CN106905191A - A kind of nitrogen mustards compound containing hydroxamic acid group and its production and use - Google Patents

A kind of nitrogen mustards compound containing hydroxamic acid group and its production and use Download PDF

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CN106905191A
CN106905191A CN201710125667.8A CN201710125667A CN106905191A CN 106905191 A CN106905191 A CN 106905191A CN 201710125667 A CN201710125667 A CN 201710125667A CN 106905191 A CN106905191 A CN 106905191A
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cancer
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alkyl
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CN106905191B (en
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袁其朋
谢瑞
邓炳华
李岩
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Beijing University of Chemical Technology
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
    • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
    • C07C259/06—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to hydrogen atoms or to acyclic carbon atoms
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol

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Abstract

一种含有羟肟酸基团的氮芥类化合物及其制备方法和用途涉及药物化学领域。该类化合物具有抑制癌细胞增殖的能力，达到治疗癌症的目的。尤其对抑制人皮肤黑色素瘤A375、人宫颈癌细胞HeLa、人肝癌细胞HepG2、人肺癌细胞A549、人结肠癌细胞HCT116具有优异的抑制癌细胞增殖的活性。该化合物具有下列通式I所示的结构,其中X1、X2、X3、X4和Z如本申请说明书中所定义。

A nitrogen mustard compound containing a hydroxamic acid group and its preparation method and use relate to the field of medicinal chemistry. The compounds have the ability to inhibit the proliferation of cancer cells and achieve the purpose of treating cancer. Especially for human skin melanoma A375, human cervical cancer cell HeLa, human liver cancer cell HepG2, human lung cancer cell A549, and human colon cancer cell HCT116, it has excellent activity of inhibiting cancer cell proliferation. The compound has the structure shown in the following general formula I, wherein X 1 , X 2 , X 3 , X 4 and Z are as defined in the specification of this application.

Description

Nitrogen mustard compound containing hydroximic acid group and preparation method and application thereof
Technical Field
The invention relates to the field of medicines, in particular to a nitrogen mustard compound containing hydroximic acid groups, and a preparation method and application thereof.
Background
Nitrogen mustard drugs, such as chlorambucil, bendamustine hydrochloride, and the like, are alkylating antineoplastic drugs which are the earliest in clinical use and have outstanding curative effects. Such drugs have highly active properties. Because chlorine atoms in chloroethyl are easy to be removed to form carbonium ions, intramolecular cyclization reaction is further carried out to form highly active ethylene imine ions. The nitrogen mustard can react with nucleophilic groups of other macromolecules in vivo, such as carboxyl, amino and sulfhydryl of protein, amino and hydroxyl of nucleic acid, phosphate radical and the like, and is subjected to alkylation modification. Nitrogen mustards readily covalently bind to the seventh nitrogen of guanine, creating cross-links within double strands of DNA or cross-links of different bases within the same strand of DNA. The nitrogen mustard is a cell cycle nonspecific medicine, has poor selectivity when being used as an antitumor medicine, has serious side effects and influences clinical use.
Currently, targeted therapy has become an important direction for cancer therapy, and a single-drug and target-target therapy mode is mostly adopted. However, unlike general diseases, tumors depend not only on the transmission of a receptor or a signaling pathway for their growth and survival, which makes strategies that act solely on a target do not completely kill tumor cells and are prone to drug resistance. Therefore, the combination of multiple drugs is a main means of clinical cancer treatment, and although the expected therapeutic effect can be achieved to a certain extent, the multiple drugs are easy to interact with each other, such as affecting the absorption and metabolism of the drugs, and even if the drugs do not interact with each other, the drugs cannot be combined together with the dosage when the drugs are applied independently.
The search and discovery of effective non-cytotoxic antitumor drugs is a key point of the current antitumor drug chemical research, and in recent years, Histone Deacetylase (HDAC) becomes a new target for designing antitumor drugs. Histone Deacetylases (HDACs) and Histone Acetyltransferases (HATs) are a broad class of mutually antagonistic proteases found in eukaryotic cells, which together regulate acetylation of terminal amino acid residues of histones to equilibrium. Acetylation and deacetylation of histones is a means of regulating gene transcription, and the degree of acetylation of histones affects gene expression by affecting chromatin structure. HDACs are overexpressed in tumor cells, thereby inhibiting the expression of certain cancer suppressor genes. A number of documents indicate that inhibiting HDAC activity is effective in inhibiting tumor cell growth, metastasis and invasion. HDAC inhibitors have become important targets for anti-tumor effects.
