阅读说明：本技术 组蛋白去乙酰酶抑制剂及其制备和应用 (Histone deacetylase inhibitor and preparation and application thereof ) 是由 徐莉英 吴斐斐 景永奎 张美慧 王林 包宇 董金华 于 2019-10-24 设计创作，主要内容包括：本发明涉及组蛋白去乙酰酶抑制剂及其制备和应用,具体涉及基于β-榄香烯结构的新型组蛋白去乙酰酶抑制剂及其制备方法,还涉及合成所述组蛋白去乙酰酶抑制剂的中间体及其制备方法,还涉及所述组蛋白去乙酰酶抑制剂在制备抗肿瘤药物中的应用,属于药物化学合成技术领域。本发明所述的锌离子结合型化合物及其盐或溶剂化物如通式(I)或(II)所示：其中R、X、n、i如权利要求书和说明书所述。(The invention relates to a histone deacetylase inhibitor, a preparation method and an application thereof, in particular to a novel histone deacetylase inhibitor based on a beta-elemene structure and a preparation method thereof, an intermediate for synthesizing the histone deacetylase inhibitor and a preparation method thereof, and an application of the histone deacetylase inhibitor in preparing antitumor drugs, belonging to the technical field of chemical synthesis of drugs. The zinc ion binding compound and the salt or solvate thereof are shown as the general formula (I) or (II): wherein R, X, n, i are as described in the claims and the description.)

1. A zinc ion-binding compound represented by the general formula (I) or (II):

in the general formula (I),

r is a group capable of complexing zinc ions together with carbonyl groups: hydroxy, hydroxyamino, C1-C4 alkylamino, N-methylhydroxylamino, substituted or unsubstituted phenylamino, said substituents being amino, halogen, nitro, C1-C4 alkyl, wherein the cyclohexane skeleton has three chiral centers;

in the general formula (II),

n is an integer of 0 to 1; i is an integer of 0 to 1.

X is COO, CONH, COCH ═ CH;

r is a group capable of complexing zinc ions together with carbonyl groups: hydroxyl, hydroxyamino, C1-C4 alkylamino, N-methylhydroxylamino, substituted or unsubstituted phenylamino, amino, halogen, nitro, C1-C4 alkyl, where the cyclohexane skeleton has three chiral centers.

2. The zinc ion-binding compound represented by the general formula (I) or (II) according to claim 1:

in the general formula (I),

r is hydroxyl, hydroxyamino, methylamino, N-methylhydroxylamino, phenylamino, o-aminophenylamino, o-chlorophenylamino, wherein the cyclohexane skeleton has three chiral centers;

in the general formula (II),

r is hydroxyl, hydroxyamino, methylamino, N-methylhydroxylamino, phenylamino, o-aminophenylamino, o-chlorophenylamino, wherein the cyclohexane skeleton has three chiral centers.

3. The zinc ion-binding compound represented by the general formula (I) or (II) according to claim 1 or 2:

4. the zinc ion-binding compound of the general formula (I) or (II) according to any one of claims 1 to 3, wherein the salt is a salt with a suitable non-toxic organic acid or inorganic acid, and a salt or solvate thereof.

5. A method for producing a zinc ion-binding compound represented by the general formula (I) or (II) and a salt or solvate thereof according to claim 3, wherein: a and an intermediate A1 are subjected to N-alkylation reaction to obtain an intermediate A2, and A2 is subjected to hydrolysis and aminolysis to obtain compounds LDX-A-1 and LDX-A-2; the LDX-A-1 reacts with arylamine after being acylated by oxalyl chloride to obtain compounds LDX-A-3, LDX-A-4 and LDX-A-5 ', and the LDX-A-5' reduces nitro by iron powder/ammonium chloride to obtain a compound LDX-A-5;

6. a method for producing a zinc ion-binding compound represented by the general formula (I) or (II) and a salt or solvate thereof according to claim 3, wherein:

a and anhydrous piperazine are subjected to nucleophilic substitution reaction to obtain D-10, and terephthalaldehyde is reduced by sodium borohydride to obtain B1; b1 and D-10 react in the presence of N, N' -carbonyldiimidazole to obtain an intermediate B2, B2 and malonic acid react in a Knoevenagel-Doebner to obtain a compound LDX-B-1; LDX-B-1 reacts with hydroxylamine hydrochloride and methylamine hydrochloride in the presence of N, N' -carbonyldiimidazole to obtain compounds LDX-B-2 and LDX-B-5; under the existence of DCC and DMAP, LDX-B-1 and aniline react to obtain a compound LDX-B-3; the LDX-B-1 is acylated by oxalyl chloride and then reacts with o-chloroaniline to obtain a compound LDX-B-4;

7. a method for producing a zinc ion-binding compound represented by the general formula (I) or (II) and a salt or solvate thereof according to claim 3, wherein:

4-formyl methyl benzoate is reacted by Knoevenagel-Doebner to obtain C1, an intermediate C1 is acylated by oxalyl chloride and then reacts with D-10 to obtain an intermediate C2, C2 is hydrolyzed to obtain a compound LDX-C-1, and the LDX-C-1 reacts with hydroxylamine hydrochloride and methylamine hydrochloride in the presence of N, N' -carbonyldiimidazole to obtain compounds LDX-C-2 and LDX-C-6; under the existence of DCC and DMAP, LDX-C-1 and aniline react to obtain a compound LDX-C-3; the LDX-C-1 is acylated by oxalyl chloride and then reacts with o-chloroaniline to obtain a compound LDX-C-5; the LDX-C-1 is acylated by oxalyl chloride and then reacts with o-nitroaniline to obtain an intermediate LDX-C-4 ', and the LDX-C-4' is subjected to nitro reduction by iron powder/ammonium chloride to obtain a compound LDX-C-4;

4-bromomethyl phenylpropenoic acid methyl ester and phthalimide potassium salt are taken as raw materials, intermediate D1 is obtained through drape Rayle synthesis reaction, D1 reacts with D-10 in the presence of solid phosgene to obtain D2, D2 is subjected to alkaline hydrolysis to obtain LDX-D-1, and LDX-D-1 reacts with hydroxylamine hydrochloride in the presence of N, N' -carbonyldiimidazole to obtain LDX-D-2.

8. A pharmaceutical composition comprising a zinc ion-binding compound of formula (I) or (II) as defined in any one of claims 1 to 4, and a salt or solvate thereof, and a pharmaceutically acceptable excipient.

9. Use of a zinc ion binding compound of formula (I) or (II) as defined in any one of claims 1 to 4 and salts or solvates thereof or a pharmaceutical composition as defined in claim 7 for the manufacture of a medicament for the treatment of cancer.

10. The use of claim 9, wherein the cancer is leukemia, multiple myeloma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, acute promyelocytic lymphoma, breast cancer, cervical cancer, melanoma, liver cancer, lung cancer, ovarian cancer, or prostate cancer.

The technical field is as follows:

the invention belongs to the technical field of chemical synthesis of medicines, and relates to a histone deacetylase inhibitor, a preparation method and an application thereof, in particular to a novel histone deacetylase inhibitor based on a beta-elemene structure and a preparation method thereof, an intermediate for synthesizing the histone deacetylase inhibitor and a preparation method thereof, and an application of the histone deacetylase inhibitor in preparing antitumor drugs.

