CN117736208A - A class of pyrrole [2,3-d] pyrimidine derivatives and their preparation methods and applications - Google Patents

Abstract

The invention belongs to the field of organic chemistry, and particularly relates to a pyrrole [2,3-d ] pyrimidine derivative, and a preparation method and application thereof. The specific technical scheme is as follows: pyrrole [2,3-d ] pyrimidine derivative shown in structural formula (I). The invention provides a novel class of pyrrole [2,3-d ] pyrimidine derivatives, and in particular provides 70 novel compounds. The derivative has good CDK9 inhibition activity, can be used for treating cancers mediated by highly dependent CDK9, provides a novel targeted treatment strategy for anti-tumor treatment, and has great pharmaceutical potential.

Description

A class of pyrrole [2,3-d] pyrimidine derivatives and preparation methods and applications thereof

Technical Field

The invention belongs to the field of organic chemistry, and specifically relates to a class of pyrrole [2,3-d] pyrimidine derivatives and a preparation method and application thereof.

Background Art

Cyclin-dependent kinases (CDKs) belong to serine/phosphorylase kinases, which play an important role in biological processes such as cell proliferation, apoptosis, and transcription under the regulation of intracellular and extracellular signals. CDK9 is an important member of the CDK family and is mainly involved in the transcriptional regulation process. The heterodimer composed of CSK9 and cyclin (T1, T2a, T2b, K) participates in the formation of the positive transcription elongation factor (p-TEFb) complex and regulates the transcriptional elongation process by phosphorylating the carboxyl-terminal domain of RNA polymerase II, mainly Ser-2 phosphorylation, as a subunit. The inhibition and transcriptional blockage of CDK9 lead to the rapid consumption of RNA transcription of Myc protein and short-lived anti-apoptotic proteins, thereby leading to the death of tumor cells that are highly dependent on these proteins. Therefore, CDK9 can be used as an anti-tumor drug target, and inhibition of CDK9 is expected to inhibit the invasion and metastasis of tumor cells.

The development of CDK9 inhibitors has become one of the most important research areas in the industry to expand the scope of CDK family inhibitors. In terms of drug molecules, small molecule chemical drugs are currently the main focus; in terms of indications, solid tumors and blood cancers are still in the exploratory stage as a whole, and no highly targeted tumor type has been formed. Moreover, most of the current candidate CDK9 inhibitors are non-selective, such as Palbociclib, Flavopiridol, Roscovitine, Roniciclib, Ribociclib, etc. These compounds have defects such as poor selectivity, large side effects, and unknown mechanisms, which limit their further development and application.

Therefore, the development of new, highly selective and active CDK9 inhibitors is of great significance for both basic research and clinical application.

Summary of the invention

The purpose of the present invention is to provide a new type of pyrrole [2,3-d] pyrimidine derivatives and a preparation method and application thereof.

In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a pyrrole [2,3-d] pyrimidine derivative, the general structural formula of which is as follows:

Wherein, _R1 is selected from any one of methyl, ethyl, and isopropyl;

R ₂ is selected from Any of the following;

R ₃ to R ₆ are independently selected from H, substituted or unsubstituted C1-C8 alkyl, Any one of, n = 0 ~ 1;

_R7 is selected from any one of substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted 3-8 membered cycloalkyl, and substituted or unsubstituted C2-C8 alkenyl.

Preferably, _R2 is any one of the following structural formulas:

Preferably, the structural formula of the derivative is any one of those in Table 1.

Accordingly, the preparation method of the derivative comprises the following steps:

a. Preparation of intermediate SM2: 5-bromo-2-hydroxypyridine, acetonitrile and Cs ₂ CO ₃ are mixed, and 2-iodopropane or iodoethane or iodomethyl is added to obtain a mixture; the mixture is heated and stirred, and after the reaction is completed, the mixture is filtered and the solvent is drained to obtain SM2;

b. Preparation of intermediate SM3: SM2, bis(pinacol)diboron, potassium acetate and _PdCl2 were mixed, 1,4-dioxane was added as solvent, and the mixture was heated under the protection of inert gas until the reaction was completed, cooled to room temperature, extracted, the organic phases were combined, concentrated, pulped, filtered, the upper filter cake was retained, and dried to obtain SM3;

