CN115572297A

CN115572297A - O-phenylenediamine derivative containing pyrazolopyrimidine and preparation method and application thereof

Info

Publication number: CN115572297A
Application number: CN202110686133.9A
Authority: CN
Inventors: 赵桂森; 袁文娟; 禚慧君; 冉凡胜; 刘洋; 景永奎
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2021-06-21
Filing date: 2021-06-21
Publication date: 2023-01-06
Anticipated expiration: 2041-06-21
Also published as: CN115572297B

Abstract

The application provides an o-phenylenediamine derivative containing pyrazolopyrimidine, and a preparation method and application thereof, wherein the structure of the o-phenylenediamine derivative containing pyrazolopyrimidine is shown as a formula I:

wherein X is selected from oxygen, amide and methylene; y is selected from carbon or nitrogen; when Y is carbon, the hydrogen thereof is unsubstituted or substituted by C ₁ ‑C ₅ Alkyl substitution of (a); z is selected from the group consisting of methylene, benzyl, piperidinyl, pyridinyl, pyrrolidinyl, pyrimidinyl, imidazolyl and oxadiazolyl, and Z is unsubstituted or substituted with C _1‑5 Alkyl or carbonyl substituted; r is selected from hydrogen, halogen, nitro, amino, substituted amino, cyano, methyl, methoxy and trifluoroA methyl group; the substituted amino is represented by C ₁ ‑C ₅ Amino substituted with an alkyl group of (1); n is any integer from 0 to 7; wherein R is ₁ Is composed of

Description

O-phenylenediamine derivative containing pyrazolopyrimidine and preparation method and application thereof

Technical Field

The application relates to the field of organic compound synthesis and medical application, in particular to an o-phenylenediamine derivative containing pyrazolopyrimidine and a preparation method and application thereof.

Background

The information in this background section is disclosed only to enhance understanding of the general background of the application and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

BTK Kinase (Bruton's tyrosine Kinase) is the Tec Kinase family member that is currently reported to be most closely associated with human diseases. BTK is expressed in most hematopoietic cells, particularly in B cells, myeloid cells and platelets, is a key regulator of the BCR (B Cell Receptor) signaling pathway, and has a close relationship with the proliferation, differentiation and apoptosis of B-lymphomas. Ibrutinib (IBN) is the first irreversible BTK inhibitor on the market, and although it exerts important therapeutic effects in clinical research and treatment, it is of great significance to develop novel BTK inhibitors due to adverse reactions and drug resistance caused by off-target action. The epigenetic regulator Histone Deacetylase (HDAC) can catalyze the acetylation state at lysine residues of proteins, thereby regulating chromatin structure and transcriptional activity, involving a variety of pathophysiological states in vivo. The HDAC inhibitor can inhibit the expression level of Cyclin D1 protein, and regulate the expression level of oncostatin p53 and NF-kB signal pathway conduction. Meanwhile, the HDAC regulates the binding capacity of HSP90 and the drug-resistant mutant protein, and the stability of the mutant protein is increased. This provides a theoretical basis for solving the primary and secondary drug resistance of ibrutinib. The combination of HDAC inhibitor and BTK inhibitor shows good synergistic inhibition in several lymphomas. However, poor pharmacokinetic changes or unexpected toxicity limit the use of combinations. Therefore, the BTK/HDAC double-target inhibitor with double inhibition effects is designed and synthesized to act on multiple pathogenesis to play a synergistic effect, simultaneously alleviate the adverse reaction of the medicament, and provide a new treatment strategy for the curative effect of the B cell malignant tumor.

Disclosure of Invention

The invention provides an o-phenylenediamine derivative containing pyrazolopyrimidine, and a preparation method and application thereof, wherein the derivative has the activity of inhibiting BTK and/or HDAC enzyme and the activity of resisting cell proliferation, and can inhibit the growth of mantle cell lymphoma cells and be used for preparing antitumor drugs.

Specifically, the technical scheme of the invention is as follows:

in a first aspect of the present invention, the present invention provides an o-phenylenediamine derivative containing pyrazolopyrimidine, or a pharmaceutically acceptable salt or isomer thereof, wherein the structure of the derivative is shown in formula I:

wherein X is selected from oxygen, amide and methylene;

y is selected from carbon or nitrogen; when Y is carbon, the hydrogen thereof is unsubstituted or substituted by C ₁ -C ₅ Alkyl substitution of (a);

z is selected from the group consisting of methylene, benzyl, piperidinyl, pyridinyl, pyrrolidinyl, pyrimidinyl, imidazolyl and oxadiazolyl, and Z is unsubstituted or substituted with C _1-5 Alkyl or carbonyl substituted;

r is selected from hydrogen, halogen, nitro, amino, substituted amino, cyano, methyl, methoxy and trifluoromethyl; the substituted amino is substituted by C ₁ -C ₅ Amino substituted with the alkyl group of (1);

n is any integer from 0 to 7;

wherein Z is

When n is not 1.

In some embodiments of the invention, the derivative further conforms to the structure shown in formula II or formula III:

wherein Z, R and n are as defined above.

Further, in some embodiments of the present invention, Z is selected from methylene, benzyl, piperidinyl, and pyrrolidinyl; r is selected from hydrogen, methyl and trifluoromethyl; n is any integer from 0 to 5.

In particular, in some embodiments, when Z is benzyl, piperidinyl, or pyrrolidinyl, the benzyl group is attached at its methylene end to the nitrogen end of the pyrazole, the piperidinyl group is attached at its N-terminus to the carbon chain of formula (I), and the pyrrolidinyl group is attached at its N-terminus to the carbon chain of formula (I) (- (CH) in formula (II) ₂ ) _n -a structure.

In particular, in some embodiments, preferred piperidinyl groups are selected from

Preferred pyrrolidinyl radicals are

In some embodiments of the present invention, of the structures of formula II above, preferred structures of Z are selected from methylene, piperidinyl, and pyrrolidinyl.

Further, in some preferred embodiments of these embodiments, the derivatives further conform to the following structure:

wherein n is any integer of 1-5, for example, n can be 1, 2, 3,4 or 5.

In some embodiments of the invention, the inventors have found that the choice of piperidinyl group, particularly in the structure of formula II, where Z is found to be during the course of the experiment, and the choice of the structure of the piperidinyl group and the value of n have a greater effect on the activity

When n is 1, it has poor effect on drug-resistant strains such as IBN primary drug-resistant MCL cell strain Maver-1, and under the same test conditions, Z is

n is 1 or Z is

When n is greater than 1, for example, 3, the compound has better antiproliferative activity on the drug-resistant MCL cell strain.

In some embodiments of the present invention, in the structures of formula III above, preferred structures of Z are selected from methylene, benzyl, and piperidinyl.

wherein, in IIID, the letter indicates chiral carbon, the wavy line indicates a carbon-carbon single bond, including

Three cases; r and n are as defined above. Preferably, R is selected from hydrogen, methyl and trifluoromethyl, and n is any integer from 0 to 4, such as n is 0, 1, 2, 3 or 4. Wherein, the structure of formula IIIB' is the case that n is 0 in the structure of formula IIIB.

In an embodiment of the present invention, the substrate is,

is a key structure for maintaining activity, especially when the general structure also has

When the structure is adopted, the compound can be ensured to have BTK and/or HDACs enzyme inhibiting activityAnd antiproliferative activity against mantle cell lymphoma, e.g., in the course of the study, the inventors found that when Z is benzyl, if not

Group, the compound hardly retains its antitumor activity while having the same

The compounds show excellent antiproliferative activity against mantle cell lymphoma, especially those with R being trifluoromethyl, when Z is benzyl and n is 0, according to the general structure of formula III, and particularly excellent antiproliferative activity against drug-resistant cells such as IBN primary drug-resistant MCL cell line Maver-1, IC thereof ₅₀ Can be as low as 0.7. Mu.M.

Specifically, the present invention provides a series of examples of pyrazolopyrimidine-containing o-phenylenediamine derivatives, or a pharmaceutically acceptable salt thereof, or an isomer thereof, which are selected from the following compounds (Z1) to (Z33):

(Z1): 2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidin-1-yl) -N- (2-aminophenyl) acetamide;

(Z2): 2- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide;

(Z3): 4- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) -4-oxobutanamide;

(Z4): 4- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) butanamide;

(Z5): 3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N- (2-aminophenyl) propionamide;

(Z6): 4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N- (2-aminophenyl) butanamide;

(Z7): 5- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N- (2-aminophenyl) pentanamide;

(Z8): 6- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N- (2-aminophenyl) hexanamide;

(Z9): 5- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) pentanamide;

(Z10): 6- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) hexanamide;

(Z11): 4- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) butanamide;

(Z12): 5- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) pentanamide;

(Z13): 6- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) hexanamide;

(Z14): 5- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidin-1-yl) -N- (2-aminophenyl) pentanamide;

(Z15): 6- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidin-1-yl) -N- (2-aminophenyl) hexanamide;

(Z16): 4- (4-amino-1- (4- ((2-aminophenyl) carbamoyl) benzyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (pyridin-2-yl) benzamide;

(Z17): 4- (4-amino-1- (4- ((2-aminophenyl) carbamoyl) benzyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide;

(Z18): 4- (4-amino-1- (4- ((2-aminophenyl) carbamoyl) benzyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide;

(Z19): 4- (4-amino-1- (1- (2- ((2-aminophenyl) amino) -2-oxoethyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (pyridin-2-yl) benzamide;

(Z20): 4- (4-amino-1- (1- (2- ((2-aminophenyl) amino) -2-oxoethyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide;

(Z21): (R) -4- (4-amino-1- (1- (2- ((2-aminophenyl) amino) -2-oxoethyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide;

(Z22): 4- (4-amino-1- (1- (2- ((2-aminophenyl) amino) -2-oxoethyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide;

(Z23): 4- (4-amino-1- (3- ((2-aminophenyl) amino) -3-oxopropyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide;

(Z24): 4- (4-amino-1- (3- ((2-aminophenyl) amino) -3-oxopropyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide;

(Z25): 4- (4-amino-1- (4- ((2-aminophenyl) amino) -4-oxobutyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide;

(Z26): 4- (4-amino-1- (4- ((2-aminophenyl) amino) -4-oxobutyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide;

(Z27): 4- (4-amino-1- (5- ((2-aminophenyl) amino) -5-oxopentyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide;

(Z28): 4- (4-amino-1- (5- ((2-aminophenyl) amino) -5-oxopentyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide;

(Z29): 4- (4-amino-1- (6- ((2-aminophenyl) amino) -6-oxohexyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide;

(Z30): 4- (4-amino-1- (6- ((2-aminophenyl) amino) -6-oxohexyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide;

(Z31): 4- (4-amino-1- (1- (4- ((2-aminophenyl) amino) -4-oxobutyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide;

(Z32): 4- (4-amino-1- (1- (4- ((2-aminophenyl) amino) -4-oxobutyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide;

(Z33): 4- (4-amino-1- (1- (5- ((2-aminophenyl) amino) -5-oxopentyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide.

