CN101648921B - Benzamide compound used as histone deacetylase inhibitor and application thereof - Google Patents

Abstract

The invention discloses a compound in the formula (I) or pharmaceutically acceptable salt thereof. Ar is aryl or heterocyclic group and is substituted by optional one or more of the following groups: C1-8 alkyl, C1-8 alkoxy, halogen, nitryl, C1-8 aminoalkyl, C1-8 alkyl amino group, C1-8 thio-alkyl, C1-8 halogenated alkyl, C1-8 halogenated alkoxy, C1-8 ester group, phenyl or heterocyclic group. n is an integer between 0 and 8. The drugs prepared by the compound can be used for treating solid tumors or leukemia correlating with cell differentiation or proliferation.

Description

Be used as benzamide compounds of NSC 630176 and uses thereof

Technical field

The present invention relates to a kind of synthetic preparing technical field of NSC 630176, a kind of benzamide compounds that is used as NSC 630176 of specific design and uses thereof.

Background technology

The formation that comprises the tumour numerous disease is a quite complicated process, relates to the variation how range gene responds internal and external environment, thereby is implemented in the regulation and control of expressing on time and the space.The field, a forward position of rising in the genetics in recent years--epigenetics (Epigenetics) provides new approaches for answering these problems.The molecular basis of epigenetic is mainly concerned with two aspects: one is the modification that methylates at DNA, and another is the acetylation modification at the chromatin histone.Except dna sequence dna as the genetic coding, apparent gene mechanism is the most basic adjusting factor of genetic expression and protein synthesis subsequently.In eukaryotic cell, in the G0 phase, DNA is closely filled by nucleosome, so that DNA can not be near transcribing element.Acetylation of histone reaction is a conclusive apparent gene process, can reinvent transcribe element and DNA near ability, thereby initiation genetic expression.There is a large amount of evidences to show now; the chromatin albumen reconstruction of surrounding DNA is the basic mechanism of gene regulating epigenetics; this comprises histone afterbody reversible post transcriptional modificaiton, as: the methylating of lysine residue acetylize, lysine residue and arginine residues, the phosphorylation of serine residue, the ubiquitinization and the ubiquitin-likeization of lysine residue.Wherein, the acetylation modification of histone is the most general.The acetylation modification of histone plays a role in gene expression regulation.Histone acetylizing in vivo occurs on the conservative Methionin amino of protein N terminal heredity, but the acetylation modification of histone H 3 and H4 is more extensive than H2A and H2B.The acetylizad important site of histone H 3 is Lys9 and Lysl4, and the acetylize site of histone H 4 is Lys5, Lys8, Lysl2 and Lysl6.

(Histone deacetylase is HDAC) with histone acetyl based transferase (histone aceyl transferase, HAT) acetylize of decision histone for histon deacetylase (HDAC).The acetylize level of core histones is the result of HDACs and the common regulation and control of HATs.HDAC is the catalytic subunit of polyprotein matter mixture, participates in histone and nonhistones protein deacetylation.HDAC mediation nucleosomal structure changes and regulatory gene is expressed, and participates in cell cycle progression and differentiation, and relevant with development with the generation of multiple disorders such as cancers, acute myeloid leukaemia, virus and infection etc.Generally speaking, it is relevant with the active enhancing of genetic transcription that the acetylation of histone level strengthens, and that the acetylize level is crossed is low relevant with the genetic expression inhibition.

By chemically modified, the configuration of nucleosome is changed to histone.Modification to histone occurs on the conservative Methionin amino of protein N terminal heredity.These lysine residues are positively charged under the physiological pH condition, and the negative oxygen ion of positively charged lysine residue and DNA phosphate radical skeleton interacts, and makes DNA be surrounded closelyr by histone.HAT partly transfers to acetyl-CoA (acetyl-CoA) ethanoyl the epsilon-amino group of specific Lys residue on the core histones N-terminal.Positive charge on the amino is eliminated, and at this moment the negative charge that had of dna molecular itself helps the expansion of DNA conformation, and the structure of nucleosome becomes lax.This lax structure has promoted contacting of transcription factor and collaborative transcription factor and dna molecular, so acetylation of histone can activate the transcription of specific gene.HDAC then removes the ethanoyl on the histone Lys residue; recover the positive polarity of histone, positively charged Lys residue is electrical opposite with dna molecular, has increased the magnetism between DNA and the histone; make the not accessible transcriptional regulatory element of promotor, thereby suppress to transcribe.

HDAC and HAT directly do not combine with DNA.HAT and other acetylation of histone enzymes and transcription factor form mixture, and HDAC is as being raised with other mixture of transcribing common inhibition.The activity of histone is regulated and control by post transcriptional modificaiton.The phosphorylation reaction of HDAC1 and HDAC2 causes the active increase of HDAC.HDAC not only makes histone generation deacetylation, and can make nonhistones generation deacetylation, as p53 and GATA-1.Acetylizad p53 shows that itself and target sequence have stronger avidity.

Many researchs have confirmed that the high/low acetylize of histone plays an important role in tumour takes place.On the one hand, it is found that acetylation of histone is relevant with the metamorphosis of tumour cell with deacetylated variation; On the other hand, HAT (for example p300/CBP, pCAF, ACTR etc.) or HDAC can interact with some oncogenes and cancer suppressor gene (cancer suppressor genes such as P53, Rb, p16, p21, p27 being arranged as what participate in cell cycle regulating) product, thereby modify or mediate the effect of these products pair genetic transcription relevant with cytodifferentiation and cell proliferation.By restraining effect to deacetylation, can cause chromatin to cross acetylize, promote the gene activation of cancer cells, thereby cause cytodifferentiation or death.

