CN115572297B - Pyrazolopyrimidine-containing o-phenylenediamine derivative, and preparation method and application thereof - Google Patents

Pyrazolopyrimidine-containing o-phenylenediamine derivative, and preparation method and application thereof Download PDF

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CN115572297B
CN115572297B CN202110686133.9A CN202110686133A CN115572297B CN 115572297 B CN115572297 B CN 115572297B CN 202110686133 A CN202110686133 A CN 202110686133A CN 115572297 B CN115572297 B CN 115572297B
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amino
drying
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CN115572297A (en
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赵桂森
袁文娟
禚慧君
冉凡胜
刘洋
景永奎
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Shandong University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The application provides a pyrazolopyrimidine-containing o-phenylenediamine derivative, and a preparation method and application thereof, wherein the pyrazolopyrimidine-containing o-phenylenediamine derivative has a structure shown in a formula I: Wherein X is selected from oxygen, amido and methylene; y is selected from carbon or nitrogen; when Y is carbon, hydrogen is unsubstituted or substituted by C 1-C5 alkyl; z is selected from methylene, benzyl, piperidinyl, pyridinyl, pyrrolidinyl, pyrimidinyl, imidazolyl, and oxadiazolyl, Z being unsubstituted or substituted with C 1‑5 alkyl or carbonyl; r is selected from hydrogen, halogen, nitro, amino, substituted amino, cyano, methyl, methoxy and trifluoromethyl; the substituted amino group is an amino group substituted with an alkyl group of C 1-C5; n is any integer from 0 to 7; wherein R 1 is

Description

Pyrazolopyrimidine-containing o-phenylenediamine derivative, and preparation method and application thereof
Technical Field
The application relates to the fields of organic compound synthesis and medical application, in particular to an o-phenylenediamine derivative containing pyrazolopyrimidine as well as a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the 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 already known to those of ordinary skill in the art.
BTK kinase (Bruton's tyrosine Kinase) is the most closely reported Tec kinase family member associated with human disease. BTK is expressed in most hematopoietic cells, particularly B cells, myeloid cells and platelets, is a key regulator of BCR (B Cell Receptor) signaling pathway, and has a close relationship with proliferation, differentiation and apoptosis of B lymphomas. Ibrutinib (Ibrutinib, IBN) is the first irreversible BTK inhibitor on the market, and although it plays an important role in clinical research and treatment, development of a novel BTK inhibitor is significant due to adverse reactions and drug resistance caused by off-target actions. Epigenetic regulator histone deacetylases (Histone deacetylase, HDAC) can catalyze the acetylation of protein lysine residues to regulate chromatin structure and transcriptional activity, involving multiple pathophysiological states in vivo. The HDAC inhibitor can inhibit the expression level of Cyclin D1 protein, regulate the expression level of oncostatin p53 and conduct NF- κB signal path. Meanwhile, HDAC regulates the binding capacity of HSP90 and drug-resistant mutant proteins, and increases the stability of the mutant proteins. The method provides a theoretical basis for solving the primary and secondary drug resistance of ibrutinib. The current combination of HDAC inhibitors and BTK inhibitors shows good synergistic inhibition in various lymphomas. However, poor pharmacokinetic changes or unexpected toxicity limit the use of combination therapies. Therefore, the BTK/HDAC double-target inhibitor with double inhibition effect is designed and synthesized to act on various pathogenesis to play a synergistic effect, and meanwhile, adverse reaction of medicines is relieved, so that a new treatment strategy is provided for the curative effect of B cell malignant tumor.
Disclosure of Invention
The invention provides an o-phenylenediamine derivative containing pyrazolopyrimidine, a preparation method and application thereof, wherein the derivative has an inhibitory activity and an anti-cell proliferation activity on BTK and/or HDAC enzyme, and can inhibit the growth of mantle cell lymphoma cells for preparing anti-tumor drugs.
Specifically, the technical scheme of the invention is as follows:
In a first aspect of the present invention, there is provided an o-phenylenediamine derivative containing pyrazolopyrimidine or a pharmaceutically acceptable salt thereof or an isomer thereof, wherein the derivative has the structure shown in formula I:
Wherein X is selected from oxygen, amido and methylene;
Y is selected from carbon or nitrogen; when Y is carbon, hydrogen is unsubstituted or substituted by C 1-C5 alkyl;
Z is selected from methylene, benzyl, piperidinyl, pyridinyl, pyrrolidinyl, pyrimidinyl, imidazolyl, and oxadiazolyl, Z being unsubstituted or substituted with C 1-5 alkyl or carbonyl;
R is selected from hydrogen, halogen, nitro, amino, substituted amino, cyano, methyl, methoxy and trifluoromethyl; the substituted amino group is an amino group substituted with an alkyl group of C 1-C5;
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 corresponds to the structure of formula II or formula III:
wherein Z, R, n is as defined above.
Further, in some embodiments of the 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, and pyrrolidinyl, the benzyl is attached at its methylene terminus to the nitrogen terminus of the pyrazole, the piperidinyl is attached at its N terminus to the carbon chain of the formula, and the pyrrolidinyl is attached at its N terminus to the carbon chain of the formula, i.e., - (CH 2)n -structure in the formula.
In particular, in some embodiments, the preferred piperidinyl group is selected fromPreferred pyrrolidinyl groups are/>
In some embodiments of the present invention, in the above structure of formula II, the preferred structure of Z is selected from methylene, piperidinyl and pyrrolidinyl.
Further, in some more excellent examples of these embodiments, the derivatives also conform to the following structure:
where n is any integer from 1 to 5, for example n may be 1,2,3, 4 or 5.
In some embodiments of the invention, the inventors have found that the choice of piperidinyl group, especially in the structure of formula II, where Z is found during the course of the experiment to beWhen n is 1, the effect is poor 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 anti-proliferative activity of the drug-resistant MCL cell strain is better.
In some embodiments of the invention, in the above structure of formula III, the preferred structure of Z is selected from methylene, benzyl, and piperidinyl.
Further, in some more excellent examples of these embodiments, the derivatives also conform to the following structure:
Wherein IIID represents chiral carbon, the wavy line represents a single carbon-carbon bond, including Three cases; r, n are as defined above. Preferably R is selected from hydrogen, methyl and trifluoromethyl, 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 where n is 0 in the structure of formula IIIB.
In an embodiment of the present invention,Is a key structure for maintaining activity, especially when the general structure is simultaneously provided with/>Structurally, the compounds are guaranteed to have BTK and/or HDACs inhibitory activity and antiproliferative activity against mantle cell lymphoma, e.g., during research, the inventors found that when Z is benzyl, if notA group, the compound has difficulty in maintaining its antitumor activity while having/>The compounds exhibit excellent antiproliferative activity against mantle cell lymphomas, i.e., when Z is benzyl and n is 0, and especially when R is trifluoromethyl, in the general structure according to formula III, particularly excellent antiproliferative activity against drug resistant cells such as IBN primary drug resistant MCL cell line Maver-1, with IC 50 as low as 0.7 μm.
