阅读说明：本技术 二苯甲烷-4,4’-二酰腙类化合物及其应用 (Diphenylmethane-4, 4' -diacyl hydrazone compound and application thereof ) 是由 杨鹏 陈岩 于 2018-09-07 设计创作，主要内容包括：本发明属于医药技术领域,涉及二苯甲烷-4,4’-二酰腙类化合物及其应用,具体涉及以二苯甲烷-4,4’-二甲酸为原料,制备的二苯甲烷-4,4’-二酰腙一系列化合物,并测试了它们与DNA的结合、荧光响应性能以及对四种肿瘤细胞生长的抑制性能。结果表明,该系列分子与双螺旋DNA均具有较好的结合,且对AT碱基对数量有明显的荧光响应：表现为375nm处出现较强的荧光,520nm处荧光猝灭；此外,该类化合物也具有较高的体外抗肿瘤活性。可以用于制备抗肿瘤药物。(The invention belongs to the technical field of medicines, relates to a diphenylmethane-4, 4 ' -diacylhydrazone compound and an application thereof, and particularly relates to a series of diphenylmethane-4, 4 ' -diacylhydrazone compounds prepared by taking diphenylmethane-4, 4 ' -dicarboxylic acid as a raw material, and tests the combination of the compounds and DNA, the fluorescent response performance and the inhibition performance on the growth of four tumor cells. The result shows that the series of molecules and the double-helix DNA have better combination and obvious fluorescent response to the number of AT base pairs: the fluorescence is shown to be stronger at 375nm and quenched at 520 nm; in addition, the compounds also have high in-vitro anti-tumor activity. Can be used for preparing antitumor drugs.)

1. Diphenylmethane-diacyl hydrazone compounds with the following general structure and pharmaceutically acceptable salts or hydrates thereof,

x is nitrogen or carbon atom;

y is a nitrogen or carbon atom;

z is a nitrogen or carbon atom;

r is hydrogen, amino, halogen, nitro, hydroxyl, C1-C6 ester group, substituted or unsubstituted C1-C4 amine, substituted or unsubstituted 5-10 membered heterocyclyl or heteroaryl, said heterocyclyl or heteroaryl containing 1-3 heteroatoms N, O or S, said substituents being: C1-C10 alkyl, C1-C10 alkoxy, C1-C10 alkylamino;

n is 0 to 5.

2. The diphenylmethane-bisacylhydrazone compound of claim 1, or a pharmaceutically acceptable salt or hydrate thereof,

wherein the content of the first and second substances,

r is hydrogen, amino, nitro, hydroxyl, C1-C6 ester group, halogen, substituted or unsubstituted C1-C4 amine, substituted or unsubstituted 5-10 member heterocyclic group containing 1-3 heteroatoms N, O or S, and the substituents are: C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino.

3. The diphenylmethane-bisacylhydrazone compound of claim 1 or 2, or a pharmaceutically acceptable salt or hydrate thereof,

wherein the content of the first and second substances,

r is hydrogen, hydroxyl, C1-C6 ester group, halogen, substituted or unsubstituted C1-C4 amine, substituted or unsubstituted 5-6 membered heterocyclic group containing 1-3N atoms, the substituents are: C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino.

4. The diphenylmethane-bisacylhydrazone compound of any one of claims 1 to 3, or a pharmaceutically acceptable salt or hydrate thereof,

wherein the content of the first and second substances,

when X, Y or Z is N atom, R is hydrogen, methyl, ethyl, tert-butyl, dimethylamino, diethylamino, chlorine, bromine, iodine, fluorine, BOC protected piperazine, piperazine-N hydrochloride, methoxyacyl, ethoxyacyl, piperazine-N bromate, piperazine-N iodate;

when X, Y and Z are C atoms at the same time, R is hydrogen, methyl, ethyl, tertiary butyl, dimethylamino, diethylamino, chlorine, bromine, iodine, fluorine, BOC protected piperazine, 4-piperazine-N hydrochloride, methoxyacyl and ethoxyacyl.

5. The following diphenylmethane-diacyl hydrazone compounds and pharmaceutically acceptable salts or hydrates thereof:

6. the diphenylmethane-bisacylhydrazone compound of any one of claims 1 to 5, wherein the pharmaceutically acceptable salt is a salt formed between the compound and an acid, the acid is an organic acid or an inorganic acid, the inorganic acid is hydrochloric acid, sulfuric acid, hydrobromic acid or phosphoric acid, the organic acid is an organic acid selected from acetic acid, citric acid, oxalic acid, tartaric acid, benzoic acid or malic acid, and the hydrate has a real number of crystal water in the range of 0 to 4.