The antitumor mechanism of hdac inhibitor depends on the regulation and control of genetic expression, in a large amount of bodies and experiment in vitro confirm its antitumor mechanism have following some:

Hdac inhibitor can inducing apoptosis of tumour cell, its therapeutic action is optionally inducing apoptosis of tumour cell, a large amount of clinical experiment results and clinical preceding experimentation on animals prove that hdac inhibitor can be realized selectivity between normal cell and tumour cell.Dose limit toxicity comprises, platelet injury, feels sick, makes symptoms such as fatigue, but under many circumstances, these side effects can be controlled clinically.Experiment in vitro shows, transformant for the reaction of hdac inhibitor mainly by the type decided of cell, rather than by the structure and the concentration decision of hdac inhibitor.Hdac inhibitor can produce cytotoxicity to the tumour cell of hyperplasia and stagnation, and normal cell can be resisted the necrocytosis of hdac inhibitor inductive.Transformant can produce different phenotypic alternations to hdac inhibitor, comprises that the G1 phase stagnates, the caspase of eventually end differentiation, plastosome mediation relies on accent is died.In normal cell and transformant, all there is acetylation of histone, so the cytotoxicity that appears in the transformant is not because due to the active difference of hdac inhibitor in them.But for different transformants, the mechanism of hdac inhibitor inducing cell death is different.

Hdac inhibitor can also block the cell cycle and promote cytodifferentiation.Hdac inhibitor is that the cell cycle arrest on the G1/S border of this effect and retinoblastoma albumen (pRb) and associated protein mediation is closely related because it has the inducing cell differentiation and found at first.The rise of hdac inhibitor by CDKN1A (p21waf1/cip1) or the downward modulation by cyclin, thereby at G1/S border inducing cell cycle arrest, the cessation of growth cessation of hdac inhibitor mediation changes by directly influencing the chromatin recurring structure, causes specific site genetic expression.Although 2% of the not enough gene number of affected gene has special effect.

Hdac inhibitor can also be stagnated with mediation G2/M mutually by activating the G2 associated card.Under given conditions, losing of G2 associated card can be used for determining the apoptotic sensitivity degree of tumour cell to hdac inhibitor.The G2 outpost of the tax office relevant with hdac inhibitor may and arm between heterochromatic highly acetylated relevant, the forfeiture at the G2 outpost of the tax office can cause the segmentization of chromosomal separation and nuclear.In addition, in the reaction of study of tumor suppressor genes Rb mediation, hdac inhibitor also plays an important role.

A large amount of experiments show that different tumor necrosis factor receptor super family members and its aglucon all are activated after hdac inhibitor is handled.Studies have shown that, hdac inhibitor inductive apoptosis with induce one or more death receptors and/or aglucon relevant.Yet whether the signal conduction by death receptor is necessary for hdac inhibitor inductive apoptosis, and current still the existence disputes on.

Some independently the strong Support Line plastochondria of result of study apoptosis pathway in the vital role of the death of neoplastic cells of hdac inhibitor mediation.They think that BCL2 or BCL-XI have blocked apoptosis pathway, have suppressed different hdac inhibitor mediated Apoptosis.But how on earth hdac inhibitor excites inner cell mechanism of apoptosis also to require further study.Think excitation mechanism and selectively activate or to induce BH3-only albumen relevant with regulation activity oxyradical (ROS) at present.

Hdac inhibitor can suppress tumor-blood-vessel growth, and this reduces relevant with short angiogenesis gene expression degree.Most of hdac inhibitors can be reduced vascular endothelial growth factor (VEGF), Prostatropin (bFGF), HIF-1 α (HIF1 α), angiogenin, inner membrance endothelium kinases-2 (TIE2) and endothelial nitric oxide synthase synthetic enzyme (eNOS).Hdac inhibitor can be reduced the expression of Chemokine Receptors 4 (C-X-C motif CXCR4), it is very important that Chemokine Receptors is circulated to neovascularity generation site for going back to the nest of bone marrow precursor with endotheliocyte, and these two kinds of situations all exist in non-conversion endotheliocyte and tumor cell line.In addition, hdac inhibitor can suppress the differentiation of endothelial progenitor cells.Above result of study shows that hdac inhibitor can pass through to change angiogenesis gene, thereby suppresses the generation of blood vessel, particularly for infantile tumour, can block nutrition supply, suppresses the diffusion of tumour.

Evidence shows more and more, and hdac inhibitor can strengthen the antineoplastic immune ability thereby perhaps improve activity of immune cells by directly acting on malignant cell.I and the II proteinoid of hdac inhibitor by raising main histocompatibility complex (MHC), promote the expression of adhesion molecule (as CD40, CD80, CD86) and cell-cell adhesion molecule 1 (ICAM1) to strengthen the immunogenicity of tumour cell altogether.Nearest result of study shows that the MHC I class chain associated molecule MICA on hdac inhibitor inducing tumor cell surface and MICB express.SAHA can as tumor necrosis factor-α (TNF α), plain-1 (IL-1) of white (cell) Jie and interferon-(IFN γ), induce acute graft versus host disease (GVHD) by suppressing the promoting immunity factor.Hdac inhibitor has regulating effect to the various kinds of cell factor, might be as the immunological rejection effect of novel immunological reagent treatment autoimmune disorder and histoorgan transplanting.

Hdac inhibitor is to the therapeutic action that has of nervous system disorders.The small molecules hdac inhibitor can be to the neuronic balance of transcribing, regulate cytoskeletal function, influence immunne response and strengthen the proteolytic degradation path, be applied to clinical treatment human brain disease at present, as Rubinstein-Taybi syndromes, Rett syndrome, Friedreich ataxia, Huntington Chorea and encephalitis periaxialis scleroticans.

In addition, hdac inhibitor also has radiosensitizing effect.

Had been found that the NSC 630176 of multiple structure type at present, comprised (1) short chain fatty acid and salt thereof, as butyric acid and isovaleric acid sodium; (2) hydroxamic acid is as SAHA (Vorinostat) and LBH-589 (Panobinostat); (3) benzamide is as MS-275 (Entinostat) and MGCD0103; (4) cyclic tetrapeptide is as trapoxin and FK-228; Also has the NSC 630176 as pharmacophore such as ethyl ketone, trifluorumethylketone, keto-amide, 2-amino aniline, mercaptan and acetyl derivatives thereof.Yet these drug candidate major parts also rest on the preclinical study state, and a part has entered clinical study.Wherein the SAHA structure obtained FDA approval listing suc as formula shown in the X in 2006, only was approved for treatment skin T-cell lymphoma--treat a kind of rare cancer.

