Targeting DNA substitution 1h-pyrazolo[3,4-b]quinoline class hdac inhibitors and preparation method and application thereof

Abstract

The application discloses a DNA-targeted substitution 1H-pyrazolo[3,4-b]quinoline HDAC inhibitor and a preparation method and application thereof. The compound is a compound with a general formula (I) structure or a pharmaceutically acceptable salt. In the general formula (I), R is hydrogen, an alkyl group, halogen, a nitro group, an alkoxy group or a trifluoromethyl group; a linker is a substituted C1-C9 alkyl group, a C3-C8 cycloalkyl group, a C5-C10 aryl group, a single heterocyclic aryl group containing 5 or 6 ring atoms or a double heterocyclic aryl group having 8 to 15 ring atoms; wherein the heterocyclic aryl group contains 1-4 heteroatoms independently selected from O, S, N, oxidized S or oxidized N; a carbon atom or a nitrogen atom is a connecting point of a heteroaromatic ring structure and maintains a stable aromatic ring. The compound has strong inhibitory activity on HDAC and can effectively target DNA, and can prevent or treat related mammalian diseases caused by abnormal expression of HDAC and abnormal biological functions of DNA. The application also relates to a pharmaceutical use of a composition of the compound with the general formula I.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitor, its preparation method, and its application. Background Technology

[0002] Histone deacetylases (HDACs) and histone acetyltransferases (HATs) work synergistically to regulate the acetylation modification level of histones, representing a crucial regulatory mechanism in epigenetics. HDACs catalyze the deacetylation of histone lysine residues, restoring the positive charge of histones, increasing the attraction between DNA and histones, and tightening loose chromatin, thereby blocking the expression of tumor suppressor genes. HDACs are overexpressed in various tumor cells (such as colon cancer, liver cancer, breast cancer, lung cancer, and gastric cancer) and play a vital role in tumor cell cycle, differentiation, apoptosis, invasion, metastasis, and angiogenesis, making them an important target in current anti-tumor drug research. Due to the compensatory behavior of tumors, developing multi-target drugs based on HDACs has become an effective means to improve tumor treatment efficacy and reduce tumor drug resistance.

[0003] As a crucial biological macromolecule, deoxyribonucleic acid (DNA) plays a vital role in the storage of genetic information, protein synthesis, and the maintenance of life activities. In the DNA double helix structure, paired base pairs form two "grooves" with different geometric features on its surface: the deeper and wider "greater groove" is the main region for DNA-DNA and DNA-protein interactions; the narrower and shallower "smaller groove" is the main region for DNA-small molecule interactions. Most proteins interact with the greater groove through structural and functional complementarity, while a few proteins can induce conformational changes in DNA, exposing the smaller groove and thus binding. Small molecules that can specifically bind to DNA grooves (such as natural products and synthetic compounds) can competitively inhibit DNA-protein binding and regulate gene expression. Furthermore, small molecules can directly or allosterically prevent DNA-protein interactions by inserting between DNA base pairs, affecting the biological function of DNA. DNA-targeting anticancer drugs are among the first-line drugs in clinical treatment, and their combination with other drugs can improve the survival rate of cancer patients, attracting significant attention in drug development and clinical treatment.

[0004] As cancer treatment becomes more sophisticated, the limitations of single-target drugs are becoming increasingly apparent. While combination therapies offer significant efficacy, their complex dosage ratios and potential interactions between different drugs severely restrict their widespread application. Compared to combination therapies, multi-target drugs exhibit more predictable pharmacokinetic characteristics, higher patient compliance, and can achieve simultaneous distribution and synergistic effects in target tissues. Chemotherapy drugs are crucial for cancer treatment; however, the inherent DNA repair capabilities of cancer cells often increase patient mortality and render these drugs ineffective. Research indicates that HDAC activity is critical to the expression of genes involved in DNA synthesis and repair. Therefore, developing DNA-targeting HDAC inhibitors holds promise for overcoming the repair capabilities of cancer cells, offering new opportunities for improving cancer treatment. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitor, its preparation method and application.

[0006] To solve the above-mentioned technical problems, the basic concept of the technical solution adopted by the present invention is as follows:

[0007] I. DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitors

[0008] A DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitor is a compound having the general formula (I) or a pharmaceutically acceptable salt thereof;

[0009] ;

[0010] (I)

[0011] In general formula (I), R is hydrogen, alkyl, halogen, nitro, alkoxy, or trifluoromethyl; Linker is a substituted C1-C9 alkyl, C3-C8 cycloalkyl, C5-C10 aryl, monoheterocyclic aryl containing 5 or 6 ring atoms, or diheterocyclic aryl having 8 to 15 ring atoms.

[0012] The heterocyclic aryl group contains 1-4 heteroatoms, which are independently selected from O, S, N, oxidized S or oxidized N; the carbon atom or nitrogen atom is the connecting point of the heterocyclic aromatic ring structure, maintaining a stable aromatic ring.

[0013] Preferably, R is hydrogen, alkyl, halogen, nitro, or alkoxy.

[0014] Linker is a halogenated C1-C7 alkyl, C1-C7 alkyl, C3-C8 cycloalkyl, morpholine group substituted with 1-2 hydroxyl groups, halogen, nitro, cyano substituents or unsubstituted aromatic group Ar, aromatic group Ar or -NH-R1 linked to piperazine group;

[0015] Ar is a phenyl, naphthyl, pyridinyl, pyridazinyl, pyrazinyl, indene, quinazolinyl, purinyl, indoleyl, quinolinyl, pyrimidinyl, pyrroleyl, pyrazolyl, thiazolyl, benzo[b]thiazolyl, isoxazolyl, oxathiadiazolyl, isoxathiazolyl, tetrazolyl, imidazolyl, triazinyl, furanyl, benzofuranyl, and indoleyl group containing one substituent or without a substituent.

[0016] R1 is a C1-C7 alkyl group with 1-2 hydroxyl, halogen, nitro, or cyano substituents, or without substitution, and an aromatic group Ar connected to a C1-C3 alkylene group; the substituent is a hydroxyl, halogen, nitro, cyano, guanidinium, carboxyl, halogenated C1-C7 alkyl, C1-C7 alkoxy, C1-C7 alkyl, C3-C8 cycloalkyl, C5-C10 aryl, or a heteroaryl group containing 1-2 heteroatoms and having a ring number of 5-10.

[0017] Further preferably, the aforementioned DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitor is one of the following:

[0018] N-hydroxy-2-(1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-1);

[0019] N-hydroxy-4-(1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-2);

[0020] N-hydroxy-5-(1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-3);

[0021] N-hydroxy-6-(1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-4);

[0022] N-hydroxy-7-(1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-5);

[0023] N-hydroxy-8-(1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-6);

[0024] N-hydroxy-2-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-7);

[0025] N-hydroxy-4-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-8);

[0026] N-hydroxy-5-(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-9);

[0027] N-hydroxy-6-(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-10);

[0028] N-hydroxy-7-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-11);

[0029] N-hydroxy-8-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-12);

[0030] N-hydroxy-2-(6-bromo-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-13);

[0031] N-hydroxy-4-(6-bromo-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-14);

[0032] N-hydroxy-5-(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-15);

[0033] N-hydroxy-6-(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-16);

[0034] N-hydroxy-7-(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)heptanamide (HDNI-17);

[0035] N-hydroxy-8-(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)octamide (HDNI-18);

[0036] N-hydroxy-2-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-19);

[0037] N-hydroxy-4-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-20);

[0038] N-hydroxy-5-(6-methoxy-1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-21);

[0039] N-hydroxy-6-(6-methoxy-1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-22);

[0040] N-hydroxy-7-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-23);

[0041] N-hydroxy-8-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-24);

[0042] N-hydroxy-2-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-25);

[0043] N-hydroxy-4-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-26);

[0044] N-hydroxy-5-(6-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-27);

[0045] N-hydroxy-6-(6-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-28);

[0046] N-hydroxy-7-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-29);

[0047] N-hydroxy-8-(6-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)octamide (HDNI-30);

[0048] 4-[(1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]-N-hydroxybenzamide (HDNI-31);

[0049] (E)-3-[4-[(1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]-N-hydroxyacrylamide (HDNI-32);

[0050] 4-[(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]-N-hydroxy-benzamide (HDNI-33);

[0051] (E)-3-[4-[(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]-N-hydroxyacrylamide (HDNI-34);

[0052] 4-[(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]-N-hydroxybenzamide (HDNI-35);

[0053] (E)-3-[4-[(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]-N-hydroxyacrylamide (HDNI-36);

[0054] 4-[(6,7-dimethyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]-N-hydroxybenzamide (HDNI-37);

[0055] (E)-3-[4-[(6,7-dimethyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]-N-hydroxyacrylamide (HDNI-38)

[0056] The preferred compounds listed above are numbered in parentheses to correspond to the reaction routes below and the compound structures in Table 1.

[0057] The terms and definitions used in this article have the following meanings:

[0058] "Aryl" refers to an aromatic hydrocarbon containing a ring system, such as phenyl or naphthyl, which is optionally fused with a cycloalkyl group, preferably having 5-7 ring atoms, more preferably 5-6 ring atoms. Preferred aryl groups contain 5-15 carbon atoms.

[0059] "Heteroaryl" is an aromatic heterocyclic group, which can be monocyclic or bicyclic. They contain one or more, preferably 1-4, more preferably 1-3, and even more preferably 1-2 heteroatoms, independently selected from O, S, and N. Heteroaryl groups include oxidized S or N, such as sulfinyl, sulfonyl, and N oxides of tricyclic nitrogen. A carbon or nitrogen atom serves as a bonding point in the heteroaryl ring structure, thereby maintaining a stable aromatic ring. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyrazinyl, indene, benzothiophene, quinazolinyl, purinyl, indole, quinolinyl, pyrimidinyl, pyrroleyl, oxazolyl, thiazolyl, thiophene, isoxazolyl, oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazole, triazinyl, furanyl, benzofuranyl, and indoleyl.

[0060] "Arylalkyl" refers to an aryl group linked by a C1-C6 alkylene group.

[0061] "Heteroarylene alkyl" refers to a heteroarylene group linked by a C1-C6 alkylene group.

