CN1724516A - A kind of natural and synthetic isothiocyanate compound and its application in the treatment and prevention of cancer - Google Patents

Abstract

The invention relates to naturally or artificially synthesized iso-thiocyanates i.e. JC-5411 compounds and their use in treating and preventing malignant tumor, wherein the compounds can directly suppress the methylation of the gene promoter / 5-UTR area CpG DNA, directly inhibit the reactivity of histone deacethylases (HADC), selectively promote histone acetylation and methylation, then further recover the expression of genes with important functions that are shut abnormally in cancer cells, thus control the malignant proliferation of cancer cells.

Description

A kind of isosulfocyanate compound of natural and synthetic and the application in treatment and preventing cancer thereof

Technical field

The invention belongs to medical applications (or chemical pharmacy) field; relate to the JC-5411 series compound; with the lsothiocyanates is mother nucleus structure; it no matter is natural or synthetic; can directly suppress dna methylation and histone deacetylase (histone deacetylase unusual in the tumour cell; HADC) activity; promote acetylation of histone and methylating selectively, and then make some of being closed unusually in the cancer cells have the gene such as the cancer suppressor gene of critical function, regulate the cell cycle; apoptotic gene; the gene of dna damage and reparation, the pernicious gene of invading profit and shifting of anticancer etc. recovers to express, and brings into play its normal function; so making the malignant proliferation of cancer cells is controlled; thereby can suppress to comprise colorectal carcinoma, cancer of the stomach, lung cancer effectively; prostate cancer; liver cancer, mammary cancer, colorectal carcinoma; cervical cancer, multiple human body tumour cell growths such as melanoma and neuroblastoma.Compare with known natural or dna methylation of synthetic and histone deacetylase inhibitors; this series compound is the unique unusual dna methylation but also the double inhibitor of inhibition of histone deacetylase of not only having suppressed; and it is strong to have curative effect; toxic side effect is humble; characteristics such as chemicobiology stable in properties can be used for treatment and prevention malignant tumour.

Background technology

Though because EPDML progress, the further improvement of early diagnosis and therapy method, obtained significant achievement in treatment for cancer and prevention in the past more than ten years, yet tumour remains one of principal disease that threatens people's life, although and surgical operation and radiocurable continuous maturation, the continuous appearance of new methods of treatment such as gene therapy etc., but up to now still none method can replace chemotherapy.Advance over year, the progress of oncomolecularbiology research, some good effects, the medicine of high specificity such as Avastin (Avastin) (anti-angiogenic formation, U.S. Genentech company) and Iressa (Urogastron pathway inhibitor, AstraZeneca) increased new powerful mean for the treatment of malignant tumour.But, can not reach the purpose of healing tumour far away at the medicine of a certain gene or path owing to tumour is the disease that a polymolecular (gene) pathology causes.Existing cancer therapy drug, though have certain curative effect, they are cell toxicity medicament mostly, have serious toxic side effect.So the PTS of excavating new and effective low toxicity remains the important topic of drug research.

Now generally acknowledged, the mechanism that two kinds of main non-genetic genes are transcribed control is the covalent modification of methylating of DNA-CpG site and histone ^(1,2)Big quantity research shows that nearly all malignant tumour all has some important function of gene to methylate owing to DNA-CpG and/or the covalent modification of histone causes genetic transcription to be closed unusually unusually.These genes comprise the DNA repair enzyme, as MGMT and hMLH1 or separate toxenzyme (GSTP1).Transcribing of they closed and will be caused dna mutation ^(3-5)Other gene of closing unusually comprises the gene of regulating the cell cycle, and cancer suppressor gene influences apoptosis, the vascularization gene, and invade profit with cancer cells and shift relevant gene, as p16 ^INK4a, BRCA1, Rb, RAR2, RAR4, RASSF1A, CDH13, APC, CDH1, and FHIT ^(3,4,6-11)So, close with the relevant gene that methylates, become the target spot of diagnosing tumor and treatment ⁽¹²⁾

On the other hand, high organism comprises human body cell, and the gene transcription strictness is subjected to the control of chromatin density.Any gene that is in high-density chromatin site then is in closing condition.As everyone knows, the cell chromosome of storage genetic information mainly is made up of DNA and histone.Around DNA, the protein spheres of forming by histone.They and DNA closely encircle, and form nucleus.As far back as 1964, people such as Allfrey proved that first the methylating of histone/acetylize is synthetic relevant with RNA's.People found the covalent modification and the chromatinic structure of histone in succession afterwards, the adjusting of genetic transcription, and the transmission of non-hereditary signal, and malignant tumour etc. is relevant.Nucleome and nucleome are directly regulated in the chemically modified of reversible histone, nucleome and DNA, and the interaction between DNA and transcription factor ⁽¹³⁾Histone chemically modified after the translation comprises acetylize, methylates and phosphorylation.They realize by the antipodal enzyme of corresponding effect, that is: acetyltransferase and deacetylase (HDAC); Methyltransgerase and demethylation enzyme; Starch phosphorylase and Phospholipid hydrolase ⁽¹⁴⁾

DNA CpG site methylate and histone HDAC together fellowship some have the closing of gene of important physiological function in the cancer cells.Binding site (MBD) albumen that methylates can combine with methylated CpG site specifically, thereby suppressor gene is transcribed ⁽¹⁵⁾Importantly in the MBD protein family, all members have identical MBD, wherein, MeCP2, MBD2 and MBD3 and huge albumen composition interrelate.These huge albumen compositions contain histone deacetylase HDACl, and HDAC2, and have the chromatin reconstitution function.This chromatin reconstitution active result hinders transcription factor to combine with DNA, and then the transcribing of suppressor gene.Obviously; because dna methylation and histone deacetylase interact in closing the normal gene transcription; use dna methylation inhibitor and histone deacetylase inhibitors simultaneously; then can block transcribing of normal gene synergistically and close, thereby play the effect that stops canceration and treatment tumour.