Background art:

histone deacetylase inhibitors (HDACIs) activate cancer suppressor genes, inhibit cancer genes, and further inhibit tumor cell growth and induce apoptosis of tumor cells by regulating the level of acetylation and deacetylation of lysine residues at the N-terminus of histone (Armeanus S, Pathil A, Venturi S, et al. By far, HDAC-targeted drugs have 6 worldwide approved for marketing, 5 in the third clinical stage, 32 in the second clinical stage, and 22 in the first clinical stage. These HDACi, which are already on the market and are being clinically tested, are mainly classified into low-toxicity fatty acids, strong Zinc ion-binding hydroxamic acids, highly selective amides and others according to the type of Zinc-binding group (ZBG). The structure of HDAC inhibitors is generally composed of a surface recognition group (Cap group) that interacts with amino acids at the entrance of the N-acetylated lysine binding channel, a linking unit (CU), a zinc ion binding group (ZBG), and a linker four parts fitting into a narrow hydrophobic pocket, which links together the Cap part, the CU part, and the ZBG part (Mark L, Hamacher A, Hansen F K, et al. Histone Deacylase (HDAC) inhibitor with a non-binding unit linking molecule for HDAC4 and HDAC5 with an engineered active ingredient catalytic organic reactive cells [ J ]. 427 ]. The hydrophobic regions of CAP have been reported to be primarily benzene rings and aromatic heterocycles that interact with amino acid residues surrounding the cavity of the CAP region of the HDAC active site. Strebl M G et al found that the selectivity for class I HDACs can be enhanced by exchanging the aromatic ring of the water-transport region of HDACi for a nonaromatic hydrophobic group with a steric structure (Strebl M G, Wang C, Schroeder F A, et al. development of a fluorinated class-I HDAC radiotherapeutic derivatives key chemical inhibitors of broad spectrum [ J ]. ACS Chem neurosis, 2015, 7(5): 528-533.).

The beta-elemene is a hydrophobic sesquiterpenoid compound with a certain space structure, contains three pi bonds, has higher electron cloud density, the space structure volume is similar to the reported CAP hydrophobic region, and the beta-elemene has anticancer activity and is an active component of II-type broad-spectrum non-cytotoxic antitumor drug elemene emulsion developed by China. In order to improve the water solubility of beta-elemene and improve the anticancer activity, a series of beta-elemene piperazine derivatives were designed and synthesized in the subject group, wherein some compounds have IC (integrated Circuit) of cell strains such as leukemia K562, HL-60, cervical cancer HeLa, gastric cancer SGC-7901, prostate cancer PC3, LNCap and the like₅₀IC of value ratio beta-elemene₅₀The value is 1 to 2 orders of magnitude lower (IC)₅₀A value of about 1 to 10 μ M); has no obvious bone marrow inhibition effect, and has lower inhibition rate on normal bone marrow cells5 times of tumor cells; the breast cancer MCF-7/Adr cell line which is resistant to the chemotherapeutic drug has the same sensitivity. Further in vivo anticancer activity test results show that the beta-elemene 3-methylpiperazine and 3, 5-dimethylpiperazine derivatives can significantly inhibit the transplanted Lewis lung cancer LL/2 and liver cancer H in SPF-grade BALB/C nude mice, Kunming mice, C57BL/6 mice and ICR mice₂₂Osteosarcoma S₁₈₀The in-situ growth of tumor in mouse has higher or equal inhibiting activity than that of lead compound beta-elemene, and its antitumor effect may be related to the activation of in-vivo immune system of tumor bearing animal. Anticancer mechanism research shows that the two compounds can inhibit the growth of HL-60 and HeLa cells and induce apoptosis, mainly reduces the level of c-FLIP (FLICE-like inhibition protein) protein and induces the generation of active oxygen, and further activates Caspase-8 to activate exogenous way to induce HL-60 cells to generate apoptosis. The compound can also inhibit the activity of AKT (down-regulated P-mTOR) and mTOR (down-regulated P-P70S6K), and is likely to be a novel mTOR inhibitor (Xueliying, Dongjin, Jingyongquine, Wanyiwei. beta-Elemene nitrogenous derivatives, and a preparation method and application thereof. patent No. ZL 200610081625.0; Yu Zhiying, Wang Rui, Xu Liying, et al. beta-Elemene piperizine derivative induced apoptosis in human leukaemis cells through medication downlink regulation of c-FLIP and generation of ROS. PLoS ONE.2011,6(1): 15843; Ding Xiaoei, ShenMao, Xu Liying, et al.

13,14-bis (cis-3,5-dimethyl-1-piperazinyl) -beta-elemene, alpha novel beta-elemene derivative, brown post inhibitor or activity vitamins via inhibition of mTOR in human breast cells, ONCOLOGY LETTERS.2013,5: 1554-1558; effect of elemene piperazine on immune function of tumor-bearing mice [ J]Shenyang pharmaceutical university, 2007, 24 (4): 238-241; experimental study on induction of HeLa cell apoptosis by aged light, Dingxiaofeng, Xiaojin elemene derivatives [ J]Chinese pharmacological report, 2007, 23 (2): 246-250). However, the toxicity of the derivatives is relatively high in vivo tests, so that the amide-forming modification method is adopted in the department of king's worship in 2013, and substituted benzoyl or substituted phenylpropenoyl is introduced to the nitrogen at the 4-position of the piperazinyl of the beta-elemene substituted piperazine derivatives for synthesisBeta-elemene substituted piperazine amide derivatives are adopted, MTT method is adopted to test the in vitro anti-tumor activity of the synthesized compounds on 10 tumor cells such as human cervical cancer cells, human liver cancer cells, human fibrosarcoma cells and the like, initial in vitro experiments show that the anti-tumor effect of the beta-elemene substituted piperazine amide derivatives is mostly stronger than that of beta-elemene, and the target compounds have IC (integrated Circuit) of human melanoma cells A375-S2₅₀The values are mostly less than 10 mu M, which indirectly proves that the introduction of an amido bond in the beta-elemene piperazine derivative can enhance the specific selectivity of the beta-elemene piperazine derivative on certain tumor cells. The invention relates to a new application of beta-elemene substituted piperazine amide derivatives, in particular to a new application of beta-elemene substituted piperazine amide derivatives, a new Chinese patent number ZL 201210502358.5, a new drug for treating pain, a good looking, a summer-Ming Yu and the like, a synthesis and antitumor activity research of the beta-elemene substituted piperazine amide derivatives, and a new application of the beta-elemene substituted piperazine amide derivatives, wherein the new application is as shown in the specification, namely the application of the beta-elemene substituted piperazine amide derivatives in the technical fields of medicine chemistry, 2013, 23.

Therefore, in the present invention, by applying the pharmacophore drug design method, the replacement of the aromatic hydrophobic structure in the HDAC inhibitor by the β -elemene structure is tried for the first time; the ZBG part adopts fatty acids, hydroxamic acids and amide pharmacophores as zinc ion complex structures according to the types of HDACI on the market, and four series of novel HDAC inhibitors with beta-elemene structures are designed.