[0062] "Aryl alkenyl" refers to an aryl group linked by a C2-C6 alkenyl group.

[0063] "Heteroarylene" refers to a heteroarylene group linked by a C2-C6 alkenyl group.

[0064] "Alkyl" refers, alone or in combination, to a group derived from an alkane containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms (unless otherwise specified). It is a straight-chain alkyl or branched alkyl group, and includes straight-chain or branched alkyl groups containing a cycloalkyl moiety or interrupted by a cycloalkyl moiety. The straight-chain or branched alkyl groups are linked at any available point to produce a stable compound. Examples include, but are not limited to, 4-(isopropyl)-cyclohexylethyl or 2-methyl-cyclopropylpentyl. In many embodiments, the alkyl group is a straight-chain or branched alkyl group containing 1 to 15 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, 1 to 4 carbon atoms, or 1 to 2 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, and similar alkyl groups.

[0065] "Alkylene" is a divalent alkane-derived carbon group that is straight-chain or branched, in which two hydrogen atoms are removed from the same or different carbon atoms. Examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, and -CH2CH(CH3)-.

[0066] "Alkenyl" refers, alone or in combination, as used herein, to a straight-chain or branched hydrocarbon containing 2 to 6, preferably 2 to 4, carbon atoms and 1 to 2, preferably, a carbon-carbon double bond. Examples of alkenyl include, but are not limited to, vinyl, propenyl, isopropenyl, and butenyl.

[0067] "Cycloalkyl" is a substituted or unsubstituted, saturated or unsaturated cyclic group containing a carbon atom and / or one or more heteroatoms. The ring can be a monocyclic or fused ring, a bridged ring or a spirocyclic ring system. The number of ring atoms in each ring is 3 to 8, more preferably 3 to 6, such as cyclopropyl, cyclopentyl, cyclohexyl, adamantyl, and similar groups.

[0068] "Alkoxy" indicates the -O-alkyl group.

[0069] "Halogen" alone or in combination refers to all halogens, namely chlorine (Cl), fluorine (F), bromine (Br) or iodine (I).

[0070] "Pharmaceutically acceptable salt" refers to a salt form of a compound of general formula (I) that is both therapeutically effective and non-toxic. It can be an anionic salt formed by any acidic group (such as a carboxyl group) or a cationic salt formed by any basic group (such as an amino group). Many such salts are known in the art. These include cationic salts formed on any acidic group (such as a carboxyl group) or anionic salts formed on any basic group (such as an amino group). Many of these salts are known in the art, such as cationic salts including salts of alkali metals (such as sodium and potassium) and alkaline earth metals (such as magnesium and calcium), as well as organic salts (such as ammonium salts). Anionic salts can also be conveniently obtained by treating the basic form of I with appropriate acids, including inorganic acids such as sulfuric acid, nitric acid, and phosphoric acid; or organic acids such as acetic acid, propionic acid, glycolic acid, 2-hydroxypropionic acid, 2-oxopropionic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, 2-hydroxy-1,2,3-triamnic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, cyclohexylsulfinic acid, 2-hydroxybenzoic acid, and 4-amino-2-hydroxybenzoic acid. These salts are well known to those skilled in the art, who can prepare any salt provided by the knowledge in this field. Furthermore, those skilled in the art can choose one salt over another based on factors such as solubility, stability, and ease of preparation. The determination and optimization of these salts are within the experience of those skilled in the art.

[0071] As used herein, “stereoisomer” defines all possible stereoisomers of the compounds of the present invention or their physiological derivatives. Unless otherwise indicated, the chemical nomenclature of the compounds of the present invention includes mixtures of all possible stereochemical forms, comprising all diastereomers and enantiomers of the basic structural molecule, as well as a single isomer of the substantially pure compound of the present invention, i.e., containing less than 10%, preferably less than 5%, particularly less than 2%, and most preferably less than 1% of other isomers. All stereoisomers of the peptide-like compounds of the present invention are clearly included within the scope of the present invention.

[0072] Compounds of general formula (I) may also exist in other protected forms or derivatives, which are obvious to those skilled in the art and should be included within the scope of this invention.

[0073] The substituents described above can themselves be replaced by one or more substituents. Such substituents include those listed in C. Hansch and A. Leo, Substituent Constants for Correlation Analysis in Chemistry and Biology (1979). Preferred substituents include alkyl, alkenyl, alkoxy, hydroxy, oxy, nitro, amino, aminoalkyl (e.g., aminomethyl), cyano, halogen, carboxyl, carbonylalkoxy (e.g., carbonylethoxy), thio, aryl, cycloalkyl, heteroaryl, heterocycloalkyl (e.g., piperidinyl, morpholinyl, pyrroleyl), imino, hydroxyalkyl, aryloxy, arylalkyl, and combinations thereof.

[0074] "Pharmaceutical composition" refers to a preparation containing a therapeutically significant amount of an active pharmaceutical agent, prepared in a form suitable for administration to a patient. Therefore, the preparation does not contain any one or more components in amounts that a reasonably prudent medical practitioner would find unsuitable for administration to a general population. In many cases, such pharmaceutical compositions are sterile preparations.

[0075] Room temperature refers to the ambient temperature during experimental operations, which is controlled within the range of 10~30ºC.

[0076] II. Preparation method of DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitors

[0077] A method for preparing DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitors includes the following steps: using acetanilides 1a-1f with different substitutions as starting materials, firstly, under the action of phosphorus oxychloride and N,N-dimethylformamide (DMF), cyclization is carried out to generate substituted quinolines 2a-2f; then, under the catalysis of p-toluenesulfonic acid monohydrate, it reacts with hydrazine hydrate to generate substituted 1H-pyrazolo[3,4-b]quinolines 3a-3f; then, it undergoes a nucleophilic substitution reaction with methyl or ethyl bromocarboxylate of different chain lengths at the N-position of 1H-pyrazolo[3,4-b]quinoline to generate key intermediates 4a-4z and 4aa-4al; finally, it reacts with hydroxylamine hydrochloride to obtain the target compound HDNI-1-HDNI-38.

[0078] The synthesis route is as follows:

[0079] ;

[0080] The definitions of R and Linker are the same as those described in general formula (I) above;

[0081] Reagents and conditions: a) Phosphorus oxychloride, N,N-dimethylformamide (DMF), 79℃; b) Hydrazine hydrate, p-toluenesulfonic acid monohydrate, dimethyl sulfoxide (DMSO), 130℃; c) Methyl or ethyl bromocarboxylic acid, potassium carbonate, DMF, 80℃; d) Hydroxylamine hydrochloride, potassium hydroxide, methanol, room temperature.

[0082] The structural formulas of the target compounds in the synthetic route are shown in Table 1 below:

[0083] Table 1 Structural formulas of the target compounds

[0084] HDNI-1 -H <![CDATA[-CH2- <!-- 5 -->]]> HDNI-2 -H <![CDATA[-(CH2)3-]]> HDNI-3 -H <![CDATA[-(CH2)4-]]> HDNI-4 -H <![CDATA[-(CH2)5-]]> HDNI-5 -H <![CDATA[-(CH2)6-]]> HDNI-6 -H <![CDATA[-(CH2)7-]]> HDNI-7 <![CDATA[7-CH3]]> <![CDATA[-CH2-]]> HDNI-8 <![CDATA[7-CH3]]> <![CDATA[-(CH2)3-]]> HDNI-9 <![CDATA[7-CH3]]> <![CDATA[-(CH2)4-]]> HDNI-10 <![CDATA[7-CH3]]> <![CDATA[-(CH2)5-]]> HDNI-11 <![CDATA[7-CH3]]> <![CDATA[-(CH2)6-]]> HDNI-12 <![CDATA[7-CH3]]> <![CDATA[-(CH2)7-]]> HDNI-13 6-Br <![CDATA[-CH2-]]> HDNI-14 6-Br <![CDATA[-(CH2)3-]]> HDNI-15 6-Br <![CDATA[-(CH2)4-]]> HDNI-16 6-Br <![CDATA[-(CH2)5-]]> HDNI-17 6-Br <![CDATA[-(CH2)6-]]> HDNI-18 6-Br <![CDATA[-(CH2)7-]]> HDNI-19 <![CDATA[6-OCH3]]> <![CDATA[-CH2-]]> HDNI-20 <![CDATA[6-OCH3]]> <![CDATA[-(CH2)3-]]> HDNI-21 <![CDATA[6-OCH3]]> <![CDATA[-(CH2)4-]]> HDNI-22 <![CDATA[6-OCH3]]> <![CDATA[-(CH2)5-]]> HDNI-23 <![CDATA[6-OCH3]]> <![CDATA[-(CH2)6-]]> HDNI-24 <![CDATA[6-OCH3]]> <![CDATA[-(CH2)7-]]> HDNI-25 <![CDATA[6-CH3]]> <![CDATA[-CH2-]]> HDNI-26 <![CDATA[6-CH3]]> <![CDATA[-(CH2)3-]]> HDNI-27 <![CDATA[6-CH3]]> <![CDATA[-(CH2)4-]]> HDNI-28 <![CDATA[6-CH3]]> <![CDATA[-(CH2)5-]]> HDNI-29 <![CDATA[6-CH3]]> <![CDATA[-(CH2)6-]]> HDNI-30 <![CDATA[6-CH3]]> <![CDATA[-(CH2)7-]]> HDNI-31 -H <![CDATA[-CH2-Ph-]]> HDNI-32 -H <![CDATA[-CH2-Ph-CH=CH-]]> HDNI-33 <![CDATA[7-CH3]]> <![CDATA[-CH2-Ph-]]> HDNI-34 <![CDATA[7-CH3]]> <![CDATA[-CH2-Ph-CH=CH-]]> HDNI-35 6-Br <![CDATA[-CH2-Ph-]]> HDNI-36 6-Br <![CDATA[-CH2-Ph-CH=CH-]]> HDNI-37 <![CDATA[6,7-di-CH3]]> <![CDATA[-CH2-Ph-]]> HDNI-38 <![CDATA[6,7-di-CH3]]> <![CDATA[-CH2-Ph-CH=CH-]]>

[0085] The specific steps for operating the compound will be described in detail in the embodiments.