c. Preparation of intermediate SM5: 4-chloro-6-iodo-7H-pyrrolo[2,3-d]pyrimidine, SM3, potassium carbonate and _PdCl2 were mixed, and a mixture of dioxane/ethanol/water was added as a solvent. After the oil bath reaction was completed, the reaction solution was chromatographed, gradient eluted, and the fractions were collected and concentrated to obtain SM5;

d. Preparation of intermediate SM6: Mix SM5, N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester or (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-en-1-yl)carbamic acid tert-butyl ester or 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester or N-tert-butyloxycarbonylpiperidine-4-boronic acid pinacol ester, potassium carbonate and _PdCl2 , add a mixed solution of dioxane/ethanol/water as a solvent, and after the oil bath reaction is complete, perform chromatographic separation on the reaction solution, perform gradient elution, collect fractions, and concentrate to obtain SM6;

e. Dissolve SM6 in dichloromethane and add trifluoroacetic acid. Complete the reaction at room temperature. Concentrate the reaction solution and disperse the concentrate in water. Adjust the pH value to pH>9. During the precipitation, a large amount of solid is precipitated. Filter and rinse the filter cake to obtain the pyrrole [2,3-d] pyrimidine derivative SM7.

Preferably, after step e, the method further comprises step f: dissolving SM7 in dichloromethane, and then adding acid anhydride or isocyanate respectively; after the reaction is complete, filtering with suction, rinsing the filter cake to obtain the pyrrole [2,3-d] pyrimidine derivative; or; dissolving SM7 and polyformaldehyde in methanol, after the reaction is complete, adding NaBH ₄ , and after the reaction is complete again, separating to obtain the pyrrole [2,3-d] pyrimidine derivative.

Correspondingly, the derivative or the derivative prepared by the preparation method, and/or the use of the pharmaceutically acceptable salt, solvate, hydrate, isomer, polymorph of the derivative in the preparation of drugs.

Preferably, the application is application in the preparation of CDK9 inhibitors.

Preferably, the application is application in the preparation of anti-tumor drugs.

Correspondingly, a pharmaceutical composition comprising the derivative or the derivative prepared by the preparation method, and/or the pharmaceutically acceptable salt, solvate, hydrate, isomer, polymorph of the derivative.

Preferably, the pharmaceutical composition comprises pharmaceutically acceptable excipients.

Beneficial effects of the invention: The invention provides a new class of pyrrole [2,3-d] pyrimidine derivatives, and specifically provides 70 new compounds. The derivatives have good CDK9 inhibitory activity, can be used to treat cancers that are highly dependent on CDK9 mediation, provide a new targeted therapy strategy for anti-tumor therapy, and have great pharmaceutical potential.

DETAILED DESCRIPTION

1. The present invention provides a new type of pyrrole [2,3-d] pyrimidine derivatives. The general structural formula of the derivatives is shown in formula (I).

Wherein, _R1 is selected from any one of methyl, ethyl and isopropyl.

R ₂ is selected from wherein R ₃ to R ₆ are independently selected from H, substituted or unsubstituted C1 to C8 alkyl, Any one of (n=0～1);

_R7 is selected from any one of substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted 3-8 membered cycloalkyl, and substituted or unsubstituted C2-C8 alkenyl.

A more preferred solution is: R ₂ is any one of the following structural formulas:

A more preferred embodiment is that the structural formula of the pyrrole[2,3-d]pyrimidine derivative is any one of those in Table 1.

Table 1 Structural formula comparison table of pyrrole [2,3-d] pyrimidine derivatives

2. The present invention also provides a method for preparing the pyrrole [2,3-d] pyrimidine derivative. The preparation method is divided into five categories, as follows:

(I) The first type of preparation method, the reaction equation is as follows:

The specific steps include:

(a) Preparation of intermediate SM2. SM1 (5-bromo-2-hydroxypyridine), acetonitrile and Cs ₂ CO ₃ were mixed, and then 2-iodopropane was added to obtain a mixture. The mixture was heated to 70-80°C and stirred for 4 h. After TLC showed that the reaction was complete, it was filtered, and the filter cake was treated with acetonitrile. The solvent was drained to obtain a gray solid, namely SM2.