The invention content is as follows:

according to the invention, a beta-elemene structure is introduced into a histone deacetylase inhibitor (HDACI) for the first time to serve as a CAP hydrophobic region of the HDACI, and a ZBG part adopts pharmacophores of fatty acids, hydroxamic acids and amides to serve as zinc ion complex structures according to the types of the HDACI on the market, so that a series of novel histone deacetylase inhibitors with novel structures based on the beta-elemene structure are synthesized, and pharmacological tests prove that the novel histone deacetylase inhibitors have good anticancer activity and HDAC inhibitory activity.

The invention aims to provide a novel histone deacetylase inhibitor based on a beta-elemene structure with a novel structure, a salt thereof or a solvate of the histone deacetylase inhibitor and the salt.

Another object of the present invention is to provide a method for preparing the novel histone deacetylase inhibitor based on the beta-elemene structure.

The third object of the present invention is to provide a pharmaceutical composition containing the novel histone deacetylase inhibitor based on the beta-elemene structure.

The fourth purpose of the invention is to provide the application of the novel histone deacetylase inhibitor based on the beta-elemene structure, and the novel histone deacetylase inhibitor or the composition based on the beta-elemene structure can be used for preparing various antitumor drugs.

Specifically, the novel beta-elemene structure-based novel histone deacetylase inhibitor provided by the invention has the following structure:

a. the connecting chain Linker is a zinc ion combined compound of an aliphatic chain and a salt or solvate thereof:

r is a group capable of complexing zinc ions together with carbonyl groups: hydroxyl, hydroxyamino, C1-C4 alkylamino, N-methylhydroxylamino, substituted or unsubstituted phenylamino, amino, halogen, nitro, C1-C4 alkyl, where the cyclohexane skeleton has three chiral centers.

Further, R is hydroxyl, hydroxyamino, methylamino, N-methylhydroxylamino, phenylamino, o-aminophenylamino, o-chlorophenylamino, wherein the cyclohexane skeleton has three chiral centers.

The invention provides a preparation method of a zinc ion combined compound with a connecting chain Linker as a fatty chain, which is characterized by comprising the following steps: a and an intermediate A1 are subjected to N-alkylation reaction to obtain an intermediate A2, and A2 is subjected to hydrolysis and aminolysis to obtain compounds LDX-A-1 and LDX-A-2; the LDX-A-1 is acylated by oxalyl chloride and then reacts with arylamine to obtain compounds LDX-A-3, LDX-A-4 and LDX-A-5 ', and the LDX-A-5' is subjected to nitro reduction by iron powder/ammonium chloride to obtain a compound LDX-A-5. Wherein the cyclohexane skeleton has three chiral centers.

b. The connecting chain Linker is a zinc ion combined compound of an aromatic chain and a salt or solvate thereof:

wherein n is an integer from 0 to 1; i is an integer of 0 to 1.

X is COO, CONH, COCH ═ CH;

r is a group capable of complexing zinc ions together with carbonyl groups: hydroxyl, hydroxyamino, C1-C4 alkylamino, N-methylhydroxylamino, substituted or unsubstituted phenylamino, amino, halogen, nitro, C1-C4 alkyl, where the cyclohexane skeleton has three chiral centers.

Further, R is hydroxyl, hydroxyamino, methylamino, N-methylhydroxylamino, phenylamino, o-aminophenylamino, o-chlorophenylamino, etc., wherein the cyclohexane skeleton has three chiral centers.

The invention provides a preparation method of a zinc ion combined compound with an aromatic chain as a connecting chain Linker, which is characterized by comprising the following steps: a and anhydrous piperazine are subjected to nucleophilic substitution reaction to obtain D-10, and terephthalaldehyde is reduced by sodium borohydride to obtain B1. B1 and D-10 react in the presence of N, N' -Carbonyldiimidazole (CDI) to obtain intermediate B2, and B2 and malonic acid react in the presence of Knoevengel-Doebner to obtain compound LDX-B-1. LDX-B-1 reacts with hydroxylamine hydrochloride and methylamine hydrochloride in the presence of CDI to obtain compounds LDX-B-2 and LDX-B-5; under the existence of DCC and DMAP, LDX-B-1 and aniline react to obtain a compound LDX-B-3; the LDX-B-1 is acylated with oxalyl chloride and then reacts with o-chloroaniline to obtain a compound LDX-B-4.

The invention provides a preparation method of a zinc ion combined compound with an aromatic chain as a connecting chain Linker, which is characterized by comprising the following steps: 4-formyl methyl benzoate is reacted by Knoevenagel-Doebner to obtain C1, an intermediate C1 is acylated by oxalyl chloride and then reacts with D-10 to obtain an intermediate C2, C2 is hydrolyzed to obtain a compound LDX-C-1, and the LDX-C-1 is reacted with hydroxylamine hydrochloride and methylamine hydrochloride in the presence of CDI to obtain compounds LDX-C-2 and LDX-C-6; under the existence of DCC and DMAP, LDX-C-1 and aniline react to obtain a compound LDX-C-3; the LDX-C-1 is acylated by oxalyl chloride and then reacts with o-chloroaniline to obtain a compound LDX-C-5; the LDX-C-1 is acylated by oxalyl chloride and then reacts with o-nitroaniline to obtain an intermediate LDX-C-4 ', and the LDX-C-4' is subjected to nitro reduction by iron powder/ammonium chloride to obtain a compound LDX-C-4.

The invention provides a preparation method of a zinc ion combined compound with an aromatic chain as a connecting chain Linker, which is characterized by comprising the following steps: 4-bromomethyl phenylpropenoic acid methyl ester and phthalimide potassium salt are taken as raw materials, intermediate D1 is obtained through drape Rayle synthesis reaction, D1 reacts with D-10 in the presence of solid phosgene to obtain D2, D2 is subjected to alkaline hydrolysis to obtain LDX-D-1, and LDX-D-1 reacts with hydroxylamine hydrochloride in the presence of CDI to obtain LDX-D-2.

The solvent used in the preparation process of the novel histone deacetylase inhibitor based on the beta-elemene structure is a common solvent, such as glacial acetic acid, methanol, ethanol, acetone, dichloromethane, DMF and the like.

In the novel histone deacetylase inhibitor based on the beta-elemene structure and the medicinal salt thereof, the medicinal salt is formed by a proper non-toxic organic acid or inorganic acid.

The invention provides a pharmaceutical composition, which comprises the novel histone deacetylase inhibitor based on a beta-elemene structure, a medicinal salt thereof or a solvate thereof and a pharmaceutically acceptable excipient.

Furthermore, the invention provides a novel histone deacetylase inhibitor based on a beta-elemene structure, a medicinal salt thereof or a solvate thereof and application of a medicinal composition thereof in preparing various anti-cancer medicaments.

The cancer is leukemia, multiple myeloma, peripheral T cell lymphoma, cutaneous T cell lymphoma, acute promyelocytic lymphoma, breast cancer, cervical cancer, melanoma, liver cancer, lung cancer, ovarian cancer or prostatic cancer, etc.

The specific implementation mode is as follows:

the following examples are provided to illustrate the applicability of the present invention, and it will be understood by those skilled in the art that various modifications and substitutions can be made to the corresponding technical features according to the teachings of the prior art, and still fall within the scope of the present invention as claimed.