The inventor creatively introduces a hydroximic acid structure with HDAC inhibitory activity into a chlorambucil structure, and synthesizes a series of novel nitrogen mustard compounds containing hydroximic acid groups, wherein the nitrogen mustard compounds have the antitumor activity of the nitrogen mustard group and the inhibitory activity on HDAC. The results of evaluation on the activity of in vitro anti-tumor cells, the influence on the monoclonal formation of the tumor cells, the influence on the tumor cell cycle and the influence on the apoptosis of the tumor cells show that the anti-tumor activity of the compound in the patent is improved by 5 to 18 times compared with that of the parent drug chlorambucil.
Disclosure of Invention
The invention relates to design and synthesis of a class of compounds, and aims to overcome the defects in the prior art, the invention provides a nitrogen mustard compound with better antitumor activity and containing hydroximic acid groups, and a preparation method and application thereof. The invention can solve the problems by adopting the following technical scheme:
in a first aspect of the invention, there is provided a compound having the general formula I or a pharmaceutically acceptable salt thereof or a solvate thereof or a prodrug molecule thereof:
wherein,
X1、X2、X3、X4each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, amino, hydroxy, mercapto, carboxyl, alkoxy, cycloalkoxy, haloaryl, alkoxycarbonyl, halogen, cyano, amido, thiocyano, isothiocyanato, ureido, sulfo;
z is selected from alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, said alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl being optionally unsubstituted or substituted with one or more substituents each independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, amino, hydroxy, mercapto, carboxyl, alkoxy, cycloalkoxy, halogen, cyano, nitro, nitroso, sulfo;
in a preferred embodiment of the first aspect of the invention, the compound is preferably selected from the following features:
X1、X2、X3、X4each independently selected from the group consisting of hydrogen, alkyl, alkoxy, cycloalkoxy, alkoxycarbonyl, halogen, cyano, nitro, nitroso, amido.
Z is selected from alkyl, aryl, alkenyl, alkyne, said alkyl, aryl, alkenyl being optionally unsubstituted or substituted by one or more substituents each independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, amino, hydroxy, mercapto, carboxy, alkoxy, cycloalkoxy, halogen, cyano, sulfo;
in a most preferred embodiment of the first aspect of the invention, there are provided the following specific compounds:
table 1 compounds of which the invention is a part of the synthesis
In a second aspect of the present invention, there is provided a pharmaceutical composition comprising: a compound according to the first aspect of the invention or a pharmaceutically acceptable salt thereof or a solvate thereof or a prodrug molecule thereof.
In a third aspect of the present invention, there is provided a use of the compound of the above general formula i and its pharmaceutically acceptable salt or solvate or prodrug molecule in the preparation of a medicament for treating or preventing tumor and cancer.
In a preferred embodiment of the third aspect of the present invention, the tumor or cancer is selected from melanoma, gastric cancer, cervical cancer, ovarian cancer, liver cancer, lung cancer, nasopharyngeal cancer, colon cancer, rectal cancer, lymphatic cancer, blood cancer, bone marrow cancer, brain cancer, skin cancer, bone cancer, nasopharyngeal cancer, pancreatic cancer, kidney cancer, thyroid cancer, prostate cancer, bladder cancer, esophageal cancer, breast cancer.
In a fourth aspect of the invention, there is provided a process for the preparation of a compound of formula I as defined above, which process comprises the steps of:
(a) stirring the compound 1 and diethanol amine at a high temperature to react to obtain a compound 2;
(b) reacting the compound 2 with a chlorinating agent to obtain a compound 3
(c) Reducing the nitro group of the compound 3 to obtain a compound 4
(d) Reacting the compound 4 with the compound 5 under the action of a condensation reagent to obtain a compound 6
(e) Reacting the compound 6 with hydroxylamine hydrochloride and potassium hydroxide to obtain a compound 7
Z, X therein1、X2、X3And X4The first aspect of the invention provides a compound as defined in formula I.
In a preferred embodiment of the fourth aspect of the present invention, the reaction temperature of the reaction step (a) is from 50 to 250 degrees celsius; the chlorination reagent used in the reaction step (b) is selected from thionyl chloride, oxalyl chloride, phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride, sulfuryl chloride and phosphorus pentachloride; the nitro reducing reagent used in reaction step (c) is selected from the group consisting of an active metal and an acid, wherein the active metal is selected from the group consisting of: zinc, iron and tin, the acid being selected from: hydrochloric acid, sulfuric acid, propionic acid, acetic acid, formic acid, phosphoric acid; the condensation reagent used in the reaction step (d) is selected from boric acid, sulfuric acid, N' -dicyclohexylcarbodiimide; the reaction temperature in the reaction step (e) is 0 to 50 ℃.