Yet, up to the present, it is also fewer that the SAHA medicine can better be treated the clinical evidence of solid tumor, people are in detail and the abundant deep layer mechanism of understanding SAHA medicine and treatment thereof and unusual to the effect of different tumours still, so this medicine is in Application Areas can only rest on experience and limited zone.In addition, the SAHA medicine has the side reaction of heart aspect.And show that by structural analysis of X ray crystalline diffraction and structure activity relationship SAHA has 3 zones: melts combine district (metal binding), form hydrogen bond with the amino-acid residue of enzyme active sites, and and Zn ²⁺Chelating; Connect sequence (Iinker), occupy the throat of enzyme, the length decision melts combine district and the Zn of its chain ²⁺Bonding state; Surface cog region (surface recognition) combines with the amino-acid residue in the enzyme active sites outside, and the decision inhibitor molecules is to the identification and the combination degree of enzyme, assistant metal land and Zn ²⁺Chelating.Owing to directly act on zine ion, make it have certain toxic action.

MS-275 is a kind of derivative of benzamide suc as formula shown in the XI, is effective histone deacetylase inhibitor, shows anti-tumor activity in preclinical study.Experiment confirm is arranged, can suppress the activity of the intravital histone deacetylase of acute myeloid leukemia patient in late period effectively, and not have significant cardiac toxic.

And current tumor incidence is more and more higher, has pressed for a kind of medicine for treating tumor thing with low toxicity, also needs more alternative NSC 630176 medicines.The present invention comes therefrom.

Summary of the invention

The object of the invention is to provide a kind of novel inhibitors of histone deacetylase, problem such as solved in the prior art that medicine inhibitors of histone deacetylase kinds such as treatment tumour and leukemia are less, curative effect is definite inadequately and toxicity is bigger.

In order to solve these problems of the prior art, technical scheme provided by the invention is:

The compound of a kind of formula (I) or its pharmacy acceptable salt,

Wherein, Ar is for for aryl or heterocyclic radical, and it is optional to be selected from following group and to replace by one or more: C1-8 alkyl, C1-8 alkoxyl group, halogen, nitro, C1-8 aminoalkyl group, C1-8 alkylamino, C1-8 alkylthio, C1-8 haloalkyl, C1-8 halogenated alkoxy, C1-8 ester group, phenyl or heterocyclic radical; N is selected from 0～8 integer.

Preferably, described aryl is a phenyl.

Preferably, described heterocyclic radical is selected from pyridine, thiophene, furans, pyrroles or imidazoles.

Preferably, described aryl or heterocyclic radical are optional is selected from following group and replaces by one or more: C1-8 alkyl, halogen, nitro or C1-8 haloalkyl.

Preferably, described aryl or heterocyclic radical are optional is selected from following group and replaces by one or more: the haloalkyl of the alkyl of C1-4, halogen, nitro or C1-4.

Preferably, n is 6.

Each group among the present invention generally has following meaning:

Term " alkyl " refers to that straight or branched C1-n alkyl then represents the saturated aliphatic radical of 1-n carbon atom, comprise straight chain and branched group (for example " C1-20 alkyl ", be meant that this group is an alkyl, and the carbochain amount of carbon atom of alkyl is between 1～20, promptly contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms etc., until the alkyl that comprises 20 carbon atoms.And the restriction of this 1-20 does not comprise the carbonatoms of the replacement on the alkyl, as replace " alkyl " in the alkylamino, when being not particularly limited its carbonatoms, only refer to that wherein the carbonatoms of indicated moieties is 1-20, and do not comprise substituent carbonatoms on the alkyl and other the substituent carbonatomss on the amino.Adopt the statement of " C1-8 alkyl " then to represent to contain in this alkyl the alkyl of 1～8 carbon atom.)

Term " alkoxyl group " is the alkyl that connects by Sauerstoffatom; Comprise methoxyl group, oxyethyl group, propoxy-as the C1-8 alkoxyl group; The C1-8 alkoxyl group, the carbonatoms that is meant the alkyl in the alkoxyl group is 1～8.

Haloalkyl, the alkyl that the expression halogen atom replaces, this replacement comprises single replacement and polysubstituted, wherein the notion of alkyl is as mentioned above.C1～8 haloalkyls, the carbonatoms that is meant the alkyl in the haloalkyl is 1～8.Haloalkyl refers to the group that the H atom is replaced by halogen atom on the alkyl; Be meant the group that the H on the alkyl is all replaced by F as perfluoroalkyl.

The structure of alkylamino is: alkyl-NH-.The structure of aminoalkyl group is: NH ₂-alkyl-.Alkylthio is meant to have the alkyl that sulphur atom replaces.

Heterocyclic radical is meant heteroatomic cyclic groups by 3 to 8 annular atomses such as containing N, O, S, and in this group, heteroatoms can only contain the N atom, also can contain O or S atom.Wherein heteroatomic number can be one, also can be for a plurality of.This heterocycle can be saturated class cycloalkanes structure, also can be undersaturated aromatic ring class formation.More specifically, this term nitrogen heterocycle includes but not limited to pyrryl, Pyrrolidine base, piperidyl, piperazinyl, morpholinyl, piperazinyl, pyrimidyl, imidazolyl etc.

Should be clear that some formula (I) compound can present tautomerism.Formula (I) compound can exist with the form of solvation not, also can exist with the form of solvation.Even can there be heteromorphism in some formula of the present invention (I) compound.