[0086] Those skilled in the art can modify the above steps to improve the yield. They can determine the synthetic route based on basic knowledge in the field, such as selecting reactants, solvents, and temperatures. Yields can be improved by using various conventional protecting groups to avoid side reactions. These conventional protection methods can be found, for example, in T. Greene's *Protecting Groups in Organic Synthesis*.

[0087] III. Application of DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitors

[0088] This invention also provides the use of these compounds in the preparation of drugs for the prevention or treatment of mammalian diseases caused by abnormal HDAC expression and DNA biological function. The mammalian diseases associated with abnormal HDAC expression and DNA biological function include cancer, neurodegenerative diseases, viral infections, inflammation, leukemia, malaria, and diabetes.

[0089] Furthermore, the present invention also includes a pharmaceutical composition suitable for oral administration to mammals, comprising any compound of general formula I above, and a pharmaceutically acceptable carrier, optionally comprising one or more pharmaceutically acceptable excipients.

[0090] Furthermore, the present invention also includes a pharmaceutical composition suitable for parenteral administration to mammals, comprising any compound of general formula I or general formula II described above, and a pharmaceutically acceptable carrier, optionally comprising one or more pharmaceutically acceptable excipients.

[0091] The inhibitory activity of the compound against HDAC was evaluated using fluorescence analysis. HDAC activates the fluorescent substrate (Ac-Leu-Gly-Lys(Ac)-AMC) by hydrolyzing the acetyl group on the lysine residue. The activated substrate is then hydrolyzed by trypsin, releasing a fluorophore that fluoresces at 390 / 460 nm. The HDAC inhibitory activity of the test compound can be calculated by measuring the fluorescence values ​​of the control and experimental groups.

[0092] The changes in UV absorption and fluorescence intensity of compounds with good HDAC activity before and after the addition of calf thymus DNA (CT-DNA) were measured using UV-Vis spectroscopy and fluorescence spectroscopy to determine the targeting binding of the test compounds to DNA.

[0093] The cellular activity of the compounds was tested using the CCK-8 assay. Tumor cell suspensions (human non-small cell lung cancer cell line A549 and human erythroleukemia cell line HEL) and human embryonic kidney cell line HEK293T were seeded into 96-well plates, and culture medium containing different concentrations of the compounds was added to each well. After incubation, CCK-8 solution was added, and after further incubation, the absorbance (OD) value of each well was measured at 450 nm using a microplate reader. The cell growth inhibition rate was calculated to determine the activity of the compounds.

[0094] In vitro enzyme inhibition experiments showed that most compounds exhibited good inhibitory activity against HDAC1, superior to the positive control drug SAHA, such as compounds HDNI-4, HDNI-5, HDNI-6, HDNI-9, HDNI-10, HDNI-11, HDNI-12, HDNI-16, HDNI-17, HDNI-18, HDNI-22, HDNI-23, HDNI-24, HDNI-27, HDNI-28, HDNI-29, HDNI-30, HDNI-31, HDNI-32, HDNI-33, HDNI-34, HDNI-35, HDNI-36, HDNI-37, and HDNI-38. In particular, compounds HDNI-17 and HDNI-23 showed inhibitory activity against HDAC1 that was more than 40 times greater than that of SAHA. Therefore, compounds HDNI-17 and HDNI-23, with better activity, were selected for further investigation into their targeted binding to DNA.

[0095] UV absorption spectroscopy showed that the UV absorption of CT-DNA at 260 nm was enhanced upon the addition of HDNI-17 and HDNI-23, respectively, but the UV absorption did not change significantly upon the addition of SAHA. Fluorescence spectroscopy showed that the fluorescence intensity decreased significantly when HDNI-17 and HDNI-23 were incubated with CT-DNA. However, SAHA did not cause any change. These results indicate that compounds HDNI-17 and HDNI-23 have significant DNA-targeting binding activity.

[0096] In in vitro cell proliferation assays, compounds HDNI-17 and HDNI-23 showed good inhibitory activity against A549 and HEL tumor cells, significantly superior to the positive control drug SAHA (approximately 10-fold). Furthermore, compared to tumor cells, these two compounds exhibited weaker inhibitory activity against normal HEK293T cells. In particular, compound HDNI-23 showed a selectivity of 101-1607 times against tumor cells, significantly superior to the positive control drug SAHA (6.7-87.7 times).

[0097] In summary, these substituted 1H-pyrazolo[3,4-b]quinoline compounds, especially HDNI-17 and HDNI-23, hold promise for the further development of more active DNA-targeting HDAC inhibitors, which is of great significance for the preparation of drugs to prevent or treat related mammalian diseases caused by abnormal HDAC expression and DNA biological function.

[0098] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art.

[0099] The compounds of this invention exhibit strong inhibitory activity against HDAC and can effectively target DNA, thus preventing or treating related mammalian diseases caused by abnormal HDAC expression and DNA biological function. This invention also relates to the pharmaceutical use of compositions having compound I.

[0100] The specific embodiments of the present invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0101] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention, but do not constitute an undue limitation of the invention. Obviously, the drawings described below are merely some embodiments, and those skilled in the art can obtain other drawings based on these drawings without creative effort. In the drawings:

[0102] Figure 1This is a synthetic route for DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitors.

[0103] Figure 2 These are the UV-Vis absorption spectra of compounds HDNI-17, HDNI-23, and SAHA, and their incubation with CT-DNA.

[0104] Figure 3 These are the fluorescence spectra of compounds HDNI-17, HDNI-23, and SAHA, and their incubation with CT-DNA.

[0105] It should be noted that these accompanying drawings and textual descriptions are not intended to limit the scope of the invention in any way, but rather to illustrate the concept of the invention to those skilled in the art by referring to specific embodiments. Detailed Implementation

[0106] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0107] Example 1. Synthesis of 2-chloro-3-quinoline carbaldehyde (2a)

[0108] Phosphorus oxychloride (6.53 mL, 70 mmol) was slowly added dropwise to DMF (2.32 mL, 30 mmol) under ice bath conditions. After stirring for 15 min, acetanilide (1.35 g, 10 mmol) was added, and the reaction flask was transferred to an oil bath at 79 °C. After stirring for 19 h, heating was stopped, and the reaction solution was cooled to room temperature. Subsequently, it was poured into ice water, and a yellow solid precipitated by stirring. After filtration, washing with distilled water, and drying, 1.51 g of a pale yellow solid was obtained, yield: 79%. This intermediate did not require purification and was used directly in the next step.

[0109] Synthesis of 1H-pyrazolo[3,4-b]quinoline (3a)

[0110] Hydrazine hydrate (1.1 mL, 18 mmol) was added to a DMSO solution of compound 2a (0.96 g, 5 mmol) and p-toluenesulfonic acid monohydrate (0.48 g, 2.5 mmol), and the mixture was transferred to an oil bath at 130 °C and reacted for 1.5 h. The reaction was monitored by TLC. After the reaction was completed, the reaction solution was cooled to room temperature. Subsequently, it was slowly added to ice water and stirred with a glass rod, resulting in the precipitation of a yellow solid. After standing for a period of time, the solid was filtered and dried to obtain 0.70 g of yellow solid, yield: 83%, mp: 211-213 °C. 1H NMR(400 MHz, DMSO-d6), δ 13.62 (s, 1H), 8.97 (s, 1H), 8.48 (s, 1H), 8.18 (d, J =8.0 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.83-7.79 (m, 1H), 7.53-7.49 (m, 1H).

[0111] Synthesis of methyl 2-(1H-pyrazolo[3,4-b]quinoline-1-yl)acetate (4a)

[0112] Methyl bromoacetate (0.2 mL, 2 mmol) was added to a DMF solution of compound 3a (0.17 g, 1 mmol) and potassium carbonate (1.38 g, 10 mmol), and the solution was transferred to an oil bath at 80 °C. After 1 h of reaction, the reaction was monitored by TLC. Once the reaction was complete, the reaction solution was cooled to room temperature, and 1 mol / L hydrochloric acid and distilled water were added sequentially. The mixture was stirred thoroughly, then extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. Purification was performed by silica gel column chromatography (petroleum ether / ethyl acetate) to obtain 0.17 g of a milky white solid, 70% yield, mp: 121–123 °C. 1 H NMR (400 MHz, CDCl3), δ 8.65 (s,1H), 8.31 (s, 1H), 8.11 (d, J = 8.8 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.78-7.74 (m, 1H), 7.49-7.45 (m, 1H), 5.44 (s, 2H), 3.77 (s, 3H).

[0113] Synthesis of N-hydroxy-2-(1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-1)

[0114] On one hand, hydroxylamine hydrochloride (4.67 g, 67 mmol) was dissolved in 24 mL of methanol under ice bath conditions to obtain solution A; on the other hand, KOH (5.6 g, 100 mmol) was added to 14 mL of methanol and stirred in an oil bath at 40 °C to dissolve it, obtaining solution B. Subsequently, solution B was slowly added dropwise to solution A under ice bath conditions. After the addition was complete, stirring was continued in an ice bath for 30 min. The mixture was then filtered to obtain a clear and transparent potassium hydroxylamine solution. The key intermediate 4a (0.24 g, 1 mmol) was dissolved in 8 mL of methanol, and then 10 mL of potassium hydroxylamine solution was added to react. After the reaction was complete, the pH was adjusted to 7 with 1 mol / L hydrochloric acid solution, and the mixture was filtered. The filter cake was recrystallized from methanol to give 0.18 g of a pale yellow solid, yield 74%, mp: 217-218 °C. 1 H NMR(400 MHz, DMSO-d6), δ 10.91 (s, 1H), 9.06 (s, 1H), 8.99 (s, 1H), 8.50 (s,1H), 8.19 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.84-7.81 (m, 1H),7.54-7.50 (m, 1H), 5.13 (s, 2H). 13 HRMS (ESI) m / z Calcd for C 12 H 11 N4O2 [M+H] + Found: 243.0882, Found: 243.0885.