Drawings
FIG. 1: inhibitory Activity of Compounds on HeLa Nuclear HDAC enzymes
FIG. 2: effect of Compounds on the Monoclonogenic Capacity of tumor cells
Detailed Description
The term "alkyl" as used herein refers to a group consisting of only carbon and hydrogen atoms, and having no unsaturation (e.g., double bonds, triple bonds, or rings), which encompasses a wide variety of possible geometric and stereoisomeric groups. The group is attached to the rest of the molecule via a single bond. By way of non-limiting examples of alkyl groups, mention may be made of the following linear or branched groups: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl and seven further isomers thereof, n-hexyl and sixteen further isomers thereof, n-heptyl and respective isomers thereof, n-octyl and respective isomers thereof, n-nonyl and respective isomers thereof.
The term "cycloalkyl" as used herein refers to a saturated non-aromatic ring system of at least 3 carbon atoms which may be monocyclic, bicyclic, polycyclic, fused, bridged, or spiro. As non-limiting examples of cycloalkyl groups, the following groups may be cited: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl; and fused, bridged, or spiro ring groups formed from two or more of the above-described monocyclic rings via a common side and a common carbon atom.
The term "alkenyl" as used herein refers to a group formed in the presence of one or more double bonds (other than methyl) in the above alkyl group.
The term "alkynyl" as used herein refers to a group formed when one or more triple bonds (other than methyl) are present in the alkyl group described above.
The term "alkoxy" as used herein refers to a group having an oxygen atom attached to the alkyl group and a single bond through the oxygen atom to the rest of the molecule, and encompasses a wide variety of possible geometric and stereoisomeric groups. As non-limiting examples of alkoxy radicals, the following straight-chain or branched radicals may be cited: methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy and further seven isomers, n-hexoxy and further sixteen isomers, n-heptoxy and various isomers, n-octoxy and various isomers, n-nonoxy and various isomers.
The term "aryl" as used herein refers to an aromatic ring system consisting of at least 6 carbon atoms, which may be monocyclic, bicyclic, polycyclic, wherein bicyclic and polycyclic rings may be formed from a single ring by single bond linkages or by fusion. As non-limiting examples of aryl groups, the following groups may be cited: phenyl, naphthyl, anthryl, phenanthryl, indenyl, pyrenyl, perylenyl, pentalenyl, heptalenyl, triphenylenyl, tetracenyl, pentalenyl, pentacenyl, tetrao-phenylene, hexaphenyl, hexacenyl, coronenyl, trinaphthyl, heptenyl, heptaphenyl, egg phenyl, biphenyl, binaphthyl.
The term "heteroaryl" as used herein refers to a 5-14 membered aromatic heterocyclic ring system having one or more heteroatoms independently selected from N, O or S, which may be monocyclic, bicyclic, polycyclic, wherein bicyclic and polycyclic rings may be formed from a single ring by single bond linkages or fused. As non-limiting examples of heteroaryl groups, the following groups may be cited: oxazolyl, isoxazolyl, imidazolyl, furyl, indolyl, isoindolyl, pyrrolyl, triazolyl, triazinyl, tetrazolyl, thienyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzofuryl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzothienyl, benzofuryl, carbazolyl, isoquinolyl, quinazolinyl, naphthyridinyl, purinyl, thiadiazolyl, indolizinyl, phenazinyl, coumarinyl, pyridopyridyl, pyridopyridazinyl, imidazopyridyl, imidazopyridazinyl; and a group formed by the above-mentioned heteroaryl group by a single bond connection or a fusion connection.
The term "heterocyclyl" as used herein, means a non-aromatic 3-18 membered ring system consisting of carbon atoms and heteroatoms independently selected from N, O or S, which ring system may be monocyclic, bicyclic, or polycyclic, and may be fused, bridged, or spiro, and may optionally contain one or more double bonds. As non-limiting examples of heterocyclyl groups, the following groups may be mentioned: acridinyl, benzodioxacyclohexyl, benzopyranyl, chromanyl, dioxolanyl, decahydroisoquinolinyl, indanyl, indolinyl, isoindolinyl, isochromanyl, morpholinyl, piperazinyl, 2-oxopiperazinyl, octahydroindolyl, octahydroisoindolyl, 4-piperidinonyl, dihydroquinolinyl, dihydroisoquinolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, 1- (diphenylmethyl) piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, and combinations thereof.