The pharmacy acceptable salt that is fit to of formula (I) compound can be the acid salt of formula (I) compound, can be as following inorganic or acid-additive salt that organic acid generated: hydrochloric acid, Hydrogen bromide, sulfuric acid, trifluoroacetic acid, citric acid or toxilic acid; Can be salt, as an alkali metal salt or alkaline earth salt (calcium salt, magnesium salts or ammonium salt etc.) with enough tart formulas (I) compound.The pharmacy acceptable salt that the another kind of formula (I) compound is fit to can be the salt that forms in human or animal body behind giving construction (I) compound.

Another purpose of the present invention is to provide a kind of pharmaceutical composition, it is characterized in that described pharmaceutical composition comprises compound or its pharmacy acceptable salt and pharmaceutically acceptable diluent or carrier.

Can be with the form administration of compound of the present invention with prodrug.Prodrug is meant through just having the compound of pharmacological action after transforming in the organism.Can use prodrug to change the physicochemical property or the pharmacokinetics aspect character of The compounds of this invention.When compound of the present invention contains can connect the suitable group that changes the character group or substituted radical the time, can form prodrug.

Another purpose of the present invention has been to provide compound or the application of its pharmacy acceptable salt aspect the preparation medicine, it is characterized in that described medicine is used for the treatment of and cytodifferentiation or relevant solid tumor or the leukemia of propagation.

Another purpose of the present invention is to provide a kind of method for preparing compound or its pharmacy acceptable salt, it is characterized in that said method comprising the steps of:

(1) makes the compound or its salt of formula (II)

Obtain the compound of formula (III) with acyl chloride reaction,

(2) compound of the formula (III) that (1) is obtained and O-Phenylene Diamine reaction back deprotection obtain the compound of formula (I); Wherein the ortho position amino of O-Phenylene Diamine is protected;

Ar is for for aryl or heterocyclic radical, and it is optional to be selected from following group and to replace by one or more: C1-8 alkyl, C1-8 alkoxyl group, halogen, nitro, C1-8 aminoalkyl group, C1-8 alkylamino, C1-8 alkylthio, C1-8 haloalkyl, C1-8 halogenated alkoxy, C1-8 ester group, phenyl or heterocyclic radical n are selected from 0～8 integer.

More specifically, cyanobenzene or heterocyclic aryl formonitrile HCN that compound of the present invention can replace by difference are starting raw material, make amidoxime with the oxammonium hydrochloride reaction; Do solvent and acid binding agent with pyridine then, under refluxad make 3,5-is dibasic 1,2,4-Evil ribavirin derivative; With the hydrolysis of synthetic methyl esters, acidifying, make the intermediate carboxylic acid; Through becoming acyl chlorides, acid amides and deprotection steps, finally obtain target molecule then.

For the preparation corresponding pharmaceutical compositions, except at least a active substance of the present invention, also use solid support material, filler, solvent, thinner, tinting material or binding agent.The selection of auxiliary agent and consumption thereof depend on the route of administration of this medicine, use as in oral, intravenous injection, abdominal injection, intracutaneous, muscle, the nose or part.The preparation of forms such as tablet, coating tablet, capsule, granule, drops, liquid dosage form and syrup is suitable for oral, and solution, suspension agent, sprays etc. are applicable to non-enteron aisle, part and inhalation.Give patient or laboratory animal dosage body weight, method of application, refer to disease and the administration of illness situation according to patient or animal.

Compound of the present invention studies confirm that through pharmaceutical activity this compound has and positive drug SAHA and MS-275 similar activity, can be applied to treat and cytodifferentiation or relevant solid tumor or the leukemic medicine aspect of propagation.

Description of drawings

Below in conjunction with drawings and Examples the present invention is further described:

Fig. 1 is The compounds of this invention figure that influences to the survival rate of A549 when 10 μ M;

Fig. 2 is The compounds of this invention figure that influences to the survival rate of NCI-H661 when 10 μ M.

Embodiment

Below in conjunction with specific embodiment such scheme is described further.Should be understood that these embodiment are used to the present invention is described and are not limited to limit the scope of the invention.The implementation condition that adopts among the embodiment can be done further adjustment according to the condition of concrete producer, and not marked implementation condition is generally the condition in the normal experiment.

The preparation of embodiment 1 N-(2-aminophenyl)-7-(3-phenyl-1,2,4-Evil ribavirin-5-yl)-heptamide (compound 1)

With aniline oxime (5g, 36.7mmol) be dissolved in the 15mL pyridine, under the room temperature condition, dropwise add 8-chloro-8-oxo methyl caprylate (9.11g in 30min, 44.1mmol), finish back flow reaction, react completely to the aniline oxime, be cooled to room temperature, adding ethyl acetate and water distributes, organic layer is used aqueous hydrochloric acid, saturated sodium bicarbonate solution and the saturated common salt water washing of 2M successively, and the organic layer dried over mgso obtains colourless oil liquid through silica gel column chromatography.Productive rate 65%.Other substituent Methylheptanoate is synthetic by the same method, and productive rate is 52-70%.

¹H-NMR(500Hz，CDCl ₃)δ：8.06-8.08(m，2H)，7.28-7.49(m，3H)，3.66(s，CO ₂CH ₃，3H)，2.94(t，J＝7.5Hz，het-CH ₂CH ₂，2H)，2.31(t，J＝7.4Hz，CH ₂CH ₂CO ₂Me，2H)，1.85-1.91(m，CH ₂，2H)，1.62-1.68(m，CH ₂，2H)，1.37-1.47(m，2CH ₂，4H)。MS：(M+H) ⁺289.1，(M+Na) ⁺311.1。

7-(3-phenyl-1,2, the 4-Evil ribavirin-5-yl) Methylheptanoate of 15mmol is dissolved in the tetrahydrofuran (THF) of 30mL, adds the lithium hydroxide aqueous solution 30mL of 0.6M, stirred overnight at room temperature.Add 150mL water, ethyl acetate extraction discards organic layer, lower aqueous solution is acidified to pH and equals 3, ethyl acetate extraction (3x50mL) merges organic layer, the saturated common salt washing, anhydrous magnesium sulfate drying, the pressure reducing and steaming solvent promptly obtains 7-(3-phenyl-1,2,4-Evil ribavirin-5-yl) enanthic acid, yield is 70-95%.Mp：100-101℃。

At 0 ℃, (0.42mL 4.8mmol) is added drop-wise in the tetrahydrofuran (THF) (40mL) of 7-(3-substituted-phenyl-1,2,4-Evil ribavirin-5-yl) enanthic acid (1.0mmol) with DMF (40 μ L) and oxalyl chloride.Finish, continue reaction 1h in room temperature.Decompression steams solvent, obtains acyl chlorides and directly drops into the next step.