[0115] Example 2. Synthesis of N-hydroxy-4-(1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-2)

[0116] The intermediate and target compound were prepared as described in Example 1. The product was a milky white solid, 76% yield, mp: 189-190℃. 1H NMR (400 MHz, DMSO-d6), δ 10.40 (s, 1H), 8.98 (s, 1H), 8.75 (s, 1H), 8.49(s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 8.8 Hz, 1H), 7.84 (t, J = 7.6Hz, 1H), 7.53-7.49 (m, 1H), 4.58 (t, J = 6.8 Hz, 2H), 2.20-2.13 (m, 2H), 2.06-2.00 (m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 168.45, 149.58, 147.55,133.08, 131.24, 130.74, 129.71, 127.75, 124.04, 123.48, 116.37, 45.91, 29.64,25.30. HRMS (ESI) m / z Calcd for C 14 H 15 N4O2 [M+H] + Found: 271.1195, Found: 271.1199.

[0117] Example 3. Synthesis of N-hydroxy-5-(1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-3)

[0118] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 77% yield, mp: 176-178℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.37 (s, 1H), 8.97 (s, 1H), 8.68 (s, 1H), 8.48(s, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 8.8 Hz, 1H), 7.83-7.80 (m,1H), 7.53-7.49 (m, 1H), 4.58-4.55 (m, 2H), 2.04 (t, J = 7.2 Hz, 2H), 1.94-1.87 (m, 2H), 1.55-1.48 (m, 2H). 13C NMR (100 MHz, DMSO-d6), δ 168.82, 149.56,147.55, 132.91, 131.17, 130.69, 129.69, 127.77, 124.00, 123.44, 116.29,45.97, 31.83, 28.75, 22.55. HRMS (ESI) m / z Calcd for C 15 H 17 N4O2 [M+H] + :285.1352, Found: 285.1355.

[0119] Example 4. Synthesis of N-hydroxy-6-(1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-4)

[0120] The intermediate and target compound were prepared as described in Example 1. The product was a milky white solid, 53% yield, mp: 136-137℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.33 (s, 1H), 8.97-8.96 (m,1H), 8.67 (s, 1H), 8.47 (d, J = 1.2 Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 8.8 Hz, 1H),7.83-7.79 (m, 1H), 7.52-7.49 (m, 1H), 4.56-4.53 (m, 2H), 1.96-1.89 (m, 4H),1.59-1.52 (m, 2H), 1.31-1.23 (m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 168.98,149.56, 147.55, 132.86, 131.16, 130.69, 129.69, 127.79, 124.00, 123.43,116.30, 46.12, 32.11, 28.82, 25.84, 24.74. HRMS (ESI) m / z Calcd for C 16 H 19 N4O2[M+H] + Found: 299.1508, Found: 299.1509.

[0121] Example 5. Synthesis of N-hydroxy-7-(1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-5)

[0122] The intermediate and target compound were prepared as in Example 1. A pale yellow solid, 41% yield, mp: 134-136℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.38 (s, 1H), 8.97 (s, 1H), 8.72 (s, 1H), 8.48(s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.84-7.80 (m,1H), 7.53-7.49 (m, 1H), 4.57-4.54 (m, 2H), 1.96-1.89 (m, 4H), 1.50-1.43 (m,2H), 1.34-1.29 (m, 4H). 13 C NMR (100 MHz, DMSO-d6), δ 169.09, 149.57, 147.56,132.85, 131.16, 130.70, 129.70, 127.79, 124.00, 123.44, 116.31, 46.16, 32.22,28.97, 28.17, 25.93, 25.05. HRMS (ESI) m / z Calcd for C 17 H 21 N4O2 [M+H] + 313.1665, Found: 313.1664.

[0123] Example 6. Synthesis of N-hydroxy-8-(1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-6)

[0124] The intermediate and target compound were prepared as described in Example 1. The product was a pale yellow solid, 20% yield, mp: 128-130℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.36 (s, 1H), 8.97 (s, 1H), 8.70 (s, 1H), 8.48(s, 1H), 8.18 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.84-7.80 (m,1H), 7.53-7.49 (m, 1H), 4.57-4.54 (m, 2H), 1.94-1.88 (m, 4H), 1.49-1.42 (m,2H), 1.31-1.15 (m, 6H). 13C NMR (100 MHz, DMSO-d6), δ 169.11, 149.57, 147.55,132.84, 131.15, 130.69, 129.70, 127.78, 123.99, 123.43, 116.30, 46.16, 32.25,29.04, 28.49, 28.29, 26.12, 25.08. HRMS (ESI) m / z Calcd for C 18 H 23 N4O2 [M+H] + :327.1821, Found: 327.1822.

[0125] Example 7. N-hydroxy-2-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-7) synthesis

[0126] The intermediate and target compound were prepared as in Example 1. White solid, 78% yield, mp: 216-218℃. 1 HNMR (400 MHz, DMSO-d6), δ 10.94 (s, 1H), 9.09 (s, 1H), 8.88 (s, 1H), 8.44 (s,1H), 8.06 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.36 (d, J = 8.8 Hz, 1H), 5.11(s, 2H), 2.55 (s, 3H). 13 C NMR (100 MHz, DMSO-d6), δ 163.94, 150.41, 147.86,140.93, 133.75, 130.79, 129.31, 126.22, 126.11, 122.43, 115.91, 47.06, 21.74.HRMS (ESI) m / z Calcd for C 13 H 13 N4O2 [M+H] + Found: 257.1039, Found: 257.1043.

[0127] Example 8. N-hydroxy-4-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-8) synthesis

[0128] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 54% yield, mp: 201-203℃. 1H NMR (400 MHz, DMSO-d6), δ 10.38 (s, 1H), 8.89 (s, 1H), 8.74 (s, 1H), 8.43(s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 4.56 (t, J = 6.8 Hz, 2H), 2.55 (s, 3H), 2.19-2.12 (m, 2H), 2.05-2.01 (m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 168.44, 149.72, 147.90, 140.85, 133.00, 130.79,129.29, 126.34, 126.03, 122.35, 115.82, 45.88, 29.66, 25.30, 21.72. HRMS(ESI) m / z Calcd for C 15 H 17 N4O2 [M+H] + Found: 285.1352, Found: 285.1353.

[0129] Example 9. N-hydroxy-5-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)pentanamide (HDNI-9) synthesis

[0130] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 37% yield, mp: 203-205℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.35 (s, 1H), 8.88 (s, 1H), 8.67 (s, 1H), 8.41(s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 4.55 (t, J = 6.8 Hz, 2H), 2.55 (s, 3H), 2.03 (t, J = 7.2 Hz, 2H), 1.93-1.85(m, 2H), 1.54-1.46 (m, 2H). 13C NMR (100 MHz, DMSO-d6), δ 168.82, 149.70,147.90, 140.80, 132.84, 130.73, 129.28, 126.36, 125.99, 122.31, 115.75,45.92, 31.84, 28.75, 22.56, 21.71. HRMS (ESI) m / z Calcd for C 16 H 19 N4O2 [M+H] + :299.1508, Found: 299.1508.

[0131] Example 10. N-hydroxy-6-(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-10) Synthesis

[0132] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 51% yield, mp: 173-175℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.31 (s, 1H), 8.88 (s, 1H), 8.66 (s, 1H), 8.41(s, 1H), 8.05 (d, J = 8.8 Hz, 1H), 7.83 (s, 1H), 7.35 (d, J = 7.6 Hz, 1H), 4.54-5.50 (m, 2H), 2.55 (s, 3H), 1.94-1.88 (m, 4H), 1.58-1.51 (m, 2H), 1.30-1.22 (m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 168.97, 149.69, 147.90, 140.78,132.77, 130.69, 129.27, 126.37, 125.98, 122.30, 115.75, 46.06, 32.10, 28.81,25.83, 24.73, 21.71. HRMS (ESI) m / z Calcd for C 17 H 21 N4O2 [M+H] + Found: 313.1665, Found: 313.1668.

[0133] Example 11. N-hydroxy-7-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-11) Synthesis

[0134] The intermediate and target compound were prepared as in Example 1. Yellow solid, yield 32%, mp: 147-149℃. 1 HNMR (400 MHz, DMSO-d6), δ 10.33 (s, 1H), 8.88 (s, 1H), 8.66 (s, 1H), 8.41 (s,1H), 8.05 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.35 (dd, J1 = 1.2 Hz, J2 = 8.4Hz, 1H), 4.54-4.50 (m, 2H), 2.55 (s, 3H), 1.94-1.89 (m, 4H), 1.49-1.42 (m,2H), 1.29-1.27 (m, 4H). 13 C NMR (100 MHz, DMSO-d6), δ 169.05, 149.70, 147.90,140.79, 132.76, 130.69, 129.27, 126.36, 125.97, 122.29, 115.75, 46.10, 32.20,28.93, 28.14, 25.91, 25.01, 21.71. HRMS (ESI) m / z Calcd for C 18 H 23 N4O2 [M+H] + :327.1821, Found: 327.1817.

[0135] Example 12. N-hydroxy-8-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-12) Synthesis

[0136] The intermediate and target compound were prepared as in Example 1. A pale yellow solid, 64% yield, mp: 169-171℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.32 (s, 1H), 8.88 (s, 1H), 8.66 (s, 1H), 8.41(s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.35 (d, J = 8.4 Hz, 1H), 4.54 (t, J = 6.8 Hz, 2H), 2.55 (s, 3H), 1.93-1.87 (m, 4H), 1.48-1.41 (m, 2H), 1.30-1.15 (m, 6H). 13C NMR (100 MHz, DMSO-d6), δ 169.09, 149.70, 147.90,140.78, 132.75, 130.68, 129.27, 126.35, 125.97, 122.29, 115.74, 46.10, 32.23,29.00, 28.47, 28.27, 26.11, 25.06, 21.70. HRMS (ESI) m / z Calcd for C 19 H 25 N4O2[M+H] + Found: 341.1978.

[0137] Example 13. N-hydroxy-2-(6-bromo-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-13) synthesis

[0138] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 94% yield, mp: 237-239℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.94 (s, 1H), 9.09 (s, 1H), 8.95 (s, 1H), 8.54(s, 1H), 8.47 (s, 1H), 7.97 (d, J = 9.2 Hz, 1H), 7.90-7.87 (m, 1H), 5.13 (s, 2H). 13 HRMS (ESI) m / z Calcdfor C 12 H 10 BrN4O2 [M+H] + : 320.9987, Found: 320.9991.