The term "halogen" or "halo" as used herein refers to fluorine, chlorine, bromine or iodine.
The foregoing and other objects, features and advantages of the invention will be apparent to those skilled in the art from the following detailed description, wherein it is shown and described that the invention is susceptible to more complete understanding by reference to the following examples, and are not to be construed as limiting the scope of the claimed subject matter of the invention to any or all of the following examples and limitations, except as described and illustrated in the accompanying drawings.
Example 1: synthesis of a portion of the Compounds of the invention
Preparation of N- {4- [ bis (2-chloroethyl) amino ] phenyl } -N' -hydroxyoctanediamide (H101).
Step (1): weighing parafluoronitrobenzene (2.80g, 20mmol) and diethanolamine (10.50g, 100mmol) in a 50ml single-neck round-bottom flask, stirring at 118 deg.C, confirming completion of reaction by thin layer chromatography, cooling the reaction solution to room temperature, adding into ice water under stirring, precipitating a large amount of solid, and filtering to obtain yellow solid 3.94g (yield 87%)
Step (2): weighing the yellow solid (2.76g, 12.2mmol) obtained in the step (1), dichloromethane (27.5ml) and pyridine (10ml) into a 100ml three-neck round-bottom flask, placing the whole reaction system in an ice bath to cool to 0 ℃, dropwise adding thionyl chloride (3.27g, 27.5mmol) by using a constant pressure dropping funnel, adding 400ml of dichloromethane into the reaction liquid after confirming the completion of the reaction by using a thin layer chromatography dot plate, placing the mixture in a separating funnel, washing for 3 times by water, drying the dichloromethane phase by using anhydrous magnesium sulfate, filtering and carrying out rotary evaporation to obtain 2.93g of yellow solid (yield 91%)
And (3): weighing the yellow solid (2.71g, 10.3mmol) obtained in the step (2), zinc powder (2.68g, 41.2mmol), methanol (25ml) and water (5ml) into a 100ml three-neck round-bottom flask, dropwise adding dilute hydrochloric acid (10.3ml, 1mol/L) by using a constant-pressure dropping funnel at normal temperature, continuing stirring at normal temperature after dropwise adding, confirming the completion of the reaction by using a thin layer chromatography dot plate, filtering the reaction solution, spin-drying the filtrate, extracting with ethyl acetate, washing with water for three times, drying with anhydrous potassium carbonate, filtering, and spin-drying to obtain 1.87g of gray solid (yield 78%)
And (4): monomethyl suberate (0.58g, 3.1mmol) was weighed into a 100ml single neck round bottom flask, dissolved by adding 15ml dichloromethane, 2 drops of N, N-dimethylformamide were added, the entire reaction system was iced with oxalyl chloride, after the addition, the ice was removed, and after stirring at room temperature for 1 hour, the reaction system was spin-dried to give an oil. Adding gray solid (0.6g, 2.51mmol) obtained in the step (3), tetrahydrofuran (15ml) and triethylamine (0.34g, 3.34mmol) into another 100ml three-neck flask, dissolving the oily substance obtained in the previous step with 5ml of tetrahydrofuran, dropwise adding the tetrahydrofuran solution of the oily substance into the 100ml three-neck flask by using a constant pressure dropping funnel under ice bath, continuing stirring at normal temperature after dropwise adding, after confirming that the reaction is finished by using a thin layer chromatography dot plate, after spin-drying the reaction liquid, extracting with 400ml of ethyl acetate, washing with water for 2 times, 100ml each time, washing with saturated sodium carbonate aqueous solution twice, 100ml each time, then drying the ethyl acetate phase with anhydrous magnesium sulfate, filtering, spin-drying to obtain oily substance, and purifying by using a silica gel column chromatography to obtain solid 0.64g (the yield is 64%)
And (5): potassium hydroxide (2.47g, 44mmol) was weighed into a 100ml single neck round bottom flask, 15ml methanol was added thereto, and the mixture was stirred at room temperatureUntil the potassium hydroxide is completely dissolved, adding hydroxylamine hydrochloride (3.5g, 50.4mmol), placing the reaction system in a water bath at 35 ℃ and stirring for 0.5 hour, then placing the reaction system in an ice bath, adding 0.5g of the solid obtained in the step 4 (0.5g, 1.2mmol), continuing stirring in the ice bath, after confirming that the reaction is completed by a thin layer chromatography dot plate, adding the reaction liquid into the ice water in stirring, immediately precipitating a large amount of solid, filtering the solid, and recrystallizing and purifying acetonitrile to obtain 0.28g of the solid (yield 57%).1H NMR(400MHz,DMSO-d6):＝10.35(s,1H),9.58(s,1H),8.66(s,1H),7.42(d,J＝8.9Hz,2H),6.70(d,J＝8.9Hz,2H),3.70(m,8H),2.24(t,J＝7.4Hz,2H),1.95(t,J＝7.4Hz,2H),1.53(m,4H),1.28(m,4H)；13C NMR(400MHz,DMSO-d6):＝25.0,25.1,28.4,32.2,36.2,41.2,52.3,112.1,120.9,129.7,142.3,170.4ppm；C18H27Cl2N3O3,MS(ES+)m/z:406.39(M+H)+.