2-(N-t-butoxycarbonyl amino) aniline (1.1mmol) is dissolved in the tetrahydrofuran (THF), adds N-methylmorpholine (1.0mmol),, rise to room temperature reaction 4h then at 0 ℃ of tetrahydrofuran solution (10mL) that drips above-mentioned acyl chlorides.Remove solvent under reduced pressure, add ethyl acetate (100mL), through 1M aqueous hydrochloric acid, 1M aqueous sodium hydroxide solution and saturated common salt washing, anhydrous magnesium sulfate drying obtains intermediate 3 successively.

Be dissolved in the 10mL methylene dichloride 3, add the 1.5mL trifluoroacetic acid, the room temperature deprotection, reaction finishes, and drips the unsaturated carbonate aqueous solutions of potassium and does not emit to there being no bubble.With methylene dichloride or ethyl acetate extraction, through 1M aqueous sodium hydroxide solution and saturated common salt washing, anhydrous magnesium sulfate drying obtains target compound then, and productive rate is 71%.

Mp：115-117℃。 ¹H-NMR(300Hz，CDCl ₃)δ：8.07(m，2H)，7.45-7.51(m，3H)，7.22(br?s，1H)，7.17(d，J＝7.57Hz，1H)，7.03-7.07(m，1H)，6.78-6.81(m，2H)，3.86(br，2H)，2.96(t，J＝7.48Hz，het-CH ₂CH ₂，2H)J＝7.48Hz，，2H)，2.40(t，J＝7.39Hz，CH ₂CH ₂CO ₂NH，2H)，1.87-1.92(m，CH ₂，2H)，1.76-1.79(m，CH ₂，2H)，1.47-1.49(m，2CH2，4H)。13C-NMR(75Hz，CDCl ₃)δ：179.87(C-5)，171.71(C-3)，168.27(C＝O)，131.0，129.30，128.83，127.39，127.19，126.90，125.62，125.23，124.44，119.71，118.34，114.23，113.88，36.76，32.03，28.62，26.49，26.40，25.46，24.40。MS：(M+H) ⁺365.2，(M+Na) ⁺387.2；(M-H) ^-363.2，(M+Cl) ^-399.1。

The preparation of 2-(N-t-butoxycarbonyl amino) aniline

(30g 0.278mol) is dissolved in the 500mL chloroform, and reaction mixture is cooled to 0 ℃ with O-Phenylene Diamine.In mixture, add sodium bicarbonate (23.5g, 0.278mol) and sodium-chlor (mixture is at 0 ℃ for 16.3g, aqueous solution 0.278mol). stir 30min.(61g, 150mL chloroformic solution 0.278mol) slowly drops in the above-mentioned reaction mixture with the Boc acid anhydrides.Finish, mixture continues 10min at 0 ℃, rises to room temperature, and 12h then refluxes.The 3x chloroform extraction is used in the mixture cooling, merges organic layer, uses saturated sodium bicarbonate aqueous solution, saturated common salt water washing then successively, the organic layer anhydrous magnesium sulfate drying.Filter, remove solvent under reduced pressure, obtain crude product, crude product is through silica gel column chromatography, and developping agent is petrol ether/ethyl acetate (2: 1), obtains 2-(N-t-butoxycarbonyl amino) aniline, and product is off-white color solid 46g, and productive rate is 80%. ¹H-NMR(300MHz，CDCl ₃)：δ1.53(s，9H，t-C ₄H ₉)，3.81(br?s，2H，NH ₂)，6.36(br?s，1H，NH)，6.9-6.7(m，2H，CH)，7.00(m，1H，CH)，7.28(d，J＝7.5Hz，CH)。

The preparation of embodiment 2 N-hydroxyl-7-(3-p-methylphenyl-1,2,4-Evil ribavirin-5-yl) heptamide (compound 2)

The preparation method: the aniline oxime among the embodiment 1 is replaced with the para-totuidine oxime, and other preparation method is identical.

Mp：122-123℃。 ¹H-NMR(300Hz，CDCl ₃)δ：7.95(d，J＝8.11Hz，2H)，7.27(d，J＝8.11Hz，2H)，7.26(br?s，1H)，7.15(d，J＝7.71Hz，1H)，7.02-7.07(m，1H)，6.76-6.80(m，2H)，3.86(br，2H)，2.94(t，J＝7.48Hz，het-CH ₂CH ₂，2H)J＝7.48Hz，，2H)，2.41(s，3H)，2.38(t，J＝7.39Hz，CH ₂CH ₂CO ₂NH，2H)，1.87-1.91(m，CH ₂，2H)，1.73-1.78(m，CH ₂，2H)，1.47-1.48(m，2CH ₂，4H)。 ¹³C-NMR(75Hz，CDCl ₃)δ：179.67(C-5)，171.62(C-3)，168.24(C＝O)，141.38，140.79，129.52，129.26，127.28，127.13，125.20，124.34，124.04，119.50，118.20，36.72，29.66，26.46，26，38，25.43，21.51。MS：(M+H) ⁺379.2，(M+Na) ⁺401.2；(M-H) ^-377.2，(M+Cl) ^-413.3。IR(cm-1)：3400.16，3327.08，1655.41，1642.42，1592.82，1527.71，1460.21，1412.83，1355.08，783.69，746.37，703.35。

The preparation of embodiment 3 N-(2-aminophenyl)-7-(3-p-methoxyphenyl-1,2,4-Evil ribavirin-5-yl)-heptamide (compound 3)

The preparation method: the aniline oxime among the embodiment 1 is replaced with 4-anisole amidoxime, and other preparation method is identical.