EXAMPLE 1 monochloro beta-elemene intermediatesPreparation of

In a 100mL three-necked bottle, 5.0g (24.47mmol, 1eq.) of beta-elemene, 3.5mL (61.17mmol, 2.50eq.) of glacial acetic acid and 40mL of dichloromethane are added, an ice water bath is cooled to 5 ℃, 15mL (25.45mmol, 1.04eq.) of sodium hypochlorite solution is slowly dropped under stirring, the reaction is continued for 4 hours after 15 minutes. After the reaction, 30mL of water was added, the aqueous layer was extracted with dichloromethane, the organic phases were combined, washed with saturated brine, the organic layer was retained, dried over anhydrous sodium sulfate, filtered, and concentrated by distillation under reduced pressure to give a crude product. The monochloro beta-elemene oily substance is obtained by column chromatography, 2.7g, and the yield is 46.2%.

Example 2 beta-elemene piperazinePreparation of

Dissolving 2.0g (8.38mmol, 1eq.) of monochloro beta-elemene mixture and 1.8g (20.94 mmol, 2.5eq.) of anhydrous piperazine in 40mL of anhydrous ethanol, and reacting at 20-80 ℃ for 7 h. After completion of the reaction, distillation was performed under reduced pressure, water was added to the residue, and the mixture was extracted with dichloromethane, the organic layer was retained, the organic layer was washed three times with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated by distillation under reduced pressure to give 1.2g of a pale yellow oily substance with a yield of 49.7%.

EXAMPLE 3 intermediate methyl 6-aminocaproate hydrochloridePreparation of

Adding 120mL of anhydrous methanol into a 250mL three-necked bottle, reducing the temperature of a reaction system to-10 ℃ under the protection of nitrogen, slowly and dropwise adding 10.9mL (152.47mmol, 5eq.) of thionyl chloride into the reaction solution, keeping the temperature of the reaction solution at-10 ℃ for reaction for 10min, then adding 4g (30.49mmol, 1eq.) of 6-aminocaproic acid into the reaction solution, and finally moving the reaction solution to room temperature for reaction for 24 h. After the reaction, the mixture was concentrated by distillation under reduced pressure, and then 25mL of anhydrous methanol was added to the residue to dissolve it, and 100mL of anhydrous ether was added to precipitate a product which was then filtered to give 3.2g of methyl 6-aminocaproate hydrochloride A1 in a yield of 57.8%, mp: 118-. ESI-MS, M/z 146.3[ M + H-Cl]⁺。

EXAMPLE 4 intermediate hydroxymethylbenzaldehydePreparation of

Dissolving 2.0g (14.90mmol, 1eq.) of terephthalaldehyde in 25mL of anhydrous ethanol and 35mL of tetrahydrofuran, adding 140mg (3.72mmol, 0.25eq.) of sodium borohydride in four batches at the temperature of-10 to-5 ℃ under stirring, after 1h, moving the reaction system to room temperature, and continuing to react for 5 h. After the reaction is finished, 1mol/L diluted hydrochloric acid is added into the reaction liquid to adjust the pH value to 5, the solvent is removed by reduced pressure distillation, water is added into the residue and then ethyl acetate is used for extraction, an organic layer is reserved, the residue is washed by saturated saline solution, anhydrous sodium sulfate is dried, filtration is carried out, a crude product is obtained by reduced pressure distillation, column chromatography is carried out to obtain a white solid 1.6g, the yield is 83.0 percent, and m.p.33-35 ℃.

EXAMPLE 5 intermediate Methylparaben Phenylpropanoic acidPreparation of

2.0g (12.18mmol, 1eq.) of methyl 4-formylbenzoate and 3.8g (36.55mmol, 3eq.) of malonic acid were dissolved in 10mL of pyridine, and 3.4mL (3.99mmol, 3eq.) of hexahydropyridine was added to the reaction mixture at 80-100 deg.CAnd reacting for 4 h. After the reaction is finished, the pH is adjusted by using 1mol/L dilute hydrochloric acid until no solid is separated out, and the product is filtered and dried to obtain 2.3g of product, the yield is 91.6 percent, and the m.p.246-248 ℃.¹H-NMR (600MHz,DMSO-d₆)δ12.56(brs,1H),7.97(d,J＝8.3Hz,1H),7.83(d,J＝8.2Hz, 1H),7.64(d,J＝16.1Hz,1H),6.66(d,J＝16.0Hz,1H),3.87(s,2H)。

EXAMPLE 6 intermediate (E) -3- [4- (aminomethyl) phenyl]Acrylic acid methyl esterPreparation of

0.10g (0.39mmol) of methyl 4-bromomethyl phenylpropenoate and 0.0726g (0.39mmol) of phthalimide potassium salt are added into a 50mL three-necked flask, 5mL of acetone is added as a solvent, and the mixture is stirred and reacted at 20-55 ℃ for about 14 h. After the reaction, the solvent was distilled off under reduced pressure, a suitable amount of water was added to the reaction solution, extraction was performed with ethyl acetate, the organic phases were combined, washed with saturated brine, dried with anhydrous sodium sulfate, suction-filtered, and concentrated to obtain 0.12g of a white solid with a yield of 96.0%. Adding the product obtained in the step into a 50mL reaction bottle, dissolving the product with 4mL methanol, then adding 0.1mL (1.56 mmol) of 80% hydrazine hydrate into the reaction solution, stirring the mixture at room temperature for 4 hours to react, wherein the reaction solution begins to become clear, monitoring by TLC that the intermediate 2 of the raw material is obviously reduced, continuing the reaction for 2 hours until the raw material is completely reacted, adding 8 drops of glacial acetic acid, and stirring the mixture for 5 hours. The solvent was distilled off under reduced pressure, an appropriate amount of dichloromethane was added to the reaction solution to dissolve all of it, an appropriate amount of water was added to the reaction solution to extract it, the aqueous layer was retained, anhydrous sodium carbonate solid was added to the aqueous layer to adjust the pH of the solution to 9, and extraction was performed with dichloromethane, and the product was transferred to the organic layer. The organic layer was washed with saturated brine, and dried over anhydrous sodium sulfate. Suction filtration and concentration gave 0.05g of a white solid in 67.2% yield.

¹H-NMR(600MHz,DMSO-d₆)δ8.61(brs,2H),7.67(d,J＝7.9Hz,2H),7.48(d, J＝15.4Hz,1H),7.11(d,J＝8.0Hz,2H),6.31(d,J＝15.4Hz,1H),4.31(s,2H),3.6(s,3H)； ESI-MS m/z:192.1[M+H]⁺.