Because CpG methylates and the effect of histone chemically modified in the deterioration process of canceration and tumour is clear and definite day by day, seeking dna methylation inhibitor and histone deacetylation inhibitor has become the new direction that anticarcinogen is developed ^(12,16)

Common dna methylation inhibitor inhibition dnmt rna (DNA methyltransferases, DNMTs).Adopt the gene knockout technology, make DNMT1 and DNMT3b deactivation, then make all CpG of cancer suppressor gene site reverse that methylates that methylates.So methyltransgerase is the important target enzyme of methylation inhibitor.Methyltransgerase has two substrate binding sites, cytosine(Cyt) residue and S-VITAMIN B4-methionine(Met) (S-adenosyl-methionine).So any chemical structure is similar to then might combining with DNMTs of cytosine(Cyt) or S-VITAMIN B4-methionine(Met), and becomes competitive inhibitor.In fact, first competitive inhibitor of DNMTs is the 5-aza-cytidine ⁽¹⁷⁾This medicine once was used for leukemic treatment.Owing to it is easily degraded and lose the effect of DNMTs inhibitor by the sweet deaminase of nuclear, this compound is seldom used.

The normal clinically dna methylation inhibitor that uses be decitabing (5 '-Aza) (and Decitabine, or 5 '-Aza, Super-Gen Inc., CA, USA).Prove that in the cell in vitro system this compound can suppress dna methylation effectively.But, liver and spleen tissue have only 15 to 20 minutes effective half-life in the decitabing body because having high-caliber cytosine(Cyt) deaminase.Can participate in the dna molecular owing to the sweet derivative of this nucleoid simultaneously, and then disturb normal DNA metabolism, so toxic side effect is big.

What is interesting is that being used as clinically that the medicine procainamide (Procainamide) of anti-heart disorder and local anesthetic PROCAINE HCL, PHARMA GRADE (Procaine) be considered to is the dna methylation inhibitor of the sweet class of non-nuclear ⁽¹⁸⁾, in prostate cancer cell, can suppress to separate toxenzyme, the i.e. dna methylation of GSTP1 effectively ⁽¹⁹⁾Although it is to be evaluated that these medicine antitumor actions have, undoubtedly, excavate during novel dna methylation inhibitor carries out in full preparation ⁽¹²⁾

That meanwhile, another interrupts the non-hereditary canceration and the process of deterioration thereof is inhibition of histone deacetylase (HADC).As previously mentioned; HADC's is unusual in the tumour cell; make the methylating of histone; acetylize descends unusually, thereby has increased combining and block the effect of transcription factor and target gene between histone and DNA, makes the gene with important physiological function; as cancer suppressor gene; regulate the cell cycle, apoptotic gene, the gene of dna damage reparation etc. is closed unusually.Thereby histone deacetylase inhibitors becomes one of focus in the antitumour drug research field in recent years.So far, existing ten kinds of hdac inhibitors are in clinic trial.Though still have various problems to need to solve, clinic trial result still other people rouses oneself.

In order to fill up the research blank of China in this field, our Jiexi Medical Science ﹠ Technology Co., Ltd., Wuxi City is on big quantity research basis, find lsothiocyanates (JC-5411 series compound), no matter be natural, still synthetic can suppress methylating of DNA-CpG site, HDAC can be suppressed again, thereby the gene of being closed unusually in cancer cells restore funcitons (up to now, still the none compound has such dual function) again can be made effectively.

Our a large amount of experiments show that also this compounds has very strong antitumour activity.With existing methylate and hdac inhibitor compare, the JC-5411 series compound has following characteristics:

1, the JC-5411 series compound not only suppresses dna methylation but also suppress HADC, and this is unique in present all known these class medicines.Singlely handle cancer cells, just can reach and make important function of gene such as the cancer suppressor gene recovery normal function of closing unusually after the canceration with the JC-5411 compounds.

2, compare with present widely used dna methylation inhibitor decitabing, JC-5411 is that effect of DNA demethylation or antitumous effect all are better than decitabing.

3, HADC inhibitor, chemistry and the drug metabolism character instability of present clinic trial.Though many these compounds are very strong in vitro inhibition HADC activity, whole antitumour activity is not strong, or invalid.And our experiment has proved the JC-5411 series compound, and its cylinder metabolism-ure PEITC-NAC still has the activity that is equal to.

4, (SuberoylanilideHydroxamic Acid SAHA) compares, and JC-5411 series compound antitumour activity is better than SAHA with HADC inhibitor N-Vorinostat that present clinic trial is best.

5, to compare with histone deacetylase inhibitors Sodium propanecarboxylate on probation clinically, the activity intensity of JC-5411 approximately is 200 times of Sodium propanecarboxylate.

6, no matter be the traditional sweet class dna methylation inhibitor of nuclear or lignocaine of existing discovery, the anti-heart disorder or the local anesthetic of clinical use such as PROCAINE HCL, PHARMA GRADE all have serious toxic side effect, have limited the clinical use of its treatment malignant tumour.Just the opposite, the JC-5411 series compound comes from natural food, and we the experiment proved that JC-5411 series compound selectivity is extremely strong, under active drug dosage, the normal human cell is not had any undesirable action.

Summary of the invention

The objective of the invention is to seek a kind of isosulfocyanate compound and the application in treatment and preventing cancer thereof of natural and synthetic, this compound can restart normal gene function, can be used for treatment and prevents malignant tumour.

Isothiocyanic acid compounds (Isothoicyanates), i.e. JC-5411 series compound, natural or synthetic, be representative with JC-5411, be dna methylation and histone deacetylase inhibitors, its structural formula is as follows:

The tumour of indication comprises various leukemia and various solid tumor.

In use, can be single therapy, can also be combination therapy.Combination therapy can be and other chemotherapy coupling, can also with the herbal medicine coupling, or with operation, radiotherapy, immunotherapy, hormonotherapy, couplings such as gene therapy.Combination therapy can be to treat simultaneously, can also be different precedence treatments.

The JC-5411 series compound can adopt any medicine-feeding way as medicinal, any administering mode.Promptly can be oral, injection sucks, and Transdermal absorption is implanted, cavity, mucous membrane, and intravenous drip etc.

The JC-5411 series compound can be made tablet as medicinal, pill, and capsule, paste, creme, patch, pulvis, injection, sprays, implant is fastened agent, multiple formulation such as drops.

The present invention has worldwide proved that JC-5411 can suppress GSTP1 gene promoter (Promoter) dna methylation effectively first by dna methylation specific DNA polymerase chain reaction,PCR (MS-PCR) and quantitative dna methylation specific DNA polymerase chain reaction,PCR (Pyro-sequencing) in hormone-dependent type and hormone independent form people prostatitis cancer cells.Its effect is better than the positive drug decitabing.The GSTP1 gene almost completely is closed by abnormal methylation owing to starting the subarea in the cancer cells of people prostatitis.

The present invention has proved first worldwide that by Western blotting JC-5411 can activate people prostatitis cancer cells effectively and express GSTP1 albumen again.