Mp：130-131℃。 ¹H-NMR(300Hz，CDCl ₃)δ：8.00(d，J＝8.81Hz，2H)，7.26(br?s，1H)，7.16-7.18(m，1H)，7.04-7.07(m，1H)，6.98(d，J＝8.84Hz，2H)，6.77-6.80(m，2H)，3.85(s，3H)，3.82(br，2H)，2.94(t，J＝7.48Hz，het-CH ₂CH ₂，2H)J＝7.48Hz，，2H)，2.40(t，J＝7.39Hz，CH ₂CH ₂CO ₂NH，2H)，1.87-1.91(m，CH ₂，2H)，1.73-1.78(m，CH ₂，2H)，1.47-1.49(m，2CH ₂，4H)。 ¹³C-NMR(75Hz，CDCl ₃)δ：179.55(C-5)，171.55(C-3)，167.97(C＝O)，161.87，158.80，128.98，127.14，119.58，118.27，114，24，55.35，36.79，28.60，26.46，26，38，25.45，24.38。MS：(M+H) ⁺395.2，(M+Na) ⁺417.2；(M-H) ^-393.2，(M+Cl) ^-429.2。IR(cm-1)：3401.41，3321.15，1654.60，1615.21，1592.55，1527.03，1461.82，1414.351362.51，1256.33，1176.13，1030.11，842.48，753.83。

The preparation of embodiment 4 N-(2-aminophenyl)-7-(3-p-trifluoromethyl phenyl-1,2,4-Evil ribavirin-5-yl)-heptamide (compound 4)

The preparation method: the aniline oxime among the embodiment 1 is replaced with 4-trifluoromethylbenzene amidoxime, and other preparation method is identical.

Mp：178-180℃。 ¹H-NMR(300Hz，DMSO-d6)δ：9.07(br?s，1H)，7.15(d，J＝7.04Hz，2H)，6.86-6.91(m，2H)，6.71(d，J＝7.9Hz，2H)，6.50-6.56(m，2H)，4.79(br，2H)，2.29-2.34(m，het-CH ₂CH ₂，CH ₂CH ₂CO ₂NH，4H)1.61(m，CH ₂，4H)，1.36(m，2CH ₂，4H)。 ¹³C-NMR(75Hz，DMSO-d6)δ：180.02(C-5)，169.01(C-3)，167.24(C＝O)，161.67，158.80，128.64，127.14，119.58，118.69，114.63，36.79，28.60，26.46，26，38，25.45，24.38。

The preparation of embodiment 5 N-(2-aminophenyl)-7-(3-rubigan-1,2,4-Evil ribavirin-5-yl)-heptamide (compound 5)

The preparation method: the aniline oxime among the embodiment 1 is replaced with 4-chlorobenzene amidoxime, and other preparation method is identical.

Mp：125-127℃。 ¹H-NMR(300Hz，CDCl ₃)δ：8.01(d，J＝8.53Hz，2H)，7.45(d，J＝8.54Hz，2H)，7.26(br?s，1H)，7.15(d，J＝7.69Hz，1H)，7.02-7.07(m，1H)，6.76-6.80(m，2H)，3.85(br，2H)，2.94(t，J＝7.48Hz，het-CH ₂CH ₂，2H)2.39(t，J＝7.39Hz，CH ₂CH ₂CO ₂NH，2H)，1.87-1.91(m，CH ₂，2H)，1.73-1.78(m，CH ₂，2H)，1.47-1.48(m，2CH2，4H)。 ¹³C-NMR(75Hz，CDCl ₃)δ：180.07(C-5)，171.58(C-3)，167.46(C＝O)，140.75，137.23，129.13，128.68，127.15，125.39，125.17，124.34，119.54，118.24，36.72，29.66，26.46，26，38，25.43，21.51。MS：(M+H) ⁺399.2，(M+Na) ⁺421.2；(M-H) ^-397.2。

Embodiment 6 N-(2-aminophenyl)-7-[3-(2-chloro-phenyl-)-1,2,4-Evil ribavirin-5-yl]-preparation of heptamide (compound 6)

The preparation method: the aniline oxime among the embodiment 1 is replaced with 2-chlorobenzene amidoxime, and other preparation method is identical.Mp：80-82℃。MS：(M+H) ⁺399.2，(M+Na) ⁺421.2；(M-H) ^-397.2。

The preparation of embodiment 7 N-(2-aminophenyl)-7-(3-p-nitrophenyl-1,2,4-Evil ribavirin-5-yl)-heptamide (compound 7)

The preparation method: the aniline oxime among the embodiment 1 is replaced with 4-oil of mirbane amidoxime, and other preparation method is identical.

Mp：151-153℃。 ¹H-NMR(300Hz，CDCl ₃)δ：8.34(d，J＝8.9Hz，2H)，8.25(d，J＝8.9Hz，2H)，7.26(br?s，1H)，7.15(d，J＝7.69Hz，1H)，7.03-7.08(m，1H)，6.77-6.81(m，2H)，3.85(br，2H)，2.97(t，J＝7.48Hz，het-CH ₂CH ₂，2H)2.41(t，J＝7.39Hz，CH ₂CH ₂CO ₂NH，2H)，1.90-1.95(m，CH ₂，2H)，1.76-1.81(m，CH ₂，2H)，1.48-1.51(m，2CH ₂，4H)。 ¹³C-NMR(75Hz，CDCl ₃)δ：180.78(C-5)，171.50(C-3)，166.80(C＝O)，149.43，140.72，132.86，129.37，128.39，127.22，125.12，124.40，124.10，119.66，118.36，36.81，29.71，28.68，26，51，26.35，25.45。MS：(M+H) ⁺410.2，(M+Na) ⁺432.2；(M-H) ^-408.2。IR(cm-1)：3401.41，3321.15，16447.73，1529.32，13447.63。

Embodiment 8 N-(2-aminophenyl)-7-[3-(3-nitrophenyl)-1,2,4-Evil ribavirin-5-yl]-preparation of heptamide (compound 8)

The preparation method: the aniline oxime among the embodiment 1 is replaced with 3-oil of mirbane amidoxime, and other preparation method is identical.

