 1mmol) in 10mL of n-butanol, adding triethylamine (0.7mL, 5mmol) and piperazine-2, 2, 3, 3, 5, 5, 6, 6-d8 hydrochloride (332mg, 2mmol), reacting at 90 ℃ for 6 hours, cooling to room temperature, adding 20mL of water, extracting with ethyl acetate, washing the organic phase with 20mL of saturated saline solution, drying over anhydrous sodium sulfate, concentrating, and separating by a silica gel column to obtain 175mg of a yellow solid with a yield of 81%. ESI-MS: 217[ M ]⁺+1]。

Step 2.4- (6-nitropyridin-3-yl) piperazine-2, 2, 3, 3, 5, 5, 6, 6-d 8-1-carboxylic acid tert-butyl ester (compound 11) synthesis.

1- (6-Nitropyridin-3-yl) piperazine-2, 2, 3, 3, 5, 5, 6, 6-d8(175mg, 0.8mmol) was dissolved in 10mL of dichloromethane and triethylamine (0.22mL, 1.6mmol) and (Boc) were added sequentially₂O (210mg, 0.96mmol), stirred at room temperature overnight, after completion of the reaction 10mL of water was added, the layers were separated, the aqueous phase was extracted with dichloromethane, the organic phases were combined, washed with 20mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to give 230mg of a yellow solid with a yield of 90%.¹H NMR(400MHz，CDCl₃)8.18(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 3.4- (6-aminopyridin-3-yl) piperazine-2, 2, 3, 3, 5, 5, 6, 6-d 8-1-carboxylic acid tert-butyl ester (compound 12) synthesis.

Tert-butyl 4- (6-nitropyridin-3-yl) piperazine-2, 2, 3, 3, 5, 5, 6, 6-d 8-1-carboxylate (230mg, 0.73mmol) was dissolved in 10mL of isopropanol, 30mg of 10% palladium on carbon was added, replaced with hydrogen three times, and the mixture was stirred at room temperature under a hydrogen atmosphere of 1 atm overnight. After the reaction is completed, palladium-carbon is filtered, the filtrate is concentrated, and a reddish brown solid of 165mg is obtained by silica gel column separation, with the yield of 80%.¹H NMR(400MHz，CDCl₃)7.73(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 Synthesis of tert-butyl 4- (6- ((7-cyclopentyl-6-dicarboxamide-7-H-pyrrolo [2, 3-d ] pyrimidin-2-yl) amino) -pyridin-3-yl) piperazine-2, 2, 3, 3, 5, 5, 6, 6-d 8-1-carboxylate (Compound 13).

2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2, 3-d ]]Pyrimidine-6-carboxamide (90mg, 0.3mmol) was dissolved in 5mL of methyl isobutyl ketone, palladium acetate (3mg, 0.015mmol), 1, 1 '-binaphthyl-2, 2' -bis-diphenylphosphine (BINAP, 18mg, 0.03mmol), cesium carbonate (146mg, 0.45mmol) and tert-butyl 4- (6-amino-pyridin-3-yl) -piperazine-2, 2, 3, 3, 5, 5, 6, 6-d 8-1-carboxylate (97mg, 3.3mmol) were added in this order, nitrogen was replaced three times, and the reaction mixture was reacted at 110 ℃ overnight. After the reaction, the temperature was reduced to room temperature, 10mL of water was added, extraction was performed with ethyl acetate, the organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to obtain 110mg of a pale yellow oily substance with a yield of 66%.¹H NMR(300MHz，CDCl₃)8.71(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＝8.8Hz，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-Synthesis of cyclopentyl-N, N-dimethyl-2- ((5- (piperazin-1-yl-2, 2, 3, 3, 5, 5, 6, 6-d8) pyridin-2-yl) amino) -7H-pyrrolo [2, 3-d ] pyrimidine-6-carboxamide (Compound 14).

4- (6- ((7-cyclopentyl-6-dicarboxamide-7-H-pyrrolo [2, 3-d)]Pyrimidin-2-yl) amino) -pyridin-3-yl) piperazine-2, 2, 3,tert-butyl 3, 5, 5, 6, 6-d 8-1-carboxylate (110mg, 2mmol) was dissolved in 5mL of dichloromethane, 2mL of trifluoroacetic acid was added, stirring was carried out at room temperature for 2 hours, the solvent was evaporated under reduced pressure, 10mL of saturated sodium bicarbonate solution was added, stirring was carried out for 10 minutes, extraction was carried out with dichloromethane, the organic phase was washed with 10mL of saturated saline, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to obtain 80mg of a pale yellow solid with a yield of 90%.¹H NMR(400MHz，CDCl₃)8.68(s，1H)，8.35(d，J＝9.1Hz，1H)，7.99(d，J＝2.9Hz，1H)，7.81(s，1H)，7.32(dd，J＝9.1，3.0Hz，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-pyrrolo [2, 3-d]Pyrimidine-6-carboxamide (Compound 19)

The specific synthesis steps are as follows:

step 1.2 Synthesis of amino-5- (piperazin-1-yl) pyridine-3, 4, 6-d3 (Compound 16).