In a second aspect of the present invention, the present invention provides a method for producing the pyrazolopyrimidine-containing o-phenylenediamine derivative or a pharmaceutically acceptable salt thereof or an isomer thereof described in the first aspect above, which comprises:

reacting a compound 1, namely 1H-pyrazolo [3,4-d ] pyrimidine-4-amine, serving as a starting material with N-bromosuccinimide under the heating condition to obtain an intermediate 2, namely 3-bromo-1H-pyrazolo [3,4-d ] pyrimidine-4-amine; carrying out Mitsunobu reaction on the intermediate 2 and N-Boc-3/4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine to obtain an intermediate 3; removing the Boc protecting group from the intermediate 3 to obtain an intermediate 4; the intermediate 2 or the intermediate 4 and methyl ester substituted by different bromine are subjected to nucleophilic substitution to obtain an intermediate 5; the intermediate 5 and the intermediate 8 are subjected to a Suzuki reaction to obtain an intermediate 9; hydrolyzing the intermediate 9 to obtain an intermediate 10; the intermediate 10 and o-phenylenediamine are subjected to amide condensation to obtain a compound shown in a general formula (I);

wherein the reaction route is shown as follows:

wherein the content of the first and second substances,

r' is

or-B (OH) ₂ (ii) a R, X, Y, Z, n are as defined above.

Further, the method of the present invention comprises: taking the compound 1 as a starting material, and reacting the starting material with N-bromosuccinimide in DMF under a heating condition to obtain an intermediate 2; the intermediate 2 is reacted with N-Boc-3/4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine by Mitsunobu reactionTo obtain an intermediate 3; removing Boc protecting group from the intermediate 3 under concentrated hydrochloric acid to obtain an intermediate 4; the intermediate 2 or 4 and methyl ester substituted by different bromine are subjected to nucleophilic substitution to obtain an intermediate 5; the intermediate 5 and the intermediate 8 are subjected to a Suzuki reaction under the catalysis of palladium tetrakis (triphenylphosphine) to obtain an intermediate 9; hydrolyzing the intermediate 9 under alkaline conditions to obtain an intermediate 10; intermediate 10 with o-phenylenediamine in N ₂ Amide condensation is carried out under protection to obtain the compound in the general formula I.

In a third aspect of the present invention, the present invention provides a pharmaceutical composition comprising the pyrazolopyrimidine-containing o-phenylenediamine derivative described in the first aspect above, or a pharmaceutically acceptable salt thereof, or an isomer thereof.

The "composition" as used herein refers to a pharmaceutical product comprising a therapeutically effective amount of the specified ingredients, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

In a fourth aspect of the present invention, the present invention provides a pharmaceutical preparation comprising the pyrazolopyrimidine-containing o-phenylenediamine derivative or a pharmaceutically acceptable salt thereof or an isomer thereof described in the first aspect above and at least one pharmaceutically acceptable adjuvant or carrier.

The pyrazolopyrimidine-containing o-phenylenediamine derivative of the present invention or a pharmaceutical composition or pharmaceutical preparation containing the same can be administered in the form of a unit dose. The administration dosage form can be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or mixed suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill agent, patch, liniment, etc.

The pharmaceutical combination or pharmaceutical preparation of the present invention may further comprise conventional carriers, including but not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances (e.g. phosphates, glycerol, sorbitan esters, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts) or electrolytes, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The amount of carrier in the pharmaceutical composition or formulation may be from 1% to 98% by weight, usually about 80% by weight. For convenience, the local anesthetic, preservative, buffer, etc. may be dissolved directly in the vehicle.

The pharmaceutically acceptable excipients include, but are not limited to, excipients which may be binders, fillers, lubricants, disintegrants, buffers, stabilizers, preservatives, and the like. The auxiliary material refers to a component except for an effective component in the pharmaceutical composition or the pharmaceutical preparation, is nontoxic to a subject, and can stably coexist with a pharmaceutical active component or stably coexist after adopting a proper means.

Oral tablets and capsules may contain binders such as syrup, acacia, sorbitol, tragacanth or polyvinylpyrrolidone; fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine; lubricants such as magnesium stearate, talc, polyethylene glycol, silica; a disintegrant such as potato starch, or an acceptable humectant such as sodium lauryl sulfate may be present. The tablets may be coated by methods known in the art of pharmacy.

The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dried product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, hydrogenated edible fats and oils, emulsifying agents, such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.

Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.

For parenteral administration, liquid dosage forms are generally prepared from the compound and a sterile carrier. The carrier is preferably water. The compound can be dissolved in the carrier or made into suspension solution according to the concentration of the carrier and the drug, and the compound is firstly dissolved in water when made into the solution for injection, filtered and sterilized and then filled into a sealed bottle or ampoule.

Some embodiments of the invention include a method of producing a pharmaceutical composition or pharmaceutical formulation comprising mixing at least one compound according to any of the present disclosure with a pharmaceutically acceptable adjuvant or carrier. The formulations are prepared by any suitable method, usually by uniformly mixing the active compound with liquid and/or finely divided solid excipients in the desired ratio, and then, if desired, shaping the resulting mixture into the desired shape. Of course, one skilled in the art can formulate the compounds of the invention into pharmaceutical compositions or formulations using techniques well known in the art. For example, the pharmaceutical preparation may be prepared according to the modern pharmaceutical preparation series compiled by Shenyang pharmaceutical university. In addition to those mentioned herein, other suitable pharmaceutical excipients are known in the art, see for example the 2005 edition handbook of pharmaceutical excipients (fourth edition of original works), authors (en) r.c. ro (Raymond C Rowe), (american) p.j. sertbasis (Paul J Sheskey).

In a fifth aspect of the present invention, there is provided the use of the pyrazolopyrimidine-containing o-phenylenediamine derivative according to the first aspect, or a pharmaceutically acceptable salt or isomer thereof, or the pharmaceutical composition according to the third aspect, or the pharmaceutical formulation according to the fourth aspect, for the preparation of a BTK modulator (especially inhibitor) drug and/or a HDACs modulator (especially inhibitor) drug or an antitumor drug. Particularly, the compound has BTK/HDACs double-target inhibitory enzyme activity, and can be used for preparing BTK/HDACs double-target inhibitory drugs. The tumor of the invention is lymphoma, in particular mantle cell lymphoma.

In a sixth aspect of the present invention, the present invention also provides a method for treating a tumor, which comprises administering to a subject a therapeutically effective dose of the pyrazolopyrimidine containing o-phenylenediamine derivative according to the first aspect described above or a pharmaceutically acceptable salt or isomer thereof, or the pharmaceutical composition according to the third aspect described above or the pharmaceutical formulation according to the fourth aspect described above.

Wherein, the tumor especially refers to lymphoma, especially mantle cell lymphoma; the subject refers to an animal, preferably a mammal, most preferably a human, who has been the object of treatment, observation or experiment. By therapeutically effective amount is meant an amount of active compound or pharmaceutical agent, including a compound of the present invention, that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other medical professional, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It will be appreciated that the optimum dosage and interval for administration of the active ingredients of the invention will be determined by the nature and external conditions, such as form, route and site of administration, and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It should also be recognized that the optimal course of treatment, i.e., the daily dosage of the compound over the nominal time period, may be determined by methods known in the art.

The invention has the following beneficial effects: the o-phenylenediamine derivative containing pyrazolopyrimidine or the pharmaceutically acceptable salt or isomer thereof has the activity of inhibiting BTK and HDAC enzyme and the activity of resisting cell proliferation, can inhibit the growth of mantle cell lymphoma cells, and can be used for preparing antitumor drugs.

Detailed Description

The present application is further illustrated with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present application. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The reagents or starting materials used in the present application can be purchased from conventional sources, and unless otherwise specified, the reagents or starting materials used in the present application can be used in a manner conventional in the art or in accordance with the product specifications. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present application. The preferred embodiments and materials described herein are exemplary only.

In an embodiment of the present invention, there is provided a process for the preparation of a compound of formula I, comprising carrying out according to the following reaction scheme:

wherein R' is

or-B (OH) ₂ (ii) a X is selected from oxygen or amide group; y is selected from carbon or nitrogen; z is selected from the group consisting of methylene, benzyl, piperidinyl, and pyrrolidinyl; r is selected from hydrogen, methyl and trifluoromethyl; n is any integer from 0 to 5.

Specifically, the preparation method comprises the following steps: taking the compound 1 as a starting material, and reacting the starting material with N-bromosuccinimide in DMF under a heating condition to obtain an intermediate 2; carrying out Mitsunobu reaction on the intermediate 2 and N-Boc-3/4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine to obtain an intermediate 3; removing Boc protecting group from the intermediate 3 under concentrated hydrochloric acid to obtain an intermediate 4; the intermediate 2 or 4 and methyl ester substituted by different bromine are subjected to nucleophilic substitution to obtain an intermediate 5; the intermediate 5 and the intermediate 8 are subjected to a Suzuki reaction under the catalysis of palladium tetrakis (triphenylphosphine) to obtain an intermediate 9; hydrolyzing the intermediate 9 under an alkaline condition to obtain an intermediate 10; intermediate 10 with o-phenylenediamine in N ₂ Amide condensation is carried out under protection to obtain the compound with the general formula (I).

And, further, when the compound of the present invention conforms to the general structure of formula II, the present invention provides a process for the preparation of a compound of formula II, which comprises carrying out the following reaction scheme:

the combinationThe reagents and conditions in the synthetic route are (a) NBS, DMF,85 ℃,4h; (b) N-Boc-3-hydroxypiperidine/N-Boc-4-hydroxypiperidine/1-Boc-3-hydroxypyrrolidine, diisopropyl azodicarboxylate, triphenylphosphine, anhydrous tetrahydrofuran, 0 ℃ for 5min; (c) tetrahydrofuran: hydrochloric acid =4, 1,r.t.,5h; (d) Different methyl bromocarboxylates, K ₂ CO ₃ DMF, r.t.,5h; (h) Tetrakis (triphenylphosphine) palladium, potassium phosphate trihydrate, 1, 4-dioxane, water =4, microwave, 120 ℃,15min; (i) 3MNaOH, etOH, r.t.,4h; (g) O-phenylenediamine, HATU, DIEPA, dark, 0 ℃ to r.t.,8h.

Specifically, the preparation method of the compound of the formula II comprises the following steps:

(1) Dissolving the compound 1 in DMF, adding NBS, carrying out oil bath at 85 ℃, carrying out heating reflux reaction for 4h, carrying out TLC detection to detect that the reaction is basically complete, naturally cooling the reaction solution to room temperature, pouring into ice water, stirring, precipitating a large amount of yellow solid, carrying out suction filtration, washing a filter cake with water, and drying to obtain an intermediate 2.

(2) The intermediate 2, N-Boc-3-hydroxypiperidine or N-Boc-4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine and triphenylphosphine were dissolved in anhydrous Tetrahydrofuran (THF), cooled in an ice bath, diisopropyl azodicarboxylate (DIAD) was slowly added dropwise, stirred in an ice bath for 5min, and the solution became clear from turbid. TLC detecting reaction completely, adding ethyl acetate for extraction, combining organic phases, adding salt water for washing, and adding anhydrous Na ₂ SO ₄ Drying, filtering, distilling off the solvent under reduced pressure, and separating by silica gel column chromatography to obtain an intermediate 3.

(3) And dissolving the intermediate 3 in THF, dropwise adding concentrated hydrochloric acid, reacting at room temperature for 5h, separating out a white solid, and detecting the reaction by TLC. And (4) carrying out suction filtration, washing a filter cake by using ethyl acetate, and drying to obtain an intermediate 4.

(4) Dissolving intermediate 2 or 4 in DMF, adding different methyl bromocarboxylates, K ₂ CO ₃ Stirring at room temperature for 5h, extracting with ethyl acetate, mixing organic phases, and adding anhydrous Na ₂ SO ₄ Drying, filtering, evaporating the solvent under reduced pressure, and separating by silica gel column chromatography to obtain intermediate 5.