[0139] Example 14. N-hydroxy-4-(6-bromo-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-14) synthesis

[0140] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 83% yield, mp: 200-202℃. 1H NMR (400 MHz, DMSO-d6), δ 10.36 (s, 1H), 8.97-8.93 (m, 1H), 8.73 (d, J =2.4 Hz 1H), 8.53-8.50 (m, 1H), 8.48-8.42 (m, 1H), 8.01-7.95 (m, 1H), 7.90-7.84 (m, 1H), 4.56 (t, J = 6.8 Hz, 2H), 2.19-2.12 (m, 2H), 2.04-2.01 (m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 168.37, 149.64, 145.98, 133.55, 133.31, 131.26,130.65, 129.94, 125.11, 116.74, 115.81, 45.96, 29.57, 25.20. HRMS (ESI) m / zCalcd for C 14 H 14 BrN4O2 [M+H] + : 349.0300, Found: 349.0303.

[0141] Example 15. N-hydroxy-5-(6-bromo-1H-pyrazolo[3,4-b]quinoline-1-yl)pentanamide (HDNI-15) synthesis

[0142] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, yield 67%, mp: 199-201℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.36 (s, 1H), 8.96 (s, 1H), 8.67 (s, 1H), 8.52(s, 1H), 8.46 (d, J = 2.4 Hz, 1H), 8.01 (d, J = 9.2 Hz, 1H), 7.89-7.87 (m,1H), 4.57-4.53 (m, 2H), 2.03 (t, J = 7.2 Hz, 2H), 1.94-1.86 (m, 2H), 1.55-1.47 (m, 2H). 13C NMR (100 MHz, DMSO-d6), δ 168.79, 149.60, 145.98, 133.53,133.16, 131.26, 130.62, 129.95, 125.07, 116.66, 115.78, 46.04, 31.79, 28.68,22.51. HRMS (ESI) m / z Calcd for C 15 H 16 BrN4O2 [M+H] + : 363.0457, Found: 363.0461.

[0143] Example 16. N-hydroxy-6-(6-bromo-1H-pyrazolo[3,4-b]quinoline-1-yl)hexanoamide (HDNI-16) synthesis

[0144] The intermediate and target compound were prepared as in Example 1. Yellow solid, yield 34%, mp: 210-212℃. 1 HNMR (400 MHz, DMSO-d6), δ 10.31 (s, 1H), 8.96 (s, 1H), 8.66 (s, 1H), 8.51 (s,1H), 8.47 (d, J = 2.0 Hz, 1H), 8.01 (d, J = 9.2 Hz, 1H), 7.89 (dd, J1 = 2.0Hz, J2 = 9.2 Hz, 1H), 4.55-4.52 (m, 2H), 1.96-1.89 (m, 4H), 1.59-1.51 (m,2H), 1.30-1.23 (m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 168.95, 149.60, 145.97,133.51, 133.11, 131.25, 130.59, 129.97, 125.06, 116.67, 115.77, 46.18, 32.09,28.75, 25.80, 24.70. HRMS (ESI) m / z Calcd for C16H18BrN4O2 [M+H]+: 377.0613, Found: 377.0614.

[0145] Example 17. N-hydroxy-7-(6-bromo-1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-17) synthesis

[0146] The intermediate and target compound were prepared as in Example 1. Yellow solid, yield 87%, mp: 205-207 °C. 1H NMR (400 MHz, DMSO-d6), δ 10.33 (s, 1H), 8.96 (s, 1H), 8.66 (s, 1H), 8.51(s, 1H), 8.46 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 9.2 Hz, 1H), 7.89 (dd, J1 =2.0 Hz, J2 = 9.2 Hz, 1H), 4.55 (t, J = 6.8 Hz, 2H), 1.93 (t, J = 7.2 Hz, 2H), 1.49-1.42 (m, 2H), 1.33-1.25 (m, 4H). 13 C NMR (100 MHz, DMSO-d6), δ 169.03,149.60, 145.97, 133.51, 133.09, 131.25, 130.59, 129.96, 125.06, 116.67,115.76, 46.21, 32.18, 28.87, 28.11, 25.88, 24.99. HRMS (ESI) m / z Calcd forC 17 H 20 BrN4O2 [M+H] + : 391.0770, Found: 391.0771.

[0147] Example 18. N-hydroxy-8-(6-bromo-1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-18) synthesis

[0148] The intermediate and target compound were prepared as in Example 1. Yellow solid, 70% yield, mp: 184-186℃. 1 HNMR (400 MHz, DMSO-d6), δ 10.35 (s, 1H), 8.95 (s, 1H), 8.70 (s, 1H), 8.51 (s,1H), 8.46 (d, J = 2.0 Hz, 1H), 8.00 (d, J = 9.2 Hz, 1H), 7.89 (dd, J1 = 2.0Hz, J2 = 9.2 Hz, 1H), 4.55-4.52 (m, 2H), 1.94-1.87 (m, 4H), 1.49-1.41 (m,2H), 1.31-1.15 (m, 6H). 13C NMR (100 MHz, DMSO-d6), δ 169.06, 149.61, 145.97,133.51, 133.09, 131.25, 130.59, 129.94, 125.05, 116.66, 115.75, 46.22, 32.21,28.94, 28.45, 28.23, 26.07, 25.04. HRMS (ESI) m / z Calcd for C 18 H 22 BrN4O2 [M+H] + Found: 405.0926, Found: 405.0927.

[0149] Example 19. N-hydroxy-2-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-) Synthesis of 19)

[0150] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 52% yield, mp: 219-221℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.90 (s, 1H), 9.05 (s, 1H), 8.81 (s, 1H), 8.42 (s, 1H), 7.95 (d, J = 8.8 Hz, 1H), 7.52-7.48 (m, 2H), 5.10 (s, 2H), 3.92(s, 3H). 13 HRMS (ESI) m / z Calcdfor C 13 H 13 N4O3 [M+H] + Found: 273.0988, Found: 273.0992.

[0151] Example 20. N-hydroxy-4-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-) Synthesis of 20)

[0152] The intermediate and target compound were prepared as in Example 1. Yellow solid, yield 43%, mp: 181-183 °C. 1H NMR (400 MHz, DMSO-d6), δ 10.38 (s, 1H), 8.81 (d, J = 3.6 Hz, 1H), 8.40 (s,1H), 7.97 (d, J = 9.2 Hz, 1H), 7.50-7.47 (m, 2H), 4.55-4.52 (m, 2H), 3.92 (s,3H), 2.18-2.11 (m, 2H), 2.05-2.01 (m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ168.45, 154.94, 148.84, 144.02, 132.24, 129.14, 128.98, 124.81, 124.64,116.34, 105.74, 55.43, 45.89, 29.65, 25.32. HRMS (ESI) m / z Calcd for C 15 H 17 N4O3[M+H] + : 301.1301, Found: 301.1305.

[0153] Example 21. N-hydroxy-5-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)pentanamide (HDNI-) Synthesis of 21)

[0154] The intermediate and target compound were prepared as described in Example 1. Yellow solid, 51% yield, mp: 153-155 °C. ¹H NMR (400 MHz, DMSO-d6), δ 10.42 (s, 1H), 8.82 (s, 1H), 8.40 (s, 1H), 7.99 (d, J = 9.2 Hz, 1H), 7.51-7.47 (m, 2H), 4.55 (t, J = 6.8 Hz, 2H), 3.92 (s, 3H), 2.03-2.00 (m, 2H), 1.92-1.85 (m, 2H), 1.54-1.47 (m, 2H). 13 C NMR (100MHz, DMSO-d6), δ 168.85, 154.93, 148.70, 143.88, 132.16, 129.11, 129.04,124.76, 124.67, 116.34, 105.79, 55.45, 46.02, 31.83, 28.79, 22.57. HRMS (ESI)m / z Calcd for C 16 H 19N4O3 [M+H] + Found: 315.1457, Found: 315.1460.

[0155] Example 22. N-hydroxy-6-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)hexanoamide (HDNI-) Synthesis of 22)

[0156] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 30% yield, mp: 189-191℃. ¹H NMR (400 MHz, DMSO-d6), δ 10.32 (s, 1H), 8.80 (s, 1H), 8.39 (s, 1H), 7.98 (d, J = 9.2 Hz, 1H), 7.50-7.47 (m, 2H), 4.53-4.50 (m, 2H), 3.92 (s, 3H), 1.95-1.88 (m, 4H), 1.59-1.51 (m, 2H), 1.31-1.23 (m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 168.98, 154.91, 148.82, 144.02, 132.01, 129.18, 128.89, 124.75,124.59, 116.28, 105.72, 55.42, 46.11, 32.12, 28.86, 25.86, 24.75. HRMS (ESI)m / z Calcd for C 17 H 21 N4O3 [M+H] + Found: 329.1614, Found: 329.1616.

[0157] Example 23. N-hydroxy-7-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-) Synthesis of 23)

[0158] The intermediate and target compound were prepared as in Example 1. Yellow solid, yield 39%, mp: 163-165℃. 1 HNMR (400 MHz, DMSO-d6), δ 10.35 (s, 1H), 8.80 (s, 1H), 8.69 (s, 1H), 8.39 (s,1H), 7.98 (d, J = 8.8 Hz, 1H), 7.50-7.46 (m, 2H), 4.53- 4.50 (m, 2H), 3.92 (s, 3H), 1.94-1.87 (m, 4H), 1.49-1.42 (m, 2H), 1.31-1.25 (m, 4H).13 C NMR (100MHz, DMSO-d6), δ 169.06, 154.90, 148.83, 144.02, 131.99, 129.17, 128.88,124.74, 124.59, 116.28, 105.71, 55.42, 46.13, 32.20, 28.98, 28.15, 25.93,25.03. HRMS (ESI) m / z Calcd for C 18 H 23 N4O3 [M+H] + Found: 343.1770, Found: 343.1768.