7. A process for the preparation of diphenylmethane-4, 4' -bisacylhydrazone compounds as claimed in claims 1 to 3, comprising the steps of: the diphenylmethane-4, 4 '-dicarboxylic acid firstly generates esterification reaction with ethanol, then generates ammonolysis reaction with hydrazine hydrate to prepare the diphenylmethane-4, 4' -dihydrazide, and finally generates aldehyde-amine condensation reaction with aldehyde to prepare the target acylhydrazone compound.

Wherein n and R are as defined in claim 1.

8. A pharmaceutical composition comprising the diphenylmethane-bisacylhydrazone compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier.

9. Use of the diphenylmethane-bisacylhydrazone compound of any one of claims 1 to 6, or a pharmaceutically acceptable salt or hydrate thereof, or the composition of claim 8, in a fluorescent molecular probe technology.

10. Use of the diphenylmethane-bisacylhydrazone compound or the pharmaceutically acceptable salt or hydrate thereof according to any one of claims 1 to 6 or the composition according to claim 8 in the preparation of an antitumor drug.

Technical Field

The invention belongs to the technical field of biology, and relates to a diphenylmethane-4, 4 '-diacylhydrazone compound, a preparation method and application thereof, in particular to diphenylmethane-4, 4' -disalicylaldehyde-diacylhydrazone, a series of derivatives thereof, application thereof in a fluorescent molecular probe technology, and antitumor activity of the compound.

Background

Common DNA fluorescent probes are divided into quaternary ammonium salts and non-quaternary ammonium salts, wherein the quaternary ammonium salts cannot stain living cells due to poor cell membrane permeability, and the current widely-used DNA fluorescent probes only have Hoechst. Diphenylmethane-4, 4-diacyl hydrazone compounds are a DNA fluorescent molecular probe with a brand new structure. In the absence of the double helix DNA, the two benzene ring planes at both ends overlap each other, so that there is a fluorescence (eximer) peak at 520 nm. When double-helix DNA exists, the molecules of the compound are combined with the minor groove of the DNA, hydrogen bonds are formed between phenolic hydroxyl groups on the compound and hydroxyl oxygen which is not combined on a T basic group, the overlapped conformation of salicylaldehyde parts at two ends of the compound is released, the original push-pull electron system is changed, the fluorescence at 520nm is quenched, and the fluorescence at 375nm is enhanced strongly.

The obvious difference of fluorescence before and after the diphenyl methane diacyl hydrazone DNA fluorescent probe is combined with the double-helix DNA makes the diphenyl methane diacyl hydrazone DNA fluorescent probe have the potential of becoming a novel DNA fluorescent probe. Meanwhile, the fluorescent probe is a non-quaternary ammonium salt, is not limited by the permeability of cell membranes, and can possibly dye living cells, thereby bringing a new choice for the in vitro staining technology of living cells.

The acylhydrazone bond of the acylhydrazone compound is hydrolyzed under the acidic environment of the tumor cells to generate the hydrazide which has stronger effect of inhibiting proliferation on the tumor cells, so the acylhydrazone compound can be used as an anti-tumor active group for application.

Diphenylmethane-4, 4' -disalicylaldehyde-diacylhydrazone

Disclosure of Invention

The invention provides a series of diphenylmethane-4, 4' -diacyl hydrazone compounds, which have a chemical structural general formula as follows:

x is nitrogen or carbon atom;

y is a nitrogen or carbon atom;

z is a nitrogen or carbon atom;

r is hydrogen, amino, nitro, hydroxyl, C1-C6 ester group, halogen, substituted or unsubstituted C1-C4 amine, substituted or unsubstituted 5-10 membered heterocyclyl or heteroaryl, said heterocyclyl or heteroaryl containing 1-3 heteroatoms N, O or S, said substituents being: C1-C10 alkyl, C1-C10 alkoxy, C1-C10 alkylamino;

n is 0 to 5.