(5) The intermediate 5, 4-phenoxyphenylboronic acid (8 d), palladium tetratriphenylphosphine and potassium phosphate trihydrate were placed in a microwave tube, and 1, 4-dioxane and water (4)And carrying out microwave reaction at 120 ℃ for 15min. TLC detection shows that the reaction is basically complete, ethyl acetate is added into the reaction liquid for extraction, organic phases are combined, and anhydrous Na is added ₂ SO ₄ Drying, filtering, evaporating the solvent under reduced pressure, and separating by silica gel column chromatography to obtain intermediate 9a.

(6) And (3) dissolving the intermediate 9a in absolute ethyl alcohol, adding 3M NaOH, stirring for 4h at normal temperature, and detecting by TLC to complete the reaction. And (3) decompressing and distilling to remove ethanol, adjusting the pH value to 5-6 by using 1M HCl, separating out a solid, performing suction filtration, retaining a filter cake, and drying to obtain an intermediate 10a.

(7) Dissolving the intermediate 10a, HATU, DIEPA and DMF, clarifying the solution, and stirring for 20-30min under ice bath condition. After TLC detection activation is completed, o-phenylenediamine and N are added ₂ Protecting, reacting in dark, and stirring at normal temperature overnight. The reaction was essentially complete by TLC. Pouring the reaction solution into ice water, separating out solid, and filtering. Extracting the filtrate with ethyl acetate, combining the organic phases, anhydrous Na ₂ SO ₄ Drying and filtering. Separating by silica gel column chromatography, evaporating under reduced pressure to remove solvent, and drying to obtain target compounds Z1-Z15.

And, further, when the compounds of the present invention conform to the general structure of formula III, the present invention provides a process for the preparation of a compound of formula III, comprising the following reaction scheme:

the reagents and conditions in the synthetic route are (a) NBS, DMF,85 ℃,4h; (b) N-Boc-3-hydroxypiperidine/N-Boc-4-hydroxypiperidine/1-Boc-3-hydroxypyrrolidine, diisopropyl azodicarboxylate, triphenylphosphine, anhydrous tetrahydrofuran, 0 ℃ for 5min; (c) tetrahydrofuran: hydrochloric acid = 4; (d) Different methyl bromocarboxylates, K ₂ CO ₃ DMF, r.t.,5h; (e) 4-Bromobenzoic acid, HBTU, et ₃ N, DMF, r.t.,12h; (f) 4-bromobenzoic acid, phosphorus oxychloride, pyridine, at 0 ℃ for 5min; (g) Pinacol ester diborate, potassium acetate, tetrakis (triphenylphosphine) palladium, 1, 4-dioxane, N ₂ Microwave at 120 deg.C for 15min; (h) Tetrakis (triphenylphosphine) palladium, potassium phosphate trihydrate, 1, 4-dioxane, water =4, microwave, 120 ℃,15min; (i) 3MNaOH, etOH, r.t.,4h; (j) O-phenylenediamine, HATU, DIEPA, dark, 0 ℃ to r.t.,8h.

Specifically, the method comprises the following steps:

(2) The intermediate 2, N-Boc-3-hydroxypiperidine or N-Boc-4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine and triphenylphosphine were dissolved in anhydrous Tetrahydrofuran (THF), cooled in an ice bath, diisopropyl azodicarboxylate (DIAD) was slowly added dropwise, stirred in an ice bath for 5min, and the solution became clear from turbid. TLC detecting reaction, adding ethyl acetate for extraction, combining organic phases, adding saturated saline solution for washing, and anhydrous Na ₂ SO ₄ Drying, filtering, evaporating the solvent under reduced pressure, and separating by silica gel column chromatography to obtain intermediate 3.

(3) And dissolving the intermediates 3a-3c in THF, dropwise adding concentrated hydrochloric acid, reacting at room temperature for 5h, precipitating a white solid, and detecting the reaction by TLC (thin layer chromatography). And (4) carrying out suction filtration, washing a filter cake by using ethyl acetate, and drying to obtain an intermediate 4.

(4) Dissolving intermediate 2 or 4 in DMF, adding different methyl bromo-carboxylates, K ₂ CO ₃ Stirring at room temperature for 5h, extracting with ethyl acetate, mixing organic phases, and adding anhydrous Na ₂ SO ₄ Drying, filtering, evaporating the solvent under reduced pressure, and separating by silica gel column chromatography to obtain intermediate 5.

(5) Dissolving 4-bromobenzoic acid in DMF, adding HBTU and triethylamine, activating at room temperature for half an hour, then adding 2-aminopyridine compounds (6 a-6 b), reacting at room temperature overnight, detecting by TLC to obtain a complete reaction, pouring the reaction solution into ice water, stirring, precipitating a light yellow solid, performing suction filtration, washing a filter cake with water, and drying to obtain an intermediate 7a-7b; placing 2-aminopyridine compound (6 c), 4-bromobenzoic acid and phosphorus oxychloride in a 100mL eggplant-shaped bottle, adding pyridine to dissolve under stirring in an ice bath, continuing stirring for 5min, detecting by TLC that the reaction is complete basically, pouring the reaction liquid into ice water, stirring, precipitating light yellow solid, performing suction filtration, washing filter cake with water, and drying to obtain an intermediate 7c.

(6) Placing the intermediate 7a-7c, pinacol ester diboron, potassium acetate and palladium tetratriphenylphosphine in a 100ml eggplant-shaped bottle, adding 1, 4-dioxane for dissolution, carrying out microwave reaction at 120 ℃ for 15min, detecting by TLC to ensure that the reaction is complete basically, adding ethyl acetate into the reaction liquid for extraction, combining organic phases, adding salt water for washing, and adding anhydrous Na ₂ SO ₄ Drying, filtering, evaporating the solvent under reduced pressure, and separating by silica gel column chromatography to obtain intermediates 8a-8c.

(7) Putting the intermediate 5, the intermediates 8a to 8c, the palladium tetratriphenylphosphine and the potassium phosphate trihydrate into a microwave tube, adding 1, 4-dioxane and water (4) for dissolving, and performing microwave reaction at 120 ℃ for 15min. TLC detection shows that the reaction is basically complete, ethyl acetate is added into the reaction liquid for extraction, organic phases are combined, and anhydrous Na is added ₂ SO ₄ Drying, filtering, evaporating the solvent under reduced pressure, and separating by silica gel column chromatography to obtain intermediate 9b.

(8) And (3) dissolving the intermediate 9b in absolute ethyl alcohol, adding 3M NaOH, stirring for 4 hours at normal temperature, and detecting complete reaction by TLC. And (3) decompressing and distilling to remove ethanol, adjusting the pH value to 5-6 by using 1M HCl, separating out a solid, performing suction filtration, retaining a filter cake, and drying to obtain an intermediate 10b.

(9) Dissolving the intermediate 10b, HATU, DIEPA and DMF, clarifying the solution, and stirring for 20-30min under ice bath condition. After TLC detection activation is completed, o-phenylenediamine and N are added ₂ Protecting and protecting the reaction from light, and stirring the mixture at normal temperature overnight. The reaction was essentially complete by TLC. Pouring the reaction solution into ice water, separating out solid, and filtering. Extracting the filtrate with ethyl acetate, combining the organic phases, anhydrous Na ₂ SO ₄ Drying and filtering. Separating by silica gel column chromatography, evaporating the solvent under reduced pressure, and drying to obtain the target compound Z16-Z33.

The following examples refer to the above methods for the specific preparation of compounds and for the characterization of related compounds:

example 1Preparation of intermediate 2

Taking 4-amino pyrazolo [3,4-d]Pyrimidine (1,5.0 g, 0.037mol) and NBS (7.9 g, 0.0444mol) were placed in 250mL eggplant-shaped bottles, dissolved in 25mL DMF, the solution was cloudy, heated at 85 ℃ and refluxed in an oil bath. The reaction solution dissolved to brownSolution, TLC (PE: EA = 1) after 4h monitored the reaction complete. After the reaction is finished, cooling to room temperature, pouring into 100mL of ice water, precipitating a large amount of yellow solid, performing suction filtration, washing a filter cake (30 mL multiplied by 3) with water, and drying to obtain an intermediate 2, wherein the yellow solid is weighed to be 7.496g, and the yield is 94.59%; ¹ H NMR(400MHz,DMSO-d ₆ )δ13.73(s,1H),8.15(s,1H),7.76(s,1H),6.82(s,1H)。

example 2Preparation of intermediate 3

Taking 3-bromo-1H-pyrazolo [3,4-d]Pyrimidin-4-amine (9.34 mmol) and triphenylphosphine (PPh) ₃ 28.03 mol) into a 100mL eggplant-shaped bottle, respectively adding N-Boc-3-hydroxypiperidine, N-Boc-4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine (14.02 mol), adding anhydrous tetrahydrofuran (THF, 20 mL), stirring for dissolving, cooling in an ice bath, slowly adding diisopropyl azodicarboxylate (DIAD, 28.03 mol) dropwise, stirring in the ice bath for 5min, and clarifying the solution from turbidity. TLC detected the reaction was complete, ethyl acetate (30 mL × 3) was added for extraction, the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and silica gel column chromatography was performed using ethyl acetate: petroleum ether =4 to obtain intermediate 3.

Example 3Preparation of intermediate 4

The intermediate 3 was taken out and dissolved in a 250mL dry eggplant-shaped bottle with an appropriate amount of tetrahydrofuran, and concentrated hydrochloric acid was added to the solution in accordance with V (THF) = V (concentrated HCl) = 4. The reaction was carried out at room temperature for 5h, a white solid precipitated, and the reaction was monitored by TLC (PE: EA = 1). Suction filtration and ethyl acetate washing of the filter cake (20 mL × 0, drying, weighing gave intermediate 4.

Example 4Preparation of intermediate 5

Taking intermediate 2 (5.99 mmol), adding methyl bromomethylbenzoate/methyl 4-bromobutyrate/methyl 5-bromovalerate/methyl 6-bromohexanoate (7.19 mmol) or intermediate 4, adding methyl 4-bromobutyrate/methyl 5-bromovalerate/methyl 6-bromohexanoate (7.19 mmol) respectively, and adding K ₂ CO ₃ The powder (14.99 mmol) was put in a 100mL eggplant-shaped bottle, DMF (20 mL) was added thereto, and the mixture was dissolved by stirring and reacted at room temperature for 5 hours. TLC detection reaction is complete, the reaction solution is poured into cold water (50 mL), ethyl acetate (30 mL. Times.3) is added for extraction, the organic phases are combined, and saturation is addedWashing with saline (20 mL), drying over anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, and subjecting to silica gel column chromatography with CH ₂ Cl ₂ :CH ₃ OH =120, to give intermediate 5.

Example 5Preparation of intermediate 7

1. Preparation of intermediate 7a

P-bromobenzoic acid (4.70g, 23.38mmol) was taken out and dissolved in DMF (30 mL) in a 250mL eggplant-shaped bottle, HBTU (8.86g, 23.38mmol) and triethylamine (6.45g, 63.75mmol) were added thereto, and the mixture was stirred at room temperature for half an hour, followed by addition of 2-aminopyridine (6 a,2.00g, 21.25mmol) and allowed to react at room temperature overnight. TLC detection is carried out to ensure that the reaction is complete basically, the reaction solution is poured into ice water (250 mL), stirred, light yellow solid is separated out, filtered, washed by water and dried to obtain an intermediate 7a; 4.12g of white solid, yield 70%.