[0159] Example 24. N-hydroxy-8-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-) Synthesis of 24)

[0160] The intermediate and target compound were prepared as in Example 1. Yellow solid, yield 64%, mp: 171-173℃. 1 HNMR (400 MHz, DMSO-d6), δ 10.32 (s, 1H), 8.80 (s, 1H), 8.39 (s, 1H), 7.97 (d,J = 9.2 Hz, 1H), 7.50-7.47 (m, 2H), 4.54 (t, J = 6.8 Hz, 2H), 3.92 (s, 3H), 1.93-1.87 (m, 4H), 1.48-1.41 (m, 2H), 1.30-1.15 (m, 6H). 13 C NMR (100 MHz, DMSO-d6), δ 169.09, 154.90, 148.83, 144.02, 131.98, 129.16, 128.88, 124.74,124.59, 116.26, 105.72, 55.42, 46.14, 32.23, 29.05, 28.48, 28.27, 26.11,25.06. HRMS (ESI) m / z Calcd for C 19 H 25 N4O3 [M+H] + : 357.1927, Found: 357.1927.

[0161] Example 25. N-hydroxy-2-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-25) Synthesis

[0162] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 80% yield, mp: 230-232℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.90 (s, 1H), 9.05 (s, 1H), 8.84 (s, 1H), 8.44(s, 1H), 7.94-7.91 (m, 2H), 7.67-7.64 (m, 1H), 5.10 (s, 2H), 2.52 (s, 3H). 13 CNMR (100 MHz, DMSO-d6), δ 163.97, 150.08, 146.24, 133.58, 133.33, 132.72,130.13, 127.84, 127.40, 124.18, 116.44, 47.09, 20.99. HRMS (ESI) m / z Calcdfor C 13 H 13 N4O2 [M+H] + Found: 257.1039, Found: 257.1043.

[0163] Example 26. N-hydroxy-4-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-26) Synthesis

[0164] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 48% yield, mp: 206-208℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.41 (s, 1H), 8.83 (s, 1H), 8.76 (s, 1H), 8.44(s, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.89 (s, 1H), 7.66 (dd, J1 = 2.0 Hz, J2 =8.8 Hz, 1H), 4.57-4.53 (m, 2H), 2.51 (s, 3H), 2.20-2.13 (m, 2H), 2.07-2.03(m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 168.45, 149.38, 146.30, 133.25, 132.80,132.59, 130.08, 127.76, 127.51, 124.08, 116.33, 45.88, 29.65, 25.30, 20.98.HRMS (ESI) m / z Calcd for C15 H 17 N4O2 [M+H] + Found: 285.1352, Found: 285.1355.

[0165] Example 27. N-hydroxy-5-(6-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-27) Synthesis

[0166] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 60% yield, mp: 190-192℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.36 (s, 1H), 8.83 (s, 1H), 8.67 (s, 1H), 8.42 (s, 1H), 7.97 (d, J = 8.8 Hz, 1H), 7.89 (s, 1H), 7.66-7.63 (m, 1H), 4.56-4.52 (m, 2H), 2.51 (s, 3H), 2.03-2.00 (m, 2H), 1.93-1.86 (m, 2H), 1.55-1.47 (m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 168.82, 149.36, 146.30, 133.22,132.64, 132.55, 130.04, 127.76, 127.54, 124.05, 116.26, 45.94, 31.83, 28.76,22.55, 20.98. HRMS (ESI) m / z Calcd for C 16 H 19 N4O2 [M+H] + : 299.1508, Found:299.1508.

[0167] Example 28. N-hydroxy-6-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)hexanoamide (HDNI-28) Synthesis

[0168] The intermediate and target compound were prepared as in Example 1. Yellow solid, 90% yield, mp: 184-186℃. 1HNMR (400 MHz, DMSO-d6), δ 10.31 (s, 1H), 8.83 (s, 1H), 8.66 (s, 1H), 8.42 (s,1H), 7.97 (d, J = 8.8 Hz, 1H), 7.89 (s, 1H), 7.66 (dd, J1 = 1.6 Hz, J2 = 8.8Hz, 1H), 4.54-4.50 (m, 2H), 2.51 (s, 3H), 1.94-1.88 (m, 4H), 1.59-1.51 (m,2H), 1.31-1.23 (m, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 168.96, 149.36, 146.29,133.20, 132.58, 132.54, 130.00, 127.74, 127.55, 124.04, 116.26, 46.08, 32.10,28.82, 25.84, 24.73, 20.98. HRMS (ESI) m / z Calcd for C 17 H 21 N4O2 [M+H] + 313.1665, Found: 313.1669.

[0169] Example 29. N-hydroxy-7-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-29) Synthesis

[0170] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 81% yield, mp: 180-182℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.33 (s, 1H), 8.83 (s, 1H), 8.66 (s, 1H), 8.42(s, 1H), 7.97 (d, J = 9.2 Hz, 1H), 7.89 (s, 1H), 7.66-7.63 (m, 1H), 4.54-4.51(m, 2H), 2.51 (s, 3H), 1.93-1.88 (m, 4H), 1.48-1.42 (m, 2H), 1.29-1.26 (m,4H). 13C NMR (100 MHz, DMSO-d6), δ 169.04, 149.37, 146.29, 133.20, 132.56,132.52, 129.99, 127.74, 127.54, 124.02, 116.26, 46.11, 32.19, 28.94, 28.13,25.91, 25.01, 20.97. HRMS (ESI) m / z Calcd for C 18 H 23 N4O2 [M+H] + Found: 327.1821, Found: 327.1823.

[0171] Example 30. N-hydroxy-8-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-30) Synthesis

[0172] The intermediate and target compound were prepared as in Example 1. White solid, yield 59%, mp: 169-171℃. 1 HNMR (400 MHz, DMSO-d6), δ 10.33 (s, 1H), 8.82 (s, 1H), 8.67 (s, 1H), 8.42 (s,1H), 7.97 (d, J = 9.2 Hz, 1H), 7.89 (s, 1H), 7.66-7.63 (m, 1H), 4.54 (t, J =6.8 Hz, 2H), 2.51 (s, 3H), 1.93-1.87 (m, 4H), 1.49-1.41 (m, 2H), 1.32-1.17(m, 6H). 13 C NMR (100 MHz, DMSO-d6), δ 169.08, 149.37, 146.29, 133.20, 132.56,132.52, 129.98, 127.74, 127.53, 124.02, 116.25, 46.12, 32.22, 29.02, 28.47,28.26, 26.11, 25.05, 20.97. HRMS (ESI) m / z Calcd for C 19 H 25 N4O2 [M+H] + Found: 341.1978, Found: 341.1982.

[0173] Example 31. 4-[(1H-pyrazolo[3,4-b]quinoline-1-yl)methyl]-N-hydroxybenzamide (HDNI-31) Synthesis

[0174] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 96% yield, mp: 187-189℃. 1 H NMR (400 MHz, DMSO-d6), δ 11.16 (s, 1H), 9.02 (s, 2H), 8.54 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.07 (d, J = 8.8 Hz, 1H), 7.85-7.81 (m, 1H), 7.70-7.67(m, 2H), 7.55-7.51 (m, 1H), 7.33 (d, J = 8.0 Hz, 2H), 5.84 (s, 2H). 13 C NMR(100 MHz, DMSO-d6), δ 163.95, 149.74, 147.70, 140.73, 133.90, 132.01, 131.56,130.94, 129.77, 127.75, 127.30, 127.17, 124.18, 123.67, 116.36, 49.45. HRMS(ESI) m / z Calcd for C 18 H 15 N4O2 [M+H] + Found: 319.1195, Found: 319.1196.

[0175] Example 32. (E)-3-[4-[(1H-pyrazolo[3,4-b]quinoline-1-yl)methyl]phenyl]-N-hydroxypropene Synthesis of amide (HDNI-32)

[0176] The intermediate and target compound were prepared as in Example 1. A pale yellow solid, 83% yield, mp: 145-146℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.74 (s, 1H), 9.02 (s, 2H), 8.53 (s, 1H), 8.20 (d, J = 10.0 Hz, 1H), 8.07 (d, J = 8.8 Hz, 1H), 7.85-7.81 (m, 1H), 7.54-7.49(m, 3H), 7.42 (d, J = 15.6 Hz, 1H), 7.30 (d, J = 8.4 Hz, 2H), 6.43 (d, J =15.6 Hz, 1H), 5.81 (s, 2H). 13C NMR (100 MHz, DMSO-d6), δ 162.58, 149.72,147.69, 138.95, 137.77, 134.05, 133.80, 131.49, 130.89, 129.74, 127.93,127.76, 127.69, 124.16, 123.64, 119.08, 116.35, 49.51. HRMS (ESI) m / z Calcdfor C 20 H 17 N4O2 [M+H] + Found: 345.1352, Found: 345.1353.

[0177] Example 33. 4-[(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]-N-hydroxybenzamide Synthesis of (HDNI-33)

[0178] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 96% yield, mp: 187-189℃. 1 H NMR (400 MHz, DMSO-d6), δ 11.17 (s, 1H), 9.03 (s, 1H), 8.94 (s, 1H), 8.48(s, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.38 (dd, J1 = 1.6 Hz, J2 = 8.4 Hz, 1H), 7.32 (d, J = 8.4 Hz, 2H), 5.81 (s,2H), 2.55 (s, 3H). 13 C NMR (100 MHz, DMSO-d6), δ 164.01, 149.89, 148.08,141.19, 140.83, 133.87, 132.01, 131.17, 129.40, 127.36, 127.20, 126.37,126.27, 122.52, 115.85, 49.48, 21.75. HRMS (ESI) m / z Calcd for C 19 H 17 N4O2 [M+H] + Found: 333.1352, Found: 333.1357.

[0179] Example 34. (E)-3-[4-[(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]-N-hydroxy Synthesis of hydroxyacrylamide (HDNI-34)

[0180] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 63% yield, mp: 147-149℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.72 (brs, 1H), 9.17 (brs, 1H), 8.93 (s, 1H), 8.47 (s, 1H), 8.08 (d, J = 8.8 Hz, 1H), 7.83 (s, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.42-7.35 (m, 2H), 7.30 (d, J = 7.6 Hz, 2H), 6.43 (d, J = 15.6 Hz, 1H), 5.78 (s, 2H), 2.55 (s, 3H). 13 C NMR (100 MHz, DMSO-d6), δ 162.58, 149.86,148.06, 141.11, 139.04, 137.83, 134.04, 133.75, 131.08, 129.37, 127.98,127.71, 126.37, 126.22, 122.49, 119.05, 115.83, 49.52, 21.73. HRMS (ESI) m / zCalcd for C 21 H 19 N4O2 [M+H] + Found: 359.1508.