Example 7 intermediatesPreparation of

Weighing the intermediate a (1.5g, 6.28mmol, 1eq.), the intermediate A1(1.71g, 9.42mmol, 1.5eq.) and 2.6mL triethylamine (18.84mmol, 3eq.) in a 100mL eggplant-shaped bottle, adding 10mL N, N-Dimethylformamide (DMF), heating to 70-150 ℃ and reacting for 5 h. After the reaction is finished, cooling to room temperature, precipitating triethylamine hydrochloride crystals in reaction liquid, filtering, adding 60mL of water into filtrate, extracting with ethyl acetate, combining organic layers, washing with saturated saline solution, reserving the organic layer, adding anhydrous sodium sulfate for drying, filtering, distilling under reduced pressure to obtain a crude product, and carrying out column chromatography to obtain 0.7g of yellow oily matter with the yield of 32.1%.¹H-NMR(600MHz, DMSO-d₆)δ5.83(dd,J＝17.9,10.4Hz,1H),5.03–4.54(m,6H),4.09(br s,1H), 3.57(s,3H),3.14(m,2H),2.42(m,2H),2.39–1.17(m,19H),0.95(s,3H)； ESI-MS,m/z 348.2[M+H]⁺。

Example 8 intermediatesPreparation of

0.5g (3.67mmol, 1eq.) of intermediate B1 was dissolved in 4mL of anhydrous DMF, and 0.715g (4.41mmol, 1.2eq.) of CDI was added to the reaction mixture under nitrogen protection, and the mixture was reacted at room temperature for 2 hours. Subsequently, 1.27 g (4.41mmol, 1.2eq.) of intermediate D-10 was dissolved in 6mL of anhydrous DMF and added to the reaction system to react at room temperature for 12 hours. After the reaction, 30mL of saturated saline was added to the reaction solution, extraction was performed with ethyl acetate, the organic layer was retained, the organic layer was washed with saturated saline, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to obtain a crude product, which was subjected to column chromatography to obtain 0.8g of pale yellow oily substance with a yield of 48.3%.¹H-NMR(600MHz,DMSO-d₆)δ10.02 (s,1H),7.92(d,J＝8.2Hz,2H),7.57(d,J＝8.1Hz,2H),5.83(dd,J＝17.9,10.4Hz, 1H),5.19(s,2H),4.96–4.58(m,6H),3.42(m,4H),2.92(m,2H),2.32(m,4H),2.13 –1.97(m,2H),1.74–1.31(m,9H),0.98(s,3H)；ESI-MS,m/z 451.1[M+H]⁺。

Example 9 intermediatesPreparation of

0.3g (1.45mmol, 1eq.) is weighed out and dissolved in 4mL of anhydrous dichloromethane, 0.37mL (4.36mmol, 3eq.) of oxalyl chloride is added under nitrogen protection, then 2 drops of anhydrous DMF are added dropwise, and the reaction is carried out at room temperature for 30 min. Then, the residue was distilled under reduced pressure, and 4mL of anhydrous methylene chloride was added to dissolve the residue, and then 0.5g (1.75 mmol, 1.2eq.) of intermediate D-10 was dissolved in 5mL of anhydrous methylene chloride and added to the reaction system to react at room temperature for 6 hours. After the reaction is finished, slowly adding water, extracting by using dichloromethane, combining and reserving organic layers, washing by using saturated saline solution, reserving the organic layers, drying by using anhydrous sodium sulfate, filtering, and concentrating by reduced pressure distillation to obtain a crude product. Column chromatography to obtain 0.5g pale yellow solid with yield of 72.1%, m.p.137-139 ℃.¹H-NMR(600MHz,DMSO-d₆) δ7.96(d,J＝8.5Hz,2H),7.86(d,J＝8.5Hz,2H),7.52(d,J＝15.4Hz,1H),7.40(d, J＝15.5Hz,1H),5.83(dd,J＝17.9,10.4Hz,1H),4.97–4.59(m,6H),3.86(s, 3H),3.64(m,4H),2.93(m,2H),2.38–2.31(m,4H),2.11–1.98(m,2H),1.68– 1.33(m,9H),0.98(s,3H)；ESI-MS,m/z 477.4[M+H]⁺。

Example 10 intermediatesPreparation of

170mg (888.98. mu. mol) of intermediate D1 was charged into a 50mL single-necked flask, dissolved in 3.0mL of anhydrous dichloromethane, 263mg (888.98. mu. mol) of phosgene solution as a solid was weighed, dissolved in 3mL of dichloromethane, and then added dropwise to the reaction mixture, and finally 0.12mL (888.98. mu. mol) of triethylamine solution diluted with 3.0mL of dichloromethane was added dropwise to the reaction mixture. Stirring and reacting for 1h at normal temperature under the protection of nitrogen. The solvent was distilled off under reduced pressure to give a yellow oil. The yellow oil was dissolved in 2.0mL of dichloromethane, 256.45mg of β -elemene piperazine (D-10) (888.98 μmol) was added, the reaction was stirred at room temperature for 4h, and the progress of the reaction was followed by TLC. After completion of the reaction, the solvent was distilled off, an appropriate amount of water was added thereto, followed by extraction with dichloromethane, the organic layer was retained, and the organic layer was washed with saturated brine, washed 3 times with 1mol/L hydrochloric acid, and finally with 2% NaHCO₃Washing the solution until the organic phase solution is neutral, and adding anhydrous sodium sulfate for drying. DrawerFiltration and concentration gave 0.31g of a white solid in 68.9% yield. ESI-MS M/z 506.2[ M + H ]]⁺。

EXAMPLE 11 general procedure for the hydrolysis of methyl ester

1.44mmol of methyl ester and 1.5mL of methanol were put into a 50mL eggplant-shaped bottle, and 4.4mL of a 1mol/L (4.32mmol, 3eq.) sodium hydroxide solution and then 2mL of tetrahydrofuran were added to react at room temperature for 24 hours. After the reaction is finished, adding 1mol/L diluted hydrochloric acid to adjust the pH value to 7, carrying out reduced pressure distillation to obtain a residue, adding water into the residue, extracting with dichloromethane, combining and reserving an organic layer, washing with saturated saline solution, reserving the organic layer, drying with anhydrous sodium sulfate, filtering, carrying out reduced pressure distillation and concentration to obtain a crude product, and carrying out column chromatography on the crude product to obtain a pure product.

EXAMPLE 12 general method for Synthesis of Compound having o-chloroaniline Structure

0.15mmol of substituted carboxylic acid was dissolved in 1.5mL of anhydrous dichloromethane, 0.04 mL (0.45mmol, 3eq.) of oxalyl chloride was added dropwise to the reaction mixture under nitrogen protection, and one drop of anhydrous DMF was added dropwise for reaction for 30 min. The residue was then distilled under reduced pressure, and 1.5mL of anhydrous methylene chloride was added to dissolve the residue, followed by addition (0.22 mmol, 1.5eq.) of o-chloroaniline and reaction at room temperature for 5 hours. After the reaction is finished, slowly adding water, extracting by dichloromethane, combining and reserving organic layers, washing by saturated saline solution, reserving the organic layers, drying by anhydrous sodium sulfate, filtering, distilling and concentrating under reduced pressure to obtain a crude product, and separating by column chromatography to obtain the target compound.

EXAMPLE 13 general Synthesis of Compounds containing hydroxamic acid, N-methylformamide Structure

0.20mmol of substituted carboxylic acid was dissolved in 2mL of anhydrous DMF, and 49.4mg (0.30 mmol, 1.5eq.) of CDI was added to the reaction solution under nitrogen protection, and the mixture was reacted at room temperature for 2 hours. Then, 0.41mmol (2eq.) of hydroxylamine hydrochloride or methylamine hydrochloride was added to the reaction system, and the reaction was continued for 12 hours. After the reaction is finished, adding 30mL of saturated saline solution into the reaction solution, extracting with ethyl acetate, retaining the organic layer, washing the organic layer with the saturated saline solution, drying with anhydrous sodium sulfate, filtering, distilling under reduced pressure to obtain a crude product, and carrying out column chromatography to obtain a pure product.