The present invention has worldwide proved JC-5411 and cylinder metabolism-ure JC-5411-NAC thereof inhibition of histone demethylation enzyme effectively first by the fluorescence enzyme linked immunosorbent assay.

The present invention has proved first worldwide that by Western blotting JC-5411 regulates the chemically modified of histone significantly selectively, promptly promotes the histone H 3 acetylize, and histone H 3 K4 methylates (result of this chemically modified helps genetic transcription).In contrast, under the equal experiment condition, JC-5411 inhibition of histone H3K9 methylates (this proteic methylating is unfavorable for gene transcription).Experiment also shows that under the same conditions, JC-5411 descends the protein level of HDACl.With histone deacetylase inhibitors Sodium propanecarboxylate on probation clinically ⁽²⁰⁾Compare, the activity intensity of JC-5411 approximately is 200 times (effect that JC-5411 produced of 10 μ M is equivalent to the effect that Sodium propanecarboxylate produced of 2mM) of Sodium propanecarboxylate.

The present invention shows Anticancer Activities by MTT and srb assay, and JC-5411 is to mammary cancer, liver cancer, lung cancer, cancer of the stomach, colorectal carcinoma, cervical cancer, melanoma, hormone-dependent type and hormone independent form prostate cancer and neuroblastoma all have good antitumous effect.Particularly importantly JC-5411 also has the restraining effect identical with hormone-dependent prostate cancer to the hormone independent form prostate cancer of feeling simply helpless clinically.

The present invention proves that by the human prostata cancer model that reaches nude mouse JC-5411 has stronger antitumour activity to the human prostata cancer of the non-dependence of hormone.

Being noted that JC-5411 in tumor therapeutic procedure, can be single therapy, can also be and 2 kinds 3 kinds or more multiple medicines thing combination therapy, administration simultaneously, or different precedence administration.Here said combination therapy can be and other Western medicine that herbal medicine merges use, or and hormonotherapy, radiotherapy, immunotherapy, chemotherapy, cold therapy and gene therapy combined treatment.

The medicament production treatment tumour that this invention provides, these products contain the JC-5411 of effective dose and all suitable additives that other pharmaceutical industry is adopted, and various suitable medicine-feeding way.Consummate drug manufacture technology comprises that the prodrug that uses nontoxic pharmaceutical industry to accept is raw material, adopts several different methods to extract from natural materials or synthetic JC-5411.Consummate drug manufacture technology also comprises uses solvent that pharmaceutical industry the accepts solvent as JC-5411, water for example, and mineral oil, edible oil, dimethyl sulfoxide (DMSO), etc.

JC-5411 can be made into oral administration, local application, inhalation, formulations such as anum administration.Use material that nontoxic pharmaceutical industry accepts as carrier, additives, excipient.Be pointed out that once more administering mode can be a drug combination.Oral administered dosage form comprises: tablet, capsule, tincture, lozenge, oral syrup or other all suitable formulations.

JC-5411 can be made into the formulation that is administered systemically, and comprising: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraspinal injection, intrathecal injection, or other any drug administration by injection and instillation.In addition, the JC-5411 preparation still can contain other pharmaceutically all available non-toxic substances except that the JC-5411 that contains effective dose.Comprise one or more carriers, thinner, additives, and if necessary, other medicinal component.

JC-5411 can be made into any type of oral dosage form, comprising: tablet, and lozenge, the soft or hard capsule can be obeyed aqua, tincture, oral administration mixed suspension, pulvis, emulsifying agent etc.Oral dosage form can contain one or more sweeting agents, correctives, and color agent and sanitas are to guarantee that color is perfect and to be convenient to take.

The excipient that is used for the JC-5411 tablet can be the inert thinner, as lime carbonate, and yellow soda ash, calcium lactate, calcium phosphate, sodium phosphate; System granule: W-Gum, alginic acid; Tackiness agent: starch, gelatin or gum arabic; Slipping agent: stearic acid, Magnesium Stearate or talcum powder.Adopt all known technologies, make dressing or the tablet of having no clothes.Coated dosage form comprises enteric coating and slow release formulation.The material that is used for slow release formulation can be glycerine monoester or dialycerides salt.

The JC-5411 capsule can be a hard capsule, can also be soft capsule.The auxiliary material that is used for hard capsule can be lime carbonate, calcium phosphate or kaolin.Be used for the auxiliary material such as the water of soft capsule, oils comprises peanut oil, sweet oil or liquid paraffin.

The oral water type of JC-5411 suspension can be formulated with the JC-5411 and the suitable diluent of effective dose.Its dilution suspensoid can be sodium cellulose glycolate or methylcellulose gum, Vltra tears, alginic acid sodium salt, polyvinylpyrrolidone, tragcanth, gum arabic.Dispersion agent or wetting agent comprise: natural phospholipid, as Yelkin TTS, or the condenses of alkene oxide and lipid acid, as 17 alkyl oxidation of ethylene alkanols, or ethene and fatty acid part esterification condensation thing, or hexitol such as the single oleate of polyoxyethylene sorbitol, or ethylene oxide and fatty acid part fat condensation product, and alcohol anhydride, as polyethylene sorbyl alcohol methyl esters hydrochlorate.

The oral water type of JC-5411 suspension also may contain one or more sanitass, as ethyl to or o-hydroxy st-yrax salt; Contain one or more sweeting agents, as sucrose or asccharin.

JC-5411 oil type suspension can be suspended in the JC-5411 of effective dose in the vegetables oil, as soybean oil, and peanut oil, Semen Maydis oil, sweet oil, sesame oil, theobroma oil.Or mineral oil, as liquid paraffin.Said preparation also can contain thinner, as beeswax, and wax, or cetyl alcohol, sweeting agent mentioned above, and correctives and stablizer are as antioxidant vitamin C (xitix).

Adopt aforesaid dispersion agent, wetting agent, suspensoid, sanitas, correctives, sweeting agent, and tinting material, JC-5411 also can be made into oil-in-water preparation.JC-5411 makes oil-in-water emulsifiers.Its oil phase can be aforesaid vegetables oil, as soybean oil, and sweet oil, peanut wet goods.Can also be liquid paraffin, mineral oil, or the mixture of two classes.Emulsifying agent can be natural emulsifying agent, as kordofan gum, or tragcanth, natural phospholipid such as soybean phospholipid, Yelkin TTS, lipid acid, the fat of hexitol or acid anhydride or half resin are as the sorbose monoester.Also artificial chemical materials, as ethylene oxide half fat condenses, polyethylene sorbyl alcohol monoester salt.The oil-in-water type emulsion preparation of JC-5411 also can contain sweeting agent and correctives etc.