[0181] Example 35. 4-[(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]-N-hydroxybenzamide Synthesis of (HDNI-35)

[0182] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 93% yield, mp: 216-217℃. 1 H NMR (400 MHz, DMSO-d6), δ 11.13 (brs, 1H), 9.01 (brs, 2H), 8.58 (s, 1H), 8.50 (d, J = 2.0 Hz, 1H), 8.01 (d, J = 9.2 Hz, 1H), 7.91 (dd, J1 = 2.0 Hz, J2= 9.2 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 5.83 (s,2H). 13C NMR (100 MHz, DMSO-d6), δ 163.93, 149.81, 146.16, 140.53, 134.16,133.79, 132.06, 131.36, 131.03, 129.96, 127.35, 127.21, 125.29, 116.76,116.05, 49.53. HRMS (ESI) m / z Calcd for C 18 H 14 BrN4O2 [M+H] + : 397.0300, Found:397.0301.

[0183] Example 36. (E)-3-[4-[(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]N-hydroxy Synthesis of Acrylamide (HDNI-36)

[0184] The intermediate and target compound were prepared as described in Example 1. A pale yellow solid, 75% yield, mp: 146-148℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.04 (brs, 2H), 9.01 (s, 1H), 8.58 (s, 1H), 8.50 (s, 1H), 8.01 (d, J = 9.2 Hz, 1H), 7.91 (dd, J1 = 2.4 Hz, J2 = 9.2 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 15.6 Hz, 1H), 7.29 (d, J = 8.0Hz, 2H), 6.43 (d, J = 15.6 Hz, 1H), 5.80 (s, 2H). 13 C NMR (100 MHz, DMSO-d6), δ 162.57, 149.77, 146.14, 138.64, 137.45, 134.20, 134.04, 133.74, 131.33,130.94, 129.95, 127.97, 127.68, 125.25, 119.37, 116.75, 116.00, 49.59. HRMS(ESI) m / z Calcd for C 20 H 16 BrN4O2 [M+H] + : 423.0457, Found: 423.0457.

[0185] Example 37. 4-[(6,7-dimethyl-1H-pyrazolo[3,4-b]quinoline-1-yl)methyl]-N-hydroxybenzoyl Synthesis of amine (HDNI-37)

[0186] The intermediate and target compound were prepared as in Example 1. A pale yellow solid, 63% yield, mp: 197-199℃. 1 H NMR (400 MHz, DMSO-d6), δ 11.17 (s, 1H), 9.07 (s, 1H), 8.82 (s, 1H), 8.44(s, 1H), 7.88 (s, 1H), 7.83 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.32 (d, J =8.0 Hz, 2H), 5.79 (s, 2H), 2.47 (s, 3H), 2.43 (s, 3H). 13 C NMR (100 MHz, DMSO-d6), δ 163.85, 149.57, 147.08, 141.57, 140.83, 133.59, 133.38, 131.99,129.93, 128.07, 127.31, 127.14, 126.74, 123.04, 115.77, 49.43, 20.30, 19.47.HRMS (ESI) m / z Calcd for C 20 H 19 N4O2 [M+H] + : 347.1508, Found: 347.1508.

[0187] Example 38. (E)-3-[4-[(6,7-dimethyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]- Synthesis of N-hydroxyacrylamide (HDNI-38)

[0188] The intermediate and target compound were prepared as in Example 1. A pale yellow solid, 84% yield, mp: 183-185℃. 1 H NMR (400 MHz, DMSO-d6), δ 10.75 (s, 1H), 9.04 (s, 1H), 8.81 (s, 1H), 8.43(s, 1H), 7.88 (s, 1H), 7.83 (s, 1H), 7.50 (d, J = 7.6 Hz, 2H), 7.42 (d, J =15.6 Hz, 1H), 7.30 (d, J = 7.6 Hz, 2H), 6.43 (d, J = 15.6 Hz, 1H), 5.76 (s,2H), 2.47 (s, 3H), 2.43 (s, 3H). 13C NMR (100 MHz, DMSO-d6), δ 162.65, 149.56,147.08, 141.54, 139.10, 137.84, 134.02, 133.50, 133.35, 129.88, 128.07,127.96, 127.69, 126.75, 123.02, 119.03, 115.78, 49.50, 20.30, 19.47. HRMS(ESI) m / z Calcd for C 22 H 21 N4O2 [M+H] + 373.1665, Found: 373.1668

[0189] Activity evaluation of target compounds

[0190] Experimental Example 1: HDAC1 Enzyme Inhibition Test of Target Compound

[0191] Experimental reagents:

[0192] HDAC1 enzyme solution, HDAC buffer, fluorescent test substrate (Ac-Leu-Gly-Lys(Ac)-AMC), trypsin stop solution, etc.

[0193] Experimental apparatus:

[0194] Thermo Varioskan Flash Full-Wavelength Multifunctional Microplate Reader.

[0195] Experimental steps:

[0196] (1) Set up experimental group, 100% group and blank group

[0197] Experimental group: 10 µL compound solution + 50 µL enzyme solution + 40 µL fluorescent substrate;

[0198] 100% group: 10 µL HDAC buffer + 50 µL enzyme solution + 40 µL fluorescent substrate;

[0199] Blank group: 10 µL HDAC buffer + 50 µL HDAC buffer + 40 µL fluorescent substrate;

[0200] (2) Incubate 10 µL of compound solutions or HDAC buffer of different concentrations with 50 µL of enzyme solution or HDAC buffer for 10 min (experimental group: 10 µL compound solution + 50 µL enzyme solution; 100% group: 10 µL HDAC buffer + 50 µL enzyme solution; blank group: 10 µL HDAC buffer + 50 µL HDAC buffer). Add 40 µL of fluorescent substrate to each well, and then incubate at 37°C for 60-120 min. Then add 100 µL of trypsin stop solution to each well, continue incubation for 30 min, and then measure the fluorescence value of the sample.

[0201] (3) Calculate the inhibition rate of the compound at each concentration according to the following formula, and then fit the standard curve using Graphpad Prism software to obtain the IC50 of the compound. 50 value.

[0202]

[0203] The experimental results are shown in Table 2.

[0204] Table 2. Results of the test for inhibition of HDAC1 enzyme by the target compound.

[0205] HDNI-1 305.25 HDNI-14 194.65 HDNI-27 54.02 HDNI-2 147.85 HDNI-15 84.97 HDNI-28 23.95 HDNI-3 86.87 HDNI-16 19.70 HDNI-29 3.85 HDNI-4 50.39 HDNI-17 1.65 HDNI-30 5.83 HDNI-5 2.12 HDNI-18 10.71 HDNI-31 35.84 HDNI-6 19.14 HDNI-19 388.80 HDNI-32 17.14 HDNI-7 470.80 HDNI-20 271.70 HDNI-33 50.03 HDNI-8 221.75 HDNI-21 112.85 HDNI-34 14.33 HDNI-9 56.01 HDNI-22 16.24 HDNI-35 32.06 HDNI-10 16.04 HDNI-23 1.41 HDNI-36 9.51 HDNI-11 3.56 HDNI-24 23.52 HDNI-37 33.73 HDNI-12 51.57 HDNI-25 366.35 HDNI-38 24.05 HDNI-13 328.45 HDNI-26 220.55 SAHA 67.90

[0206] The values ​​in the table are the average of the results of three trials.

[0207] As shown in the table, most compounds exhibited good inhibitory activity against HDAC1, superior to the positive control drug SAHA, such as compounds HDNI-4, HDNI-5, HDNI-6, HDNI-9, HDNI-10, HDNI-11, HDNI-12, HDNI-16, HDNI-17, HDNI-18, HDNI-22, HDNI-23, HDNI-24, HDNI-27, HDNI-28, HDNI-29, HDNI-30, HDNI-31, HDNI-32, HDNI-33, HDNI-34, HDNI-35, HDNI-36, HDNI-37, and HDNI-38. In particular, compounds HDNI-17 and HDNI-23 showed more than 40 times the inhibitory activity against HDAC1 enzyme compared to SAHA. Therefore, compounds HDNI-17 and HDNI-23, with better activity, were selected to evaluate their binding to DNA.

[0208] Experimental Example 2: Detection of the binding of active target compounds to DNA (In vitro)

[0209] (1) Ultraviolet-Visible Spectroscopy

[0210] Experimental reagents:

[0211] Calf thymus DNA (CT-DNA);

[0212] Test buffer: Tris-HC1 buffer;

[0213] Experimental apparatus:

[0214] Ultraviolet-visible spectrophotometer (UV-2600i).

[0215] Experimental steps:

[0216] The baseline was calibrated using Tris-HCl buffer (20 mM, pH = 7.4), and the measurement range was 200-400 nm. The CT-DNA concentration was fixed at 0.2 mg / mL, and a certain concentration of the compound (20 μM) was added. The UV-Vis absorption spectrum of the mixture was measured. Three groups were set up: ① 0.2 mg / mL CT-DNA group; ② 20 μM compound group; ③ 0.2 mg / mL CT-DNA + 20 μM compound group. The positive control drug SAHA was also set up with the same three groups. The UV-Vis absorption spectra were plotted using Graphpad Prism software. Experimental results are attached. Figure 2 .

[0217] from Figure 2 It can be seen that when the active compounds HDNI-17 and HDNI-23 were added to the CT-DNA solution, the UV absorption of CT-DNA at 260 nm was significantly enhanced, but SAHA did not cause a significant change. This indicates that these compounds have a certain DNA binding ability.

[0218] (2) Fluorescence spectroscopy

[0219] Experimental reagents:

[0220] Calf thymus DNA (CT-DNA);

[0221] Test buffer: Tris-HC1 buffer;

[0222] Experimental apparatus:

[0223] Fluorescence spectrophotometer (Shimadzu, RF-6000).

[0224] Experimental steps:

[0225] The baseline was corrected using Tris-HCl buffer (20 mM, pH = 7.4), and the measurement range was 300-600 nm. The CT-DNA concentration was fixed at 0.2 mg / mL, and a certain concentration of the compound (20 μM) was added. The fluorescence spectrum of the mixture was measured. Three groups were set up: ① 0.2 mg / mL CT-DNA group; ② 20 μM compound group; ③ 0.2 mg / mL CT-DNA + 20 μM compound group. The positive control drug SAHA was also set up with the same three groups. Fluorescence spectra were plotted using Graphpad Prism software. Experimental results are attached. Figure 3 .