Specifically, the present invention provides a series of examples of pyrazolopyrimidine-containing o-phenylenediamine derivatives or pharmaceutically acceptable salts thereof or isomers thereof 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) butyramide;
(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) valeramide;
(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) butyramide;
(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, there is provided a process for preparing pyrazolopyrimidine-containing o-phenylenediamine derivative as described in the first aspect above or a pharmaceutically acceptable salt or isomer thereof comprising:
Taking a compound 1, namely 1H-pyrazolo [3,4-d ] pyrimidine-4-amine, as an initial raw material, and reacting with N-bromosuccinimide under a heating condition to obtain an intermediate 2, namely 3-bromo-1H-pyrazolo [3,4-d ] pyrimidine-4-amine; intermediate 2 and N-Boc-3/4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine are subjected to Mitsunobu reaction to obtain intermediate 3; removing the Boc protecting group from the intermediate 3 to obtain an intermediate 4; intermediate 2 or intermediate 4 and methyl ester substituted by different bromine are nucleophilic substituted to obtain intermediate 5; intermediate 5 and intermediate 8 undergo a Suzuki reaction to obtain intermediate 9; intermediate 9 is hydrolyzed to obtain intermediate 10; the intermediate 10 and o-phenylenediamine undergo amide condensation to obtain a compound of a general formula (I);
wherein, the reaction route is as follows:
wherein,
R' isOr-B (OH) 2; r, X, Y, Z, n are as defined above.
Further, the method of the present invention comprises: taking a compound 1 as a starting material, and reacting with N-bromosuccinimide in DMF (dimethyl formamide) under a heating condition to obtain an intermediate 2; intermediate 2 and N-Boc-3/4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine are subjected to Mitsunobu reaction to obtain intermediate 3; removing the Boc protecting group of the intermediate 3 under concentrated hydrochloric acid to obtain an intermediate 4; intermediate 2 or 4 and methyl ester substituted by different bromine are nucleophilic substituted to obtain intermediate 5; intermediate 5 and intermediate 8 undergo a Suzuki reaction under the catalysis of tetrakis (triphenylphosphine) palladium to obtain intermediate 9; intermediate 9 is hydrolyzed under alkaline condition to obtain intermediate 10; the intermediate 10 and o-phenylenediamine are subjected to amide condensation under the protection of N 2 to obtain the compound shown in the general formula I.
In a third aspect of the present invention, there is provided a pharmaceutical composition comprising a pyrazolopyrimidine-containing o-phenylenediamine derivative described in the first aspect above or a pharmaceutically acceptable salt or isomer thereof.
The term "composition" as used herein refers to a pharmaceutical product comprising a therapeutically effective amount of the specified ingredients, as well as any product that results, directly or indirectly, from the combination of the specified ingredients in the specified amounts.
In a fourth aspect, the present invention provides a pharmaceutical formulation comprising a pyrazolopyrimidine-containing o-phenylenediamine derivative as described in the first aspect or a pharmaceutically acceptable salt or isomer thereof 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 formulation containing the same may be administered in unit dosage form. The administration dosage form may be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, and mixed rotation. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.
The pharmaceutical combinations or formulations of the present invention may also contain conventional carriers, including but not limited to: ion exchangers, alumina, aluminium stearate, lecithin, serum proteins such as human serum proteins, buffer substances (e.g. phosphates, glycerol, sorbitol, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts) or electrolytes, protamine sulphate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, wool esters, etc. The carrier may be present in the pharmaceutical composition or formulation in an amount of from 1% to 98% by weight, typically about 80% by weight. For convenience, local anesthetics, preservatives, buffers, and the like may be directly dissolved in the carrier.
The pharmaceutically acceptable excipients, including but not limited to excipients, may be binders, fillers, lubricants, disintegrants, buffers, stabilizers, preservatives, and the like. The auxiliary materials refer to components except effective components in the pharmaceutical composition or the pharmaceutical preparation, are nontoxic to a subject, and can be stably coexistent with the pharmaceutical active components or stably coexistent after adopting proper means.
Oral tablets and capsules may contain binders such as syrup, acacia, sorbitol, tragacanth or polyvinylpyrrolidone; may contain fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine; may contain lubricants such as magnesium stearate, talc, polyethylene glycol, silica; disintegrants, such as potato starch, or acceptable wetting agents, such as sodium lauryl sulfate, may be included. The tablets may be coated by methods known in the pharmaceutical arts.
The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dry product, and can be 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 (possibly containing edible oils) such as almond oil, fats and oils such as glycerin, ethylene glycol, or ethyl alcohol; preservatives, such as methyl or propyl parahydroxybenzoate, sorbic acid. Flavoring or coloring agents may be added as desired.
Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.
For parenteral administration, liquid dosage forms are typically made of the compound and a sterile carrier. The carrier is water. Depending on the carrier and drug concentration selected, the compound may be dissolved in either the carrier or in suspension, and when preparing an injectable solution, the compound is first dissolved in water, filtered and sterilized, and filled into sealed bottles or ampoules.
Some embodiments of the invention include a method of producing a pharmaceutical composition or pharmaceutical formulation comprising admixing at least one compound according to any of the disclosure herein with a pharmaceutical excipient or carrier. Formulations are prepared by any suitable method, typically by uniformly mixing the active compound with liquid and/or finely divided solid auxiliary materials in the desired proportions, and then, if desired, shaping the resulting mixture into the desired shape. Of course, the compounds of the present invention may be formulated into pharmaceutical compositions or formulations by those skilled in the art using techniques well known in the art. For example, the preparation of the pharmaceutical preparation can be carried out according to a modern pharmaceutical preparation book of the university of Shenyang pharmacy. Other suitable pharmaceutical excipients are known in the art, in addition to those mentioned herein, for example see the pharmaceutical excipient handbook of 2005 edition (original fourth edition), authors (english) r.c. ro (Raymond C Rowe), (m) p.j. schencki (Paul J Sheskey).
In a fifth aspect of the present invention there is provided the use of a pyrazolopyrimidine-containing o-phenylenediamine derivative as described in the first aspect or a pharmaceutically acceptable salt or isomer thereof as described in the third aspect or a pharmaceutical composition as described in the fourth aspect or a pharmaceutical formulation as described in the fourth aspect for the manufacture of a BTK modulator (especially inhibitor) medicament and/or an HDACs modulator (especially inhibitor) medicament or an anti-tumour medicament. In particular, the compound provided by the invention has BTK/HDACs double-target enzyme inhibition activity, and can be used for preparing BTK/HDACs double-target inhibitor drugs. The tumor of the invention is lymphoma, in particular mantle cell lymphoma.