Mp：126-128℃。 ¹H-NMR(300Hz，CDCl ₃)δ：8.93(s，1H)，8.34-8.42(m，2H)，7.65-7.70(m，1H)，7.26(br?s，1H)，7.15-7.22(m，1H)，7.03-7.07(m，1H)，6.77-6.80(m，2H)，3.84(b?r，2H)，2.98(t，J＝7.48Hz，het-CH ₂CH ₂，2H)2.40(t，J＝7.39Hz，CH ₂CH ₂CO ₂NH，2H)，1.90-1.95(m，CH ₂，2H)，1.76-1.81(m，CH ₂，2H)，1.48-1.51(m，2CH ₂，4H)。 ¹³C-NMR(75Hz，CDCl ₃)δ：180.75(C-5)，171.59(C-3)，166.72(C＝O)，148.64，140.77，130.02，129.32，128.79，127.18，125.61，125.18，124.40，122.56，119.60，118.30，36.78，29.70，28.67，26，51，26.35，25.45。MS：(M+H) ⁺410.2，(M+Na) ⁺432.2；(M-H) ^-408.2。IR(cm-1)：3401.41，3321.15，1648.66，1595.69，1527.03，1533.62，1515.83，1461.80，1369.06，1349.24，757.29，718.58。

Embodiment 9 N-(2-aminophenyl)-7-[3-(pyridin-3-yl)-1,2,4-Evil ribavirin-5-yl]-preparation of heptamide (compound 9)

The preparation method: the aniline oxime among the embodiment 1 is replaced with 3-pyridine amidoxime, and other preparation method is identical.

Mp：85-87℃。 ¹H-NMR(300Hz，CDCl ₃)δ：8.72-8.74(m，H)，8.33(d，J＝7.94，1H)，7.39-7.43(m，2H)，7.26(br?s，1H)，7.16(d，J＝7.68，1H)，7.03-7.06(m，1H)，6.76-6.79(m，2H)，3.84(br，2H)，2.96(t，J＝7.48Hz，het-CH ₂CH ₂，2H)2.39(t，J＝7.39Hz，CH ₂CH ₂CO ₂NH，2H)，1.89-1.92(m，CH ₂，2H)，1.75-1.78(m，CH ₂，2H)，1.47-1.48(m，2CH ₂，4H)。 ¹³C-NMR(75Hz，CDCl ₃)δ：180.42(C-5)，171.66(C-3)，166.33(C＝O)，151.89，148.57，140.82，134.67，127.12，125.23，124.39，123.64，123.25，119.46，118.18，36.69，29.70，28.64，26，45，26.33，25.44。MS：(M+H) ⁺366.2，(M+Na) ⁺388.2；(M-H) ^-364.2，(M+Cl) ^-400.2。

Embodiment 10 N-(2-aminophenyl)-7-[3-(thiophene-2-yl)-1,2,4-Evil ribavirin-5-yl]-preparation of heptamide (compound 10)

The preparation method: the aniline oxime among the embodiment 1 is replaced with 3-thiophene amidoxime, and other preparation method is identical.

Mp：82-85℃。 ¹H-NMR(300Hz，CDCl ₃)δ：7.77(m，1H)，7.49((m，1H)，7.26(br?s，1H)，7.15-7.20(m，1H)，7.03-7.07(m，1H)，6.77-6.79(m，2H)，3.84(br，2H)，2.92(m，2H)2.34-2.40(m，2H)，1.66-1.88(m，2CH ₂，4H)，1.48-1.51(m，2CH ₂，4H)。 ¹³C-NMR(75Hz，CDCl ₃)δ：179.86(C-5)，171.69(C-3)，164.29(C＝O)，140.71，130.02，129.36，128.84，127.93，127.14，125.23，124.37，122.40，119.55，118.59，118.21，114.53，36.70，33.87，28.69，26.37，26.32，25.41。MS：(M+H) ⁺371.2，(M+Na) ⁺393.2；(M-H) ^-369.(M+Cl) ^-405.1。

The preparation of embodiment 11 N-(2-aminophenyl)-7-(3-to fluorophenyl-1,2,4-Evil ribavirin-5-yl)-heptamide (compound 11)

The preparation method: the aniline oxime among the embodiment 1 is replaced with 4-fluorobenzene amidoxime, and other preparation method is identical.

Mp：128-129℃。 ¹H-NMR(300Hz，CDCl ₃)δ：8.04-8.09(m，2H)，7.26(brs，1H)，7.13-7.18(m，3H)，7.02-7.07(m，1H)，6.74-6.79(m，2H)，3.84(br，2H)，2.93(t，J＝7.48Hz，het-CH ₂CH ₂，2H)2.36(t，J＝7.39Hz，CH ₂CH ₂CO ₂NH，2H)，1.85-1.90(m，CH ₂，2H)，1.71-1.75(m，CH ₂，2H)，1.45-1.46(m，2CH ₂，4H)。 ¹³C-NMR(75Hz，CDCl ₃)δ：179.96(C-5)，171.69(C-3)，166.13(C＝O)，171.69，167.42，162.80，140.82，129.43，127.12，125.25，119.43，118.13，116.11，115.82，36.63，29.70，28.59，26，42，26.33，25.41。MS：(M+H) ⁺383.2，(M+Na) ⁺405.2；(M-H) ^-381.2，(M+CD ^-427.2。

The bioactivity research of embodiment 12 The compounds of this invention

The different concns drug effect of the present invention that obtains with the foregoing description behind people's non-small cell lung adenocarcinoma cell strain (A549) and National People's Congress's cell lung cancer cell (NCI-H661) 72h, the influence of WST-8 method detection of drugs on cell proliferation.