Tert-butyl 4- (6-aminopyridin-3-yl) piperazine-1-carboxylate (556mg, 2mmol) was added to 10mL of a 10% aqueous solution of sodium deuteroxide and reacted at 200 ℃ for 5 hours under nitrogen. After cooling to room temperature, 10mL of heavy water was added, extraction was performed with dichloromethane, and the organic phase was washed with 10mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to obtain 145mg of a pale yellow oil with a yield of 40%.¹H NMR(300MHz，CDCl₃)5.62(s，2H)，3.40-3.44(m，4H)，2.83-2.89(m，4H)；ESI-MS：182[M⁺+1]。

Step 2.4- (6-aminopyridin-3-yl-2, 4, 5-d3) Synthesis of tert-butyl 4- (6-aminopyridin-3-yl) -piperazine-1-carboxylate (Compound 17).

Will 2-amino-5- (piperazin-1-yl) pyridine-3, 4, 6-d3(145mg, 0.8mmol) was dissolved in 10mL of dichloromethane and triethylamine (0.22mL, 1.6mmol) and (Boc) were added sequentially₂O (210mg, 0.96mmol), stirring at room temperature overnight, adding 10mL of water after the reaction is complete, stirring for layering, extracting the aqueous phase with dichloromethane, combining the organic phases, washing with 20mL of saturated saline solution, drying over anhydrous sodium sulfate, concentrating, and separating by a silica gel column to obtain 200mg of brown solid with a yield of 90%.¹H NMR(300MHz，CDCl₃)5.65(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-dicarboxamide-7-H-pyrrolo [2, 3-d ] pyrimidin-2-yl) amino) -pyridin-3-yl-2, 4, 5-d3) synthesis of tert-butyl 4- (6- ((7-cyclopentyl-6-dicarboxamide) -2, 4, 5-d) piperazine-1-carboxylate (compound 18).

2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2, 3-d ]]Pyrimidine-6-carboxamide (90mg, 0.3mmol) was dissolved in 5mL of methyl isobutyl ketone, palladium acetate (3mg, 0.015mmol), BINAP (18mg, 0.03mmol), cesium carbonate (146mg, 0.45mmol) and tert-butyl 4- (6-aminopyridin-3-yl-2, 4, 5-d3) (93mg, 0.33mmol) were added in this order, the mixture was replaced with nitrogen three times, and the reaction mixture was reacted at 110 ℃ overnight. After the reaction, 10mL of water was added, extraction was performed with ethyl acetate, and the organic phase was washed with 10mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to obtain 116mg of a pale yellow oily substance with a yield of 70%.¹H NMR(300MHz，CDCl₃)8.71(s，1H)，7.98(s，1H)，6.46(s，1H)，4.77(t，J＝8.8Hz，1H)，3.60(t，J＝5.1Hz，4H)，3.12(d，J＝17.7Hz，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-Synthesis of cyclopentyl-N, N-dimethyl-2- ((5- (piperazin-1-yl) pyridin-2-yl-3, 4, 6-d3) amino) -7H-pyrrolo [2, 3-d ] pyrimidine-6-carboxamide (Compound 19).

4- (6- ((7-cyclopentyl-6-dicarboxamide-7-H-pyrrolo [2, 3-d)]Pyrimidin-2-yl) amino) -pyridin-3-yl-2, 4, 5-d3) piperazine-1-carboxylic acid tert-butyl ester (116mg, 2.2mmol) was dissolved in 5mL of dichloromethane, 2mL of trifluoroacetic acid was added, stirring was carried out at room temperature for 2 hours, and the solution was evaporated under reduced pressure10mL of saturated sodium carbonate solution is added into the solvent, the mixture is stirred for 10 minutes, dichloromethane is used for extraction, an organic phase is washed by 10mL of saturated saline solution, anhydrous sodium sulfate is dried, concentration is carried out, and light yellow solid 85mg is obtained by silica gel column separation, and the yield is 90%.¹H NMR(300MHz，CDCl₃)8.69(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-cyclopentenamine (1.66g, 20mmol) was dissolved in 20mL CH₃OD, 160mg of 10% palladium on carbon was added, and deuterium gas was substituted three times, and the mixture was stirred overnight at room temperature under a deuterium atmosphere of 1 atm. After the reaction is completed, palladium-carbon is filtered out, and the filtrate is concentrated to obtain a reddish brown solid 1.57g with the yield of 90%. ESI-MS: 88[ M ]⁺+1]。

Step 2.5-bromo-2-chloro-N- (cyclopentyl-3, 4-d2) pyrimidin-4-amine (compound 23) synthesis.