In a sixth aspect of the invention, the invention also provides a method of treating a tumour, comprising administering to a subject a therapeutically effective dose of a pyrazolopyrimidine-containing o-phenylenediamine derivative as described in the first aspect above or a pharmaceutically acceptable salt or isomer thereof or a pharmaceutical composition as described in the third aspect above or a pharmaceutical formulation as described in the fourth aspect above.
Wherein the tumor is in particular a lymphoma, in particular a mantle cell lymphoma; the subject is an animal, preferably a mammal, most preferably a human, who has been the subject of treatment, observation or experiment. By a therapeutically effective amount is meant an amount of an 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 staff, which includes alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated. It must be recognized that the optimal dosage and spacing of the active ingredients of the present invention is determined by its nature and external conditions such as the form, route and site of administration and the particular mammal being treated, and that such optimal dosage may be determined by conventional techniques. It must also be appreciated that the optimal course of treatment, i.e. the daily dosage of the simultaneous compounds over the nominal time period, can be determined by methods well known in the art.
The invention has the following beneficial effects: the pyrazolopyrimidine-containing o-phenylenediamine derivative or pharmaceutically acceptable salt or isomer thereof has inhibition activity and anti-cell proliferation activity on BTK and HDAC enzymes, can inhibit the growth of mantle cell lymphoma cells, and can be used for preparing anti-tumor drugs.
Detailed Description
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.
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 materials used in the present application may be purchased in conventional manners, and unless otherwise indicated, they may be used in conventional manners in the art or according to 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 methods and materials described herein are presented for illustrative purposes only.
In an embodiment of the present invention, the present invention provides a process for the preparation of a compound of formula I, which comprises following reaction scheme:
Wherein R' is Or-B (OH) 2; x is selected from oxygen or amido; y is selected from carbon or nitrogen; 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.
Specifically, the preparation method comprises the following steps: taking a compound 1 as a starting material, and reacting with N-bromosuccinimide in DMF (dimethyl formamide) under a heating condition to obtain an intermediate 2; intermediate 2 and N-Boc-3/4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine are subjected to Mitsunobu reaction to obtain intermediate 3; removing the Boc protecting group of the intermediate 3 under concentrated hydrochloric acid to obtain an intermediate 4; intermediate 2 or 4 and methyl ester substituted by different bromine are nucleophilic substituted to obtain intermediate 5; intermediate 5 and intermediate 8 undergo a Suzuki reaction under the catalysis of tetrakis (triphenylphosphine) palladium to obtain intermediate 9; intermediate 9 is hydrolyzed under alkaline condition to obtain intermediate 10; the intermediate 10 and o-phenylenediamine are subjected to amide condensation under the protection of N 2 to obtain a compound shown in 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 the compound of formula II, which comprises following reaction scheme:
Reagents and conditions in the synthetic route (a) NBS, DMF,85 ℃,4h; (b) N-Boc-3-hydroxypiperidine/N-Boc-4-hydroxypiperidine/1-Boc-3-hydroxypyrrolidine, diisopropyl azodicarboxylate, triphenylphosphine, anhydrous tetrahydrofuran, at 0deg.C for 5min; (c) tetrahydrofuran, hydrochloric acid = 4:1, r.t.,5h; (d) Different methyl bromocarboxylates, K 2CO3, DMF, r.t.,5h; (h) Tetrakis (triphenylphosphine) palladium, potassium phosphate trihydrate, 1, 4-dioxane: water=4:1, microwaves, 120 ℃,15min; (i) 3m noh, etoh, r.t.,4h; (g) O-phenylenediamine, HATU, DIEPA, protected from light, 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, detecting that the basic reaction is complete by TLC, naturally cooling the reaction liquid to room temperature, pouring the reaction liquid 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) Intermediate 2, N-Boc-3-hydroxy piperidine or N-Boc-4-hydroxy piperidine or 1-Boc-3-hydroxy pyrrolidine and triphenylphosphine are dissolved in anhydrous Tetrahydrofuran (THF), cooled in ice bath, and diisopropyl azodicarboxylate (DIAD) is slowly added dropwise, the ice bath is stirred for 5min, and the solution becomes clear from turbidity. TLC detection reaction is complete, ethyl acetate is added for extraction, the organic phases are combined, brine is added for washing, anhydrous Na 2SO4 is used for drying, filtration, reduced pressure evaporation is carried out to remove the solvent, and silica gel column chromatography is carried out for separation, thus obtaining an intermediate 3.
(3) Intermediate 3 is dissolved in THF, concentrated hydrochloric acid is added dropwise, the reaction is carried out for 5 hours at room temperature, white solid is separated out, and TLC detection reaction is complete. Suction filtration, washing the filter cake with ethyl acetate and drying to obtain intermediate 4.
(4) Intermediate 2 or 4 is dissolved in DMF, different methyl bromocarboxylate, K 2CO3 and stirred at room temperature for 5 hours, the reaction is completed, ethyl acetate is used for extraction, the organic phases are combined, anhydrous Na 2SO4 is dried, filtered, the solvent is distilled off under reduced pressure, and the intermediate 5 is obtained by silica gel column chromatography separation.
(5) The intermediate 5, 4-phenoxyphenylboronic acid (8 d), the tetraphenylphosphine palladium and the potassium phosphate trihydrate are placed in a microwave tube, 1, 4-dioxane and water (4:1) are added for dissolution, and the microwave reaction is carried out for 15min at 120 ℃. TLC detection of the completion of the basic reaction, extraction of the reaction solution with ethyl acetate, mixing of the organic phases, drying over anhydrous Na 2SO4, filtration, evaporation of the solvent under reduced pressure, column chromatography on silica gel, and isolation of intermediate 9a.
(6) Intermediate 9a is taken, absolute ethyl alcohol is dissolved, 3M NaOH is added, stirring is carried out for 4h at normal temperature, and TLC detection reaction is complete. Ethanol is distilled off under reduced pressure, the pH is adjusted to 5-6 by using 1M HCl, solid is precipitated, suction filtration is carried out, a filter cake is reserved, and the intermediate 10a is obtained after drying.
(7) Dissolving intermediate 10a, HATU and DIEPA in DMF, clarifying solution, and stirring under ice bath condition for 20-30min. After complete activation by TLC, o-phenylenediamine is added, N 2 is added for protection, the reaction is carried out in a dark place, and the mixture is stirred at normal temperature overnight. The reaction was essentially complete as detected by TLC. Pouring the reaction solution into ice water, precipitating solid, and suction filtering. The filtrate was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous Na 2SO4, and filtered. Separating by silica gel column chromatography, evaporating solvent under reduced pressure, and drying to obtain target compounds Z1-Z15.