2. Preparation of intermediate 7b

P-bromobenzoic acid (4.06g, 20.35mmol), triethylamine (5.61g, 50.48mmol) and HBTU (7.77g, 20.35mmol) were sequentially added to a 250mL eggplant-shaped bottle, dissolved in 25mL DMF, stirred in ice bath for 20min, and the orange clear solution was obtained. The compound 2-amino-4-methylpyridine (6b, 2g, 18.50mmol) was added and reacted overnight at room temperature, and the reaction was complete by TLC. The mixture was poured into 200mL of ice water to precipitate a solid, which was filtered off with suction and the filter cake (10 mL) was washed with water. The filtrate had a small amount of product, extracted with ethyl acetate (30 mL. Times.3), and the organic phases were combined and Na anhydrous ₂ SO ₄ Drying, filtering and evaporating the solvent. Silica gel column chromatography (ethyl acetate: petroleum ether =50 = 1), drying, to give 7b as a white solid, weighing 2.445g, 45.54% yield; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.81(s,1H),8.25(d,J＝4.8Hz,1H),8.03(s,1H),7.95(d,J＝7.6Hz,2H),7.72(d,J＝7.5Hz,2H),7.02(d,J＝4.9Hz,1H),2.36(s,3H)。

3. preparation of intermediate 7c

2-amino-4-trifluoromethylpyridine (6c, 200mg, 1.23mmol) and p-bromobenzoic acid (299mg, 1.48mmol) are sequentially added into a 100mL eggplant-shaped bottle, and 3mL pyridine is added to dissolve and clarify the solution. Slowly dropping POCl under ice bath condition ₃ (378mg, 2.46mmol), generating a large amount of white smoke by reaction exothermy, stirring for 5min under an ice bath condition, and detecting the completion of the reaction by TLC. 30mL of ice are poured inSeparating out solid in water, carrying out suction filtration, washing a filter cake (5 mL) with water, and drying to obtain a white solid 7c, weighing 286mg, and obtaining the yield of 67.29%; ¹ H NMR(400MHz,DMSO-d ₆ )δ11.40(s,1H),8.69(d,J＝5.0Hz,1H),8.52(s,1H),7.98(d,J＝7.8Hz,2H),7.75(d,J＝7.9Hz,2H),7.56(d,J＝5.0Hz,1H)。

example 6Preparation of intermediate 8

Intermediate 7a-7c (14.55 mol), pinacol diboron (18.94 mmol), potassium acetate (KOAc, 43.71 mmol) and palladium tetratriphenylphosphine (Pd (PPh) ₃ ) ₄ 0.58 mol) was put into a 100mL round bottom flask, and 1, 4-dioxane (40 mL) was added thereto and dissolved, followed by microwave reaction at 120 ℃ for 15min. TLC detection shows that the reaction is almost complete, ethyl acetate (30 mL. Times.3) is added to the reaction solution for extraction, the organic phases are combined and washed with saturated brine (20 mL) and anhydrous Na ₂ SO ₄ Drying, filtering, evaporating the solvent under reduced pressure, and performing silica gel column chromatography EA: PE = 50.

Example 7Preparation of intermediate 9

1. Preparation of intermediate 9a

Intermediate 5 (729.39. Mu. Mol), intermediate 8d (1.46 mmol) and palladium tetrakistriphenylphosphine (Pd (PPh) ₃ ) ₄ 36.47. Mu. Mol) and potassium phosphate trihydrate (K) ₃ PO ₄ ·3H ₂ O,1.46 mmol) was put in a 35mL microwave tube, dissolved by adding 1, 4-dioxane and water (10mL, 4. TLC detection to complete the reaction, adding ethyl acetate (30 mL × 3) into the reaction solution for extraction, combining organic phases, adding saline (20 mL) for washing, drying with anhydrous sodium sulfate, filtering, evaporating the solvent under reduced pressure, and performing silica gel column chromatography on CH ₂ Cl ₂ :CH ₃ OH =100, to give intermediate 9a.

2. Preparation of intermediate 9b

Intermediate 5 (729.39. Mu. Mol), intermediates 8a-8c (1.46 mmol) and palladium tetrakistriphenylphosphine (Pd (PPh) ₃ ) ₄ 36.47. Mu. Mol) and potassium phosphate trihydrate (K) ₃ PO ₄ ·3H ₂ O,1.46 mmol) was put in a 35mL microwave tube, and 1, 4-dioxane and water (10mL, 4. TLC detection shows that the reaction is basically complete, and ethyl acetate is added into the reaction liquid(30 mL. Times.3), the organic phases were combined, washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, the solvent was evaporated under reduced pressure, and subjected to silica gel column chromatography CH ₂ Cl ₂ :CH ₃ OH =100, to give intermediate 9b.

Example 8Preparation of intermediate 10

And (3) dissolving the intermediate 9 in a proper amount of absolute ethyl alcohol, adding 3M NaOH (0.5-2 mL), stirring for 4h at normal temperature, and detecting by TLC to complete the reaction. And (3) evaporating ethanol under reduced pressure, adjusting the pH value to 5-6 by using 1M HCl, separating out a solid, performing suction filtration, and dropwise adding a little 1M HCl into the filtrate until no precipitate is generated. The filter cake was retained and dried to give intermediate 10.

Example 9Preparation of target Compounds Z1 to Z33

Sequentially adding the intermediate 10 (1.0 eq), HATU (1.1 eq) and DIEPA (3.0 eq) into a 100mL eggplant-shaped bottle, adding 5-10 mL of LDMF to dissolve, clarifying the solution, and stirring for 20-30min under the ice-bath condition. After TLC detection activation was complete, o-phenylenediamine (1.1 eq), N, was added ₂ Protecting and protecting the reaction from light, and stirring the mixture at normal temperature overnight. The reaction was essentially complete by TLC and the solution was yellow. The reaction mixture was poured into 100mL of ice-water, and the solid was precipitated and filtered off. The filtrate was extracted with ethyl acetate (30 mL. Times.3), and the organic phases were combined, anhydrous Na ₂ SO ₄ Drying and filtering. The product was separated and purified by silica gel column chromatography (dichloromethane: methanol = 20).

2- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidin-1-yl) -N- (2-aminophenyl) acetamide (Z1)

White solid, yield 48%, mp 90-92 ℃; ¹ H NMR(400MHz,CDCl ₃ )δ9.59(s,1H),8.33(s,1H),7.53(d,J＝7.9Hz,2H),7.40(t,J＝7.5Hz,2H),7.19(t,J＝7.3Hz,1H),7.07(d,J＝7.5Hz,3H),7.02-6.99(m,3H),6.70(d,J＝7.9Hz,1H),6.62(t,J＝7.6Hz,1H),5.73–5.51(m,3H),3.59(d,J＝16.8Hz,1H),3.51(d,J＝9.9Hz,1H),3.35(s,2H),3.03(t,J＝8Hz,,1H),2.80(q,J＝8.1Hz,1H),2.63–2.52(m,1H),2.44-2.36(m,1H). ¹³ C NMR(100MHz,CDCl ₃ )δ169.32,158.60,157.84,156.17,155.94,154.06,144.13,140.83,129.99,129.96,127.50,126.94,125.15,124.17,123.92,119.71,119.10,118.84,117.78,98.73,59.14,57.77,56.06,53.62,32.09.HRMS(ESI):calcd for C ₂₉ H ₂₈ N ₈ O ₂ [M+H] ⁺ 521.2408,found 521.2428。

2- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide (Z2)

White solid, yield 72%, mp 217-218 deg.C; ¹ H NMR(400MHz,DMSO-d ₆ )δ1H NMR(400MHz,DMSO)δ9.25(s,1H),8.25(s,1H),7.67(d,J＝8.5Hz,2H),7.44(t,J＝7.9Hz,2H),7.26(d,J＝7.6Hz,1H),7.22–7.11(m,5H),6.92(t,J＝7.4Hz,1H),6.76(d,J＝7.7Hz,1H),6.58(t,J＝7.3Hz,1H),4.78(s,3H),3.18(d,J＝41.3Hz,5H),2.36(d,J＝11.8Hz,3H),1.96(s,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ168.81,158.65,157.53,156.78,155.97,154.15,143.36,142.15,130.61,130.53,128.59,126.20,125.32,124.29,124.26,119.46,119.43,117.17,116.84,97.94,61.95,53.02,45.96,31.49.HRMS(ESI):calcd for C ₃₀ H ₃₀ N ₈ O ₂ [M+H] ⁺ 535.2565,found 535.2574。

4- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) -4-oxobutanamide (Z3)

White solid, yield 58%, mp 124-127 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.17(s,1H),8.28(s,1H),7.68(s,2H),7.44(s,2H),7.15(s,6H),6.90(s,1H),6.71(s,1H),6.52(s,1H),4.87(d,J＝43.3Hz,2H),4.62(d,J＝22.8Hz,1H),4.19(d,J＝66.1Hz,1H),3.96(s,0.5H),3.64(s,0.5H),3.12(s,1H),2.94–2.53(m,5H),2.19(d,J＝46.7Hz,2H),1.89(s,1H),1.61(d,J＝53.4Hz,1H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.15,171.09,170.80,170.59,158.68,157.59,156.79,156.17,154.52,154.38,143.80,143.72,143.01,130.61,130.57,128.41,126.41,126.28,124.26,123.73,119.47,119.44,116.33,115.93,97.94,97.83,60.24,53.06,52.59,46.00,45.35,31.30,30.16,29.98,28.45,25.06,23.99.HRMS(ESI):calcd for C ₃₂ H ₃₂ N ₈ O ₂ [M+H] ⁺ 577.2670,found 577.2667。

4- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) butanamide (Z4)

White solid, yield 60%, mp:110-112 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.10(s,1H),8.25(s,1H),7.65(d,J＝8.6Hz,2H),7.44(t,J＝7.9Hz,2H),7.21-7.11(m,6H),6.88(t,J＝7.1Hz,1H),6.70(d,J＝7.8Hz,1H),6.52(t,J＝7.5Hz,1H),4.82(s,3H),3.05(d,J＝7.8Hz,1H),2.92(d,J＝9.2Hz,1H),2.47–2.30(m,5H),1.99(s,3H),1.83-1.74(m,4H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.49,158.65,157.54,156.82,156.07,154.29,143.49,142.40,130.59,130.54,128.54,126.15,125.81,124.23,124.05,119.47,119.42,116.62,116.33,97.85,58.11,57.69,53.68,53.07,34.11,30.00,24.60,22.88.HRMS(ESI):calcd for C ₃₂ H ₃₄ N ₈ O ₂ [M+H] ⁺ 563.2878,found 563.2872。

3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N- (2-aminophenyl) propionamide (Z5)

White solid, yield 36%, mp:181-183 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.24(s,1H,-CONH),8.28(s,1H),7.68(d,J＝8.2Hz,2H),7.45(t,J＝7.6Hz,2H),7.23–7.12(m,5H),7.08(d,J＝7.6Hz,1H),6.88(d,J＝7.3Hz,1H),6.69(d,J＝7.8Hz,1H),6.51(d,J＝7.1Hz,1H),4.83(s,2H),4.66(t,J＝6.5Hz,2H),2.96(t,J＝6.6Hz,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ168.95,158.63,157.55,156.78,156.22,154.62,143.69,142.84,130.62,130.51,128.43,126.53,126.21,124.27,123.38,119.47,119.43,116.38,116.07,97.77,43.42,36.09.HRMS(ESI):calcd for C ₂₆ H ₂₃ N ₇ O ₂ [M+H] ⁺ 466.1986,found 466.1990。