5-bromo-2, 4-dichloropyrimidine (3.6g, 16mmol) was dissolved in 20mL of absolute ethanol, triethylamine (4.4mL, 32mmol) was added, cyclopentylamine-3, 4-d2(1.57g, 18mmol) was slowly added dropwise in an ice bath, after the dropwise addition, the mixture was stirred at room temperature for 5 hours, the solvent was evaporated under reduced pressure, and the residue was separated by a silica gel column to give 3.3g of a white solid with a yield of 75%.¹H NMR(400MHz，CDCl₃)8.09(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＝7.2Hz，1H)，1.67(s，1H)，1.46(tt，J＝13.7，6.3Hz，2H)；ESI-MS：278[M⁺+1]。

Step 3.2-chloro-N- (cyclopentyl-3, 4-d2) -5- (3, 3-diethoxypropyl-1-yn-1-yl) pyrimidin-4-amine (compound 24) synthesis.

5-bromo-2-chloro-N- (cyclopentyl-3, 4-d2) pyrimidin-4-amine (2.0g, 7.3mmol) and propiolic aldehyde diethyl diacetal (1.1g, 8.6mmol), Pd (dppf) Cl₂(510mg, 0.73mmol), CuI (140mg, 0.73mmol) and 5mL of triethylamine were added to 20mL of DMF, and the mixture was purged with nitrogen three times, warmed to 100 ℃ and reacted overnight. After completion of the reaction, the reaction mixture was cooled to room temperature, 40mL of water was added, extraction was performed with ethyl acetate, and the organic phase was washed with 20mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to obtain 1.01g of a pale yellow oily substance with a yield of 43%. ESI-MS: 326[ M ]⁺+1]。

Step 4.2-chloro-7- (cyclopentyl-3, 4-d2) -6- (diethoxymethyl) -7H-pyrrolo [2, 3-d ] pyrimidine (compound 25) synthesis.

2-chloro-N- (cyclopentyl-3, 4-d2) -5- (3, 3-diethoxypropyl-1-yn-1-yl) pyrimidin-4-amine (1.01g, 3.1mmol) was dissolved in 10mL of tetrahydrofuran, tetrabutylammonium fluoride (2.4g, 9.3mmol) was added, and the reaction was carried out at 65 ℃ for 2 hours. The solvent was removed by rotary evaporation, 20mL of water was added, extraction was performed with ethyl acetate, the organic phase was washed with 20mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to give 830mg of a pale yellow oil with a yield of 82%. ESI-MS: 326[ M ]⁺+1]。

Step 5.2-chloro-7- (cyclopentyl-3, 4-d2) -7H-pyrrolo [2, 3-d ] pyrimidine-6-carbaldehyde (compound 26) synthesis.

2-chloro-7- (cyclopentyl-3, 4-d2) -6- (diethoxymethyl) -7H-pyrrolo [2, 3-d]Pyrimidine (830mg, 2.6mmol) was dissolved in 10mL dioxane, 4mL concentrated hydrochloric acid was slowly added dropwise, stirred at room temperature for 20 minutes, 15mL water was added, extraction was performed with ethyl acetate, the organic phase was washed with 10mL saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column to give 525mg of pale yellow solid with a yield of 82%. ESI-MS: 252[ M ]⁺+1]。

Step 6.2-chloro-7- (cyclopentyl-3, 4-d2) -7H-pyrrolo [2, 3-d ] pyrimidine-6-carboxylic acid (compound 27) synthesis.

2-chloro-7- (cyclopentyl-3, 4-d2) -7H-pyrrolo [2, 3-d]Pyrimidine-6-carbaldehyde (525mg, 2.1mmol) was dissolved in 5ml of DMF, and potassium peroxomonosulfonate (1.4g, 2.3mmol) was added thereto to react at room temperature for 6 hours. After the reaction, 10mL of water was added, and a large amount of solid was precipitated, which was filtered and dried to obtain 475mg of pale yellow solid in 85% yield. ESI-MS: 268[ M ]⁺+1]。

Step 7.2-chloro-7- (cyclopentyl-3, 4-d2) -N, N-dimethyl-7H-pyrrolo [2, 3-d ] pyrimidine-6-carboxamide (compound 28) synthesis.