The syrup of JC-5411 and tincture can adopt sweeting agent, as propyl alcohol, and propylene glycol, sorbyl alcohol or sucrose.Said preparation also can contain wetting agent, sanitas, correctives, color additives.

The injection of JC-5411 can adopt aseptic parenteral solution or oily suspension.It can use all dispersion agent, wetting agent or suspensoids that are suitable on the pharmaceutics.Said preparation can be aseptic transparent liquid, uses all thinners that are suitable on the pharmaceutics, as 1,3 butylene glycol, and water, Ringer's solution, or physiological saline.In addition, aseptic lipid also can be used as solvent or suspensoid.It can be synthetic glycerine monoester or two fat, and lipid acid is as oleic acid etc.

JC-5411 also can be made into and fastens agent, as supp anal etc.Suppository can be combined by a certain amount of JC-5411 and corresponding non-stimulated excipient.Excipient is solid at normal temperatures, but liquefies and the release medicine under anus body temperature.The excipient that is suitable for comprises theobroma oil and macrogol class.

The formulation that is administered systemically of JC-5411 according to used carrier concentration, can be a suspension type, also lysotype.For easy-to-use or alleviate patient's misery, can add sanitas, buffer reagent and local anesthetic.

JC-5411 also can be used as animal doctor's medication.Take for ease of animal, the preparation that contains JC-5411 can add in the animal-feed or in the drinking-water.Also can be made into easily formulation as food or the drink of animal.

For the people, during above-mentioned disease, the dosage range of JC-5411 tentatively is decided to be per kilogram of body weight 0.01mg to 20mg in treatment, or by 60 kilograms of body weight per capita about every day of 0.6mg to 1200mg.According to administering mode, JC-5411 can be made into single dose formulation or multiple agent type.Dose unit tentatively is decided to be and contains effective ingredient 1mg to 500mg.

Every day, administration number of times was decided according to sick the kind.In most cases, no more than four times of every day.Be to be noted that dosage may depend on multiple factor, as the age, body weight, healthy state, sex, diet, and administration time, approach, the state of an illness and medicine unite use etc.So final dosage regimen should be determined as the case may be by the doctor.

The priority compounds that this patent is mentioned has ideal pharmacology as superior oral administration biaavailability, hypotoxicity, low plasma protein binding ratio and ideal inside and outside transformation period.This compounds can pass through hemato encephalic barrier, and this is an advantage to the treatment central nervous system disease.

Description of drawings

Fig. 1, the chemical structure of JC-5411 and lsothiocyanates parent nucleus thereof.

Fig. 2, JC-5411 suppress the effect of GSTP1 gene methylation.Logarithm generates the Human Prostate Cancer Cells LNCAP of phase, with (0.5-2 μ M) JC-5411 of different concns and after 5 '-AZA of concentration (positive control) handles 5 days as shown in the figure, extract genomic dna, measure the DNA of methylating (M) of GSTP1 gene promoter position and non-methylating (U) form with the MS-PCR method.

Fig. 3, quantitative assay JC-5411 is to the restraining effect of GSTP1 gene methylation.With the Human Prostate Cancer Cells LNCAP of logarithmic phase, handle 5 days with JC-5411 (2 μ M) or 5 ' Aza (positive control, 5 μ M) respectively after, the methylating of quantitative assay GSTP1 gene CpG site.

Fig. 4, JC-5411 and 5 ' Aza are to the influence of GSTP1 genetic expression in the LNCAP cell.With the Human Prostate Cancer Cells LNCAP of logarithmic phase, handle 6 days with JC-5411 (2 μ M) or 5 ' Aza (positive control, 5 μ M) respectively after, extract total protein of cell, and send out the protein level of measuring GSTP1 with Western blot.β-Actin is used as interior mark among the figure, to improve the reliability of measuring.

Fig. 5, JC-5411 is to the influence of histone chemically modified.The human prostata cancer LNCAP cell that logarithm is generated the phase was handled 36 hours with the JC-5411 of concentration as shown in the figure, and discrete group albumen carries out protein blot experiment with the locus specificity HAB, with histone level shown in measuring.β-Actin is interior mark among the figure, and is identical to show the protein content that every row added.

Fig. 6, JC-5411 is to the restraining effect of HDAC.Left figure: JC-5411 is to the restraining effect of HDAC in the prostate cancer LNCAP cell.The LNCAP cell of taking the logarithm vegetative period was handled 36 hours with the JC-5411 of different concns, extracted cell protein, measured the HDAC enzymic activity with HDAC luciferase labeled kit.The diagram result is the mean+SD of three experiments.Right figure: incubated 10 minutes with the JC-5411 of commercially available HeLa nucleus extract and different concns and cylinder metabolism-ure JC-5411-NAC thereof or with TSA (as the positive control) temperature of IC50 concentration, measure the HDAC enzymic activity with HDAC luciferase labeled kit.The diagram result is the mean+SD of secondary experiment.

Fig. 7, JC-5411 inductive apoptosis.The Human Prostate Cancer Cells LNCAP of logarithmic phase, handle 48 hours with as shown in the figure JC-5411 concentration after, measure the percentage ratio of apoptotic cell respectively with TUNEL method (black) and activatory caspases enzyme fluorometry (blueness).Data are mean+SD of secondary independent experiment among the figure.

Fig. 8, JC-5411 and positive control medicine Casodex thereof, SAHA, 5 '-Aza is to people's hormone-dependent prostate cancer LNCAP cell growth-inhibiting medicine-effect curve.People's hormone-dependent prostate cancer LNCAP cell growth of logarithmic phase is inoculated in 96 orifice plates (2500 cells/well), after 24 hours, medicine shown in the figure of adding equal proportion dilution, and continue to cultivate 7 days, measure cell with mtt assay and grow.

Fig. 9, JC-5411 and positive control medicine Casodex thereof, SAHA, 5 '-Aza is to people's hormone independent form prostate cancer AI cell growth-inhibiting medicine-effect curve.The same Fig. 8 of method.

Figure 10, JC-5411 relies on AD and hormone independent form AI prostate cancer cell growth-inhibiting medicine-effect curve to people's hormone, and with the comparison of taxol (Paclitaxel).The same Fig. 8 of method.