Compounds H102 and H134 were prepared in a similar manner to compound M101 except that the starting monomethyl suberate in step (4) was replaced with monomethyl azelate (compound H102) (0.63g, 3.1mmol) and monomethyl adipate (0.50g, 3.1mmol)
The structural data for compounds H102 and H103 are as follows:
n- {4- [ bis (2-chloroethyl) amino ] phenyl } -N' -hydroxyadipicolide (H102)
1H NMR(400MHz,DMSO-d6):＝10.36(s,1H),9.59(s,1H),8.67(s,1H),7.41(d,J＝8.7Hz,2H),6.70(d,J＝8.7Hz,2H),3.70(m,8H),2.24(t,J＝6.7Hz 2H),1.97(t,J＝6.0Hz,2H),1.53(m,4H)；13C NMR(400MHz,DMSO-d6):＝24.8,24.9,32.1,36.0,41.2,52.3,112.1,120.9,129.7,142.3,168.9,170.2ppm；C16H23Cl2N3O3,MS(ES+)m/z:376.12(M+H)+.
N- {4- [ bis (2-chloroethyl) amino ] phenyl } -N' -hydroxynonanediamide (H103)
1H NMR(400MHz,DMSO-d6):＝10.33(s,1H),9.58(s,1H),7.41(d,J＝9.0Hz,2H),6.69(d, J＝9.0Hz,2H),3.70(m,8H),2.23(t,J＝7.3Hz 2H),1.94(t,J＝7.4Hz,2H),1.52(m,4H),1.27(m,6H)；C19H29Cl2N3O3,MS(ES+)m/z:418.17(M+H)+.
Example 2: compound antitumor cell proliferation assay
(1) Experimental cell lines, five cancer cells were selected: human skin melanoma A375, human cervical carcinoma cell HeLa, human liver cancer cell HepG2, human lung cancer cell A549 and human colon cancer cell HCT 116. Before experiment, five kinds of cancer cells are preserved in liquid nitrogen, firstly, the cancer cells are taken out, the temperature of the cancer cells is quickly raised to 37 ℃ in a water bath kettle at 37 ℃, cell sap is centrifuged to remove upper frozen stock solution, the cells are transferred to 24mL cell culture bottles after being resuspended by culture medium, 6mL culture medium containing 5% CO at 37 ℃ is added into each bottle2The culture box (wherein human skin melanoma A375, liver cancer cell HepG2, human cervical cancer cell HeLa, in DMEM culture medium containing 10% fetal calf serum, subcultured after conventional digestion with 0.25% trypsin. human lung cancer cell A549 and human colon cancer cell HCT116 in RPMI-1640 culture medium containing 10% fetal calf serum, subcultured after conventional digestion with 0.25% trypsin), when the cell growth reaches about 70% -80% of the culture bottle, PBS is used to wash off the culture medium, 0.25% trypsin is used to digest the cell to drop off the cell from the culture bottle, fresh culture medium is added for centrifugation, the upper layer culture medium is removed, and 1: and (5) passage 2-3.
(2) When the cells grow stably, cancer cells in logarithmic phase are digested into single cells by pancreatin, and then diluted to (3-4) × 10 by using fresh culture medium4Cell density of one/ml in 96-well plates, 90. mu.L of cell fluid containing 5% CO at 37 ℃ per well2The culture chamber of (2) was incubated for 12 hours, and drugs were added (the synthesized compounds were diluted in advance to a series of different concentrations, and compound solutions of different concentrations were added to a 96-well plate, repeating 3 wells per concentration)And after 72 hours, adding 10 mu L of CCK-8 solution into each well of a 96-well plate, culturing for 1 hour, measuring the absorbance value at the wavelength of 450nm by using a BIO-RAD (biochemical-infrared radiometric) microplate reader, fitting by Graphpad Prism 5 software to obtain an inhibition curve, and finally obtaining the IC50The value is obtained.