2-chloro-7- (cyclopentyl-3, 4-d2) -7H-pyrrolo [2, 3-d]Pyrimidine-6-carboxylic acid (475mg, 1.8mmol), HBTU (680mg, 1.8mmol) and DIEA (0.9mL, 5.4mmol) were dissolved in 10mL DMF, 2M dimethylamine methanol solution (1.1mL, 2.2mmol) was added dropwise in ice bath, reaction was performed at room temperature for 30 minutes after completion of dropwise addition, 20mL water was added, extraction was performed with ethyl acetate, the organic phase was washed with 20mL saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column to obtain 420mg of red solid with a yield of 80%. ESI-MS: 295[ M ]⁺+1]。

Step 8.4- (6- ((7- (cyclopentyl-3, 4-d2) -6-dicarboxamide-7-H-pyrrolo [2, 3-d ] pyrimidin-2-yl) amino) -pyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester (compound 29) synthesis.

2-chloro-7- (cyclopentyl-3, 4-d2) -N, N-dimethyl-7H-pyrrolo [2, 3-d)]Pyrimidine-6-carboxamide (90mg, 0.3mmol) was dissolved in 5mL of methyl isobutyl ketone, palladium acetate (3mg, 0.015mmol), BINAP (18mg, 0.03mmol), cesium carbonate (146mg, 0.45mmol) and tert-butyl 4- (6-amino-pyridin-3-yl) -piperazine-1-carboxylate (91mg, 0.33mmol) were added in that order, nitrogen was replaced three times, and the reaction mixture was reacted at 110 ℃ overnight. After the reaction, the temperature was reduced to room temperature, 10mL of water was added, extraction was performed with ethyl acetate, the organic phase was washed with 10mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to obtain 115mg of a pale yellow oily substance with a yield of 70%. 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-pyrrolo [2, 3-d ] pyrimidine-6-carboxamide (compound 30) synthesis.

Compound 4-(6- ((7- (cyclopentyl-3, 4-d2) -6-dicarboxamide-7-H-pyrrolo [2, 3-d)]Pyrimidin-2-yl) amino) -pyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester (115mg, 0.2mmol) was dissolved in 5mL of dichloromethane, 2mL of trifluoroacetic acid was added, stirred at room temperature for 2 hours, the solvent was evaporated under reduced pressure, 10mL of saturated sodium carbonate solution was added, stirred for 10 minutes, extracted with dichloromethane, the organic phase was washed with 10mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to give 85mg of a pale yellow solid with a yield of 90%.¹H NMR(300MHz，DMSO-d₆)9.40(s，1H)，8.76(s，1H)，8.18(d，J＝9.1Hz，1H)，8.04(d，J＝3.0Hz，1H)，7.48(dd，J＝9.1，3.0Hz，1H)，6.60(s，1H)，4.73(t，J＝8.6Hz，1H)，3.27(m，4H)，3.22-3.14(m，4H)，3.05(s，6H)，2.42(d，J＝9.6Hz，1H)，1.98(d，J＝6.2Hz，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 (2.24g, 20mmol), 0.2g of 10% palladium on carbon and 10mL of heavy water were added to a 20mL microwave tube, bubbled with hydrogen for 1 minute, sealed and placed in the microwave reactor. Microwave reaction is carried out for 1.5 hours at 130 ℃, taken out after the temperature is reduced to room temperature, 20mL of heavy water is added, the temperature is raised to 95 ℃, the mixture is heated for 1 hour and then filtered while the mixture is hot, and the filtrate is concentrated to obtain a white solid 1.82 with the yield of 80 percent.¹H NMR(300MHz，DMSO-d₆)10.80(s，2H)；ESI-MS：115[M⁺+1]。

Step 2.5-Bromopyrimidine-2, 4(1H, 3H) -dione-6-d (Compound 33) Synthesis.

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

Step 3.5-bromo-2, 4-dichloropyrimidine-6-d (Compound 34) synthesis.