Example 14Preparation of

Intermediate A2 and sodium hydroxide were used as raw materials to obtain 0.45g of a pale yellow solid (compound LDX-A-1) according to the general method for ester hydrolysis, yield 93.8%, m.p.79-82 ℃.¹H-NMR(600MHz,CDCl₃)δ8.95(s, 1H),5.75(dd,J＝17.9,10.4Hz,1H),5.43–4.48(m,6H),3.34(br s,1H),2.84(m, 2H),2.16–1.99(m,2H),1.66–1.28(m,19H),0.92(s,3H)；ESI-MS, m/z334.2[M+H]⁺。

Example 15Preparation of

Intermediate A2(0.2g, 0.575mmol), 2mL of 50% hydroxylamine aqueous solution, and 2mL of methanol were put into a 50mL eggplant-shaped bottle and reacted at room temperature for 72 hours. After the reaction is finished, adding 40mL of saturated saline solution, extracting with ethyl acetate, combining and reserving organic layers, washing with saturated saline solution, reserving organic layers, drying with anhydrous sodium sulfate, filtering, distilling and concentrating under reduced pressure to obtain light yellow crude product, and performing column chromatography on the crude product to obtain 0.1g of white solid (compound LDX-A-2), wherein the yield is 49.9 percent and m.p.70-74 ℃.¹H-NMR(600MHz,DMSO-d₆)δ10.51(s,1H),7.79(s, 1H),5.82(dd,J＝17.9,10.4Hz,1H),5.28–4.56(m,6H),3.81(br s,1H),2.87– 2.80(m,2H),2.36–2.02(m,2H),2.00–1.22(m,19H),0.97(s,3H)；ESI-MS,m/z 349.4[M+H]⁺。

Example 16Preparation of

Taking a compound LDX-A-1, oxalyl chloride and aniline as raw materials, and obtaining a colorless solid according to a general synthesis method of a compound containing an o-chloroaniline structure, wherein the yield is 37.5 percent, and the m.p.103-107 ℃.¹H-NMR(600MHz,DMSO-d₆) δ9.96(s,1H),7.63–6.99(m,5H),5.82(dd,J＝17.9,10.4Hz,1H),5.19–4.59(m, 6H),3.40(s,1H),2.90–2.85(m,2H),2.11–2.00(m,2H),1.74–1.22(m,19H), 0.97(s,3H)；ESI-MS,m/z 409.3[M+H]⁺。

Example 17Preparation of

The compound LDX-A-1, oxalyl chloride and o-chloroaniline are used as raw materials, and a white solid is obtained according to the general synthesis method of the compound containing the o-chloroaniline structure, wherein the yield is 38.5 percent and m.p.91-95 ℃.¹H-NMR(600MHz, DMSO-d₆)δ9.45(s,1H),7.69–7.14(m,4H),5.82(dd,J＝17.9,10.4Hz,1H),5.00 –4.57(m,6H),4.11(br s,1H),2.61(m,2H),2.12–1.89(m,2H),1.68–1.35(m, 19H),0.97(s,3H)；ESI-MS,m/z 443.4[M+H]⁺,441.3[M-H]^-。

EXAMPLE 18 general method for reduction of Nitro group

48mg (0.86mmol, 10eq.) of iron powder, 4.6mg (0.086mmol, 1eq.) of ammonium chloride and 0.01mL (0.17mmol, 2eq.) of glacial acetic acid were put in a 50mL three-necked flask, and then 2mL of water and 1mL of DMF were added to react the reaction system at 30-85 ℃ for 20 min. Then 50mg (0.086mmol, 1eq.) of intermediate LDX-A-5 'and LDX-C-4' containing nitro groups were dissolved in 1mL of DMF and added to the reaction system, and the reaction was continued at 55 ℃ for 4 h. After the reaction is finished, filtering insoluble substances by using diatomite, adding 10mL of water into the filtrate, extracting by using ethyl acetate, retaining an organic layer, washing the organic layer by using saturated saline solution, drying the organic layer by using anhydrous sodium sulfate, filtering, carrying out reduced pressure distillation and concentration to obtain a crude product, and carrying out column chromatography to obtain a pure product.

Example 19Preparation of

The compound LDX-A-1, oxalyl chloride and o-nitroaniline are used as raw materials, and the yellow solid LDX-A-5' is obtained according to the general synthesis method of the compound containing the o-chloroaniline structure, and the yield is 38.5%. LDX-A-5' is reduced by nitro to obtain light yellow LDX-A-5 with m.p. of 80-84 ℃.¹H-NMR(600MHz,DMSO-d₆)δ10.02(s, 1H),7.66–6.85(m,4H),5.82(dd,J＝17.9,10.4Hz,1H),5.5(brs,2H),5.19–4.59(m, 6H),3.40(s,1H),2.90–2.85(m,2H),2.11–2.00(m,2H),1.74–1.22(m,19H), 0.97(s,3H)；ESI-MS,m/z 424.3[M+H]⁺。

Example 20Preparation of

0.6g (1.33mmol, 1eq.) of intermediate B2, 0.42g (3.99mmol, 3eq.) of malonic acid were dissolved in 5mL of pyridine, and then 0.4mL (3.99mmol, 3eq.) of piperidine was added to the reaction mixture and reacted at 70-110 ℃ for 4 h. After the reaction is finished, adjusting the pH value to be neutral by using 1mol/L diluted hydrochloric acid, extracting by using ethyl acetate, reserving an organic layer, washing the organic layer by using saturated saline solution, drying by using anhydrous sodium sulfate, filtering, distilling under reduced pressure to obtain a crude product, and performing column chromatography on the crude product to obtain 6.4g of a white solid (a compound LDX-B-1), wherein the yield is 97.6 percent, and m.p.55-59 ℃.¹H-NMR(600MHz,DMSO-d₆)δ12.47(s,1H),7.68(d,J＝7.9Hz, 2H),7.58(d,J＝16.0Hz,1H),7.38(d,J＝8.1Hz,2H),6.53(d,J＝16.0Hz,1H), 5.82(dd,J＝17.9,10.4Hz,1H),5.10(s,2H),4.95–4.56(m,6H),3.40(d,J＝14.1 Hz,4H),2.90(m,2H),2.29(m,4H),2.11–1.96(m,2H),1.69–1.30(m,9H),0.96(s, 3H)；ESI-MS,m/z 493.4[M+H]⁺,491.4[M-H]^-。

Example 21Preparation of

The compound LDX-B-1, CDI and hydroxylamine hydrochloride are used as raw materials, 60mg of white solid is obtained according to a general method for synthesizing the compound containing hydroxamic acid and N-methylformamide structures, the yield is 58.2 percent, and m.p.59-63 ℃.¹H-NMR (600MHz,DMSO-d₆)δ10.77(s,1H),9.05(s,1H),7.56(d,J＝8.3Hz,2H),7.45(d, J＝15.8Hz,1H),7.38(d,J＝8.0Hz,2H),6.46(d,J＝15.8Hz,1H),5.82(dd,J＝17.9,10.5Hz,1H),5.08(s,2H),4.98–4.54(m,6H),3.43-3.39(m，4H)，2.91(m, 2H),2.29(m,4H),2.02–1.96(m,2H),1.69–1.33(m,9H),0.97(s,3H)；ESI-MS, m/z 508.4[M+H]⁺。

EXAMPLE 22 general procedure for the Synthesis of Compounds containing a aniline Structure

0.20mmol of substituted carboxylic acid, 50.26mg (0.24mmol, 1.2eq.) of N, N-Dicyclohexylcarbodiimide (DCC) and 2.5mg (0.02mmol, 0.1eq.) of 4-Dimethylaminopyridine (DMAP) were weighed out and dissolved in a eggplant-shaped bottle with 3mL of dichloromethane, and then 0.02mL (0.20mmol, 1eq.) of aniline was added dropwise to the reaction system and reacted at room temperature for 8 hours. After the reaction is finished, adding water into the reaction liquid, extracting by dichloromethane, combining and reserving organic layers, washing by saturated saline solution, reserving the organic layers, drying by anhydrous sodium sulfate, filtering, distilling and concentrating under reduced pressure to obtain a crude product, and separating by column chromatography to obtain the target compound.