Table 2: the compound synthesized by the invention has inhibiting effect on five different cancer cells
The results in table 2 show that: the nitrogen mustard compound containing hydroximic acid groups synthesized by the invention has strong inhibitory activity to five cancer cells, and the compound synthesized by the invention has stronger anti-tumor effect than parent drugs of chlorambucil and bendamustine hydrochloride. The nitrogen mustard compound containing hydroximic acid groups synthesized in the patent has 5 to 18 times stronger drug effect than that of the parent drug of chlorambucil and 7 to 28 times stronger drug effect than that of the parent drug of bendamustine hydrochloride.
Example 3: inhibition assay of HDAC enzymes by Compounds
Using SwissHDAC green fluorescence test kit (product number: BML-AK53R) manufactured by Life Sciences corporation was used to perform HDAC enzyme activity inhibition experiment of HeLa cell nuclear extract, HDAC1 enzyme activity inhibition experiment was performed using kit with product number BML-AK511, HDAC2 enzyme activity inhibition experiment was performed using kit with product number BML-AK512, and HDAC6 enzyme activity inhibition experiment was performed using kit with product number BML-AK 516. The procedure was performed exactly as per kit instructions, 15. mu.L of HDAC enzyme was mixed well with 10. mu.L of test compound, the substrate and the mixture were left at 37 ℃ for 5 minutes to allow the substrate and enzyme to reach the same starting temperature, then 25. mu.L of substrate was added rapidly per well, and the whole 96 wells were put togetherThe plates were incubated at 37 deg.C for 30-60 minutes and then 50. mu.L of stop solution was added to each well. After that, the 96-well plate was left at room temperature for 10 minutes, and the fluorescence intensity was measured with a microplate reader. Obtaining the inhibition rates of the compounds with different concentrations to the HDAC enzyme, obtaining an inhibition curve by Graphpad Prism 5 software fitting, and finally obtaining IC50The value is obtained.
The results of fig. 1 show that: the nitrogen mustard compound containing hydroximic acid groups synthesized by the invention has strong inhibitory activity (the inhibition rate reaches 79.8% at the concentration of 0.625 mu M) on HDAC enzyme of HeLa cell nuclear extract, and the parent compound of chlorambucil has no inhibitory activity on HDAC enzyme of HeLa cell nuclear extract.
Table 3: inhibitory Effect of the Compound synthesized according to the present invention on HDAC enzyme
The results in table 4 show that: the nitrogen mustard compound containing the hydroxamic acid group has strong inhibition activity on HDAC1, HDAC2 and HDAC6, wherein the compound H101 has selective inhibition on HDAC1 and HDAC2 enzymes, the selectivity coefficient of HDAC6/HDAC1 is 2.3, and the selectivity coefficient of HDAC6/HDAC1 is 2.6.
Example 4: monoclonal formation assay of antitumor cell of compound
Human skin melanoma cells A375 in logarithmic growth phase were plated in 6-well plates, 3500 cells were plated per well, the cells were immediately treated with 2. mu. mol/L, 8. mu. mol/L and 16. mu. mol/L chlorambucil and H101, the DMSO-treated group was used as a blank control, after 7 days, the medium in each well was removed, PBS (phosphate buffered saline) was added for gentle washing 2 times, 3.7% aqueous formaldehyde was added for fixation for 20min, and then PBS was used for gentle washing two times, 0.1% aqueous crystal violet was used for staining 30min, PBS was used for 3 times, and the experimental results were observed and photographed.
From FIG. 2, it can be seen that the compound H101 synthesized by the present invention has a stronger anti-tumor cell monoclonal formation ability than the parent drug chlorambucil.
Example 5: experiment for inducing apoptosis of tumor cells by compound
Human skin melanoma cells A375 in logarithmic growth phase are spread on a 6-well plate, 16 ten thousand cells are spread in each well, 12 hours later, chlorambucil and H101 (with the concentration of 2. mu. mol/L, 8. mu. mol/L and 16. mu. mol/L) are added to treat the cells, DMSO is used as a control group, culture medium in each well is collected after 72 hours, cells in each well are collected by trypsinization, centrifuged at 1000rpm for 5 minutes, the centrifuged supernatant is removed, washed twice with PBS, then 100. mu.L of 1 × binding buffer solution, 5. mu.L of Alexa Fluor488annexin and 1. mu.L of propidium iodide (100. mu.g/ml) are added, and incubated for 15 minutes in the absence of light, the group singly stained with Alexa Fluor488annexin and propidium iodide is used as a control group, and samples are detected by a MoFlo XDP flow cytometer.