4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N- (2-aminophenyl) butanamide (Z6)

White solid, yield 46%, mp 279-280 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.18(s,1H),8.26(s,1H),7.69(d,J＝7.6Hz,2H),7.45(t,J＝7.2Hz,2H),7.29–7.10(m,6H),6.88(t,J＝5.6Hz,1H),6.70(d,J＝7.5Hz,1H),6.51(t,J＝7.5Hz,1H),4.92(s,2H),4.41(t,J＝5.4Hz,2H),2.43–2.31(m,2H),2.26–2.06(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ170.79,158.67,157.55,156.76,156.23,154.72,143.63,142.59,130.62,130.55,128.48,126.20,125.99,124.28,123.79,119.48,119.42,116.38,116.11,97.70,46.26,33.08,25.69.HRMS(ESI):calcd for C ₂₇ H ₂₅ N ₇ O ₂ [M+H] ⁺ 480.2142,found 480.2143。

5- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N- (2-aminophenyl) pentanamide (Z7)

White solid, 43% yield, mp:152-154 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.09(s,1H),8.26(s,1H),7.67(d,J＝7.4Hz,2H),7.44(t,J＝6.8Hz,2H),7.25–7.09(m,6H),6.93–6.83(m,1H),6.70(d,J＝7.4Hz,1H),6.51(t,J＝7.0Hz,1H,),4.81(s,2H),4.46–4.33(m,2H),2.36(t,J＝6.2Hz,2H),1.98–1.86(m,2H),1.66–1.54(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.31,158.66,157.55,156.78,156.17,154.65,143.49,142.36,130.61,130.52,128.52,126.17,125.79,124.27,123.95,119.48,119.44,116.60,116.32,97.68,46.49,35.62,29.19,22.98.HRMS(ESI):calcd for C ₂₈ H ₂₇ N ₇ O ₂ [M+H] ⁺ 494.2299,found 494.2302。

6- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N- (2-aminophenyl) hexanamide (Z8)

White solid, yield 31%, mp:164-165 ℃; ¹ H NMR(400MHz,DMSO)δ9.06(s,1H),8.27(s,1H),7.68(d,J＝8.0Hz,2H),7.44(t,J＝7.4Hz,2H),7.20(d,J＝7.2Hz,1H),7.18–7.06(m,5H),6.88(t,J＝7.4Hz,1H),6.71(d,J＝7.8Hz,1H),6.52(t,J＝7.3Hz,1H),4.80(s,2H),4.36(t,J＝6.2Hz,2H),2.30(t,J＝7.0Hz),1.96–1.84(m,2H),1.72–1.58(m,2H),1.40–1.29(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.50,158.65,157.54,156.78,156.16,154.63,143.46,142.35,130.60,130.51,128.53,126.15,125.76,124.27,124.00,119.48,119.43,116.62,116.33,97.66,46.62,36.05,29.37,26.29,25.33.HRMS(ESI):calcd for C ₂₉ H ₂₉ N ₇ O ₂ [M+H] ⁺ 508.2455,found 508.2459。

5- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) pentanamide (Z9)

White solid, yield 67%, mp 101-103 deg.C; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.10(s,1H),8.25(s,1H),7.66(d,J＝8.4Hz,2H),7.44(t,J＝7.8Hz,2H),7.25–7.09(m,6H),6.88(t,J＝7.4Hz,1H),6.71(d,J＝7.8Hz,1H),6.51(t,J＝7.4Hz,1H),4.81(m,1H),3.05(d,J＝7.9Hz,1H),2.92(d,J＝10.2Hz,1H),2.43(d,J＝20.2Hz,4H),2.32(t,J＝6.9Hz,2H),2.07–1.90(m,2H),1.82(d,J＝12.0Hz,1H),1.68(d,J＝7.9Hz,1H),1.65–1.55(m,2H),1.50(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.60,158.63,157.53,156.80,156.07,154.25,143.47,142.38,130.60,130.54,128.52,126.17,125.77,124.25,124.03,119.47,119.41,116.65,116.37,97.81,58.18,57.94,53.71,53.10,36.04,30.01,26.31,24.64,23.72.HRMS(ESI):calcd for C ₃₃ H ₃₆ N ₈ O ₂ [M+H] ⁺ 577.3034,found 577.3035。

6- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) hexanamide (Z10)

White solid, yield 55%, mp 94-95 deg.C; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.11(s,1H),8.26(s,1H),7.66(d,J＝8.3Hz,2H),7.44(t,J＝7.7Hz,2H),7.25–7.14(m,4H),7.12(d,J＝7.8Hz,2H),6.88(t,J＝7.4Hz,1H),6.70(d,J＝7.8Hz,1H),6.52(t,J＝7.4Hz,1H),4.81(s,3H),3.06(d,J＝8.7Hz,1H),2.93(d,J＝9.9Hz,1H),2.47–2.35(m,4H),2.31(t,J＝7.2Hz,2H),2.04–1.96(m,2H),1.82(d,J＝11.7Hz,1H),1.67(d,J＝11.6Hz,1H),1.60(dt,J＝14.3,7.2Hz,2H),1.53–1.42(m,2H),1.36–1.27(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.63,158.63,157.53,156.80,156.07,154.25,143.47,142.34,130.60,130.53,128.51,126.15,125.74,124.25,124.04,119.47,119.41,116.64,116.35,97.81,58.19,58.10,53.66,53.14,36.19,29.98,27.03,26.46,25.72,24.62.HRMS(ESI):calcd for C ₃₄ H ₃₈ N ₈ O ₂ [M+H] ⁺ 591.3190,found 591.3191。

4- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) butanamide (Z11)

White solid, yield 32%, mp:136-138 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.12(s,1H),8.24(s,1H),7.67(d,J＝8.3Hz,2H),7.44(t,J＝7.7Hz,2H),7.22–7.14(m,4H),7.13(d,J＝7.8Hz,2H),6.88(t,J＝7.4Hz,1H),6.71(d,J＝7.8Hz,1H),6.53(t,J＝7.4Hz,1H),4.83(s,2H),4.74–4.62(m,1H),3.04(d,J＝10.0Hz,2H),2.46–2.33(m,4H),2.30–2.18(m,2H),2.17–2.08(m,2H),1.90(d,J＝10.0Hz,2H),1.84–1.73(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.58,158.64,157.53,156.79,155.92,154.11,143.28,142.43,130.60,130.50,128.61,126.16,125.84,124.26,124.05,119.45,116.63,116.30,97.92,57.55,54.51,52.79,34.17,31.48,23.25.HRMS(ESI):calcd for C ₃₂ H ₃₄ N ₈ O ₂ [M+H] ⁺ 563.2877,found 563.2878。

5- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) pentanamide (Z12)

White solid, yield 29%, mp 104-106 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.13(s,1H),8.24(s,1H),7.67(d,J＝8.6Hz,2H),7.47–7.41(m,2H),7.20(d,J＝6.5Hz,1H)7.18–7.09(m,5H),6.89(t,J＝8.2Hz,1H),6.71(dd,J＝7.9Hz,0.9Hz,1H),6.54(t,J＝8.1Hz,1H),4.83(s,2H),4.75–4.65(m,1H),3.15–3.00(m,2H),2.47–2.40(m,2H),2.29–2.17(m,4H),1.96–1.88(m,2H),1.64(dt,J＝14.4,7.0Hz,2H),1.58–1.48(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.62,158.64,157.54,156.76,155.93,154.12,143.31,142.38,130.60,130.50,128.58,126.19,125.78,124.27,124.06,119.47,119.42,116.68,116.39,97.93,57.72,54.34,52.72,36.06,31.34,26.55,23.70.HRMS(ESI):calcd for C ₃₃ H ₃₆ N ₈ O ₂ [M+H] ⁺ 577.3034,found 577.3035。

6- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) hexanamide (Z13)

White solid, yield 46%, mp 144-146 deg.C； ¹ H NMR(400MHz,DMSO-d ₆ )δ9.30(s,1H),8.27(s,1H),7.67(d,J＝8.5Hz,2H),7.44(t,J＝8.0Hz,2H)7.24–7.10(m,6H),6.88(t,J＝7.6Hz,1H),6.71(d,J＝7.9Hz,1H),6.53(t,J＝7.5Hz,1H),5.02–4.87(m,3H),2.53–2.43(m,4H),2.38(t,J＝7.3Hz,2H),2.21–2.08(m,2H),1.80–1.70(m,2H),1.64(dt,J＝14.9,7.3Hz,2H),1.42–1.33(m,2H),1.30–1.20(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.50,158.67,157.63,156.74,156.11,154.33,143.73,142.25,130.62,130.54,128.36,126.04,125.65,124.29,124.09,119.48,119.45,116.55,116.33,97.95,60.23,51.33,51.27,35.94,31.43,26.31,25.32,22.54.HRMS(ESI):calcd for C ₃₄ H ₃₈ N ₈ O ₂ [M+H] ⁺ 591.3190,found 591.3191。

5- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidin-1-yl) -N- (2-aminophenyl) pentanamide (Z14)

White solid, yield 37%, mp 91-93 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.16(s,1H),8.29(s,1H),7.69(d,J＝8.6Hz,2H),7.44(t,J＝7.9Hz,2H)7.20(d,J＝7.4Hz,1H)7.18–7.11(m,5H),6.89(td,J＝8.2Hz,1H),6.71(dd,J＝7.9,0.9Hz,1H),6.53(td,J＝8.1Hz,1H),5.65–5.56(m,3H),3.19–3.03(m,5H),2.47–2.40(m,1H),2.38(t,J＝6.1Hz,3H),1.74–1.62(m,5H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.35,158.69,157.76,156.69,156.35,154.61,144.31,142.39,130.64,128.12,126.30,125.85,124.34,123.87,119.47,116.68,116.38,98.10,57.49,55.17,54.35,53.55,35.54,30.46,25.96,22.93.HRMS(ESI):calcd for C ₃₂ H ₃₄ N ₈ O ₂ [M+H] ⁺ 563.2877,found 563.2881。

6- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidin-1-yl) -N- (2-aminophenyl) hexanamide (Z15)

White solid, yield 49%, mp 107-109 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ9.12(s,1H),8.27(s,1H),7.68(d,J＝8.5Hz,2H),7.44(t,J＝7.9Hz,2H)7.20(d,J＝7.5Hz,1H),7.18–7.10(m,5H),6.88(t,J＝7.5Hz,1H),6.71(d,J＝7.8Hz,1H),6.53(t,J＝7.5Hz,1H),5.47(dt,J＝13.5,6.9Hz,1H),4.81(s,2H),3.39–3.29(m,2H),3.00(s,3H),2.68(s,2H),2.40(dd,J＝13.7,7.4Hz,1H),2.33(t,J＝7.3Hz,2H),1.67–1.59(m,2H),1.59–1.51(m,2H),1.42–1.32(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.60,158.64,157.62,156.79,156.18,154.64,143.84,142.34,130.64,128.43,126.17,125.75,124.26,124.04,119.49,119.42,116.65,116.37,98.04,58.55,55.65,54.75,53.44,36.14,30.50,27.55,26.85.25.59.HRMS(ESI):calcd for C ₃₃ H ₃₆ N ₈ O ₂ [M+H] ⁺ 577.3034,found 577.3031。

4- (4-amino-1- (4- ((2-aminophenyl) carbamoyl) benzyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (pyridin-2-yl) benzamide (Z16)