The invention preferably selects diphenylmethane-4, 4' -diacyl hydrazone compounds with the following structure,

wherein the content of the first and second substances,

x is nitrogen or carbon atom;

y is a nitrogen or carbon atom;

z is a nitrogen or carbon atom;

r is hydrogen, amino, nitro, hydroxyl, C1-C6 ester group, halogen, substituted or unsubstituted C1-C4 amine, substituted or unsubstituted 5-10 member heterocyclic group containing 1-3 heteroatoms N, O or S, and the substituents are: C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino;

n is 0 to 5.

The invention preferably selects diphenylmethane-4, 4' -diacyl hydrazone compounds with the following structure,

wherein the content of the first and second substances,

x is nitrogen or carbon atom;

y is a nitrogen or carbon atom;

z is a nitrogen or carbon atom;

r is hydrogen, hydroxyl, C1-C6 ester group, halogen, substituted or unsubstituted C1-C4 amine, or substituted or unsubstituted 5-6 membered heterocyclic group containing 1-3 heteroatoms N, O or S, said substituents being: C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino;

n is 0 to 2.

The invention preferably selects diphenylmethane-4, 4' -diacyl hydrazone compounds with the following structure,

wherein the content of the first and second substances,

x is nitrogen or carbon atom;

y is a nitrogen or carbon atom;

z is a nitrogen or carbon atom;

r is hydrogen, hydroxyl, C1-C6 ester group, halogen, substituted or unsubstituted C1-C4 amine, substituted or unsubstituted 5-6 membered heterocyclic group containing 1-3N atoms, the substituents are: C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino;

n is 0.

The invention preferably selects diphenylmethane-4, 4' -diacyl hydrazone compounds with the following structure,

when X, Y or Z is N atom, R is hydrogen, methyl, ethyl, tert-butyl, dimethylamino, diethylamino, chlorine, bromine, iodine, fluorine, BOC protected piperazine, piperazine-N hydrochloride, methoxyacyl, ethoxyacyl, piperazine-N bromate, piperazine-N iodate;

when X, Y and Z are C atoms at the same time, R is hydrogen, methyl, ethyl, tertiary butyl, dimethylamino, diethylamino, chlorine, bromine, iodine, fluorine, BOC protected piperazine, 4-piperazine-N hydrochloride, methoxyacyl and ethoxyacyl.

The invention preferably selects diphenylmethane-4, 4' -diacyl hydrazone compounds with the following structural general formula:

the compound also comprises pharmaceutically acceptable salts formed by the compound shown in the structural formula and hydrates thereof, and the pharmaceutically acceptable salts comprise salts formed by the compound and acid. The acid can be inorganic acid of hydrochloric acid, sulfuric acid, hydrobromic acid, phosphoric acid or organic acid selected from acetic acid, citric acid, oxalic acid, tartaric acid, benzoic acid, malic acid. The number of crystal water of the hydrate is any real number in 0-4.

The preparation method of the diphenylmethane-4, 4' -diacyl hydrazone compound comprises the following steps:

(1) preparation of diphenylmethane-4, 4' -dihydrazide

The diphenylmethane-4, 4' -dicarboxylic acid firstly undergoes an esterification reaction with ethanol and then undergoes an ammonolysis reaction with hydrazine hydrate to prepare a target product:

(2) preparation of diphenylmethane-4, 4' -diacyl hydrazone

The diphenylmethane-4, 4' -dihydrazide and aldehyde are subjected to an aldehyde-amine condensation reaction, and only an esterification reaction is carried out to prepare the target product.

Specifically, the preparation method of the diphenylmethane-4, 4' -diacyl hydrazone compound comprises the following steps:

(1) preparation of diphenylmethane-4, 4' -dihydrazide

1.1 dissolving diphenylmethane-4, 4' -dicarboxylic acid in absolute ethyl alcohol, dripping thionyl chloride under ice bath condition, transferring to oil bath, heating and stirring. After the reaction, the temperature was returned to room temperature. The reaction solution was poured into dichloromethane, washed twice with saturated sodium bicarbonate solution and once with clear water. After dichloromethane was dried over anhydrous sodium sulfate, it was distilled under reduced pressure to obtain compound a.

Dissolving compound A in water methanol, adding hydrazine hydrate, stirring at room temperature, centrifuging, collecting precipitate, and washing twice with anhydrous methanol, dichloro and petroleum ether to obtain compound B.