Embodiment

Example 1, JC-5411's is synthetic

Instrument and reagent

¹H-NMR spectrum Brucker AV-300 type nmr determination, interior mark TMS, solvent are CDCl ₃MS measures with Nicolet FTMS-2000 type mass spectrograph; Ultimate analysis Elementar Vario EL III Instrument measuring.

Thin plate chromatography (TLC) adopts silica GF254 (Haiyang Chemical Plant, Qingdao's production) self-control; Reagent is commercially available chemical pure or analytical pure product, unprocessed direct use.

The preparation of JC-5411 (Phenylethyl mustard oil, Phenethyl Isothiocyanate)

In the 50mL round-bottomed flask, add 15mLCH ₂Cl ₂With thio phosgene 3mL (40mmol), stir, be cooled to 0 ℃, (4.04g is 40mmol) with phenylethylamine (4.85g, 40mmol) mixed solution of Zu Chenging (heat release of dropping process, temperature should not above 15 ℃) slowly to drip the triethylamine of equivalent with constant pressure funnel.After drip finishing, at room temperature reacted 5～6 hours, after detecting phenylethylamine and disappear with TLC, add the 10mL shrend reaction of going out.Add 5mLCH again ₂Cl ₂, separating funnel is told organic phase, after the anhydrous sodium sulfate drying organic phase, filters, and concentrates organic phase to doing.Debris is made elutriant with sherwood oil (60～90 ℃ of boiling ranges), through silicagel column wash-out purifying, after concentrating, gets colourless oil liquid 4.9g through rectification under vacuum, productive rate 75%.

¹H-NMR δ: 7.26～7.24 (m, 3H, Ph-H), 7.12 (d, J=8.5Hz, 2H, Ph-H), 3.94 (t, J=7.0Hz, 2H, CH ₂), 2.81 (t, J=7.0Hz, 2H, CH ₂); ESI-MS:164.1[M+H] ⁺, C ₉H ₉NS (163.24); Anal.Calcd for C ₉H ₉NS:C66.22, H 5.56, N8.58; Found:C 66.30, H5.42, and N 8.34; Molecular weight is 163.24.

Example 2, JC-5411 suppress GSTP1 gene promoter dna methylation:

Material and method:

Reagent: JC-5411 phenethyl isothiocyanate Phenethyl Isothiocyanate, by Jiexi Medical Science ﹠ Technology Co., Ltd., Wuxi City synthetic (seeing embodiment one).During experiment, with the DMSO wiring solution-forming.DNA polymerase chain reaction,PCR (PCR) reagent is purchased the biotech company in American I nvitrogen.The PCR primer is purchased the Operon biotech company in the U.S.. other chemical reagent of experiment, except that specifying, all purchase Sigma chemical reagents corporation in the U.S..The poly-propionic acid amide gel reagents of protein electrophoresis, SDS, electrophoretic buffer, albumen electrotransfer damping fluid is purchased the Bio-Rad life science company in the U.S..

Cell cultures: Human Prostate Cancer Cells LNCAP and PC-3 purchase the Collection in American Type Culture.Cell places 37 ℃, 5%CO ₂In the incubator, to contain the RPMI1640 culture medium culturing of 10% foetal calf serum and penicillin and Streptomycin sulphate.

Specific methylation DNA polymerase chain reaction,PCR (MS-PCR): carry out the MS-PCR experiment to measure the influence of JC-5411 to dna methylation with the described method of Singal et al ⁽⁵⁾In brief: logarithm generates the human prostata cancer LNCAP cell of phase, handle 5 days with the JC-5411 of different concns after, extract cell genomic dna.Get the DNA sample of 1 microgram, with the sodium hydroxide (NaOH) of 0.2M handle made its sex change in 10 minutes after, add the hydrogenated quinoline (hydroquinone) of the freshly prepared 10mM of 30 μ l and 3M sodium bisulfite (pH5.0) and a little mineral oil of 520 μ l.Then the DNA sample is purified after 16 hours 50 ℃ of insulations.With the DNA through the sodium bisulfite conversion is template, carries out MS-PCR, and its condition is as follows: the type that methylates (M) PCR primer: 5 '-TTC GGG GTG TAG CGG TCG TC-3 ' and 5 '-GCC CCA ATA CTAAAT CAC GAC G-3 '; The non-type that methylates (U) PCR primer: 5 '-GAT GTT TGG GGT GTA GTG GTTGTT-3 ', and 5 '-CCA CCC CAA TAC TAA ATC ACA ACA-3 '.Carry out the PCR reaction with MJ Research PTC-100 PCR instrument, its PCR program is as follows: 95 ℃/10 minutes, and then with 95 ℃/1 minute, 54 ℃/1 minute, 72 ℃/1 minute, 72 ℃ of final chain extensions, 10 minutes.Reaction is carried out 50 cycles altogether.Each sample carries out double MS-PCR reaction.Detect the PCR reaction product with 2% agarose electrophoresis.

JC-5411 suppresses the methylated quantitative assay of DNA CpG (Pyro-sequencing): adopt the described pyro-sequencing method of document ^(21,22), we are the PCR primer with 5 '-AACTCTAAACCCCATC of 5 ' GTGATTTAGTATTGG and biotin mark, and are the primer of determined dna sequence with 5 '-AGGTTTTTGTTGGA, the program that uses a computer quantitative analysis the effect of JC-5411 demethylation.

Western blotting detects GSTP1 albumen: the Human Prostate Cancer Cells LNCAP of logarithmic phase was handled 6 days with the JC-5411 or 5 μ M, the 5 '-Aza (positive control) of different concns.Collecting cell, washing is with literature method ⁽²³⁾Extract albumen, quantitatively.The albumen of getting 50 μ g carries out the poly-propionic acid amide gel electrophoresis of SDS-.Behind the electrophoresis, the albumen electricity is forwarded on the nitrocellulose membrane, detects with special GSTP1 antibody, and with β-Aetin antibody as interior mark.

Result and discussion:

Show by a large amount of experiments of adopting prostate cancer patient specimen and clone to carry out, the GSTP1 gene methylate and closing unusually of transcribing is very special and very responsive prostate cancer molecule pathology.In order to study the influence of JC-5411 to gene methylation, we have selected PC-3 LNCAP is research object, has observed JC-5411 to GSTP1 gene methylation restraining effect with methylation specific DNA polymerase chain reaction,PCR technology.As shown in Figure 2, the GSTP1 gene of LNCAP cell is almost 100% by methylated (M is the form of methylating among Fig. 1, and U is the non-form that methylates).But the JC-5411 with different concns handled the LNCAP cell after 5 days, we have observed the DNA signal of the non-form that methylates, and the demethylation effect of JC-5411 is that drug level relies on (effective concentration is 1-2 μ M), and this drug effect and unartificial experimental error are described.With same method, we have studied JC-5411 to other cancer cells such as Human Prostate Cancer Cells PC-3 and the effect of DU145 demethylation, have all obtained same result.(5 '-AZA) (5,7) are compared, and the action intensity of 5 '-AZA demethylation of 5 μ M is suitable with the JC-5411 of 1 μ M, illustrate that JC-5411 is better than 5 '-A7A with positive control medicine decitabing.