The results of the induction of tumor cell apoptosis showed that: the compound H101 synthesized by the invention can induce apoptosis of 15.6%, 49.2% and 69.6% of human skin melanoma cells A375 at the concentrations of 2 mu mol/L, 8 mu mol/L and 16 mu mol/L respectively, while the parent drug chlorambucil can only induce apoptosis of 9.9%, 12.9% and 28.9% of human skin melanoma cells A375 at the concentrations of 2 mu mol/L, 8 mu mol/L and 16 mu mol/L. Therefore, the compound synthesized by the invention has obviously stronger capacity of inducing the apoptosis of tumor cells than the parent drug chlorambucil.
Example 6: experiment for blocking tumor cell cycle by compound
A375 human skin melanoma cells in logarithmic growth phase are taken and paved on a 6-well plate, 16 ten thousand cells are paved on each well, chlorambucil and H101 (the concentration is 8 mu mol/L) are added to treat the cells after 12 hours, DMSO is used as a control group, culture medium of each well is collected after 72 hours, cells of each well are collected by trypsinization, the cells are centrifuged for 5min at 1000rpm, supernatant after centrifugation is removed, PBS is used for washing twice, the cells are fixed by 70% ethanol, PBS is used for washing twice, RNase is added for incubation for 30min in a dark place at 37 ℃, and samples are detected by a MoFlo XDP flow cytometer.
The results show that the synthesized compound H101 of the invention can remarkably inhibit the human skin melanoma cells A375 at the G2/M stage under the concentration of 8 mu mol/L, (the ratio of cancer cells in the G2/M stage of a control group treated by DMSO is 12.6 percent, the ratio of cancer cells in the G2/M stage is increased to 62.5 percent after the treatment of 8 mu mol/L compound H101, and the ratio of the human skin melanoma cells A375 at the G2/M stage is only increased to 47.7 percent after the treatment of chlorambucil).

Claims (8)

1.一种药物，为式I所示的化合物或其药学上可接受的盐或其溶剂合物或其前药分子：1. A kind of medicine, is the compound shown in formula I or its pharmaceutically acceptable salt or its solvate or its prodrug molecule: 其中，in, X1、X2、X3、X4各自独立地选自氢、烷基、环烷基、芳基、杂芳基、杂环基、烯基、炔基、氨基、羟基、巯基、羧基、烷氧基、环烷氧基、卤代芳基，烷氧基羰基、卤素、氰基、酰胺基、硫氰基、异硫氰基、脲基、磺基；X 1 , X 2 , X 3 , and X 4 are each independently selected from hydrogen, alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, amino, hydroxyl, mercapto, carboxyl, Alkoxy, cycloalkoxy, halogenated aryl, alkoxycarbonyl, halogen, cyano, amido, thiocyano, isothiocyano, ureido, sulfo; Z选自烷基、环烷基、芳基、杂芳基、杂环基、烯基、炔基，上述烷基、环烷基、芳基、杂芳基、杂环基、烯基、炔基任选不取代或被一个或多个取代基所取代，所述取代基各自独立地选自烷基、环烷基、芳基、杂芳基、杂环基、烯基、炔基、氨基、羟基、巯基、羧基、烷氧基、环烷氧基、卤素、氰基、硝基、亚硝基、磺基。Z is selected from alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, the above-mentioned alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkyne The group is optionally unsubstituted or substituted by one or more substituents independently selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, amino , hydroxyl, mercapto, carboxyl, alkoxy, cycloalkoxy, halogen, cyano, nitro, nitroso, sulfo. 2.根据权利要求1所述的药物，其特征在于，2. The medicine according to claim 1, characterized in that, X1、X2、X3、X4各自独立地选自氢、烷基、烷氧基、环烷氧基、烷氧基羰基、卤素、氰基、硝基、亚硝基、酰胺基。X 1 , X 2 , X 3 , and X 4 are each independently selected from hydrogen, alkyl, alkoxy, cycloalkoxy, alkoxycarbonyl, halogen, cyano, nitro, nitroso, and amido. Z选自烷基、芳基、烯基、炔，上述烷基、芳基、烯基任选不取代或被一个或多个取代基所取代，所述取代基各自独立地选自烷基、环烷基、芳基、杂芳基、杂环基、烯基、炔基、氨基、羟基、巯基、羧基、烷氧基、环烷氧基、卤素、氰基、磺基。