Example 23Preparation of

The compound LDX-B-1, DCC, DMAP and aniline are used as raw materials, and 95mg of white solid (the compound LDX-B-3) is obtained according to the general synthesis method of the compound containing the aniline structure, the yield is 82.4 percent, and the temperature is between m.p.184 and 188 ℃.¹H-NMR(600MHz,DMSO-d₆)δ10.21(s,1H),7.75–7.64(m,5H),7.63(d,J＝8.0 Hz,2H),7.58(d,J＝15.7Hz,1H),7.42(d,J＝8.1Hz,2H),6.84(d,J＝15.7Hz,1H), 5.82(dd,J＝17.9,10.4Hz,1H),5.11(s,2H),5.00–4.53(m,6H),3.44-3.38(m, 4H)，2.91(m,2H),2.30(m,4H),2.10–1.96(m,2H),1.68–1.22(m,9H),0.97(s, 3H)；ESI-MS,m/z 568.3[M+H]⁺。

Example 24Preparation of

The compound LDX-B-1, oxalyl chloride and o-chloroaniline are used as raw materials, 85mg of white solid is obtained according to the general synthesis method of the compound containing the aniline structure, the yield is 69.5 percent, and the temperature is m.p.58-62 ℃.¹H-NMR(600MHz, DMSO-d₆)δ9.69 (s,1H),7.92(d,J＝8.1Hz,1H),7.64(d,J＝8.0Hz,2H),7.61(d,J＝15.7Hz,1H), 7.52(dd,J＝8.0,1.5Hz,1H),7.43(d,J＝8.2Hz,2H),7.36(ddd,J＝8.6,7.5,1.5Hz, 1H),7.20(td,J＝7.7,1.6Hz,1H),7.10(d,J＝15.8Hz,1H),5.82(dd,J＝17.9,10.4 Hz,1H),5.11(s,2H),4.97–4.55(m,6H),3.40(m,4H),2.91(m,2H),2.30(m,4H), 2.09–1.99(m,2H),1.68–1.22(m,9H),0.97(s,3H)；ESI-MS,m/z 602.4[M+H]⁺, 600.2[M-H]^-。

Example 25Preparation of

To combine withThe LDX-B-1, CDI and methylamine hydrochloride are used as raw materials, 65mg of white solid is obtained according to a general method for synthesizing a compound containing hydroxamic acid and N-methylformamide structures, the yield is 63.3%, and m.p. is 70-74 ℃.¹H-NMR (600MHz,DMSO-d₆)δ8.06(d,J＝5.0Hz,1H),7.55(d,J＝8.0Hz,2H),7.40(d,J ＝16.0Hz,1H),7.38(d,J＝8.1Hz,2H),6.59(d,J＝15.8Hz,1H),5.82(dd,J＝17.9, 10.5Hz,1H),5.08(s,2H),4.96–4.57(m,6H),3.38(s,6H),2.70(d,J＝4.7Hz,3H), 2.29(t,J＝5.2Hz,4H),2.08–1.98(m,2H),1.67–1.38(m,4H),0.96(s,3H)； ESI-MS,m/z 506.4[M+H]⁺。

Example 26Preparation of

Intermediate C2 and sodium hydroxide were used as raw materials to obtain 0.44g of a white solid (compound LDX-C-1) by the general method of ester hydrolysis, yield 90.7%, m.p.90-94 ℃.¹H-NMR(600MHz,DMSO-d₆)δ13.04 (s,1H),7.97(d,J＝8.2Hz,2H),7.85(d,J＝8.0Hz,2H),7.54(d,J＝15.4Hz,1H), 7.40(d,J＝15.4Hz,1H),5.87(dd,J＝17.8,10.6Hz,1H),5.09–4.58(m,6H),3.64 (m,4H),3.05–2.88(m,2H),2.43–2.17(m,4H),2.36(d,J＝24.6Hz,4H),2.15– 1.88(m,2H),2.12–1.98(m,2H),1.68–1.23(m,9H),0.91(s,3H)；ESI-MS,m/z 463.5[M+H]⁺,461.4[M-H]^-。

Example 27Preparation of

Taking compounds LDX-C-1, CDI and hydroxylamine hydrochloride as raw materials, and obtaining 70mg of white solid according to a general method for synthesizing the compounds containing hydroxamic acid and N-methylformamide structures, wherein the yield is 67.8 percent, and m.p.115-119 ℃.¹H-NMR(600MHz,DMSO-d₆)δ11.27(s,1H),9.07(s,1H),7.79(d,J＝8.3Hz,2H), 7.77(d,J＝8.5Hz,2H),7.50(d,J＝15.4Hz,1H),7.34(d,J＝15.4Hz,1H),5.84(dd, J＝17.8,10.6Hz,1H),5.09–4.58(m,6H),3.63(m,4H),2.94(m,2H),2.34(m,4H), 2.13–1.89(m,2H),1.69–1.39(m,9H),,0.97(s,3H)；ESI-MS,m/z 478.3[M+H]⁺。

Examples28Preparation of

85mg of white solid (compound LDX-C-3) is obtained by taking compounds LDX-C-1, DCC, DMAP and aniline as raw materials according to the general synthesis method of the compound containing the aniline structure, the yield is 73.1 percent, and the temperature is m.p.192-196 ℃.¹H-NMR(600MHz,DMSO-d₆)δ10.28(s,1H),7.98(d,J＝8.2Hz,2H),7.87(d,J＝ 8.0Hz,2H),7.73–7.66(m,5H),7.55(d,J＝15.4Hz,1H),7.38(d,J＝15.4Hz,1H), 5.83(dd,J＝17.8,10.6Hz,1H),5.12–4.45(m,6H),3.65(m,4H)2.94(m,2H),2.36 (m,4H),2.12–1.98(m,2H),1.69–1.19(m,9H),0.91(s,3H)；ESI-MS,m/z 538.6[M+H]⁺,536.7[M-H]^-。