Adopt the described pyro-sequencing method of document ^(21,22), our further quantitative assay the influence of JC-5411 to the GSTP1 gene methylation.As shown in Figure 3, topped 4 CpG of our the used determined dna sequence primer site (table be 1,2,3 and 4) respectively that methylates.As expectation, in normal prostate tissue DNA, do not detect methylating of GSTP1 gene.In contrast, GSTP1 gene methylation degree is quite high in prostate cancer cell LNCAP, and as site 1 and 3, LNCAP cell GSTP1 gene methylation is respectively 89.% and 61.8% (C:89.5% and C:61.8%).After the JC-5411 of 2 μ M handled 5 days, 1 and 3 methylate then dropped to 78.3% and 6.5% respectively.JC-5411 mediates the restraining effect that methylates and is better than positive control medicine 5 '-Aza.Under 5 '-Aza effect of 5 μ M, 1 and 3 methylate then drops to 78.3% and 37.7% respectively.

For the further GSTP1 demethylase geneization of proof JC-5411 mediation, can reset this expression of gene, we have measured LNCAP cell GSTP1 protein level after JC-5411 handles with Western blotting.As shown in Figure 4, the LNCAP cell is after the JC-5411 of 2 μ M handles 6 days, and the albumen of GSTP1 increases considerably.So we prove that first JC-5411 is a dna methylation inhibitor at experiment, its result makes the gene of being closed unusually restart expression, recovers its original function.

Example 3, JC-5411 inhibition of histone deacetylase:

Material and method:

Reagent: JC-5411 is with example 2.The poly-propionic acid amide gel reagents of protein electrophoresis, SDS, electrophoretic buffer, albumen electrotransfer damping fluid is purchased the Bio-Rad life science company in the U.S..The locus specificity acetylize and the histone H 3 antibody that methylates are purchased Upstate Biochem Technology, INC. in the U.S..Other chemical reagent of experiment except that specifying, is all purchased the Sigma chemical reagents corporation in the U.S..Histone deacetylase (HDAC) determination of activity test kit is purchased Biomol Biochem Technology, INC. in the U.S..

Cell cultures: with example 2.

Acetylation of histone and methylated mensuration: with the Human Prostate Cancer Cells LNCAP of logarithmic phase, handle 48 hours with the JC-5411 of different concns after, with the described method discrete group of people such as Yoshida albumen ⁽²⁴⁾: cell is collected with ice-cold phosphate buffered saline buffer, and place the molten cell damping fluid of 1ml (to contain: the 50mM sodium bisulfite, 8.6% sucrose, 1%TritonX-100, the 10mM magnesium chloride, and 10mM Tris-HCl pH 6.5), make homogenate with homogenizer, under 700RPM speed centrifugal 5 minutes, collecting cell nuclear.Again with molten cell damping fluid washing three times, 10mM Tris-HCl pH 7.4 (containing 13mM EDTA) washs once with nucleus.Then, press the described method of document, extract histone with acidic solution ⁽²⁴⁾,, measure the acetylize of overall core histones and methylate by Western blotting again with the locus specificity acetylize and the histone H 3 antibody that methylates.

The active mensuration of histone deacetylase (HDAC): with human prostata cancer LNCAP nucleus extract after 37 ℃ of temperature are incubated several minutes; the JC-5411 and the positive control medicine Trichostatin A that add different concns; continue temperature and incubated 10 minutes, with histone deacetylase (HDAC) determination of activity kit measurement I type and II type HDAC enzymic activity.

Result and discussion: activate pent expression of gene for whether detection JC-5411 can modify histone; we handle Human Prostate Cancer Cells LNCAP 36 hours with the JC-5411 of different concns; separated histone; adopt the locus specificity acetylize and the histone H 3 antibody that methylates, measured the acetylize of overall core histones and methylate with Western blotting.As shown in Figure 5, JC-5411 regulates the chemically modified of histone significantly selectively, promptly promotes the histone H 3 acetylize, and histone H 3 K4 methylates.And histone H 3 K4 to methylate be right chromaticness activated important symbol ⁽²⁵⁾And this effect of JC-5411 is that drug level is dependent.Opposite in this, under the equal experiment condition, (modify on the contrary with histone H 3 K4, methylating of histone H 3 K9 then is the sign of transcribing of suppressor gene for the methylating of JC-5411 inhibition of histone H3K9 ⁽²⁶⁾).Experiment also shows that under the same conditions, JC-5411 descends the protein level of HDACl.What deserves to be mentioned is the histone deacetylase inhibitors Sodium propanecarboxylate of using with clinical following formula ⁽²⁰⁾Compare, the activity intensity of JC-5411 approximately is 200 times (effect that JC-5411 produced of 10 μ M is equivalent to the effect that Sodium propanecarboxylate produced of 2mM) of Sodium propanecarboxylate.

Whether in order to study chemically modified that JC-5411 regulates histone selectively is that we adopt HDAC to measure test kit, have observed the effect of JC-5411 to histone deacetylase (HDAC) by its inhibition of histone deacetylase effectively.As shown in Figure 5, cell is after JC-5411 handles, and HDAC is active obviously to descend.This presentation of results JC-5411 can directly suppress the HADC enzymic activity.

For proving that further JC-5411 can directly suppress the HDAC enzymic activity, we adopt the source of commercially available HeLa nucleus extract as the HDAC enzyme, JC-5411 and Trichostatin (TSA with itself and different concns, the positive control medicine) temperature was incubated 10 minutes, measured the HDAC enzymic activity with fluorescence enzyme linked immunosorbent assay (ELISA).As shown in Figure 5, JC-5411 can suppress the HDAC enzymic activity significantly.Compare with TSA, though the JC-5411 enzyme inhibition activity than TSA a little less than, TSA chemistry and biological property are extremely unstable, and toxicity is big, no medicinal application value.By contrast, not only JC-5411 is effective, and its cylinder metabolism-ure JC-5411-NAC and its parent compound JC-5411 have suitable enzyme inhibition activity (seeing right half figure of Fig. 6).We also prove, induce the histone chemically modified to come to the same thing with above-mentioned, and JC-5411 also is better than Sodium propanecarboxylate to HDAC enzymic activity restraining effect.