1mL of N, N-dimethylaniline is added into 5-bromopyrimidine-2, 4(1H, 3H) -diketone-6-d (2.75g, 14.4mmol), stirring is started, cold phosphorus oxychloride (7mL, 72mmol) is slowly added into the reaction mixture dropwise under ice bath, and after the dropwise addition is finished, the temperature is raised to reflux, and the reaction is carried out for 2 hours. Cooling to room temperature, evaporating under reduced pressure to remove phosphorus oxychloride, dragging with toluene for 3 times, slowly adding ice water into the concentrated residue, adjusting pH to neutrality with ammonia water, extracting with ethyl acetate, washing the organic phase with 10mL of saturated saline solution, drying with anhydrous sodium sulfate, concentrating, and separating with silica gel column to obtain light yellow solid 2.22g with yield of 68%. ESI-MS: 228[ M ]⁺+1]。

Step 4.5-bromo-2-chloro-N-cyclopentylpyrimidin-6-d-4-amine (Compound 35) synthesis.

5-bromo-2, 4-dichloropyrimidine-6-d (2.22g, 9.78mmol) is dissolved in 20mL of absolute ethanol, triethylamine (2.7mL, 19.6mmol) is added, cyclopentylamine (0.92g, 10.8mmol) is slowly added dropwise in an ice bath, stirring is carried out at room temperature for 5 hours after the dropwise addition is finished, the solvent is evaporated under reduced pressure, and the residue is separated by a silica gel column to obtain 2.02g of a white solid with a yield of 75%.¹H NMR(300MHz，DMSO-d₆)7.37(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 5.2-chloro-N-cyclopentyl-5- (3, 3-diethoxypropyl-1-yn-1-yl) pyrimidin-6-d-4-amine (compound 36) synthesis.

5-bromo-2-chloro-N-cyclopentylpyrimidin-6-d-4-amine (2.0g, 7.3mmol) and propiolic aldehyde diethyl diacetal (1.1g, 8.6mmol), Pd (dppf) Cl₂(510mg, 0.73mmol), CuI (140mg, 0.73mmol) and 5mL triethylamine were added to 20mL of DMMF under nitrogenThe displacement was carried out three times, and the temperature was raised to 100 ℃ to carry out the reaction overnight. After completion of the reaction, the reaction mixture was cooled to room temperature, 40mL of water was added, extraction was performed with ethyl acetate, and the organic phase was washed with 20mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to obtain 1.01g of a pale yellow oily substance with a yield of 43%. ESI-MS: 325[ M ]⁺+1]。

Step 6.2-chloro-7-cyclopentyl-6- (diethoxymethyl) -7H-pyrrolo [2, 3-d ] pyrimidine-4-d (Compound 37) synthesis.

2-chloro-N-cyclopentyl-5- (3, 3-diethoxypropyl-1-yn-1-yl) pyrimidin-6-d-4-amine (1.01g, 3.1mmol) was dissolved in 10mL of tetrahydrofuran, tetrabutylammonium fluoride (TBAF, 2.4g, 9.3mmol) was added, and the reaction was carried out at 65 ℃ for 2 hours. The solvent was removed by rotary evaporation, 20mL of water was added, extraction was performed with ethyl acetate, the organic phase was washed with 20mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to give 830mg of a pale yellow oil with a yield of 82%. ESI-MS: 325[ M ]⁺+1]。

Step 7.2-chloro-7-cyclopentyl-7H-pyrrolo [2, 3-d ] pyrimidine-4-d-6-carbaldehyde (compound 38) was synthesized.

2-chloro-7-cyclopentyl-6- (diethoxymethyl) -7H-pyrrolo [2, 3-d)]Pyrimidine-4-d (830mg, 2.6mmol) is dissolved in 10mL dioxane, 4mL concentrated hydrochloric acid is slowly added dropwise, the mixture is stirred at room temperature for 20 minutes, 15mL water is added, extraction is carried out with ethyl acetate, the organic phase is washed with 10mL saturated saline, dried with anhydrous sodium sulfate, concentrated and separated by a silica gel column to obtain 525mg of light yellow solid with the yield of 82%. ESI-MS: 251[ M⁺+1]。

Step 8.2-chloro-7-cyclopentyl-7H-pyrrolo [2, 3-d ] pyrimidine-4-d-6-carboxylic acid (compound 39) synthesis.

2-chloro-7-cyclopentyl-7H-pyrrolo [2, 3-d)]Pyrimidine-4-d-6-carbaldehyde (525mg, 2.1mmol) was dissolved in 5ml of DMF, and potassium peroxomonosulfonate (1.4g, 2.3mmol) was added thereto to conduct a reaction at room temperature for 6 hours. After the reaction, 10mL of water was added, and a large amount of solid was precipitated, which was filtered and dried to obtain 475mg of pale yellow solid in 85% yield. ESI-MS: 267[ M ]⁺+1]。

Step 9.2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2, 3-d ] pyrimidine-4-d-6-carboxamide (compound 40) synthesis.