[0226] from Figure 3 It can be seen that the fluorescence intensity of CT-DNA solution and SAHA at 445 nm is weak, while the fluorescence intensity of active compounds HDNI-17 and HDNI-23 at 445 nm is high. However, when they are added to CT-DNA solution separately, the fluorescence intensity of the mixed solutions containing HDNI-17 and HDNI-23 at 445 nm decreases significantly, especially for HDNI-17, where the decrease is more pronounced. The fluorescence intensity of the mixed solution containing SAHA, however, remains unchanged. This indicates that compounds HDNI-17 and HDNI-23 can target DNA, but SAHA cannot.

[0227] Based on the HDAC enzyme inhibition test and DNA binding experiment described above, it can be seen that these compounds, especially HDNI-17 and HDNI-23, have significant dual-target inhibitory activities against both HDAC and DNA. This provides important guidance for the further development of HDAC / Mcl-1 dual-target inhibitors with higher activity.

[0228] Experimental Example 3. Assay on the inhibitory activity of the target compound on cell proliferation (In vitro)

[0229] Two compounds that showed good activity against both HDAC and DNA were selected for in vitro assays to inhibit the proliferation of cancer cells and normal cells. The results are shown in Table 3.

[0230] Experimental materials and reagents:

[0231] Human non-small cell lung cancer cell line A549, human erythroleukemia cell line HEL, human embryonic kidney cell line HEK293T, CCK-8 kit, culture medium (RPMI 1640 or DMEM), penicillin-streptomycin solution, trypsin, fetal bovine serum, 96-well plate.

[0232] Experimental methods:

[0233] Cell lines were cultured using standard methods. Logarithmic growth phase cells were used in all experiments.

[0234] Cell growth assay (CCK-8 assay): Cell suspension was seeded into 96-well plates (100 μL / well), 2000-5000 cells / well. After 12 h of seeding, 100 μL of culture medium containing different concentrations of the compound was added to each well, with three replicates for each concentration. Wells without cells served as blank wells, and wells with cells but without the compound served as control wells. The plates were incubated at 37 ºC, 5% CO2 for 48 h. Then, 10 μL of CCK-8 solution was added to each well, and incubation continued. After 1-4 h, the absorbance (OD) at 450 nm was measured using a microplate reader. The cell growth inhibition rate was calculated using the following formula:

[0235]

[0236] Table 3. Results of cell proliferation inhibition experiments using compounds HDNI-17, HDNI-23, and SAHA.

[0237]

[0238] The values ​​in the table are the averages of three trials.

[0239] Two compounds (HDNI-17 and HDNI-23) exhibiting good activity against both HDAC and DNA were selected for in vitro antiproliferative activity experiments on A549 and HEL tumor cells and normal HEK293T cells. The test data showed that both target compounds had strong inhibitory effects on both A549 solid tumor cells and HEL hematologic malignancy cells, especially HEL cells, where the inhibitory activity reached single-digit nanomolar levels. Notably, the inhibitory activity of these two compounds against these two tumor cell lines was significantly superior to the positive control drug SAHA (approximately 10-fold). Furthermore, compared to tumor cells, the inhibitory activity of these two compounds against normal cells was weaker, especially compound HDNI-23, which showed a selectivity of 101-1607 times against tumor cells, significantly superior to the positive control drug SAHA (6.7-87.7 times). This indicates that DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitors have good development prospects and can be further studied to develop compounds with higher activity for the preparation of drugs to prevent and treat related mammalian diseases caused by abnormal HDAC expression and DNA biological function.

[0240] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitor, characterized in that: It is a compound having the general formula (I) or a pharmaceutically acceptable salt; ； （I） In general formula (I), R is hydrogen, alkyl, halogen, nitro, alkoxy, or trifluoromethyl; Linker is a substituted C1-C9 alkyl, C3-C8 cycloalkyl, C5-C10 aryl, monoheterocyclic aryl containing 5 or 6 ring atoms, or diheterocyclic aryl having 8 to 15 ring atoms. The heterocyclic aryl group contains 1-4 heteroatoms, which are independently selected from O, S, N, oxidized S or oxidized N; the carbon atom or nitrogen atom is the connecting point of the heterocyclic aromatic ring structure, maintaining a stable aromatic ring.

2. The DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitor according to claim 1, characterized in that: Linker is a halogenated C1-C7 alkyl, C1-C7 alkyl, C3-C8 cycloalkyl, morpholine group substituted with 1-2 hydroxyl groups, halogen, nitro, cyano substituents or unsubstituted aromatic group Ar, aromatic group Ar or -NH-R1 linked to piperazine group; Ar is a phenyl, naphthyl, pyridinyl, pyridazinyl, pyrazinyl, indene, quinazolinyl, purinyl, indoleyl, quinolinyl, pyrimidinyl, pyrroleyl, pyrazolyl, thiazolyl, benzo[b]thiazolyl, isoxazolyl, oxathiadiazolyl, isoxathiazolyl, tetrazolyl, imidazolyl, triazinyl, furanyl, benzofuranyl, and indoleyl group containing one substituent or without a substituent. R1 is a C1-C7 alkyl group with 1-2 hydroxyl, halogen, nitro, or cyano substituents, or without substitution, and an aromatic group Ar connected to a C1-C3 alkylene group; the substituent is a hydroxyl, halogen, nitro, cyano, guanidinium, carboxyl, halogenated C1-C7 alkyl, C1-C7 alkoxy, C1-C7 alkyl, C3-C8 cycloalkyl, C5-C10 aryl, or a heteroaryl group containing 1-2 heteroatoms and having a ring number of 5-10.

3. The DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitor according to claim 1, characterized in that, It is selected from: N-hydroxy-2-(1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-1); N-hydroxy-4-(1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-2); N-hydroxy-5-(1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-3); N-hydroxy-6-(1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-4); N-hydroxy-7-(1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-5); N-hydroxy-8-(1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-6); N-hydroxy-2-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-7); N-hydroxy-4-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-8); N-hydroxy-5-(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-9); N-hydroxy-6-(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-10); N-hydroxy-7-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-11); N-hydroxy-8-(7-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-12); N-hydroxy-2-(6-bromo-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-13); N-hydroxy-4-(6-bromo-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-14); N-hydroxy-5-(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-15); N-hydroxy-6-(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-16); N-hydroxy-7-(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)heptanamide (HDNI-17); N-hydroxy-8-(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)octamide (HDNI-18); N-hydroxy-2-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-19); N-hydroxy-4-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-20); N-hydroxy-5-(6-methoxy-1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-21); N-hydroxy-6-(6-methoxy-1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-22); N-hydroxy-7-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-23); N-hydroxy-8-(6-methoxy-1H-pyrazolo[3,4-b]quinoline-1-yl)octamide (HDNI-24); N-hydroxy-2-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)acetamide (HDNI-25); N-hydroxy-4-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)butyramide (HDNI-26); N-hydroxy-5-(6-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)pentanamide (HDNI-27); N-hydroxy-6-(6-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)hexanoamide (HDNI-28); N-hydroxy-7-(6-methyl-1H-pyrazolo[3,4-b]quinoline-1-yl)heptanamide (HDNI-29); N-hydroxy-8-(6-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)octamide (HDNI-30); 4-[(1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]-N-hydroxybenzamide (HDNI-31); (E)-3-[4-[(1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]-N-hydroxyacrylamide (HDNI-32); 4-[(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]-N-hydroxy-benzamide (HDNI-33); (E)-3-[4-[(7-methyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]-N-hydroxyacrylamide (HDNI-34); 4-[(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]-N-hydroxybenzamide (HDNI-35); (E)-3-[4-[(6-bromo-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]-N-hydroxyacrylamide (HDNI-36); 4-[(6,7-dimethyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]-N-hydroxybenzamide (HDNI-37); (E)-3-[4-[(6,7-dimethyl-1H-pyrazolo[3,4-b]quinolin-1-yl)methyl]phenyl]-N-hydroxyacrylamide (HDNI-38).

4. The method for preparing the DNA-targeting substituted 1H-pyrazolo[3,4-b]quinoline HDAC inhibitor as described in any one of claims 1-3, characterized in that, Starting with acetanilides 1a-1f with different substitutions, the mixture was first cyclized under heating in the presence of phosphorus oxychloride and N,N-dimethylformamide (DMF) to generate substituted quinolines 2a-2f. Then, under the catalysis of p-toluenesulfonic acid monohydrate, it reacted with hydrazine hydrate to generate substituted 1H-pyrazolo[3,4-b]quinolines 3a-3f. Next, it underwent nucleophilic substitution at the N-position of 1H-pyrazolo[3,4-b]quinolines with methyl or ethyl bromocarboxylic acids of different chain lengths to generate key intermediates 4a-4z and 4aa-4al. Finally, it reacted with hydroxylamine hydrochloride to obtain the target compound HDNI-1-HDNI-38. The reaction route is as follows: 。 5. A pharmaceutical composition suitable for oral administration to mammals, characterized in that, It comprises an inhibitor and one or more pharmaceutically acceptable carriers or excipients, wherein the inhibitor is any one of the inhibitors described in claims 1-3 or an inhibitor prepared by the method described in claim 4.

6. A pharmaceutical composition suitable for parenteral administration to mammals, characterized in that, It comprises an inhibitor and one or more pharmaceutically acceptable carriers or excipients, wherein the inhibitor is any one of the inhibitors described in claims 1-3 or an inhibitor prepared by the method described in claim 4.

7. The use of the inhibitor according to any one of claims 1-3, the inhibitor prepared by the method of claim 4, the pharmaceutical composition according to claim 5, or the pharmaceutical composition according to claim 6 in the preparation of a drug for the prevention or treatment of mammalian diseases caused by abnormal HDAC expression and DNA biological function.

8. The application according to claim 7, characterized in that, The mammalian diseases mentioned include cancer, neurodegenerative diseases, viral infections, inflammation, leukemia, malaria, and diabetes.

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