Can also be found out that by our above-mentioned experiment it is strong that JC-5411 suppresses the dna methylation effect, required drug level is low.But this must at least two dna replication dna processes of experience could the embodiment is so need certain drug treating time (more than 5 days, seeing Fig. 3).By contrast, JC-5411 is then very fast to the chemically modified of regulating histone.Cell was handled 36 hours with JC-5411, and drug effect is very obviously (Fig. 5) just.

Be noted that up to now the none known compound not only suppresses dna methylation but also regulate the histone chemically modified.And our above-mentioned experiment has proved that JC-5411 has this dual function.This is one of this patent uniqueness.

Example 4, the JC-5411 cancer cell specific induction of apoptosis:

Material and method:

Reagent: JC-5411 is with example 2.Apoptosis in-site detecting test kit is purchased in U.S. Roche molecule biochemical corp.The caspases test kit that mensuration has activated is purchased in American I ntergen molecular biology reagent company.Other chemical reagent of experiment except that specifying, is all purchased the Sigma chemical reagents corporation in the U.S..

Cell cultures: with example 2.

Apoptotic mensuration: according to DNA splitting of chain and apoptosis morphology difference, we measure JC-5411 inductive apoptosis with described TUNEL method of document and fluorized marking method ^(27-30)With the Human Prostate Cancer Cells LNCAP of logarithmic phase, handle 48 hours with the JC-5411 of different concns after, with 4% formalin fixed film-making, transparent with 0.1%Triton again.Then with cell and or not do not incubate with as negative control the terminal enzyme (DNA) temperature.The cell that contains the caspases enzyme that has activated then indicates with fam-VAD-fmk reagent, and then carries out fluorescent dye with propidium iodide, and adopts the cell after two colorimetric method for determining dyeing.Calculate caspase positive cell frequency.

Result and discussion:

We according to apoptosis morphology difference and these two basic apoptosis characteristic measurements of dna break the influence of JC-5411 to Human Prostate Cancer Cells LNCAP apoptosis.Compare with untreated contrast, JC-5411 handled after 24 hours, many agglomerating floating dead cells occurred.Handle after two days, cell density obviously descends.Handled 48 hours with the JC-5411 of 0.1 μ M, the cell density 4-8% that approximately descends, when drug level increased to 20 μ M, cell density then descended 60%.Many cells are under the JC-5411 effect, and cell diminishes, and chromatin density increases, and tangible features of apoptosis occurred.And, promote apoptotic BAX gene, after under the JC-5411 effect 4 hours, expressing significantly increases, and parallels with the DNA chain rupture.Fig. 7 is the percentage ratio with the apoptotic cell of TUNEL method mensuration.In the JC-5411 effect back 48 hours, apoptotic cell obviously increased along with the increase of drug level.

Example 5, the research of JC-5411 anti tumor activity in vitro

Material and method:

Reagent: JC-5411 is with example 2.Other chemical reagent of experiment except that specifying, is all purchased the Sigma chemical reagents corporation in the U.S..

Cell cultures: Human Prostate Cancer Cells LNCAP is with example 2.Hormone independent form prostate cancer cell AI is by colleague's present.Other human cancer cell comprises: people's hormone independent form prostate cancer cell PC-3, DU145, breast cancer cell MCF-7, colon cancer cell LOVO, liver cancer cell Bel-7402, cervical cancer cell HeLa, melanoma cell M-14, stomach cancer cell SGC-7901, rat breast cancer cell SHZ-888 are provided by Southern Yangtze University.Pressing the described method of document cultivates ⁽³¹⁾

MTT and SRB:MTT and SRB experimental technique are as described in the document ⁽³²⁾Be summarized as follows: the cancer cells in the vegetative period of taking the logarithm, be inoculated in 96 orifice plates, cell density is every hole 2500 cells/200 μ l.Adding waits the medicine of serial dilution after 24 hours.Cell continues to cultivate 72 hours under drug effect.After the nutrient solution of 100 μ l is taken out in every hole, add 50 μ l MTT[bromination 3-(4,5-dimethylthiazole-2)-2,5-phenylbenzene tetrazole 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution continue to cultivate 4 hours.Dissolve the latent throw out of atropurpureus first with 200 μ l hydrochloric acid-aqueous isopropanols, measure absorbance value in 540nm wavelength place.If adopt srb assay, then after 72 hours, remove all nutrient solutions, after cell is fixed 1 hour with 10% Tricholroacetic Acid, put in the air seasoning 24 hours, add 50 μ lSRB (lissamine rhodamine B, sulforhodamine B) dyeing is 20-30 minute, with 1% Glacial acetic acid washing 5 times, puts in the air after the seasoning, add 10mM Tris-HCl (pH 10.0) solution dissolving red-purple albumen-SRB mixture of 200 μ l, measure absorbance value in 600nm wavelength place.Try to achieve the mean value of respectively organizing testing data, with experimental group control group is calculated cell and generate inhibiting rate.With Sigma Plot mapping software paint cell growth rate-drug level semilog plot, and try to achieve the medium effective concentration (IC of medicine ₅₀).

Result and discussion:

Adopt MTT and two kinds of analytical procedures of SRB, we study the antitumour activity of JC-5411.Table 1 is listed is JC-5411 and other chemotherapeutics half effective inhibition concentration to various cancer cells.Fig. 8-the 10th, the JC-5411 that enumerates and other medicine are to the drug effect-drug level curve of hormone-dependent type and hormone independent form prostate cancer cell LNCAP effect.By table 1 and Fig. 8-9, as can be seen, JC-5411 has stronger antitumour activity.For Human Prostate Cancer Cells, the antitumour activity of JC-5411 is better than up-to-date clinically hormone antagonist medicine Casodex.Should be noted that especially Casodex is invalid to hormone independent form prostate cancer cell.Same hormone independent form prostate cancer cell is to many chemotherapeutics resistances, and as shown in figure 10, the AI cell has approximately increased by 50 times to taxol IC50.In contrast, no matter be hormone-dependent type, or hormone independent form prostate cancer cell, all very sensitive to JC-5411, its half effective inhibition concentration IC50 is less than 1 μ M (seeing Table 1).