2-chloro-7-cyclopentyl-7H-pyrrolo [2, 3-d)]Pyrimidine-4-d-6-carboxylic acid (475mg, 1.8mmol), HBTU (680mg, 1.8mmol) and DIEA (0.9mL, 5.4mmol) were dissolved in 10mL DMF, 2M dimethylamine methanol solution (1.1mL, 2.2mmol) was added dropwise under ice bath, reaction was performed at room temperature for 30 minutes after completion of dropwise addition, 20mL water was added, extraction was performed with ethyl acetate, the organic phase was washed with 20mL saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column to obtain 420mg of red solid with a yield of 80%.¹H NMR(300MHz，DMSO-d₆)6.80(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＝12.1，7.1，6.6，2.6Hz，2H)；ESI-MS：294[M⁺+1]。

Step 10.4- (6- ((7-cyclopentyl-6-dicarboxamide-7-H-pyrrolo [2, 3-d ] pyrimidin-2-yl-4-d) amino) -pyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester (compound 41) synthesis.

2-chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo [2, 3-d ]]Pyrimidine-4-d-6-carboxamide (90mg, 0.3mmol) was dissolved in 5mL of methyl isobutyl ketone, palladium acetate (3mg, 0.015mmol), BINAP (18mg, 0.03mmol), cesium carbonate (146mg, 0.45mmol) and tert-butyl 4- (6-amino-pyridin-3-yl) -piperazine-1-carboxylate (91mg, 0.33mmol) were added in that order, nitrogen was replaced three times, and the reaction mixture was reacted at 110 ℃ overnight. After the reaction, the temperature was reduced to room temperature, 10mL of water was added, extraction was performed with ethyl acetate, the organic phase was washed with 10mL of saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by a silica gel column to obtain 115mg of a pale yellow oily substance with a yield of 70%.¹H NMR(300MHz，DMSO-d₆)9.36(s，1H)，8.17(d，J＝9.1Hz，1H)，8.01(d，J＝2.9Hz，1H)，7.46(dd，J＝9.1，3.0Hz，1H)，6.60(s，1H)，4.72(q，J＝8.9Hz，1H)，3.48(t，J＝5.0Hz，4H)，3.09-3.05(d，J＝9.6Hz，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 Synthesis of 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).

Compound 4- (6- ((7-cyclopentyl)6-Dimethylamide-7-H-pyrrolo [2, 3-d ] methyl ester]Pyrimidine-2-yl-4-d) amino) -pyridin-3-yl) piperazine-1-carboxylic acid tert-butyl ester (115mg, 0.2mmol) was dissolved in 5mL dichloromethane, 2mL trifluoroacetic acid was added, stirred at room temperature for 2 hours, the solvent was evaporated under reduced pressure, 10mL saturated sodium carbonate solution was added, stirred for 10 minutes, extracted with dichloromethane, the organic phase was washed with 10mL saturated brine, dried over anhydrous sodium sulfate, concentrated, and separated by silica gel column to give 85mg of a pale yellow solid with a yield of 90%.¹H NMR(300MHz，DMSO-d₆)9.39(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]。

And (4) testing the biological activity.

(1) And (4) measuring the enzyme activity.

Experimental materials:

CDK2/cyclin A, CDK4/cyclin D1 and CDK6/cyclin D1. The polypeptide substrates of ULight marker, ULight-4E-BP1 and ULight-MBP. Europium-labeled anti-myelin basic protein antibody and europium-labeled rabbit antibody, and an Envision multi-label analyzer for detecting signals.

The experimental method comprises the following steps:

the compounds to be detected were diluted three times, including 10 concentration gradients. The initial CDK2/cyclin A test compound concentration was 10uM, CDK4/cyclin D1 and CDK6/cyclin D1 test compound concentrations were 1 uM.

Enzyme reaction system of CDK2/cyclin A:

the standard Lance Ultra method was carried out by a 10. mu.l enzyme reaction system, containing 0.5 nanomolar CDK2/cyclin A protein, 100 nanomolar ULight-MBP polypeptide, and 25. mu.l ATP. Dissolving them in enzyme buffer respectively, wherein the buffer comprises the following components: 50mM of hydroxyethyl piperazine ethyl sulfate solution with the pH of 7.5, 1 millimole of ethylene diamine tetraacetic acid, 10 millimoles of magnesium chloride, 0.01 percent of Brij-35 and 2 millimoles of dithiothreitol. After the reaction was started, the OptiPlate384 well plates were sealed with a top heat seal membrane TopSeal-A and incubated for 60 minutes at room temperature.