(b) Preparation of intermediate SM3. SM2, bis(pinacol)diboron, potassium acetate and PdCl ₂ (palladium chloride, dppf) were mixed, and 1,4-dioxane was added as a solvent. After nitrogen (or inert gas such as argon) was replaced, the mixture was heated to 95-100°C for 2-4 hours. TLC showed that the reaction was complete, and the mixture was cooled to room temperature, extracted with ethyl acetate and water, and the organic phases were combined and concentrated. The concentrated product was dispersed in ether and slurried, filtered, and the upper filter cake was retained and dried to obtain a brown solid, namely SM3.

(c) Preparation of intermediate SM5. 4-chloro-6-iodo-7H-pyrrolo[2,3-d]pyrimidine (SM4), SM3, potassium carbonate and PdCl ₂ (dppf) were mixed, and a mixture of dioxane/ethanol/water was added as a solvent (the preferred solution is: by volume ratio, dioxane:ethanol:water=7:3:4), and after replacing nitrogen (or inert gas such as argon), the mixture was reacted in an oil bath at 70-85°C for 2-4 hours. The reaction solution was added to crude silica gel and mixed with the sample, and then chromatographic separation was performed, and the mobile phase was dichloromethane/methanol (1%-5%) for gradient elution. The fractions were collected and concentrated to obtain an off-white solid, namely SM5.

(d) Preparation of intermediate SM6. SM5, N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester, potassium carbonate and PdCl ₂ (dppf) were mixed, and a dioxane/ethanol/water mixture was added as a solvent (the preferred solution is: by volume ratio, dioxane:ethanol:water=7:3:4), and after replacing nitrogen (or inert gas such as argon), the mixture was reacted in an oil bath at 70-85°C for 2-4 hours. The reaction solution was added with crude silica gel for mixing and then chromatographically separated. The mobile phase was dichloromethane/methanol (1%-5%) for gradient elution. The fractions were collected and concentrated to obtain an off-white solid SM6.

(e) Preparation of SM7 (CD1). Dissolve SM6 in dichloromethane and add trifluoroacetic acid in multiple times (e.g., three times) (avoid boiling caused by adding trifluoroacetic acid too quickly). The reaction is completed at room temperature and the reaction solution is concentrated. The concentrated solution is dispersed in water and potassium hydroxide is added to adjust the pH to >9. During this period, a large amount of solid precipitates. Filter and rinse the filter cake with ether to obtain SM7, one of the desired pyrrole [2,3-d] pyrimidine derivatives, named CD1.

(f) Preparation of other pyrrolo[2,3-d]pyrimidine derivatives (SM8 in the reaction equation). There are two methods:

(f-1) SM7 was dissolved in dichloromethane, and then different anhydrides or isocyanates were added. After the reaction was complete, the mixture was filtered and the filter cake was rinsed with ether to obtain pure other pyrrole [2,3-d] pyrimidine derivatives.

(f-2) SM7 and paraformaldehyde were dissolved in methanol. After the reaction was complete, NaBH ₄ was added. After the reaction was complete again, the sample was mixed with coarse silica gel and separated by a Flash column to obtain pure other pyrrole [2,3-d] pyrimidine derivatives.

(II) The second type of preparation method, the reaction equation is as follows:

The specific operations of steps (a) to (f) are the same as those of preparation method (i), except that the "N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester" in step (d) is replaced by "tert-butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-ene-1-yl)carbamate".

(III) The third type of preparation method, the reaction equation is as follows:

The specific operations of steps (a) to (f) are the same as those of preparation method (i), except that the "N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester" in step (d) is replaced by "3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester".

(IV) The fourth type of preparation method, the reaction equation is as follows:

The specific operations of steps (a) to (f) are the same as those of preparation method (i), except that the "N-Boc-1,2,5,6-tetrahydropyridine-4-boric acid pinacol ester" in step (d) is replaced by "N-tert-butyloxycarbonylpiperidine-4-boric acid pinacol ester".