(2) Preparation of diphenylmethane-4, 4' -disalicylaldehyde-diacylhydrazone compounds

Dissolving the compound B and salicylaldehyde in a mixed solution of methanol and diethyl ether, stirring until the reaction is finished, returning to room temperature, and centrifuging. Washing with anhydrous methanol, dichloromethane and petroleum ether twice to obtain compound 1.

Dissolving the compound B, o-hydroxybenzaldehyde, m-hydroxybenzaldehyde, 2-pyridylaldehyde, 3-pyridylaldehyde, 4-pyridylaldehyde, 5-bromosalicylaldehyde, 4-fluorosalicylaldehyde, 4-N, N-diethylaminosalicylaldehyde, methyl 3-formyl-4-hydroxybenzoate and tert-butyl 4- (3-formyl-4-hydroxyphenyl) piperazine-1-carboxylate in a mixed solution of methanol and diethyl ether, stirring until the reaction is finished, recovering to room temperature, and centrifuging. Washing with anhydrous methanol, dichloromethane and petroleum ether twice to obtain compounds 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11.

Dissolving the compound 11 in dichloromethane, dripping concentrated hydrochloric acid, and stirring at room temperature. After the reaction, white solid particles are separated out and adhered to the wall of the reaction bottle. Washing twice with dichloromethane and petroleum ether respectively to obtain a brown yellow powdery solid pure product, and obtaining the compound 12.

The diphenylmethane-4, 4' -diacyl hydrazone compound can be used in fluorescent molecular probe technology, can be used for detecting nucleic acid, and can also be used for preparing antitumor drugs.

Specifically, the diphenylmethane-4, 4' -diacylhydrazone compound can be used as a small-molecule fluorescent probe, and the small-molecule fluorescent probe can be combined with double-helix DNA and generate fluorescence selectivity of AT base pairs.

The method comprises the following steps:

(1) ultraviolet detection

Dissolving the compound in a buffer solution, gradually adding a certain proportion of DNA solution to obtain an ultraviolet absorption value of 230-500 nm, and substituting the data into a 1:1 binding equation to obtain binding energy K.

(2) Fluorescence detection

Setting the spectrum range at 300-650 nm and the exciting wavelength at 299nm, and gradually adding a certain proportion of DNA solution into a compound buffer solution with a certain concentration to obtain the fluorescence absorption spectrum. By [ C ]_DNA/C_{Compound (I)}]As the abscissa, with F₃₇₅/F₅₂₀The graph is plotted on the ordinate, and a fluorescence change rate graph with a detection limit of 0.26. mu.M can be obtained.

The diphenylmethane-4, 4' -diacyl hydrazone compound has good activity of inhibiting the proliferation of tumor cells and good development prospect in preparing antitumor drugs.

Drawings

FIG. 1 is a dibenzo-p-toluidineUV titration spectrum of alkane-4, 4' -disalicylaldehyde-diacylhydrazone (Compound 1) with DNA (AT sequence): the concentration of the compound was 20. mu.M, [ C ]_DNA/C_{Compound (I)}]Starting at 0 and increasing to 4.5 per 0.3 equivalents; the peak decrease continues to decrease as DNA increases.

FIG. 2 shows the UV titration spectrum of diphenylmethane-4, 4' -disalicylaldehyde-bisacylhydrazone (Compound 1) with DNA (GC sequence): the concentration of the compound was 20. mu.M, [ C ]_DNA/C_{Compound (I)}]Starting at 0 and increasing to 4.5 per 0.3 equivalents; the peak decrease continues to decrease as DNA increases.

FIG. 3 shows the fluorescence titration spectra of diphenylmethane-4, 4' -disalicylaldehyde-bisacylhydrazone (Compound 1) with DNA (AT sequence): the concentration of the compound was 10. mu.M, [ C ]_DNA/C_{Compound (I)}]Starting at 0 and increasing to 4.0 every 0.25 equivalents; the peak at 525nm decreased and the peak at 375nm increased with increasing DNA.

FIG. 4 is a graph showing the fluorescence titration spectra of diphenylmethane-4, 4' -disalicylaldehyde-bisacylhydrazone (Compound 1) with DNA (GC sequence): the concentration of the compound was 10. mu.M, [ C ]_DNA/C_{Compound (I)}]Starting at 0 and increasing to 4.0 every 0.25 equivalents; the peak at 525nm decreased and the peak at 375nm did not change with increasing DNA.

Detailed Description