Medicine: positive medicine SAHA and MS-275 are synthetic for this laboratory, and structure is seen Fig. 1 and Fig. 2.

Reagent and instrument: HyQR modified form RPMI 1640 substratum, DMEM substratum, WST-8 (Sigma), electron coupling reagent 1-Methoxy PMS (Sigma), pancreatin.CO2 incubator, aseptic technique platform, microplate reader, whizzer, liquid-transfering gun, transfer pipet, centrifuge tube and 96 orifice plates etc.

Method:

1) tumor cell line is cultivated

National People's Congress's cell lung cancer cell (NCI-H661): with containing RPMI 1640 substratum of 10% foetal calf serum, in 37 ℃, the incubator of 5%CO2, cultivate.People's non-small cell lung cancer cell strain (A549):, in 37 ℃, the incubator of 5%CO2, cultivate with the DMEM substratum of 10% foetal calf serum.

2) cell viability is measured

Get and be in exponential phase of growth, cell in good condition adds an amount of trypsin digestion cell, and collecting cell is centrifugal, abandons supernatant.With the nutrient solution that contains serum suspendible cell again, count then, and cell density is diluted to 2 * 104/ml density.

Obtained cell suspension is inoculated on 96 orifice plates, 100 μ l/ holes (containing 5000/hole of tumour cell).Culture plate is changed in the constant temperature CO2 incubator,, cultivated 24 hours under 5%CO2 and the saturated humidity condition at 37 ℃.

Test-compound is mixed with earlier the storage liquid of 0.1M with DMSO, again dilute sample (unit: μ M) as required.Add test-compound 50 μ l/ holes, cultivated 72 hours, establish 3 parallel holes for every group, and repeated experiments.

After the compound effects 72 hours, remove cell grown cultures liquid, WST-8 is added in 96 orifice plates, 10 μ L/ holes place incubator to hatch 1.5 hours at 37 ℃, and microplate reader detects the absorbancy (correction of 650nm wavelength place) in every hole at 450nm wavelength place.

(1) survival rate of cell: the OD value of each test hole is deducted background OD value (perfect medium+WST-8, acellular) or blank medicine hole OD value (the dilution reagent thing+WST-8 that is subjected to of perfect medium+difference, acellular), the OD value of each repeating hole is got mean ± SD.

The survival rate of cell represents that with T/C% T is the OD value of dosing cell, and C is the OD value of control cells.

Cell survival rate %=(dosing cell OD/ control cells OD) * 100

Drug level when (2) obtaining T/C=50% (IC50).Inhibiting rate is higher than 50% compound, with GraphPad Prism 5.0 computed in software IC50 values.

Experimental result

Biological activity test is the result show, The compounds of this invention all has in various degree restraining effect to the propagation of A549 and NCI-H119, but specific activity SAHA and MS-275 inequality.

Experimental result sees Table 1.

Table 1 part of compounds of the present invention is to A549 and the effect of NCI-H119 inhibition of proliferation

Above-mentioned example only is explanation technical conceive of the present invention and characteristics, and its purpose is to allow the people who is familiar with this technology can understand content of the present invention and enforcement according to this, can not limit protection scope of the present invention with this.All equivalent transformations that spirit is done according to the present invention or modification all should be encompassed within protection scope of the present invention.

Claims (7)

1. the compound of a formula (I) or its pharmacy acceptable salt,

Wherein, Ar is phenyl or heterocyclic radical, and it is optional to be selected from following group and to replace by one or more: C1-8 alkyl, C1-8 alkoxyl group, halogen, nitro, C1-8 aminoalkyl group, C1-8 alkylamino, C1-8 alkylthio, C1-8 haloalkyl, C1-8 halogenated alkoxy, C1-8 ester group; N is selected from 0～8 integer; Described heterocyclic radical is selected from pyridine, thiophene, furans, pyrroles or imidazoles.

2. compound according to claim 1 or its pharmacy acceptable salt is characterized in that described phenyl or heterocyclic radical are optional to be selected from following group and to replace by one or more: C1-8 alkyl, halogen, nitro or C1-8 haloalkyl.

3. compound according to claim 2 or its pharmacy acceptable salt is characterized in that described phenyl or heterocyclic radical are optional to be selected from following group and to replace by one or more: the haloalkyl of the alkyl of C1-4, halogen, nitro or C1-4.

4. compound according to claim 1 or its pharmacy acceptable salt is characterized in that n is 6.

5. a pharmaceutical composition is characterized in that described pharmaceutical composition comprises compound any in the claim 1～4 or its pharmacy acceptable salt and pharmaceutically acceptable diluent or carrier.

6. any described compound of claim 1～4 or its pharmacy acceptable salt application aspect the preparation medicine is characterized in that described medicine is used for the treatment of and cytodifferentiation or relevant solid tumor or the leukemia of propagation.

7. method for preparing the described compound of claim 1 or its pharmacy acceptable salt is characterized in that said method comprising the steps of:

(1) makes the compound or its salt of formula (II)

Obtain the compound of formula (III) with acyl chloride reaction,

(2) compound of the formula (III) that (1) is obtained and O-Phenylene Diamine reaction back deprotection obtain the compound of formula (I); Wherein the ortho position amino of O-Phenylene Diamine is protected;

Ar is phenyl or heterocyclic radical, and it is optional to be selected from following group and to replace by one or more: C1-8 alkyl, C1-8 alkoxyl group, halogen, nitro, C1-8 aminoalkyl group, C1-8 alkylamino, C1-8 alkylthio, C1-8 haloalkyl, C1-8 halogenated alkoxy, C1-8 ester group; N is selected from 0～8 integer; Described heterocyclic radical is selected from pyridine, thiophene, furans, pyrroles or imidazoles.

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