White solid, yield 67%, mp 228-230 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.83(s,1H),9.55(s,1H),8.35(d,J＝4.1Hz,1H),8.26(s,1H),8.15(t,J＝7.4Hz,3H),7.86(d,J＝7.8Hz,2H),7.80(t,J＝7.5Hz,1H),7.74(d,J＝8.1Hz,2H),7.35(d,J＝7.9Hz,2H),7.18–7.10(m,1H),7.08(d,J＝7.6Hz,1H),6.89(t,J＝7.5Hz,1H),6.70(d,J＝7.9Hz,1H),6.52(t,J＝7.4Hz,1H),5.62(s,2H),4.86(s,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ166.03,165.51,158.70,156.63,155.11,152.61,148.47,143.95,143.48,140.78,138.66,136.41,134.51,134.32,129.32,128.61,127.96,127.11,126.97,123.68,120.40,116.73,116.56,115.27,97.96,50.16.HRMS(ESI):calcd for C ₃₁ H ₂₅ N ₉ O ₂ [M+H] ⁺ 556.2204,found 556.2214。

4- (4-amino-1- (4- ((2-aminophenyl) carbamoyl) benzyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide (Z17)

White solid, yield 59%, mp 238-240 deg.C; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.72(s,1H),9.54(s,1H),8.26(s,1H),8.19(d,J＝4.9Hz,1H),8.13(d,J＝8.0Hz,2H),8.01(s,1H),7.86(d,J＝7.8Hz,2H),7.73(d,J＝8.0Hz,2H),7.35(d,J＝7.9Hz,2H),7.08(d,J＝7.7Hz,1H),6.96(d,J＝4.8Hz,1H),6.89(t,J＝7.6Hz,1H),6.69(d,J＝7.9Hz,1H),6.51(t,J＝7.5Hz,1H),5.62(s,2H),4.81(s,2H),2.30(s,3H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ165.92,165.50,162.31,158.72,156.64,155.13,152.66,149.32,148.09,143.95,143.56,140.78,136.39,134.53,134.40,129.27,128.60,127.96,127.10,126.95,123.66,116.65,116.52,115.65,99.99,97.97,50.16,21.43.HRMS(ESI):calcd for C ₃₂ H ₂₇ N ₉ O ₂ [M+H] ⁺ 570.2361,found 570.2358。

4- (4-amino-1- (4- ((2-aminophenyl) carbamoyl) benzyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (Z18)

White solid, yield 55%, mp 238-239 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ11.41(s,1H)9.62(s,1H),8.71(d,J＝5.0Hz,1H),8.57(d,J＝7.4Hz,1H),8.34(s,1H),8.23(d,J＝8.2Hz,2H),7.93(d,J＝7.9Hz,2H),7.83(d,J＝8.2Hz,2H),7.57(d,J＝4.8Hz,1H),7.42(d,J＝8.1Hz,2H),7.15(d,J＝7.6Hz,1H),6.96(t,J＝7.5Hz,1H),6.77(d,J＝7.9Hz,1H),6.58(t,J＝7.4Hz,1H),5.69(s,2H),4.89(s,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ166.69,165.51,158.73,156.66,155.15,153.72,150.42,143.88,143.56,140.76,138.82,136.79,134.54,133.81,129.48,128.67,128.60,127.96,127.10,126.96,123.67,123.43,116.67,116.53,115.69,110.31,110.25,98.00,50.18.HRMS(ESI):calcd for C ₃₂ H ₂₄ F ₃ N ₉ O ₂ [M+H] ⁺ 624.2078, found 624.207, purity 97.1%, retention time 3.178min,25% methanol/75% water elution.

4- (4-amino-1- (1- (2- ((2-aminophenyl) amino) -2-oxoethyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (pyridin-2-yl) benzamide (Z19)

White solid, yield 64%, mp 146-148 deg.C; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.89(s,1H),9.25(s,1H),8.42(d,J＝4.5Hz,1H),8.30(s,1H),8.27–8.18(m,3H),7.87(t,J＝7.8Hz,1H),7.80(d,J＝7.9Hz,2H),7.26(d,J＝7.8Hz,1H),7.23–7.16(m,1H),6.92(t,J＝7.5Hz,1H),6.78(d,J＝7.9Hz,1H),6.59(t,J＝7.5Hz,1H),5.06-4.99(m,1H),4.83(s,2H),3.24-3.15(m,3H),2.97(d,J＝10.5Hz,1H),2.77(t,J＝10.5Hz,1H),2.30(t,J＝8.9Hz,1H),2.05(s,2H),1.85(s,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ168.77,166.04,158.66,156.16,154.50,152.64,148.45,143.20,142.25,138.63,136.66,134.21,129.28,128.60,126.26,125.45,124.17,120.37,117.11,116.81,115.26,97.99,61.87,58.08,53.66,53.23,29.50,24.60.HRMS(ESI):calcd for C ₃₀ H ₃₀ N ₁₀ O ₂ [M+H] ⁺ 563.2626,found 563.2628。

4- (4-amino-1- (1- (2- ((2-aminophenyl) amino) -2-oxoethyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide (Z20)

White solid, yield 65%, mp 146-148 deg.C; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.71(s,1H),9.20(s,1H),8.22(s,1H),8.19(d,J＝4.9Hz,1H),8.12(d,J＝7.8Hz,2H),8.02(s,1H),7.72(d,J＝7.8Hz,2H),7.18(d,J＝7.8Hz,1H),6.96(d,J＝4.9Hz,1H),6.84(t,J＝7.6Hz,1H),6.70(d,J＝7.9Hz,1H),6.51(t,J＝7.5Hz,1H),4.96(s,1H),4.79(s,2H),3.19(s,2H),3.10(d,J＝6.4Hz,1H),2.91(d,J＝8.9Hz,1H),2.72(s,1H),2.30(s,3H),2.25(s,1H),1.98(s,2H),1.78(s,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ165.94,158.66,156.17,154.50,152.67,149.32,148.07,143.24,142.27,136.60,134.28,129.23,128.60,126.27,125.47,124.11,121.39,117.07,116.78,115.64,97.99,61.77,58.00,53.54,53.21,29.46,24.50,21.43.HRMS(ESI):calcd for C ₃₁ H ₃₂ N ₁₀ O ₂ [M+H] ⁺ 577.2725,found 577.2791。

(R) -4- (4-amino-1- (1- (2- ((2-aminophenyl) amino) -2-oxoethyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide (Z21)

White solid, yield 67%, mp 144-146 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.79(s,1H),9.25(s,1H),8.29(s,1H),8.27(d,J＝5.0Hz,1H),8.19(d,J＝8.2Hz,2H),8.09(s,1H),7.79(d,J＝8.2Hz,2H),7.23(d,J＝7.6Hz,1H),7.04(d,J＝4.9Hz,1H),6.92(t,J＝7.3Hz,1H),6.76(d,J＝7.7Hz,1H),6.57(t,J＝7.5Hz,1H),5.02(s,1H),4.84(s,2H),3.27–3.09(m,3H),2.98(s,1H),2.76(s,1H),2.38(s,3H),2.29(s,1H),2.05(s,2H),1.86(s,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ165.95,158.62,156.18,154.47,152.58,149.37,148.07,143.35,142.35,136.50,134.31,129.20,128.62,126.47,125.64,123.76,122.13,121.42,117.05,116.72,115.63,97.97,61.26,57.58,53.16,46.10,29.25,24.07,21.41.HRMS(ESI):calcd for C ₃₁ H ₃₂ N ₁₀ O ₂ [M+H] ⁺ 577.2783,found 577.2773。

4- (4-amino-1- (1- (2- ((2-aminophenyl) amino) -2-oxoethyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (Z22)

White solid, yield 49%, mp 170-172 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ11.40(s,1H),9.25(s,1H),8.71(d,J＝5.0Hz,1H),8.59(s,1H),8.31(s,1H),8.23(d,J＝7.7Hz,2H),7.82(d,J＝7.7Hz,2H),7.56(d,J＝4.9Hz,1H),7.26(d,J＝7.8Hz,1H),6.92(t,J＝7.5Hz,1H),6.78(d,J＝7.9Hz,1H),6.59(t,J＝7.4Hz,1H),5.03(d,J＝7.8Hz,1H),4.83(s,2H),3.24(d,J＝7.6Hz,2H),3.17(d,J＝10.7Hz,1H),2.97(d,J＝10.3Hz,1H),2.78(t,J＝10.5Hz,1H),2.30(t,J＝8.1Hz,1H),2.06(s,2H),1.86(s,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ168.76,166.70,158.66,156.17,154.52,153.74,150.40,143.13,142.26,138.82,137.01,133.67,129.44,128.65,126.27,125.47,124.15,123.43,117.12,116.81,115.66,110.30,98.01,61.86,58.06,53.66,53.24,29.48,24.58.HRMS(ESI):calcd for C ₃₁ H ₂₉ F ₃ N ₁₀ O ₂ [M+H] ⁺ 631.2500,found 631.2501。

4- (4-amino-1- (3- ((2-aminophenyl) amino) -3-oxopropyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide (Z23)

White solid, yield 39%, mp 285-287 deg.C; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.78(s,1H),9.27(s,1H),8.31(s,1H),8.27(d,J＝4.8Hz,1H),8.21(d,J＝7.7Hz,2H),8.09(s,1H),7.81(d,J＝7.8Hz,2H),7.08(d,J＝7.7Hz,1H),7.04(d,J＝4.6Hz,1H),6.90(t,J＝7.4Hz,1H),6.70(d,J＝7.9Hz,1H),6.52(t,J＝7.3Hz,1H),4.87(s,2H),4.70(t,J＝6.6Hz,2H),2.99(t,J＝6.5Hz,2H),2.38(s,3H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ168.93,165.96,158.62,156.27,154.82,152.67,149.34,148.08,143.36,142.78,136.58,134.30,129.23,128.58,126.54,126.22,123.42,121.40,116.45,116.12,115.65,97.95,43.58,36.07,21.43.HRMS(ESI):calcd for C ₂₇ H ₂₅ N ₉ O ₂ [M+H] ⁺ 508.2204,found 508.2207。

4- (4-amino-1- (3- ((2-aminophenyl) amino) -3-oxopropyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (Z24)

White solid, yield 30%, mp 185-187 deg.C; ¹ H NMR(400MHz,DMSO-d ₆ )δ11.39(s,1H),9.26(s,1H),8.71(d,J＝5.1Hz,1H),8.58(s,1H),8.30(s,1H),8.22(d,J＝8.2Hz,2H),7.83(d,J＝8.1Hz,2H),7.57(d,J＝5.4Hz,1H),7.07(dd,J＝7.8,0.4Hz,1H),6.89(td,J＝7.8,0.8Hz,1H),6.69(d,J＝7.8Hz,1H),6.50(t,J＝8.1Hz,1H),4.82(s,2H),4.69(t,J＝7.0Hz,2H),2.97(t,J＝6.8Hz,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ168.94,166.72,158.64,156.31,154.84,153.73,150.43,143.27,142.83,138.82,136.98,133.69,129.45,128.64,126.53,126.21,123.44,123.40,116.41,116.09,115.69,110.30,97.97,43.60,36.07.HRMS(ESI):calcd for C ₂₇ H ₂₂ F ₃ N ₉ O ₂ [M+H] ⁺ 562.1921,found 562.1923。

4- (4-amino-1- (4- ((2-aminophenyl) amino) -4-oxobutyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide (Z25)