(V) The fifth type of preparation method, the reaction equation is as follows:

The specific operations of steps (a) to (e) are the same as those of preparation method (i), except that the "2-iodopropane" in step (a) is replaced by "iodoethane" or "iodomethane".

3. The present invention also provides the application of the pyrrole [2,3-d] pyrimidine derivative in medicine and pharmacy.

The pyrrole [2,3-d] pyrimidine derivative has good CDK9 inhibitory activity, and thus has the pharmaceutical potential of CDK9 inhibitors. At the same time, since CDK9 inhibitors have tumor prevention and treatment effects, the pyrrole [2,3-d] pyrimidine derivative also has the potential to be prepared as a tumor prevention and treatment drug.

It should be understood that the pharmaceutical potential of the pyrrole [2,3-d] pyrimidine derivatives referred to in the present invention refers not only to the pharmaceutical potential of the derivatives themselves, but also to the pharmaceutical potential of the pharmaceutically acceptable salts, solvates, hydrates, isomers, and polymorphs of the derivatives. The derivatives or their pharmaceutically acceptable salts, solvates, hydrates, isomers, and polymorphs can be used as medicines alone or in combination with other pharmaceutically acceptable substances. The salts include salts formed by reaction with acids, specifically salts obtained by reaction of the free base of the parent compound with inorganic acids or organic acids; inorganic acids include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, sulfuric acid, etc.; organic acids include acetic acid, propionic acid, acrylic acid, oxalic acid, (D) or (L) malic acid, fumaric acid, etc. The solvate refers to an association formed by one or more solvent molecules and the derivative. Solvents include water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, etc. The hydrate refers to a compound formed by combining stoichiometric or non-stoichiometric water through non-covalent intermolecular forces. The isomers refer to compounds with the same chemical composition but different spatial arrangements of atoms or groups, including diastereomers, enantiomers, regioisomers, structural isomers, rotational isomers, tautomers, etc. The polymorphs refer to the solid crystalline form of the derivatives or their complexes.

The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, rather than all embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

Example 1: Preparation of pyrrole [2,3-d] pyrimidine derivatives

1. Preparation of compounds CD1 to CD17 (effect demonstration of the first type of preparation method)

The reaction equation is the same as the first type of preparation method in the specific embodiment, comprising the following steps:

Step a, preparation of intermediate SM2. Add compound 5-bromo-2-hydroxypyridine (SM1, 17.4 g, 100 mmol), acetonitrile (200 mL), Cs ₂ CO ₃ (65 g, 200 mmol) to a 500 mL three-necked flask, and then add 2-iodopropane (12 mL, 120 mmol). Heat the reaction mixture to 80°C and stir for 4 h. TLC shows that the reaction is complete. Filter, wash the filter cake twice with 50 mL of acetonitrile, and drain the solvent to obtain 19.4 g of a gray solid (SM2) with a yield of 90%. The hydrogen nuclear magnetic resonance spectrum of SM2 is as follows:

¹ H NMR (400MHz, DMSO) δ7.95 (s, 1H), 7.48 (d, J = 9.7Hz, 1H), 6.36 (d, J = 9.7Hz, 1H), 4.99 (hept, J = 6.7Hz, 1H),1.29(d,J＝6.8Hz,6H).

Step b, preparation of intermediate SM3. Add SM2 (19.4 g, 90 mmol), bis(pinacol)diboron (27.4 g, 108 mmol), potassium acetate (17.6 g, 180 mmol) and PdCl ₂ (dppf) (3.3 g, 4.5 mmol) into a 500 mL single-mouth bottle, and then add 1,4-dioxane (200 mL) as a solvent. Perform nitrogen replacement 3 times, heat to 95 ° C and react for 2 h. After TLC shows that the reaction is complete, cool to room temperature, extract with ethyl acetate and water, combine the organic phases, and concentrate. The concentrated product is dispersed in ether for pulping, and then filtered, retaining the upper filter cake, and drying to obtain 17.7 g of brown solid (SM3), with a yield of about 75%. The hydrogen nuclear magnetic resonance spectrum of SM3 is as follows:

¹ H NMR (400MHz, DMSO) δ7.95(d,J＝1.7Hz,1H),7.82(d,J＝2.0Hz,1H),7.47(dt,J＝9.0,1.9Hz,1H),6.36( dd,J＝9.1,1.6Hz,1H),5.00(pd,J＝6.7,1.6Hz,1H),1.30(d,J＝6.8Hz,6H),1.16(s,12H).