Example 29Preparation of

The compound LDX-C-1, oxalyl chloride and o-nitroaniline are used as raw materials, and 70mg of yellow oily matter LDX-C-4' is obtained according to the general synthesis method of the compound containing an o-chloroaniline structure, and the yield is 55.6%. Obtaining light yellow LDX-C-4 by a nitro reduction method, wherein m.p. is 70-73 ℃.¹H-NMR(600MHz,DMSO-d₆)δ9.70(s,1H), 8.00(d,J＝8.0Hz,2H),7.85(d,J＝7.9Hz,2H),7.54(d,J＝15.5Hz,1H),7.41(d,J ＝15.5Hz,1H),7.16(d,J＝7.8Hz,1H),6.98(t,J＝7.6Hz,1H),6.78(d,J＝8.0Hz, 1H),6.60(t,J＝7.6Hz,1H),5.84(dd,J＝17.7,10.8Hz,1H),5.32(t,J＝4.9Hz,2H), 4.98–4.59(m,6H),3.72(s,2H),3.57(s,2H),2.94(m,2H),2.39–1.96(m,6H), 1.73–1.27(m,9H),0.98(s,3H)；ESI-MS,m/z 553.3[M+H]⁺。

Example 30Preparation of

Taking a compound LDX-C-1, oxalyl chloride and o-chloroaniline as raw materials, and obtaining 70mg of white solid according to a general synthesis method of the compound containing the o-chloroaniline structure, wherein the yield is 72.8 percent and the m.p.91-95 ℃.¹H-NMR(600MHz, DMSO-d₆)δ10.11(s,1H),8.01(d,J＝8.0Hz,2H),7.88(d,J＝7.9Hz,2H),7.60(d, J＝7.9Hz,1H),7.55(d,J＝15.8Hz,2H),7.43–7.38(m,2H),7.31(td,J＝7.7,1.7 Hz,1H),5.84(dd,J＝17.6,10.7Hz,1H),5.12–4.59(m,6H),3.72(m,4H),2.92(m, 2H),2.39–2.07(m,6H),1.85–1.26(m,9H),0.98(m,3H)；ESI-MS,m/z 572.3[M+H]⁺。

Example 31Preparation of

The compound LDX-C-1, CDI and methylamine hydrochloride are used as raw materials, 80mg of white solid is obtained according to a general method for synthesizing the compound containing hydroxamic acid and N-methylformamide structures, the yield is 77.8 percent, and m.p.60-64 ℃.¹H-NMR (600MHz,DMSO-d₆)δ8.48(d,J＝4.6Hz,1H),7.84(d,J＝8.5Hz,2H),7.79(d,J ＝8.4Hz,2H),7.50(d,J＝15.4Hz,1H),7.34(d,J＝15.4Hz,1H),5.83(dd,J＝18.0, 10.4Hz,1H),4.97–4.58(m,6H),3.63(m,4H),2.94(m,2H),2.79(d,J＝4.5Hz, 3H),2.34(m,4H),2.13–1.99(m,2H),1.73–1.22(m,9H),0.96(s,3H)；ESI-MS, m/z 476.4[M+H]⁺。

Example 32Preparation of

The intermediate D2 and sodium hydroxide were used as raw materials to obtain 0.45g of a pale yellow solid (compound LDX-D-1) by the general method for ester hydrolysis, and 0.04g of a white solid was obtained with a yield of 69.2%. m.p.110-113 deg.c,¹H-NMR(600 MHz,DMSO-d₆)δ12.05(s,1H),7.67(d,J＝7.9Hz,2H),7.45(d,J＝16.0Hz,1H), 7.11(d,J＝8.1Hz,2H),6.5(brs,1H),6.27(d,J＝16.0Hz,1H),5.82(dd,J＝17.9, 10.4Hz,1H),5.10(s,2H),4.95–4.56(m,6H),3.40(d,J＝14.1Hz,4H),2.90(m, 2H),2.29(m,4H),2.11–1.96(m,2H),1.69–1.30(m,9H),0.96(s,3H)；ESI-MS m/z:492.2[M+H]⁺.

example 33Preparation of

The compound LDX-D-1, CDI and methylamine hydrochloride are taken as raw materials, and the raw materials comprise hydroxamic acid and N-The general synthesis method of the compound with the methyl formamide structure obtains 80mg of white solid with the yield of 77.8 percent, m.p.70-74 ℃,¹H-NMR (600MHz,DMSO-d₆)δ10.5(s,1H),9.0(s,1H),7.56(d,J＝8.3Hz,2H),7.37(d,J ＝15.8Hz,1H),7.38(d,J＝8.0Hz,2H),6.89(d,J＝15.8Hz,1H),6.46(brs,1H),5.82 (dd,J＝17.9,10.5Hz,1H),5.08(s,2H),4.98–4.54(m,6H),3.43-3.39(m，4H)， 2.91(m,2H),2.29(m,4H),2.02–1.96(m,2H),1.69–1.33(m,9H),0.97(s, 3H)；ESI-MS m/z:507.4[M+H]⁺。

example 34

a. Determination of cell growth inhibitory Activity of target Compounds

Tumor cell lines used in the experiments: human acute myeloid leukemia cell line HL-60 was cryopreserved in this laboratory. Culturing HL-60 cells in 10% complete culture medium of fetal bovine serum RMPI1640 at 37 deg.C under 5% CO₂Culturing in an incubator.

Adherent cells: taking human leukemia cell HL-60 cell of logarithmic growth phase, and adjusting cell concentration to 4.5 × 10 with RPMI-1640 complete culture solution containing 10% newborn calf serum⁴A/mL single cell suspension was seeded in 24-well plates at 2mL per well. Adding drugs with different concentrations at 37 ℃ and 5% CO₂After the incubator is incubated for 72 hours, a proper amount of cell suspension is taken and added into an equivalent amount of trypan blue working solution to be mixed, and a proper volume is taken to be counted with a blood counting chamber. The total number of cells in control and dosing wells was recorded. The cell growth inhibition rate calculation formula is as follows: 1- (number of cells in well/number of cells in control well) × 100%, and half the growth inhibitory concentration (GI)₅₀)。

b. Assay of the inhibitory Activity of a target Compound on HDAC enzymes

Enzyme systems used in the experiments: HDACs, HDAC 1. Diluting HDACs and HDAC1 enzyme with buffer solution to 0.5 μ g/μ L and 0.05 μ g/μ L, adding 25 μ L buffer solution into blank well of 96-well plate to remove background, adding 15 μ L buffer solution into control well and 10 μ L buffer solution into sample well, adding 5 μ L enzyme into each well except blank well, mixing 5 μ L medicine with different concentrations, adding 5 μ L0.5mM/L and 0.25mM/LBoc-Lys (AC) -AMC substrate after the medicine and enzyme fully act, mixing, and culturing at 37 deg.C and 5% CO in culture box₂Reacting for 60min, adding 25 μ L stop buffer, and mixingIt should be 15 min. The fluorescence intensity is detected by a microplate reader under the excitation wavelength of 355nm and the emission wavelength of 460 nm. And calculating the inhibition rate according to the ratio of the fluorescence intensity of the dosing hole to the fluorescence intensity of the control hole.

The growth proliferation inhibitory activity of the target compounds on human promyelocytic leukemia cell HL-60 and the inhibitory activity on HDAC1 and HDACs are shown in Table 1.

TABLE 1 growth inhibitory Activity of target Compounds on HL-60 cells and inhibitory Activity of HDACs, HDAC1

^aNot tested.