CDK4/cyclin D1 enzyme reaction system:

the standard Lance Ultra method was carried out by a 10. mu.l enzyme reaction system, containing 1 nanomole of CDK4/cyclin D1 protein, 50 nanomole of ULight-4E-BP1 polypeptide, and 350. mu.l ATP. Dissolving them in enzyme buffer respectively, wherein the buffer comprises the following components: 50mM of hydroxyethyl piperazine ethyl sulfate solution with the pH of 7.5, 1 millimole of ethylene diamine tetraacetic acid, 10 millimoles of magnesium chloride, 0.01 percent of Brij-35 and 2 millimoles of dithiothreitol. After the reaction was started, the OptiPlate384 well plates were sealed with a top heat seal membrane TopSeal-A and incubated for 90 minutes at room temperature.

CDK6/cyclin D1 enzyme reaction system:

the standard Lance Ultra method was carried out by a 10. mu.l enzyme reaction system, containing 0.8 nanomolar CDK6/cyclin D1 protein, 50 nanomolar ULight-4E-BP1 polypeptide, and 250. mu.l ATP. The enzyme buffer solution is dissolved in 50mM hydroxyethylpiperazine ethanesulfonic acid solution with pH of 7.5, 1mM ethylenediamine tetraacetic acid, 10 mM magnesium chloride, 0.01% Brij-35, and 2mM dithiothreitol. After the reaction was started, the OptiPlate384 well plates were sealed with a top heat seal membrane TopSeal-A and incubated for 180 minutes at room temperature.

An enzyme reaction termination buffer was prepared, EDTA was dissolved in a 1-fold diluted detection buffer, and the reaction was terminated at room temperature for 5 minutes. 5 microliters of assay mix (formulated with europium-labeled anti-myelin basic protein antibody and europium-labeled rabbit antibody, respectively) was added to the CDK2/cyclin A, CDK4/cyclin D1 and CDK6/cyclin D1 reactions, respectively. And (3) incubating at room temperature for 60min, and detecting a reaction signal by using an Envision instrument according to a time-resolved fluorescence resonance energy transfer principle.

And (3) data analysis:

the original data is converted into the inhibition rate by using an equation (Max-Ratio)/(Max-Min) × 100%, and the value of IC50 can be obtained by performing curve fitting through four parameters.

Examples 2-5 and their non-deuterated compounds, Ribociclib, were tested as described above and the kinase inhibition is shown in Table 1 below.

Table 1:

as shown in Table 1, the compounds of the present invention showed excellent inhibitory activity against CDK4/cyclin D1 and CDK6/cyclin D1, and very low inhibitory activity against CDK2/cyclin A. In particular, the compounds of examples 4 and 5 have better 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 MCF-7 and MDA-MB-436 cell activity was examined.

Reagent and consumable

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

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

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

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

Principle of experiment

The ATP content directly reflects the number of cells and the cell state, and the number of viable cells in the culture is determined by quantitative determination of ATP. The living cell detection kit adopts luciferase as a detection object, a stable glow type signal generated by UltraGlow luciferase is used in the kit, the luciferase needs participation of ATP in the luminescence process, and ATP can be generated by the respiration effect and other life activity processes of cells with metabolic activity. Adding CellTiter-Glo to cell culture medium^TMThe reagent measures the luminous value, the light signal is in direct proportion to the ATP amount in the system, and ATP is positively correlated with the number of living cells, so that the proliferation condition of the cells can be detected. The panels were analyzed using Envision from PE.

Experimental methods

1. Preparation of cell plates

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

2. Preparation of the Compounds

A double well experiment was set up with 3-fold dilutions of ECHO, 10 compound concentrations.

3. Compound-treated cells

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

4. Detection of

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

Examples 2-5 and their non-deuterated compounds, 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 compounds 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 had better inhibitory activity on MCF-7 and MDA-MB-436 cells than the non-deuterated compound Ribociclib. Therefore, the compound is hopeful to be used as a medicine for treating ER positive breast cancer and HER2 negative breast cancer.

(3) And (5) evaluating the metabolic stability.

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

Preparing a stock solution: an amount of compound Ribociclib and the powders of examples 2-5 were weighed precisely and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of a 0.5M dipotassium hydrogenphosphate solution prepared in advance were mixed, the pH of the mixture was adjusted to 7.4 with the 0.5M dipotassium hydrogenphosphate solution, the mixture was diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100mM) containing 100mM potassium phosphate and 3.3mM magnesium chloride at a pH of 7.4.

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

Preparing a stop solution: acetonitrile solution containing 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide (internal standard). 25057.5 mu L of phosphate buffer solution (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of human liver microsome is respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained. 25057.5 mu L of phosphate buffer (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of SD rat liver microsome is respectively added, and the mixture is mixed evenly to obtain liver microsome dilution with the protein concentration of 0.625 mg/mL.