Step c, preparation of intermediate SM5. Add 4-chloro-6-iodo-7H-pyrrolo[2,3-d]pyrimidine (SM4, 14 g, 50 mmol), SM3 (14.4 g, 55 mmol), potassium carbonate (13.8 g, 100 mmol) and PdCl ₂ (dppf) (1.8 g, 2.5 mmol) into a 500 mL three-necked flask, and then add dioxane/ethanol/water = 7:3:4 (a total of 200 mL) as a solvent. After replacing nitrogen three times, transfer to an oil bath at 80°C to react for 2 h. Add crude silica gel to the reaction solution for sample mixing and then perform chromatographic separation. The mobile phase is dichloromethane/methanol (1% to 5%). Collect the fractions and concentrate to obtain 13.3 g of an off-white intermediate (SM5) with a yield of 70%. The hydrogen nuclear magnetic resonance spectrum of SM5 is as follows:

¹ H NMR (400MHz, DMSO) δ12.90 (s, 1H), 8.56 (s, 1H), 8.38 (d, J = 2.6Hz, 1H), 8.05 (dd, J = 9.5, 2.6Hz, 1H), 6.99(d,J＝2.1Hz,1H), 6.54(d,J＝9.5Hz,1H), 5.14(p,J＝6.8Hz,1H), 1.39(d,J＝6.8Hz,6H).

Step d, preparation of intermediate SM6. Add SM5 (3.8 g, 10 mmol), N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (3.4 g, 11 mmol), potassium carbonate (5.5 g, 40 mmol) and PdCl ₂ (dppf) (368 mg, 0.5 mmol) into a 100 mL three-necked flask, add dioxane/ethanol/water = 7:3:4 (40 mL in total) as solvent, replace nitrogen three times and transfer to an 80°C oil bath for reaction for 2 h. Add crude silica gel to the reaction solution for sample mixing and then perform chromatographic separation, the mobile phase is dichloromethane/methanol (1% to 5%), collect the fractions, and concentrate to obtain 2.9 g of an off-white intermediate (SM6), with a yield of 68%. The hydrogen nuclear magnetic resonance spectrum of SM6 is as follows:

¹ H NMR (400MHz, DMSO) δ12.38 (s, 1H), 8.73 (s, 1H), 8.51 (d, J = 2.0Hz, 1H), 8.25 (dd, J = 9.5, 2.3Hz, 1H), 6.72(s,1H),6.62(s,1H),6.57(d,J＝9.5Hz,1H),5.16–5.07(m,1H),3.46(s,2H),2.97(t,J＝5.3Hz ,2H),2.46(s,2H),1.42(d,J=6.8Hz,6H),1.32(s,9H).

Step e, preparation of SM7 (CD1). Dissolve SM6 (2.9 g, 6.8 mmol) in 20 mL of dichloromethane and add 20 mL of trifluoroacetic acid in batches. React at room temperature for 0.5 hours. After the reaction is complete, concentrate the reaction solution. Disperse the concentrated solution in water, add potassium hydroxide to adjust the pH to > 9, during which a large amount of solid precipitates. Filter and rinse the filter cake with ether to obtain a high-purity compound SM7 (CD1) without further purification. The hydrogen nuclear magnetic resonance spectrum of SM7 (CD1) is as follows:

¹ H NMR (400MHz, DMSO) δ12.32 (s, 1H), 8.71 (s, 1H), 8.51 (s, 1H), 8.25 (dd, J = 9.4, 2.3Hz, 1H), 6.76 (s, 1H) ),6.63–6.54(m,1H),6.57(d,J＝9.4Hz,1H),5.19–5.11(m,1H),3.52(s,2H),2.97(s,2H),2.44(s, 2H),1.42(d,J＝6.9Hz,6H).

Step f, preparation of other derivatives. This step is divided into two different preparation methods.