White solid, yield 36%, mp:258-260 ℃; ¹ H NMR(400MHz,DMSO-d6)δ10.81(s,1H),9.32(s,1H),8.29(s,1H),8.27(d,J＝4.4Hz,1H),8.20(d,J＝7.6Hz,2H),8.08(s,1H),7.82(d,J＝7.6Hz,2H),7.16(d,J＝7.6Hz,1H),7.04(d,J＝3.5Hz,1H),6.87(t,J＝6.9Hz,1H),6.70(d,J＝7.6Hz,1H),6.50–6.44(m,1H),5.02(s,2H),4.97(s,2H),4.49–4.39(m,2H),2.38(s,3H),2.27–2.15(m,2H)； ¹³ C NMR(100MHz,DMSO-d ₆ )δ170.79,165.96,158.65,156.31,154.89,152.66,149.31,148.10,143.29,142.53,136.62,134.29,129.24,128.61,126.13,125.93,123.84,121.40,116.35,116.13,115.67,97.89,46.46,33.10,25.71,21.43.HRMS(ESI):calcd for C ₂₆ H ₂₃ N ₇ O ₂ [M+H] ⁺ 522.2360,found 522.2364。

4- (4-amino-1- (4- ((2-aminophenyl) amino) -4-oxobutyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (Z26)

White solid, yield 31%, mp 138-140 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ11.39(s,1H),9.14(s,1H),8.71(d,J＝4.6Hz,1H),8.58(s,1H),8.30(s,1H),8.23(d,J＝7.6Hz,2H),7.84(d,J＝7.7Hz,2H),7.56(d,J＝4.0Hz,1H),7.12(d,J＝7.7Hz,1H),6.98–6.86(m,1H),6.74(d,J＝7.9Hz,1H),6.57(t,J＝7.5Hz,1H),4.46(t,J＝5.8Hz,2H),2.41–2.31(m,2H),2.25–2.17(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ170.80,166.71,158.50,156.07,154.87,153.72,150.41,143.40,142.00,138.81,136.95,133.70,129.46,128.67,126.35,126.11,124.07,123.43,116.98,116.49,115.67,110.32,97,89,46.46,33.04,25.61.HRMS(ESI):calcd for C ₂₈ H ₂₄ F ₃ N ₉ O ₂ [M+H] ⁺ 576.2078,found 576.2080。

4- (4-amino-1- (5- ((2-aminophenyl) amino) -5-oxopentyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide (Z27)

White solid, yield 36%, mp 173-175 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.79(s,1H),9.12(s,1H),8.29(s,1H),8.26(d,J＝5.0Hz,1H),8.19(d,J＝8.2Hz,2H),8.09(s,1H),7.80(d,J＝8.2Hz,2H),7.14(d,J＝7.6Hz,1H),7.03(d,J＝4.8Hz,1H),6.88(t,J＝7.2Hz,1H),6.70(d,J＝7.5Hz,1H),6.52(t,J＝7.3Hz,1H),4.82(s,2H),4.42(t,J＝6.8Hz,2H),2.43–2.35(m,5H),1.98–1.88(m,2H),1.65–1.56(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.35,165.97,158.66,156.26,154.83,152.67,149.31,148.07,143.18,142.34,136.65,134.28,129.25,128.58,126.13,125.76,124.00,121.38,116.58,116.32,115.64,97.85,46.70,35.64,29.20,23.01,21.42.HRMS(ESI):calcd for C ₂₉ H ₂₉ N ₉ O ₂ [M+H] ⁺ 536.2517,found 536.2523。

4- (4-amino-1- (5- ((2-aminophenyl) amino) -5-oxopentyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (Z28)

White solid, yield 34%, mp 169-171 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ11.39(s,1H),9.12(s,1H),8.71(d,J＝4.9Hz,1H),8.58(s,1H),8.30(s,1H),8.22(d,J＝7.8Hz,2H),7.84(d,J＝7.8Hz,2H),7.57(d,J＝4.8Hz,1H),7.14(d,J＝7.7Hz,1H),6.89(t,J＝7.5Hz,1H),6.71(d,J＝7.8Hz,1H),6.53(t,J＝7.4Hz,1H),4.82(s,2H),4.42(t,J＝6.5Hz,2H),2.38(t,J＝6.8Hz,2H),2.00–1.88(m,2H),1.68–1.57(m,2H)； ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.32,166.72,158.66,156.27,154.86,153.74,150.40,143.11,142.36,138.82,137.05,133.67,129.46,128.63,126.19,125.80,123.97,123.44,116.62,116.33,115.67,110.30,97.86,46.70,35.63,29.19,22.98.HRMS(ESI):calcd for C ₂₉ H ₂₆ F ₃ N ₉ O ₂ [M+H] ⁺ 590.2234,found 590.2236。

4- (4-amino-1- (6- ((2-aminophenyl) amino) -6-oxohexyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide (Z29)

White solid, yield 42%, mp 120-122 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.79(s,1H),9.06(s,1H),8.29(s,1H),8.26(d,J＝5.1Hz,1H),8.19(d,J＝8.3Hz,2H),8.09(s,1H),7.80(d,J＝8.3Hz,2H),7.11(d,J＝6.8Hz,1H),7.03(d,J＝5.0Hz,1H),6.88(t,J＝7.0Hz,1H),6.70(dd,J＝7.9,1.0Hz,1H),6.52(t,J＝8.1Hz,1H),4.79(s,2H),4.39(t,J＝6.9Hz,2H),2.38(s,3H),2.30(t,J＝7.3Hz,2H),1.92(dt,J＝13.8,6.7Hz,2H),1.64(dt,J＝14.3,7.2Hz,2H),1.39–1.30(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.49,165.96,158.65,156.25,154.81,152.67,149.32,148.09,143.14,142.36,136.65,134.25,129.24,128.58,126.15,125.77,123.98,116.61,116.31,115.64,97.80,46.78,36.04,29.38,26.27,25.32,21.43.HRMS(ESI):calcd for C ₃₀ H ₃₁ N ₉ O ₂ [M+H] ⁺ 550.2673,found 550.2677。

4- (4-amino-1- (6- ((2-aminophenyl) amino) -6-oxohexyl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (Z30)

White solid, yield 44%, mp 104-105 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ11.39(s,1H),9.06(s,1H),8.71(d,J＝4.2Hz,1H),8.59(s,1H),8.30(s,1H),8.23(d,J＝7.6Hz,2H),7.83(d,J＝7.6Hz,2H),7.57(d,J＝3.6Hz,1H),7.12(d,J＝7.5Hz,1H),6.89(t,J＝7.0Hz,1H),6.71(d,J＝7.6Hz,1H),6.52(t,J＝7.0Hz,1H),4.79(s,2H),4.46–4.35(m,2H),2.31(t,J＝6.5Hz,2H),2.00–1.86(m,2H),1.72–1.60(m,2H),1.43–1.29(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.49,166.73,158.66,156.27,154.85,153.74,150.42,143.07,142.36,138.82,137.06,133.66,129.45,128.64,126.15,125.77,124.00,123.44,116.62,116.32,115.67,110.31,97.84,46.81,36.05,29.37,26.28,25.32.HRMS(ESI):calcd for C ₃₀ H ₂₈ F ₃ N ₉ O ₂ [M+H] ⁺ 604.2391,found 604.2393。

4- (4-amino-1- (1- (4- ((2-aminophenyl) amino) -4-oxobutyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4-methylpyridin-2-yl) benzamide (Z31)

White solid, yield 33%, mp:164-166 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ10.79(s,1H),9.12(s,1H),8.29(s,1H),8.26(d,J＝5.0Hz,1H),8.20(d,J＝8.3Hz,2H),8.09(s,1H),7.80(d,J＝8.3Hz,2H),7.16(d,J＝7.7Hz,1H),7.03(d,J＝5.0Hz,1H),6.88(t,J＝7.6Hz,1H),6.71(d,J＝7.9Hz,1H),6.53(t,J＝8.1Hz,1H),4.93–4.82(m,1H),3.13(d,J＝7.6Hz,1H),2.98(d,J＝9.9Hz,1H),2.59–2.54(m,2H),2.48–2.42(m,2H),2.41–2.32(m,5H),2.13–1.99(m,3H),1.89–1.83(m,1H),1.82–1.75(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.45,165.95,158.64,156.18,154.48,152.66,149.33,148.08,143.21,142.41,136.61,134.29,129.23,128.61,126.18,125.83,123.99,121.40,116.60,116.30,115.64,97.97,57.92,57.61,53.66,52.99,34.04,29.92,24.45,22.75,21.43.HRMS(ESI):calcd for C ₃₃ H ₃₆ N ₁₀ O ₂ [M+H] ⁺ 605.3095,found 605.3098。

4- (4-amino-1- (1- (4- ((2-aminophenyl) amino) -4-oxobutyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (Z32)

White solid, 43% yield, mp:124-126 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ11.39(s,1H),9.13(s,1H),8.70(d,J＝4.9Hz,1H),8.58(s,1H),8.29(s,1H),8.22(d,J＝8.1Hz,2H),7.82(d,J＝8.0Hz,2H),7.56(d,J＝4.6Hz,1H),7.15(d,J＝7.6Hz,1H),6.88(t,J＝7.4Hz,1H),6.71(d,J＝7.8Hz,1H),6.53(t,J＝7.4Hz,1H),4.93–4.76(m,3H),3.07(d,J＝7.2Hz,1H),2.93(d,J＝9.7Hz,1H),2.50–2.38(m,4H),2.37–2.33(m,2H),2.06–2.00(m,1H),1.98–1.91(m,1H),1.89–1.80(m,1H),1.79–1.74(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.57,166.73,158.62,156.16,154.46,153.71,150.41,143.06,142.40,138.82,137.02,133.67,129.43,128.66,126.18,125.83,124.01,123.42,116.64,116.33,115.69,110.29,97.98,58.23,57.71,53.92,53.09,34.13,30.07,24.66,22.97.HRMS(ESI):calcd for C ₃₃ H ₃₃ F ₃ N ₁₀ O ₂ [M+H] ⁺ 659.2813,found 659.2812。

4- (4-amino-1- (1- (5- ((2-aminophenyl) amino) -5-oxopentyl) piperidin-3-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3-yl) -N- (4- (trifluoromethyl) pyridin-2-yl) benzamide (Z33)

White solid, yield 22%, mp 138-140 ℃; ¹ H NMR(400MHz,DMSO-d ₆ )δ11.41(s,1H),9.19(s,1H),8.71(d,J＝5.1Hz,1H),8.58(s,1H),8.29(s,1H),8.22(d,J＝8.3Hz,2H),7.82(d,J＝8.3Hz,2H),7.57(d,J＝5.1Hz,1H),7.14(d,J＝7.8Hz,1H),6.87(t,J＝7.6Hz,1H),6.70(d,J＝7.0Hz,1H),6.51(t,J＝8.1Hz,1H),4.92–4.78(m,3H),3.06(d,J＝10.0Hz,1H),2.92(d,J＝10.2Hz,1H),2.48–2.37(m,4H),2.34(t,J＝7.2Hz,2H),2.07–1.99(m,2H),1.95(d,J＝11.2Hz,1H),1.83(d,J＝12.6Hz,1H),1.65–1.56(m,2H),1.54–1.43(m,2H). ¹³ C NMR(100MHz,DMSO-d ₆ )δ171.66,166.71,158.62,156.16,154.46,153.72,150.42,143.04,142.29,138.80,137.04,133.66,129.44,128.65,126.02,125.66,124.11,123.43,116.55,116.34,115.66,110.31,97.99,58.27,57.98,53.93,53.09,36.05,30.05,26.37,24.69,23.77.HRMS(ESI):calcd for C ₃₄ H ₃₅ F ₃ N ₁₀ O ₂ [M+H] ⁺ 673.2969,found 673.2968。

experimental example:determination of BTK and HDAC inhibitory Activity of Compounds and on mantle cell lymphoma cellsGrowth inhibitory Activity measurement experiment

1. Compound activity assay for inhibition of BTK kinase:

experimental materials: BTK, peptide FAM-P2, ATP, DMSO, 96/384-well plate, staurosporine 1 xkinase buffer (50mM HEPES, pH 7.5,0.0015% Brij-35), stop buffer (100mM HEPES, pH 7.5,0.015% Brij-35,0.2% coating reagent #3,50mM EDTA), and the like.