And, further, when the compound of the present invention conforms to the general structure of formula III, the present invention provides a process for preparing the compound of formula III, which comprises following the reaction scheme:
Reagents and conditions in the synthetic route (a) NBS, DMF,85 ℃,4h; (b) N-Boc-3-hydroxypiperidine/N-Boc-4-hydroxypiperidine/1-Boc-3-hydroxypyrrolidine, diisopropyl azodicarboxylate, triphenylphosphine, anhydrous tetrahydrofuran, at 0deg.C for 5min; (c) tetrahydrofuran, hydrochloric acid = 4:1, r.t.,5h; (d) Different methyl bromocarboxylates, K 2CO3, DMF, r.t.,5h; (e) 4-bromobenzoic acid, HBTU, et 3 N, DMF, r.t.,12h; (f) 4-bromobenzoic acid, phosphorus oxychloride, pyridine, 0deg.C, 5min; (g) Pinacol biborate, potassium acetate, tetrakis (triphenylphosphine) palladium, 1, 4-dioxane, N 2, microwaves at 120 ℃ for 15min; (h) Tetrakis (triphenylphosphine) palladium, potassium phosphate trihydrate, 1, 4-dioxane: water=4:1, microwaves, 120 ℃,15min; (i) 3m noh, etoh, r.t.,4h; (j) O-phenylenediamine, HATU, DIEPA, protected from light, 0 ℃ to r.t.,8h.
Specifically, the method 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, detecting that the basic reaction is complete by TLC, naturally cooling the reaction liquid to room temperature, pouring the reaction liquid 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) Intermediate 2, N-Boc-3-hydroxy piperidine or N-Boc-4-hydroxy piperidine or 1-Boc-3-hydroxy pyrrolidine and triphenylphosphine are dissolved in anhydrous Tetrahydrofuran (THF), cooled in ice bath, and diisopropyl azodicarboxylate (DIAD) is slowly added dropwise, the ice bath is stirred for 5min, and the solution becomes clear from turbidity. TLC detection reaction is complete, ethyl acetate is added for extraction, the organic phases are combined, saturated saline solution is added for washing, anhydrous Na 2SO4 is used for drying, filtration, reduced pressure evaporation is carried out to remove the solvent, and silica gel column chromatography is carried out for separation, thus obtaining an intermediate 3.
(3) Intermediate 3a-3c was dissolved in THF, concentrated hydrochloric acid was added dropwise, reacted for 5h at room temperature, white solid precipitated, and TLC detection was complete. Suction filtration, washing the filter cake with ethyl acetate and drying to obtain intermediate 4.
(4) Intermediate 2 or 4 is dissolved in DMF, different methyl bromocarboxylate, K 2CO3 and stirred at room temperature for 5 hours, the reaction is completed, ethyl acetate is used for extraction, the organic phases are combined, anhydrous Na 2SO4 is dried, filtered, the solvent is distilled off under reduced pressure, and the intermediate 5 is obtained by silica gel column chromatography separation.
(5) Dissolving 4-bromobenzoic acid in DMF, adding HBTU and triethylamine, activating at room temperature for half an hour, then adding 2-aminopyridine compound (6 a-6 b), reacting overnight at room temperature, detecting the basic reaction by TLC, pouring the reaction solution into ice water, stirring, separating out light yellow solid, suction filtering, washing a filter cake with water, and drying to obtain intermediate 7a-7b; placing the 2-aminopyridine compound (6 c), 4-bromobenzoic acid and phosphorus oxychloride into a 100mL eggplant-shaped bottle, adding pyridine for dissolution under ice bath stirring, continuing stirring for 5min, detecting the basic reaction by TLC, pouring the reaction solution into ice water, stirring, precipitating a pale yellow solid, carrying out suction filtration, washing a filter cake with water, and drying to obtain an intermediate 7c.
(6) Placing the intermediates 7a-7c, pinacol ester, potassium acetate and tetraphenylphosphine palladium in a 100ml eggplant-shaped bottle, adding 1, 4-dioxane for dissolution, carrying out microwave reaction at 120 ℃ for 15min, detecting that the basic reaction is complete by TLC, adding ethyl acetate into the reaction liquid for extraction, combining organic phases, adding saline solution for washing, drying with anhydrous Na 2SO4, filtering, evaporating the solvent under reduced pressure, and separating by silica gel column chromatography to obtain the intermediates 8a-8c.
(7) The intermediate 5, the intermediates 8a-8c, the tetraphenylphosphine palladium and the potassium phosphate trihydrate are placed in a microwave tube, 1, 4-dioxane and water (4:1) are added for dissolution, and the microwave reaction is carried out for 15min at 120 ℃. TLC detection of the completion of the basic reaction, extraction of the reaction solution with ethyl acetate, mixing of the organic phases, drying over anhydrous Na 2SO4, filtration, evaporation of the solvent under reduced pressure, column chromatography on silica gel, and isolation of intermediate 9b.
(8) Intermediate 9b is taken, absolute ethyl alcohol is dissolved, 3M NaOH is added, stirring is carried out for 4 hours at normal temperature, and TLC detection reaction is complete. Ethanol is distilled off under reduced pressure, the pH is adjusted to 5-6 by using 1M HCl, solid is precipitated, suction filtration is carried out, a filter cake is reserved, and the intermediate 10b is obtained after drying.
(9) Dissolving intermediate 10b, HATU, DIEPA, DMF, clarifying, and stirring under ice bath condition for 20-30min. After complete activation by TLC, o-phenylenediamine is added, N 2 is added for protection, the reaction is carried out in a dark place, and the mixture is stirred at normal temperature overnight. The reaction was essentially complete as detected by TLC. Pouring the reaction solution into ice water, precipitating solid, and suction filtering. The filtrate was extracted with ethyl acetate, the organic phases were combined, dried over anhydrous Na 2SO4, and filtered. Separating by silica gel column chromatography, evaporating solvent under reduced pressure, and drying to obtain target compound Z16-Z33.
The following examples refer to the methods described above for specific preparation of compounds and characterization of the related compounds:
Example 1 preparation of intermediate 2
4-Aminopyrazolo [3,4-d ] pyrimidine (1, 5.0g,0.037 mol) and NBS (7.9 g,0.0444 mol) were placed in a 250mL eggplant-shaped bottle, dissolved in 25mL DMF, the solution was cloudy, heated at 85℃and refluxed with an oil bath. The reaction was dissolved to a brown solution and after 4h the reaction was monitored by TLC (PE: ea=1:2) to completion. After the completion, cooling to room temperature, pouring into 100mL of ice water, precipitating a large amount of yellow solid, carrying out suction filtration, washing a filter cake (30 mL multiplied by 3), and drying to obtain an intermediate 2, wherein the yellow solid is weighed 7.496g, and the yield is 94.59%; 1H NMR(400MHz,DMSO-d6 ) Delta 13.73 (s, 1H), 8.15 (s, 1H), 7.76 (s, 1H), 6.82 (s, 1H).