Preparation method f-1, preparation of CD2~CD16. Dissolve SM7 (67 mg, 0.2 mmol) in dichloromethane, and then add different anhydrides or isocyanates. After 0.5 h of reaction, the reaction is detected to be complete and a large amount of solid is precipitated. Filter the filter cake by suction, and rinse with ether to obtain the pure final product, which is the desired derivative CD2~CD16. The structural formula, nuclear magnetic resonance data of CD2~CD16 and the corresponding anhydride or isocyanate added are shown in Table 2.

Table 2 Compounds CD2 to CD16

Preparation method f-2, preparation of CD17. Weigh CD1 (67 mg, 0.2 mmol) and paraformaldehyde (60 mg, 2 mmol), dissolve in 10 mL methanol, react for 2 h, then add NaBH ₄ (15 mg, 0.4 mmol). React for another 2 h, after the reaction is complete, mix the sample with coarse silica gel, separate with a Flash column, and obtain CD17. The H NMR spectrum data of compound CD17 are as follows:

¹ H NMR (400MHz, DMSO) δ12.31 (s, 1H), 8.71 (d, J = 1.4Hz, 1H), 8.51 (d, J = 2.5Hz, 1H), 8.25 (dt, J = 9.5, 2.0 Hz,1H),6.73(s,1H),6.60–6.53(m,2H),5.11(p,J＝6.7Hz,1H),3.08(d,J＝3.4Hz,2H),2.59(s,4H ), 2.30 (s, 3H), 1.41 (d, J = 6.6Hz, 6H).

2. Preparation of compounds CD18 to CD34 (demonstration of the effect of the second type of preparation method)

According to method 1, the "N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester" in step d is replaced by "tert-butyl (4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)cyclohex-3-en-1-yl)carbamate". The structural formulas, nuclear magnetic resonance data and the corresponding anhydrides or isocyanates added of CD18 to CD34 are shown in Table 3. Among them, the synthesis of CD18 is similar to that of CD1, and step f is not required. The synthesis of CD32 is similar to that of CD17 and is carried out according to preparation method f-2, so there is no need to add anhydrides/isocyanates to CD18 and CD32; the remaining compounds all need to undergo step f and proceed according to step f-1.

Table 3 Compounds CD18 to CD34

3. Preparation of compounds CD35~CD51 (demonstration of the effect of the third type of preparation method)

According to method 1, the "N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester" in step d is replaced by "(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-5,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester". The structural formulas, nuclear magnetic resonance data and corresponding anhydrides or isocyanates added to CD35 to CD51 are shown in Table 4. Among them, the synthesis of CD35 is similar to that of CD1, and step f is not required. The synthesis of CD51 is similar to that of CD17 and is carried out according to preparation method f-2, so no anhydride/isocyanate needs to be added to CD35 and CD51; the remaining compounds all need to undergo step f and proceed according to step f-1.

Table 4 Compounds CD35-CD51

4. Preparation of compounds CD52-CD68 (effect demonstration of the fourth type of preparation method)

According to method 1, replace "N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester" in step d with "N-tert-butyloxycarbonylpiperidine-4-boronic acid pinacol ester". The structural formulas, nuclear magnetic resonance data and corresponding anhydrides or isocyanates added to CD52~CD68 are shown in Table 5. Among them, the synthesis of CD52 is similar to that of CD1, and step f is not required. The synthesis of CD68 is similar to that of CD17 and is carried out according to preparation method f-2, so there is no need to add anhydrides/isocyanates to CD52 and CD68; the remaining compounds all need to undergo step f and proceed according to step f-1.

Table 5 Compounds CD52-CD68

5. Preparation of compounds CD69 and CD70 (effect demonstration of the fifth type of preparation method)

According to method 1, "2-iodopropane" in step a was replaced by "iodomethane", and compound CD69 was prepared through steps a to e; "2-iodopropane" in step a was replaced by "iodoethane", and compound CD70 was prepared through steps a to e. The NMR data of CD69 and CD70 are shown in Table 6.