The experimental method comprises the following steps: the compound is diluted to 50 times of the highest concentration required in the reaction, and stock solution is prepared for standby. To a 96-well plate, 10 μ L of stock solution and 90 μ L of 1 × kinase buffer were added and mixed on a shaker for 10 minutes while setting compound-free and enzyme-free controls. After shaking up, 5. Mu.l of the mixture was transferred to a 384-well plate from a 96-well intermediate plate. Kinase was added to 1 × kinase buffer to prepare a 2.5 × enzyme solution, and FAM-labeled peptide and ATP were added to prepare a 2.5 × peptide solution. Subsequently, 10 μ L of 2.5x enzyme solution was added to each well of the 384-well plate and incubated at room temperature for 10 minutes. After that, 10. Mu.L of 2.5 Xpeptide solution was added to each well of the 384-well assay plate, incubated at 28 ℃ and 25. Mu.L of stop solution was added. The experimental results were determined using Caliper software.

Conversion group measurement results of the target Compound.

Max group: and (5) determining results of the DMSO positive control group. All experimental components except the test compound were included and the buffer was used to replenish the amount of test compound.

Min group, blank control group assay results, including all experimental constituents except the test compound and enzyme, were filled with buffer to the amounts of test compound and enzyme.

TABLE 1 inhibitory Activity of Compounds on BTK kinase

IBN: ibrutinib (Ibrutinib)

As can be seen from table 1, the compounds of the present invention exhibit various degrees of inhibition on BTK, and most of the compounds exhibit strong BTK inhibitory activity, wherein the inhibitory activity of compounds Z1 to Z4, Z18, Z20, and Z22 on BTK is comparable to IBN, and particularly the inhibitory activity of compound Z2 on BTK is twice as high as IBN.

2. Inhibition activity of compounds on HDAC assay:

experimental materials: HDAC enzyme, boc-Lys (acetyl) -AMC (fluorescent substrate for Hela nucleus extraction), tris-HCl, trypsin, EDTA, TSA, glycerol, naCl, 96-hole flat-bottom fluorescent plate, MB100-2A type microplate constant temperature oscillator.

Preparation of buffer (HDAC buffer): 15mM Tris-HCl (pH 8.0), 250. Mu.M EDTA,250mM NaCl,10% glycerol.

HDAC enzyme solution: according to the following steps of 1:80 diluted with HDAC buffer for use, and stored at-80 ℃.

Preparation of a fluorescent substrate: the substrate was dissolved in DMSO to prepare a 30mM stock solution, stored at-20 ℃ and diluted to 300. Mu.M with HDAC buffer before use, so that the DMSO content was less than 0.1%.

Preparation of a stop solution: 10mg/ml Trypsin,50mM Tris-HCl (pH 8.0), 100mM NaCl, 2. Mu.M TSA. When the preparation is ready for use, trypsin and TSA are added in corresponding amounts before use.

Preparation of test compound: compounds were diluted 50-fold with DMSO to the highest concentration required to make up stock solutions for use. The assay was diluted with HDAC buffer to achieve DMSO levels below 0.1%.

The experimental method comprises the following steps: add 50. Mu.L of diluted test compound to 96-well fluorescent plate, set 100% and blank control group at the same time, add 10. Mu.L of HDACs enzyme solution to the experimental well, incubate at 37 ℃ and shake for 30min. Add 40. Mu.L of substrate to each well of the 96-well plate and continue incubation at 37 ℃ with shaking for 30min to activate the substrate. Add 100 μ L of stop buffer to 96 well plate and shake for 20min at 37 ℃. The HDAC inhibitory activity of the compounds was calculated by measuring the fluorescence intensity in the absence of light at the emission/excitation wavelength (390 nm/460 nm).

100% control group: containing all experimental constituents except the test compound, 50 μ L HDAC buffer was substituted for the test compound. The fluorescence intensity was measured at the emission wavelength/excitation wavelength (390 nm/460 nm).

Blank control group: containing all experimental components except the test compound and the HDAC enzyme solution, 60 μ L of HDAC buffer was substituted for the test compound and HDAC enzyme solution. The fluorescence intensity was measured at the emission wavelength/excitation wavelength (390 nm/460 nm).

TABLE 2 inhibitory Activity of Compounds on HDACs

IBN: ibrutinib (Ibrutinib), MS-275: an HDAC inhibitor.

As can be seen from the test results in table 2, in formula IIB, when n =3, i.e., compound Z11, there is no inhibitory activity on HDACs, and as the number of n carbons increases, n =5 (compound Z13) also increases in inhibitory activity on HDACs; in formula IIE, when n =5, i.e. compound Z15, the inhibitory activity against HDACs is strong (IC) ₅₀ The value is 1.19). In formula III, compounds Z18 and Z29 have a stronger inhibitory activity on HDAC and a weaker inhibitory activity (IC) than the positive control MS-275 (an HDAC inhibitor) ₅₀ Values of 1.51. Mu.M, 1.58. Mu.M and 0.7. Mu.M, respectively). IBN has little inhibitory activity against HDACs.

3. Test on cell growth inhibitory Activity of Compound on mantle cell lymphoma

We performed anti-proliferation experiments on MCL cell lines tested with Jeko-1, mino, maver-1 cells, where Jeko-1 and Mino are IBN-sensitive MCL cell lines and Maver-1 is an IBN primary drug-resistant MCL cell line.

TABLE 3 inhibitory Effect of Compounds on MCL cell growth

NA = Not Active; IBN: ibrutinib (Ibrutinib)

Most of the compounds showed better anti-proliferative effects on the cells tested, wherein the anti-proliferative activity of compound Z2 on Maver-1 cell line was twice that of IBN, and the anti-proliferative activity of compounds Z10 and Z13 on Jeko-1 cell line was 2-3 times that of IBN; the growth inhibitory activity of the compounds Z15 and Z18 having the double inhibitory effects on BTK and HDACs on Jeko-1 cell line and Maver-1 cell line was three times and twenty times that of IBN, respectively.

Although the present application has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An o-phenylenediamine derivative containing pyrazolopyrimidine or a pharmaceutically acceptable salt or an isomer thereof, wherein the derivative has a structure shown in formula I:

wherein X is selected from oxygen, amide and methylene;

y is selected from carbon or nitrogen; when Y is carbon, the hydrogen thereof is unsubstituted or substituted by C ₁ -C ₅ Alkyl substitution of (b);

z is selected from the group consisting of methylene, benzyl, piperidinyl, pyridinyl, pyrrolidinyl, pyrimidinyl, imidazolyl and oxadiazolyl, and Z is unsubstituted or substituted by C _1-5 Alkyl or carbonyl substituted;

n is any integer from 0 to 7;

wherein Z is

When n is not 1.

2. The pyrazolopyrimidine-containing o-phenylenediamine derivative or a pharmaceutically acceptable salt thereof or an isomer thereof according to claim 1, characterized in that the derivative corresponds to the structure represented by formula II or formula III:

wherein Z, R and n are as defined in claim 1.

3. The pyrazolopyrimidine containing o-phenylenediamine derivative or a pharmaceutically acceptable salt thereof, or an isomer thereof according to claim 1 or 2, characterized in that Z is selected from the group consisting of methylene, benzyl, piperidinyl, and pyrrolidinyl; r is selected from hydrogen, methyl and trifluoromethyl; n is any integer from 0 to 5;

preferably, when Z is benzyl, piperidinyl, or pyrrolidinyl, the benzyl group is attached at its methylene end to the nitrogen end of the pyrazole, the piperidinyl group is attached at its N-end to the carbon chain of formula, and the pyrrolidinyl group is attached at its N-end to the carbon chain of formula;

preferably, said piperidinyl group is selected from

Preferably, the pyrrolidinyl group is

4. The pyrazolopyrimidine-containing o-phenylenediamine derivative or a pharmaceutically acceptable salt thereof or an isomer thereof according to claim 1 or 2, characterized in that in the structure of formula II, Z is selected from the group consisting of methylene, piperidinyl and pyrrolidinyl;

preferably, the derivatives conform to the following structure:

wherein n is any integer of 1-5.

5. The pyrazolopyrimidine-containing o-phenylenediamine derivative or a pharmaceutically acceptable salt thereof or an isomer thereof according to claim 1 or 2, characterized in that in the structure of formula III, Z is selected from the group consisting of methylene, benzyl and piperidinyl;

preferably, the derivatives conform to the following structure:

wherein, in IID, a star represents chiral carbon, and a wavy line represents a carbon-carbon single bond, including

R and n are as defined in claim 1 or 3;

preferably, n is any integer from 0 to 4.

6. The pyrazolopyrimidine-containing o-phenylenediamine derivative or a pharmaceutically acceptable salt thereof or an isomer thereof according to any one of claims 1 to 5, characterized in that the derivative is selected from the following compounds (Z1) to (Z33):

(Z11): 4- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) butyramide;

7. A method for producing the pyrazolopyrimidine-containing o-phenylenediamine derivative or a pharmaceutically acceptable salt thereof, or an isomer thereof according to any one of claims 1 to 6, which comprises:

reacting a compound 1, namely 1H-pyrazolo [3,4-d ] pyrimidine-4-amine, serving as a starting material with N-bromosuccinimide under the heating condition to obtain an intermediate 2, namely 3-bromo-1H-pyrazolo [3,4-d ] pyrimidine-4-amine; carrying out Mitsunobu reaction on the intermediate 2 and N-Boc-3/4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine to obtain an intermediate 3; removing the Boc protecting group from the intermediate 3 to obtain an intermediate 4; the intermediate 2 or the intermediate 4 and methyl ester substituted by different bromine are subjected to nucleophilic substitution to obtain an intermediate 5; the intermediate 5 and the intermediate 8 are subjected to Suzuki reaction to obtain an intermediate 9; hydrolyzing the intermediate 9 to obtain an intermediate 10; the intermediate 10 and o-phenylenediamine are subjected to amide condensation to obtain a compound shown in a general formula (I);

wherein the reaction route is shown as follows:

wherein R' is

or-B (OH) ₂ (ii) a R, X, Y, Z, n are as defined in any one of claims 1 to 6.

8. A pharmaceutical composition comprising the pyrazolopyrimidine-containing o-phenylenediamine derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or an isomer thereof.

9. A pharmaceutical preparation comprising the pyrazolopyrimidine-containing o-phenylenediamine derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, or an isomer thereof, and at least one pharmaceutically acceptable auxiliary.

10. Use of an o-phenylenediamine pyrazolopyrimidine containing derivative according to any one of claims 1 to 6, or a pharmaceutically acceptable salt or isomer thereof, or a pharmaceutical composition according to claim 8 or a pharmaceutical formulation according to claim 9 for the preparation of a BTK modulator drug and/or an HDAC modulator drug or an antitumor drug;

preferably, the BTK modulator is a BTK inhibitor and the HDAC modulator is an HDAC inhibitor; the tumor is mantle cell lymphoma.