Example 2 preparation of intermediate 3
3-Bromo-1H-pyrazolo [3,4-d ] pyrimidin-4-amine (9.34 mmol) and triphenylphosphine (PPh 3, 28.03 mol) were taken in a 100mL eggplant-shaped bottle, N-Boc-3-hydroxypiperidine, N-Boc-4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine (14.02 mol) were added respectively, anhydrous tetrahydrofuran (THF, 20 mL) was added and stirred for dissolution, the ice bath was cooled, diisopropyl azodicarboxylate (DIAD, 28.03 mol) was slowly added dropwise, the ice bath was stirred for 5min, and the solution was clear from cloudy. TLC detection of the reaction was complete, extraction was performed by adding ethyl acetate (30 mL. Times.3), the organic phases were combined, washing was performed by adding saturated brine (20 mL), drying over anhydrous sodium sulfate, filtration, evaporation of the solvent under reduced pressure, column chromatography of ethyl acetate on silica gel: petroleum ether=4:1, to give intermediate 3.
Example 3 preparation of intermediate 4
Intermediate 3 was taken in a 250mL dry eggplant-shaped bottle, dissolved in an appropriate amount of tetrahydrofuran, concentrated hydrochloric acid was added to V (THF): V (concentrated HCl) =4:1, and the solution was clarified. The reaction was carried out at room temperature for 5h, a white solid precipitated, and the reaction was monitored by TLC (PE: EA=1:1) and was complete. Suction filtration, ethyl acetate washing of the filter cake (20 mL. Times.0, drying, weighing) gave intermediate 4.
Example 4 preparation of intermediate 5
Intermediate 2 (5.99 mmol) was taken, methyl bromomethylbenzoate/methyl 4-bromobutyrate/methyl 5-bromovalerate/methyl 6-bromohexanoate (7.19 mmol), or intermediate 4, methyl 4-bromobutyrate/methyl 5-bromovalerate/methyl 6-bromohexanoate (7.19 mmol) was added, respectively, and K 2CO3 powder (14.99 mmol) was added to a 100mL eggplant-shaped bottle, DMF (20 mL) was added, stirred and dissolved, and reacted at room temperature for 5h. TLC detection was complete, the reaction was poured into cold water (50 mL), extracted with ethyl acetate (30 ml×3), the organic phases combined, washed with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and silica gel column chromatography CH 2Cl2:CH3 oh=120:1 to afford intermediate 5.
Example 5 preparation of intermediate 7
1. Preparation of intermediate 7a
Parabromobenzoic acid (4.70 g,23.38 mmol) was taken in a 250mL eggplant bottle, DMF (30 mL) was added for dissolution, HBTU (8.86 g,23.38 mmol) and triethylamine (6.45 g,63.75 mmol) were added, stirred at room temperature for half an hour, then 2-aminopyridine (6 a,2.00g,21.25 mmol) was added and reacted overnight at room temperature. TLC detection is basically complete, the reaction solution is poured into ice water (250 mL), and is stirred, light yellow solid is separated out, suction filtration, water washing and filter cake drying are carried out, thus obtaining an intermediate 7a; white solid 4.12g, yield 70%.
2. Preparation of intermediate 7b
Parabromobenzoic acid (4.06 g,20.35 mmol), triethylamine (5.61 g,50.48 mmol) and HBTU (7.77 g,20.35 mmol) were added sequentially to a 250mL eggplant-shaped bottle, 25mL DMF was added for dissolution, and the ice bath stirred for 20min, orange clear solution. The compound 2-amino-4-methylpyridine (6 b,2g,18.50 mmol) was added and reacted overnight at room temperature, and the reaction was complete by TLC. Poured into 200mL of ice water, solid precipitated, filtered off with suction, and the filter cake (10 mL) washed with water. The filtrate was extracted with ethyl acetate (30 mL. Times.3), the organic phases were combined, dried over anhydrous Na 2SO4, filtered and the solvent was distilled off. Silica gel column chromatography (ethyl acetate: petroleum ether=50:1), drying, obtaining white solid 7b, weighing 2.445g, yield 45.54％;1H NMR(400MHz,DMSO-d6)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 (6 c,200mg,1.23 mmol) and p-bromobenzoic acid (299 mg,1.48 mmol) were sequentially added to a 100mL eggplant-shaped bottle, and 3mL pyridine was added to dissolve, and the solution was clarified. POCl 3 (378 mg,2.46 mmol) was slowly added dropwise under ice bath conditions, the reaction exothermed to give a large amount of white smoke, stirred for 5min under ice bath conditions, and TLC detection was complete. Pouring into 30mL ice water, precipitating solid, suction filtering, washing filter cake (5 mL), drying to obtain white solid 7c, weighing 286mg, yield 67.29％;1H NMR(400MHz,DMSO-d6)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 6 preparation of intermediate 8
Intermediate 7a-7c (14.55 mol), pinacol biborate (18.94 mmol), potassium acetate (KOAc, 43.71 mmol) and tetrakis triphenylphosphine palladium (Pd (PPh 3)4, 0.58 mol) were weighed into a 100mL eggplant-shaped bottle, 1, 4-dioxane (40 mL) was added to dissolve, the reaction was subjected to microwave reaction at 120℃for 15min. TLC to detect the completion of the reaction, the reaction solution was extracted with ethyl acetate (30 mL. Times.3), the organic phases were combined, washed with saturated brine (20 mL), dried over anhydrous Na 2SO4, filtered, the solvent was distilled off under reduced pressure, and silica gel column chromatography EA: PE=50:1 to obtain intermediate 8a-8c.
Example 7 preparation of intermediate 9
1. Preparation of intermediate 9a
Intermediate 5 (729.39. Mu. Mol), intermediate 8d (1.46 mmol), tetrakis triphenylphosphine palladium (Pd (PPh 3)4, 36.47. Mu. Mol) and potassium phosphate trihydrate (K 3PO4·3H2 O,1.46 mmol) were taken in a 35mL microwave tube, 1, 4-dioxane and water (10 mL, 4:1) were added to dissolve, the reaction was carried out for 15min. TLC under 120℃to detect completion of the basic reaction, the reaction solution was extracted with ethyl acetate (30 mL. Times.3), the organic phases were combined, washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and chromatography on silica gel column CH 2Cl2:CH3 OH=100:1 was carried out to obtain intermediate 9a.