Table 6 Compounds CD69, CD70

Example 2: Demonstration of the in vitro CDK9 inhibitory effect of various pyrrole [2,3-d] pyrimidine derivatives

CDK9 was incubated in a buffer (8 mM 3-morpholinepropanesulfonic acid MOPS, pH=7.0, 0.2 mM EDTA, 100 μM KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC), and 10 mM magnesium acetate and [γ- ³³ P-ATP] were added, as well as different concentrations (1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1 nM) of each pyrrole [2,3-d] pyrimidine derivative prepared in Example 1. Mg/ATP was then added to the reaction to start the enzyme reaction process, and incubated at room temperature for 40 minutes. Finally, the reaction was terminated by diluting the solution to 0.5% with phosphate buffer, and 10 μL of the reaction solution was titrated onto the P30 membrane, washed four times with 0.425% (v/v) phosphate solution for 4 minutes each time, and then washed once with methanol. Finally, the P30 membrane was dried and scintillation counted. The scintillation count value reflects the degree of phosphorylation of the substrate, thereby characterizing the inhibition of kinase activity. The IC ₅₀ value was fitted according to the inhibition rate of 9 concentrations, and the test was repeated in duplicate. Each group was repeated 3 times and the average value was taken. At the same time, a blank control group (using an equal amount of DMSO to replace pyrrole [2,3-d] pyrimidine derivatives) and a positive control group (using 12nM concentration of AZD-4573 to replace pyrrole [2,3-d] pyrimidine derivatives; AZD-4573 is a known highly active CDK9 inhibitor) were set up. The results are shown in Table 7, in nM.

Table 7 CDK9 IC ₅₀ effect of each compound

The results showed that the newly synthesized compounds CD1 to CD70 of the present invention all had good CDK9 inhibitory activity, and the CDK9 IC ₅₀ was within 50 nM. The activity of some compounds was better than that of the positive molecule AZD4573.

Example 3: Demonstration of the anti-tumor proliferation effect of various pyrrole [2,3-d] pyrimidine derivatives

MV4-11, HCT116 and SKOV3 cells were inoculated in 96-well plates, with 100 μL IMDM containing 10% fetal bovine serum, and 10,000 to 15,000 cells per well. The set gradient (1000, 300, 100, 30, 10, 3, 1, 0.3, 0.1 nM) of each pyrrole [2,3-d] pyrimidine derivative was added, and the cells were treated for 3 × 24 h. The cell viability was determined by CCK-8 method, and the IC ₅₀ value was calculated by GraphPad Prism 8.0 software according to the inhibition rate. All experiments were repeated 3 times, and the results were averaged. At the same time, a blank control group (using an equal amount of DMSO to replace the pyrrole [2,3-d] pyrimidine derivative) and a positive control group (using an equal concentration of AZD-4573 to replace the pyrrole [2,3-d] pyrimidine derivative) were set up. The results are shown in Table 8.

Table 8 Comparison of the effects of various compounds on different tumor cells

Example 4: Demonstration of metabolic stability effects of various pyrrole [2,3-d] pyrimidine derivatives

The total volume of the incubation system was 100 μL, and the system included 0.1M PBS with pH=7.4, NADPH generating system (1 mM NADP, 5 mM 6-phosphate glucose, 1 U/mL 6-phosphate glucose dehydrogenase, 3.3 mM MgCl ₂ ); 1 μL of each test compound (CD1-CD51) solution (concentration 1 μM/L) was added on an ice bath, pre-incubated in a 37°C water bath for 5 min, 2.5 μL of human liver microsome solution was added, and the reaction was terminated by adding 200 μL of ice acetonitrile containing internal standard SAHA 20 ng/mL after continuing to incubate for 0, 5, 15, 30, 45, and 60 min, vortex mixing for 30 s, centrifuged at 13000 rpm for 10 min, and the supernatant was taken for injection. The compound concentrations measured at 5, 15, 30, 45, and 60 min were compared with the compound concentration measured at 0 min to obtain the percentage of unmetabolized compounds. The natural logarithm of the incubation time and the percentage of the compound remaining at each time point was used to draw a linear regression curve to obtain the slope k. The compound half-life T _1/2 and clearance rate CL were calculated according to the following formula. The results are shown in Table 9.