Incubation of the samples: the stock solutions of the corresponding compounds were diluted to 0.25mM each with an aqueous solution containing 70% acetonitrile, and used as working solutions. 398. mu.L of human liver microsome or rat liver microsome dilutions were added to a 96-well plate (N2), 2. mu.L of 0.25mM working solution was added, and mixed well.

Determination of metabolic stability: 300. mu.L 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 are placed in a 37 ℃ water bath box, shaken at 100 rpm and pre-incubated for 5 min. 80. mu.L of the incubation solution was taken out of each well of the incubation plate, added to the stop plate, mixed well, and supplemented with 20. mu.L of NADPH regenerating system solution as a 0min sample. Then 80. mu.L of NADPH regenerating system solution was added to each well of the incubation plate, the reaction was started, and the timer was started. The reaction concentration of the corresponding compound was 1. mu.M, and the protein concentration was 0.5 mg/mL. When the reaction was carried out for 10min, 30 min and 90min, 100. mu.L of each reaction solution was added to the stop plate and vortexed for 3min to terminate the reaction. The stop plates were centrifuged at 5000 Xg for 10min at 4 ℃. And (3) taking 100 mu L of supernatant to a 96-well plate in which 100 mu L of distilled water is added in advance, mixing uniformly, and performing sample analysis by adopting LC-MS/MS.

And (3) data analysis: and detecting peak areas of the corresponding compound and the internal standard through an LC-MS/MS system, and calculating the peak area ratio of the compound to the internal standard. The slope is determined by plotting the natural logarithm of the percentage of compound remaining against time and calculating t according to the following formula_1/2And CL_intWhere V/M is equal to 1/protein concentration.

The compounds of the invention and compounds without deuteration were tested simultaneously and compared to evaluate their metabolic stability in human and rat liver microsomes. The half-life and intrinsic hepatic clearance as indicators of metabolic stability are shown in table 3. The non-deuterated 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 non-deuterated compound, Ribociclib.

TABLE 3

(4) Pharmacokinetic evaluation in rats.

8 male Sprague-Dawley rats, 7-8 weeks old, weighing about 210g, divided into 2 groups of 4 rats each, given 5mg/kg per single oral administration; (a) control group: ribociclib; (b) test groups: the compounds of the examples were compared for their pharmacokinetic differences.

Rats were fed with standard feed and given water. Fasting began 16 hours prior to the experiment. The drug was dissolved with PEG400 and dimethyl sulfoxide. Blood was collected from the orbit at 0.083 hr, 0.25 hr, 0.5 hr, 1 hr, 2 hr, 4 hr, 6 hr, 8 hr, 12 hr and 24 hr post-dose.

The rats were briefly anesthetized after ether inhalation and 300 μ L of blood was collected from the orbit into a test tube. There was 30 μ L of 1% heparin salt solution in the tube. Before use, the tubes were dried overnight at 60 ℃. After completion of blood sample collection at a subsequent time point, rats were sacrificed after ether anesthesia.

Immediately after blood collection, the tubes were gently inverted at least 5 times to ensure mixing and then placed on ice. The blood samples were centrifuged at 5000rpm for 5 minutes at 4 ℃ to separate the plasma from the erythrocytes. Pipette 100 μ L of plasma into a clean plastic centrifuge tube, designating the name of the compound and the time point. Plasma was stored at-80 ℃ before analysis. The concentration of the compounds 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 results of the experiments show that the compounds of the invention have better pharmacokinetics and thus better pharmacodynamics and therapeutic effects in animals compared to the control compounds.

It is to be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention, and that experimental procedures not specifically identified in the examples will generally be performed under conventional conditions, or under conditions recommended by the manufacturer. Parts and percentages are parts and percentages by weight unless otherwise indicated.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims (6)

1. A compound, wherein the compound is selected from the group consisting of:

2. a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmaceutical composition of the compound of claim 1 or a pharmaceutically acceptable salt thereof as an effective ingredient, together with a conventional pharmaceutical carrier.

3. Use of a compound of claim 1 in the manufacture of a medicament for the prevention or treatment of cancer.

4. Use according to claim 3, wherein the cancer is selected from breast cancer, pancreatic cancer, colorectal cancer, lung cancer, melanoma, leukemia, glioma or mantle cell lymphoma.

5. Use according to claim 4, wherein the cancer is selected from familial melanoma.

6. The use according to claim 4, wherein said cancer is selected from non-small cell lung cancer.