2. Preparation of intermediate 9b
Intermediate 5 (729.39. Mu. Mol), intermediates 8a-8c (1.46 mmol), tetrakis triphenylphosphine palladium (Pd (PPh 3)4, 36.47. Mu. Mol) and potassium phosphate trihydrate (K 3PO4·3H2 O,1.46 mmol) were taken in a 35mL microwave tube, 1, 4-dioxane and water (10 mL, 4:1) were added to dissolve, the reaction was carried out for 15min at 120℃with microwave, the reaction solution was extracted with ethyl acetate (30 mL. Times.3), the organic phases were combined, washed with brine (20 mL), dried over anhydrous sodium sulfate, filtered, the solvent was distilled off under reduced pressure, and chromatography on silica gel column CH 2Cl2:CH3 OH=100:1 to afford intermediate 9b.
Example 8 preparation of intermediate 10
Dissolving intermediate 9 in proper amount of absolute ethyl alcohol, adding 3M NaOH (0.5-2 mL), stirring at normal temperature for 4h, and detecting the reaction completely by TLC. Ethanol was distilled off under reduced pressure, the pH was adjusted to 5-6 with 1M HCl, solids were precipitated, and suction filtration was performed, and a small amount of 1M HCl was added dropwise to the filtrate until no more precipitate was produced. The filter cake was retained and dried to afford intermediate 10.
EXAMPLE 9 preparation of target Compounds Z1-Z33
Taking intermediate 10 (1.0 eq), HATU (1.1 eq) and DIEPA (3.0 eq), sequentially adding into a 100mL eggplant-shaped bottle, adding 5-10 mL LDMF for dissolution, clarifying the solution, and stirring for 20-30min under ice bath conditions. After complete activation by TLC, o-phenylenediamine (1.1 eq) was added, protected by N 2, reacted in the dark and stirred overnight at ambient temperature. The reaction was essentially complete by TLC, and the solution was yellow. The reaction solution was poured into 100mL of ice water, and a solid was precipitated and filtered off with suction. The filtrate was extracted with ethyl acetate (30 ml×3), the organic phases were combined, dried over anhydrous Na 2SO4, and filtered. Separating and purifying by silica gel column chromatography (dichloromethane: methanol=20:1), evaporating solvent under reduced pressure, and drying to obtain target compounds Z1-Z33.
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℃;1H NMR(400MHz,CDCl3)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).13C NMR(100MHz,CDCl3)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 C29H28N8O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C30H30N8O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C32H32N8O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C32H34N8O2[M+H]+563.2878,found 563.2872.
3- (4-Amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N- (2-aminophenyl) propanamide (Z5)
White solid, yield 36％,Mp:181-183℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C26H23N7O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C27H25N7O2[M+H]+480.2142,found 480.2143.
5- (4-Amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) -N- (2-aminophenyl) pentanoamide (Z7)
White solid, yield 43％,Mp:152-154℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C28H27N7O2[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℃;1H 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).13C NMR(100MHz,DMSO-d6)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 C29H29N7O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C33H36N8O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C34H38N8O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C32H34N8O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C33H36N8O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C34H38N8O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C32H34N8O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C33H36N8O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C31H25N9O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C32H27N9O2[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)
The white solid was 97.1% pure in 55％,Mp:238-239℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C32H24F3N9O2[M+H]+624.2078,found 624.207.% yield, 3.178min retention time, 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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C30H30N10O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C31H32N10O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C31H32N10O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C31H29F3N10O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C27H25N9O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C27H22F3N9O2[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℃;1H 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);13C NMR(100MHz,DMSO-d6)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 C26H23N7O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C28H24F3N9O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C29H29N9O2[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℃;1H NMR(400MHz,DMSO-d6)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);13C NMR(100MHz,DMSO-d6)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 C29H26F3N9O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C30H31N9O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C30H28F3N9O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C33H36N10O2[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, yield 43％,Mp:124-126℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C33H33F3N10O2[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℃;1H NMR(400MHz,DMSO-d6)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).13C NMR(100MHz,DMSO-d6)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 C34H35F3N10O2[M+H]+673.2969,found 673.2968.
Experimental example determination of inhibitory Activity of Compounds against BTK and HDAC and determination of growth inhibitory Activity against mantle cell lymphoma cells
1. Compound inhibition activity assay for BTK kinase:
Experimental materials: BTK, peptide FAM-P2, ATP, DMSO, 96/384-WELL PLATE, staurosporine 1x kinase buffer (50mM HEPES,pH 7.5,0.0015%Brij-35), stop solution (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 is prepared into stock solution for later use. 10. Mu.L of stock solution and 90. Mu.L of 1 Xkinase buffer were added to a 96-well plate and mixed on a shaker for 10 minutes while setting the compound-free and enzyme-free control. After shaking, 5. Mu.l of the 96-well intermediate plate mixture was transferred to 384-well plates. Kinase was added to the 1x kinase buffer to prepare a 2.5x enzyme solution, and FAM-labeled peptide and ATP were added to prepare a 2.5x peptide solution. Subsequently 10 μl of 2.5x enzyme solution was added to each well of 384 well plates and incubated for 10 minutes at room temperature. Then 10. Mu.L of 2.5 Xpeptide solution was added to each well of 384-well assay plates, incubated at 28℃and 25. Mu.L of stop solution was added. Experimental results were determined using Caliper software.
Convertion group: measurement results of target compound.
Max group: DMSO positive control assay. All experimental constituents except the tested compounds were included and the amount of test compound was made up using buffer.
Min group-blank control group measurement results, including all experimental components except the compound and enzyme to be measured, were supplemented with buffer in amounts of the compound and enzyme to be measured.
TABLE 1 inhibitory Activity of Compounds against BTK kinase
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IBN: ibrutinib (Ibrutinib)
As can be seen from table 1, the compounds of the present invention exhibited different degrees of inhibition on BTK, and most of the compounds exhibited stronger BTK inhibitory activity, wherein the inhibition activity of the compounds Z1 to Z4, Z18, Z20, Z22 on BTK was comparable to IBN, especially the inhibition activity of the compound Z2 on BTK was twice that of IBN.
2. Inhibition activity of compounds against HDAC experiments:
Experimental materials: HDAC enzyme, boc-Lys (acetyl) -AMC (HeLa cell nucleus extracted fluorogenic substrate), tris-HCl, trypsin, EDTA, TSA, glycerol, naCl, 96-well flat-bottom fluorescent plate, MB100-2A microplate thermostatted shaker.
Buffer (HDAC buffer) formulation: 15mM Tris-HCl (pH 8.0), 250. Mu.M EDTA,250mM NaCl,10% glycerol.
HDAC enzymatic solution: according to 1:80 was diluted with HDAC buffer and stored at-80 ℃.
Preparation of a fluorogenic 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 prior to use, so that the DMSO content was less than 0.1%.