Z is selected from alkyl, aryl, alkenyl, and alkyne, and the above-mentioned alkyl, aryl, and alkenyl are optionally unsubstituted or substituted by one or more substituents, and each of the substituents is independently selected from alkyl, Cycloalkyl, aryl, heteroaryl, heterocyclyl, alkenyl, alkynyl, amino, hydroxyl, mercapto, carboxyl, alkoxy, cycloalkoxy, halogen, cyano, sulfo. 3.根据权利要求1至2任意一项所述的药物，其中所述的化合物选自:3. according to the medicine described in any one of claim 1 to 2, wherein said compound is selected from: 4.一种药物组合物，所述药物组合物包含：权利要求1至3中任意一项所述的药物。4. A pharmaceutical composition comprising: the drug according to any one of claims 1 to 3. 5.权利要求1至4中任一项所述的药物或药物组合物在制备用于治疗或预防抗肿瘤、抗癌的药物方面的用途。5. Use of the medicine or pharmaceutical composition described in any one of claims 1 to 4 in the preparation of antitumor and anticancer medicines for treatment or prevention. 6.如权利要求5所述的用途，所述肿瘤为黑色素瘤，癌症选自胃癌、宫颈癌、卵巢癌、肝癌、肺癌、鼻咽癌、结肠癌、直肠癌、淋巴癌、血癌、骨髓癌、脑癌、皮肤癌、骨癌、鼻咽癌、胰腺癌、肾癌、甲状腺癌、前列腺癌，膀胱癌、食管癌、乳腺癌。6. purposes as claimed in claim 5, described tumor is melanoma, and cancer is selected from gastric cancer, cervical cancer, ovarian cancer, liver cancer, lung cancer, nasopharyngeal cancer, colon cancer, rectal cancer, lymphoma, blood cancer, bone marrow cancer , brain cancer, skin cancer, bone cancer, nasopharyngeal cancer, pancreatic cancer, kidney cancer, thyroid cancer, prostate cancer, bladder cancer, esophageal cancer, breast cancer. 7.如权利要求1至3所述化合物的制备方法，其特征在于包括以下步骤：7. The preparation method of the compound as claimed in claims 1 to 3, characterized in that it comprises the following steps: (a)将化合物1与二乙醇胺高温搅拌反应得到化合物2；(a) Compound 1 is stirred and reacted with diethanolamine at high temperature to obtain Compound 2; (b)将化合物2与氯化试剂反应得到化合物3(b) Compound 2 is reacted with a chlorination reagent to obtain compound 3 (c)将化合物3的硝基还原得到化合物4(c) Reducing the nitro group of compound 3 to obtain compound 4 (d)将化合物4与化合物5在缩合试剂作用下反应得到化合物6(d) react compound 4 and compound 5 under the action of condensation reagent to obtain compound 6 (e)将化合物6与盐酸羟胺和氢氧化钾反应得到化合物7(e) Compound 6 is reacted with hydroxylamine hydrochloride and potassium hydroxide to obtain compound 7 其中Z、X1、X2、X3和X4如权利要求1中所定义。wherein Z, X 1 , X 2 , X 3 and X 4 are as defined in claim 1 . 8.如权利要求7所述的制备方法，其特征在于：8. preparation method as claimed in claim 7, is characterized in that: 反应步骤(a)的反应温度为50至250摄氏度；The reaction temperature of reaction step (a) is 50 to 250 degrees Celsius; 反应步骤(b)所用氯化试剂选自:二氯亚砜、草酰氯、三氯氧磷、三氯化磷、五氯化磷、磺酰氯、五氯化磷；Reaction step (b) used chlorination reagent is selected from: thionyl chloride, oxalyl chloride, phosphorus oxychloride, phosphorus trichloride, phosphorus pentachloride, sulfuryl chloride, phosphorus pentachloride; 反应步骤(c)所用硝基还原试剂选自活泼金属与酸的组合，其中活泼金属选自：锌、铁和锡，酸选自：盐酸、硫酸、丙酸、乙酸、甲酸、磷酸；The nitro reducing reagent used in the reaction step (c) is selected from the combination of active metal and acid, wherein the active metal is selected from: zinc, iron and tin, and the acid is selected from: hydrochloric acid, sulfuric acid, propionic acid, acetic acid, formic acid, phosphoric acid; 反应步骤(d)所用缩合试剂选自硼酸、硫酸、N,N'-二环己基碳二亚胺；The condensation reagent used in the reaction step (d) is selected from boric acid, sulfuric acid, N,N'-dicyclohexylcarbodiimide; 反应步骤(e)的反应温度为0至50摄氏度。The reaction temperature of the reaction step (e) is 0 to 50 degrees Celsius.
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