T _1/2 (min) = -0.693/k;

CL (μL/min/mg) = Ln (2) × 1000/T _1/2 /C _{liver microsomes}

Table 9 Comparison of metabolic stability of pyrrole [2,3-d] pyrimidine derivatives

The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (10)

1. A pyrrolo [2,3-d ] pyrimidine derivative characterized by: the structural general formula of the derivative is shown as follows:

wherein R is ₁ Any one selected from methyl, ethyl and isopropyl;

R ₂ selected from the group consisting ofAny one of them;

R ₃ ～R ₆ independently selected from H, substituted or unsubstituted C1-C8 alkyl,Any one of n=n0～1；

R ₇ Selected from any one of substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted 3-8 cycloalkyl and substituted or unsubstituted C2-C8 alkenyl.

2. The derivative according to claim 1, characterized in that: r is R ₂ Is any one of the following structural formulas:

3. the derivative according to claim 1, characterized in that: the structural formula of the derivative is any one of the following:

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

a. preparation of intermediate SM2: 5-bromo-2-hydroxypyridine, acetonitrile, cs ₂ CO ₃ Mixing, adding 2-iodopropane or iodoethane or iodomethane to obtain a mixture; heating and stirring the mixture, filtering after the reaction is completed, and pumping out the solvent to obtain SM2;

b. preparation of intermediate SM3: SM2, bis (pinacolato) diboron, potassium acetate and PdCl ₂ Mixing, and adding 1, 4-dioxaneHeating under the protection of inert gas as a solvent, cooling to room temperature after the reaction is completed, extracting, combining organic phases, concentrating, pulping, filtering, reserving an upper filter cake, and drying to obtain SM3;

c. preparation of intermediate SM5: 4-chloro-6-iodo-7H-pyrrolo [2,3-d]Pyrimidine, SM3, potassium carbonate and PdCl ₂ Mixing, adding dioxane/ethanol/water mixed solution as solvent, carrying out oil bath reaction, separating the reaction solution by chromatography, gradient eluting, collecting fraction, and concentrating to obtain SM5;

d. preparation of intermediate SM6: SM5, N-Boc-1,2,5, 6-tetrahydropyridine-4-boronic acid pinacol ester or tert-butyl (4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) cyclohex-3-en-1-yl) carbamate or 3- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) -5, 6-dihydropyridine-1 (2H) -carboxylate or tert-butyl N-t-butoxycarbonylpiperidine-4-boronic acid pinacol ester, potassium carbonate and PdCl ₂ Mixing, adding dioxane/ethanol/water mixed solution as solvent, performing chromatographic separation on the reaction solution after oil bath reaction is completed, gradient eluting, collecting fractions, and concentrating to obtain SM6;

e. dissolving SM6 in dichloromethane, adding trifluoroacetic acid, reacting completely at room temperature, concentrating the reaction solution, dispersing the concentrated solution in water, adjusting pH to be more than 9, precipitating a large amount of solids during the period, filtering, and leaching the filter cake to obtain the pyrrole [2,3-d ] pyrimidine derivative SM7.

5. The method of claim 4, wherein: after the step e, the method further comprises a step f, specifically:

SM7 is dissolved in dichloromethane, and acid anhydride or isocyanate is respectively added; after the reaction is completed, leaching and leaching filter cakes to obtain the pyrrole [2,3-d ] pyrimidine derivative; or;

dissolving SM7 and paraformaldehyde in methanol, and adding NaBH after the reaction is completed ₄ After the reaction is completed again, separating to obtain the pyrrole [2,3-d ]]Pyrimidine derivatives.

6. The use of a derivative according to any one of claims 1 to 3, or a derivative obtainable by a process according to claim 4 or 5, and/or a pharmaceutically acceptable salt, solvate, hydrate, isomer, polymorph thereof for the manufacture of a medicament.

7. The use according to claim 6, characterized in that: the use is in the preparation of a CDK9 inhibitor.

8. The use according to claim 6, characterized in that: the application is the application in preparing antitumor drugs.

9. A pharmaceutical composition comprising a derivative according to any one of claims 1 to 3, or a derivative obtainable by a process according to claim 4 or 5, and/or a pharmaceutically acceptable salt, solvate, hydrate, isomer, polymorph of said derivative.

10. The pharmaceutical composition according to claim 9, wherein: the pharmaceutical composition comprises pharmaceutically acceptable auxiliary materials.