Preparing a stop solution: 10mg/ML TRYPSIN,50mM Tris-HCl (pH 8.0), 100mM NaCl, 2. Mu.M TSA. The preparation is ready to use, and the corresponding amounts of trypsin and TSA are added before use.
Preparation of test compound: the compound was diluted with DMSO to 50 times the highest concentration required to prepare stock solution for use. The HDAC buffer was diluted prior to the experiment so that the DMSO content was less than 0.1%.
The experimental method comprises the following steps: 50. Mu.L of the diluted test compound was added to a 96 Kong Yingguang plate, 100% and blank were set simultaneously, 10. Mu.L of HDACs enzyme solution was added to the wells, and incubation was performed at 37℃for 30min. To each well of the 96-well plate, 40. Mu.L of substrate was added, incubation was continued at 37℃and shaking was continued for 30min, and the substrate was activated. 100. Mu.L of the newly prepared stop solution was added to a 96-well plate, and incubated at 37℃for 20min. The HDAC inhibitory activity of the compound was calculated by measuring the fluorescence intensity at the emission wavelength/excitation wavelength (390 nm/460 nm) in the dark.
100% Control: all experimental components except the tested compounds were included, with 50 μl HDAC buffer replacing the tested compounds. Fluorescence intensity was measured at emission wavelength/excitation wavelength (390 nm/460 nm) wavelength.
Blank control group: comprises all experimental components except the tested compound and the HDAC enzyme solution, and 60 mu L of HDAC buffer replaces the tested compound and the HDAC enzyme solution. Fluorescence intensity was measured at emission wavelength/excitation wavelength (390 nm/460 nm) wavelength.
TABLE 2 inhibitory Activity of Compounds against 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, the inhibitory activity of n=5 (compound Z13) on HDACs also increases; in formula IIE, when n=5, i.e., compound Z15, the inhibitory activity on HDACs was strong (IC 50 value of 1.19). In formula III, compounds Z18 and Z29 have strong inhibition activity on HDAC, and are slightly weaker than the positive control MS-275 (an HDAC inhibitor) (IC 50 values of 1.51. Mu.M, 1.58. Mu.M, and 0.7. Mu.M, respectively). IBN has little inhibitory activity on HDACs.
3. Experiment of Compound growth inhibition Activity on mantle cell lymphoma cell
We performed MCL cell line antiproliferative experiments on compounds, and tested cell lines were Jeko-1, mino, maver-1 cells, wherein Jeko-1 and Mino are IBN-sensitive MCL cell lines, and Maver-1 is IBN primary drug resistant MCL cell line.
TABLE 3 inhibition of MCL cell growth by Compounds
/>
Na=not Active; IBN: ibrutinib (Ibrutinib)
Most of the compounds showed better antiproliferative effect on the tested cells, wherein the antiproliferative activity of the compound Z2 on Maver-1 cell lines was twice that of IBN, and the antiproliferative activity of the compounds Z10 and Z13 on Jeko-1 cell lines was 2-3 times that of IBN; compounds Z15 and Z18 having dual inhibitory effects on BTK and HDACs showed three and twenty times the growth inhibitory activity on Jeko-1 cell line and Maver-1 cell line, respectively, as compared to IBN.
The foregoing description is only a preferred embodiment of the present application, and the present application is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present application has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims (5)

1. Pyrazolopyrimidine-containing o-phenylenediamine derivative or a pharmaceutically acceptable salt or isomer thereof, wherein the derivative is selected from the group consisting of:
(Z2): 2- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) acetamide;
(Z10): 6- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) hexanamide;
(Z13): 6- (4- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) piperidin-1-yl) -N- (2-aminophenyl) hexanamide;
(Z15): 6- (3- (4-amino-3- (4-phenoxyphenyl) -1H-pyrazolo [3,4-d ] pyrimidin-1-yl) pyrrolidin-1-yl) -N- (2-aminophenyl) hexanamide;
(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.
2. A process for preparing pyrazolopyrimidine-containing o-phenylenediamine derivative according to claim 1 or a pharmaceutically acceptable salt thereof or isomer thereof, comprising:
(1) Dissolving 1H-pyrazolo [3,4-d ] pyrimidine-4-amine in DMF, adding NBS, oil bath at 85 ℃, heating and refluxing for reaction for 4 hours, detecting that the reaction is basically complete by TLC, naturally cooling the reaction liquid to room temperature, pouring the reaction liquid into ice water, stirring, precipitating a large amount of yellow solid, suction filtering, washing a filter cake with water, and drying to obtain an intermediate 2;
The structure of intermediate 2 is as follows:
；
(2) Dissolving an intermediate 2, N-Boc-3-hydroxypiperidine or N-Boc-4-hydroxypiperidine or 1-Boc-3-hydroxypyrrolidine and triphenylphosphine in anhydrous tetrahydrofuran, cooling in ice bath, slowly dropwise adding diisopropyl azodicarboxylate, stirring in ice bath for 5 min, and clarifying from turbidity; TLC detection reaction is complete, ethyl acetate is added for extraction, the organic phases are combined, saline solution is added for washing, anhydrous Na 2SO4 is used for drying, filtration, reduced pressure evaporation is carried out to remove the solvent, and silica gel column chromatography separation is carried out, thus obtaining an intermediate 3;
the structure of the intermediate 3 is as follows:
；Z= />、/>、/>；
(3) Dissolving the intermediate 3 in THF, dropwise adding concentrated hydrochloric acid, reacting at room temperature for 5h, precipitating white solid, and detecting the reaction completely by TLC; suction filtering, washing the filter cake with ethyl acetate, and drying to obtain an intermediate 4;
The structure of intermediate 4 is as follows:
；Z= />、/>、/>；
(4) Dissolving the intermediate 4 in DMF, adding different methyl bromocarboxylate, K 2CO3, stirring at room temperature for 5h, extracting with ethyl acetate after reaction, mixing organic phases, drying with anhydrous Na 2SO4, filtering, evaporating solvent under reduced pressure, and separating by silica gel column chromatography to obtain intermediate 5;
the structure of the intermediate 5 is as follows:
；n=1，5；Z=/>、/>、/>；
(5) Placing 5, 4-phenoxyphenylboronic acid, tetraphenylphosphine palladium and potassium phosphate trihydrate serving as intermediates into a microwave tube, adding 1, 4-dioxane and water in a ratio of 4:1 for dissolution, and carrying out microwave reaction at 120 ℃ for 15 min; TLC detection is carried out on the basic reaction completion, ethyl acetate is added into the reaction solution for extraction, the organic phases are combined, anhydrous Na 2SO4 is dried, the filtration is carried out, the solvent is distilled off under reduced pressure, and the silica gel column chromatography is carried out, thus obtaining an intermediate 9a;
The structure of the intermediate 9a is as follows:
；n=1，5；Z=/>、/>、/>；
(6) Dissolving intermediate 9a in absolute ethyl alcohol, adding 3M NaOH, stirring at normal temperature for 4 h, and detecting the reaction completely by TLC; evaporating ethanol under reduced pressure, adjusting pH to 5-6 with 1M HCl, precipitating solid, vacuum filtering, retaining filter cake, and drying to obtain intermediate 10a;
the structure of the intermediate 10a is as follows:
；n=1，5；Z=/>、/>、/>；
(7) Dissolving intermediate 10a, HATU and DIEPA in DMF, clarifying the solution, and stirring under ice bath condition for 20-30 min; after TLC detection and activation are completed, o-phenylenediamine is added, N 2 is added for protection, the reaction is carried out in a dark place, and stirring is carried out at normal temperature overnight; the TLC detection reaction is basically complete; pouring the reaction solution into ice water, precipitating solid, and suction filtering; extracting the filtrate with ethyl acetate, mixing the organic phases, drying with anhydrous Na 2SO4, and filtering; separating by silica gel column chromatography, evaporating solvent under reduced pressure, and drying to obtain target compounds (Z2), (Z10), (Z13) and (Z15);
Or (1) dissolving a compound 1, namely 1H-pyrazolo [3,4-d ] pyrimidine-4-amine, in DMF, adding NBS, carrying out oil bath at 85 ℃, carrying out heating reflux reaction for 4 hours, detecting that the reaction is basically complete by TLC, naturally cooling the reaction liquid to room temperature, pouring the reaction liquid into ice water, stirring, precipitating a large amount of yellow solid, carrying out suction filtration, washing a filter cake, and drying to obtain an intermediate 2;
The structure of intermediate 2 is as follows:
；
(2) Dissolving the intermediate 2 in DMF, adding different methyl bromocarboxylate, K 2CO3, stirring at room temperature for 5 h, extracting with ethyl acetate after reaction, mixing organic phases, drying with anhydrous Na 2SO4, filtering, evaporating solvent under reduced pressure, and separating by silica gel column chromatography to obtain intermediate 5;
the structure of the intermediate 5 is as follows:
；n = 0；Z =/>；
(3) Placing a 2-aminopyridine compound 6c, 4-bromobenzoic acid and phosphorus oxychloride in a 100mL eggplant-shaped bottle, adding pyridine for dissolution under ice bath stirring, continuing stirring for 5min, detecting that the basic reaction is complete by TLC, pouring the reaction solution into ice water, stirring, separating out a pale yellow solid, carrying out suction filtration, washing a filter cake with water, and drying to obtain an intermediate 7c;
the structure of 6c is as follows:
；R=-CF3；
the structure of the intermediate 7c is as follows:
；R= -CF3；
(4) Placing the intermediate 7C, the bisboronic acid pinacol ester, potassium acetate and the tetraphenylphosphine palladium in a 100 ml eggplant-shaped bottle, adding 1, 4-dioxane for dissolution, carrying out microwave reaction at 120 ℃ for 15 min, detecting that the reaction is basically complete by TLC, adding ethyl acetate into the reaction solution for extraction, merging organic phases, adding saline solution for washing, drying with anhydrous Na 2SO4, filtering, evaporating the solvent under reduced pressure, and separating by silica gel column chromatography to obtain an intermediate 8C;
the structure of intermediate 8c is as follows:
；R= -CF3；
(5) Placing the intermediate 5, the intermediate 8C, the tetraphenylphosphine palladium and the potassium phosphate trihydrate into a microwave tube, adding 1, 4-dioxane and water to dissolve in a ratio of 4:1, and carrying out a microwave reaction at 120 ℃ to obtain a product 15 min; TLC detection is carried out on the basic reaction completion, ethyl acetate is added into the reaction solution for extraction, the organic phases are combined, anhydrous Na 2SO4 is dried, the filtration is carried out, the solvent is distilled off under reduced pressure, and the silica gel column chromatography is carried out, thus obtaining an intermediate 9b;
The structure of the intermediate 9b is as follows:
；n = 0；R= -CF3；Z = />；
(6) Dissolving intermediate 9b in absolute ethyl alcohol, adding 3M NaOH, stirring at normal temperature for 4 hours, and detecting the reaction completely by TLC; evaporating ethanol under reduced pressure, regulating pH to 5-6 with 1M HCl, precipitating solid, vacuum filtering, retaining filter cake, and drying to obtain intermediate 10b;
the structure of the intermediate 10b is as follows:
；n = 0；R= -CF3；Z = />；
(7) Dissolving intermediate 10b, HATU, DIEPA, DMF, clarifying the solution, and stirring under ice bath condition for 20-30 min; after TLC detection and activation are completed, o-phenylenediamine is added, N 2 is added for protection, the reaction is carried out in a dark place, and stirring is carried out at normal temperature overnight; the TLC detection reaction is basically complete; pouring the reaction solution into ice water, precipitating solid, and suction filtering; extracting the filtrate with ethyl acetate, mixing the organic phases, drying with anhydrous Na 2SO4, and filtering; separating by silica gel column chromatography, evaporating solvent under reduced pressure, and drying to obtain target compound (Z18).
3. A pharmaceutical composition comprising the pyrazolopyrimidine-containing o-phenylenediamine derivative of claim 1 or a pharmaceutically acceptable salt thereof or isomer thereof.
4. A pharmaceutical formulation comprising the pyrazolopyrimidine-containing o-phenylenediamine derivative of claim 1 or a pharmaceutically acceptable salt or isomer thereof and at least one pharmaceutically acceptable adjuvant.
5. Use of a pyrazolopyrimidine-containing o-phenylenediamine derivative of claim 1 or a pharmaceutically acceptable salt or isomer thereof or a pharmaceutical composition of claim 3 or a pharmaceutical formulation of claim 4 for the preparation of an antitumor drug;
The tumor is mantle cell lymphoma.
CN202110686133.9A 2021-06-21 2021-06-21 Pyrazolopyrimidine-containing o-phenylenediamine derivative, and preparation method and application thereof Active CN115572297B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111171035A (en) * 2018-11-13 2020-05-19 山东大学 Preparation method and application of 4-phenoxyphenyl pyrazolopyrimidine amide derivative
CN111662296A (en) * 2020-06-02 2020-09-15 山东大学 Hydroxamic acid derivative containing pyrazolopyrimidine and preparation method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111171035A (en) * 2018-11-13 2020-05-19 山东大学 Preparation method and application of 4-phenoxyphenyl pyrazolopyrimidine amide derivative
CN111662296A (en) * 2020-06-02 2020-09-15 山东大学 Hydroxamic acid derivative containing pyrazolopyrimidine and preparation method and application thereof

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