On the other hand, as dna methylation inhibitor, JC-5411 compares with 5 ' of widespread use-Aza, and JC-5411 is strong than 5 '-Aza to the growth-inhibiting effect of hormone-dependent type and hormone independent form prostate cancer cell.And by Fig. 8, Fig. 9 can find out under 5 ' of higher concentration-Aza (25 μ M) effect, still have the cancer cells more than 20% to survive.And under identical experiment condition, the JC-5411 of 5 μ M promptly obtains 98% inhibiting rate.With present very good clinically histone deacetylase (HDAC) inhibitor, SAHA compares, and JC-5411 also is better than SAHA to the restraining effect of examination cancer cells.As seen, because JC-5411 has the dual function that suppresses dna methylation and histone deacetylase, its antitumous effect both had been better than dna methylation inhibitor, was better than histone deacetylase inhibitors again.

By MTT and SRB experiment, we are also noted that with other type anticarcinogen and compare that JC-5411 also has its superiority.More much better than than cell induction differentiation agent vitamin A acid (Retinoid acid, IC50:21.5-＞50 μ M) as it to the cancer cells restraining effect.JC-5411 also is better than clinical Casodex that uses always (IC50:37.4 μ M) and Proscar (IC50:40.6 μ M) greatly to the restraining effect of hormone-dependent prostate cancer cell LNCAP.

Table 1:JC-5411 suppresses effective concentration (IC50, μ M) to the half of prostate cancer cell

Reach comparison (mtt assay) with other chemotherapeutics.

Medicine	Clone
				LNCAP	PC-3	DU145
	JC-5411	0.61±0.11	N/A				0.72±0.34
Daunorubicin				N/A	0.24±0.02	0.11±0.01
	Vitamin A acid	15.95±3.19	＞50				＞50
Proscar				40.60±7.12	133.68±12.94	N/A
	Casodex	37.40±7.21	120.11±17.31				N/A
Taxol				0.00035±0.0001	0.00296±0.001	0.00577±0.001

Though compare with daunorubicin or taxol, the JC-5411 antitumous effect is weak, and the former is promptly producing stronger toxic side effect under effective drug level, produces serious bone marrow depression when the plasma drug level 0.05 μ M as taxol.And JC-5411 derives from food, and toxicity is very low.

It is worthy of note, be not difficult to find out that though the LNCAP cell is very much responsive to taxol, experiment is difficult to record its IC90, because no matter concentration is much, always there is the cell about 15-20% insensitive by Figure 10.Its reason may be these cells under the taxol effect, enter the GO phase and the further effect of hiding medicine.In contrast, JC-5411 has better curative effect-concentration curve.This result shows that also JC-5411 is if different sequencing administrations with taxol then may produce significant synergy.

Table 2:JC5411 reaches the inhibiting rate of colorectal carcinoma, mammary cancer, cervical cancer, melanoma, cancer of the stomach, liver cancer, rat breast cancer

Comparison (mtt assay) with the 5-fluor-uracil inhibiting rate

Clone	JC-5411 inhibiting rate (%)	5-FU inhibiting rate (%)
			Lovo	65.7(20μM)	67.6(20μg/ml)
MCF-7	85.4(40μM)	71.0(20μg/ml)
			Hela	16.0(20μM)	27.4(20μg/ml)
M-14	49.6(20μM)	29.1(20μg/ml)
			SGC-7901	85.0(20μM)	91.6(20μg/ml)
Bel-7402	93.8(40μM)	83.6(20μg/ml)
			SHZ-888	95.3(40μM)	98.2(20μg/ml)

Table 2 is JC-5411 growth-inhibiting effects to other human body tumour cell.Be not difficult to find out that by table 2 JC-5411 all has stronger restraining effect to the growth of examination cancer cells.Specifically, JC-5411 is better than positive control medicine 5-fluor-uracil to the anti tumor activity in vitro of mammary cancer, melanoma, liver cancer; But the anti tumor activity in vitro to colorectal carcinoma, cervical cancer, cancer of the stomach, rat breast cancer is poor than positive control medicine 5-fluor-uracil.It is important to point out that the concentration of positive control medicine 5-fluor-uracil is 20 μ g/ml, this is equivalent to 153 μ M, is higher than the drug level of JC-5411.In addition, positive control medicine 5-fluor-uracil is a cytotoxic drug, and significant side effects is arranged.

Because this compound is the double inhibitor of dna methylation and histone deacetylase; and the chemically modified of unusual dna methylation and histone almost is the common trait of all cancer cells; based on our theoretical principle of experiment and effect thereof; we believe; JC-5411 is a broad-spectrum anti-cancer drug; be JC-5411 not only in the example that the present invention exemplified the tumour of trying effective, also effective to other tumour.

Example 6, the active research of JC-5411 anti-tumor in vivo

Material and method:

Medicine: JC-5411 is with example 2.Ring phosphamide and experiment except that specifying, are all purchased the Sigma chemical reagents corporation in the U.S. with other chemical reagent.Tumor inoculation and drug treating: the BALA/c hairless mouse, male, in 5 ages in week, purchase Shanghai animal center in the Chinese Academy of Sciences.The animal speech is supported in the SMP laboratory.Get people's hormone independent form prostate cancer cell PC-3, add Matrigel glue, subcutaneous vaccination is in the preceding oxter of mouse then, and average every mouse is inoculation 1 * 10 approximately ⁶Cell.Inoculate after 24 hours, animal is divided into 3 groups at random, 9 every group, one group of negative contrast, one group of the 5th day and the 15th day abdominal injection ring phosphamide after inoculation, dosage is 60mg/kg, another group is JC-5411 treatment group, and orally give JC-5411 every day (filling stomach), dosage are 100mg/kg.After treating for 6 weeks altogether, per two weeks are measured gross tumor volume, calculate gross tumor volume with formula V=L * W * H * 0.5236.Wherein L is the knurl major diameter, and W is wide footpath, and H is high.Per 3 all weighing the weight of animals.After last administration 24 hours, the weighing the weight of animals, and with sacrifice of animal, take out tumour and weigh, calculate tumour and generate inhibiting rate.

Result and discussion

The immunodeficiency type mouse is widely used in the transplanting of human tumor and the research of cancer therapy drug ⁽³³⁾. have antitumour activity in order to prove JC-5411, we in the immunodeficiency type mouse, have observed the antitumous effect of JC-5411 with hormone independent form human prostata cancer PC-3 Transplanted cells.As shown in table 3, JC-5411 has stronger restraining effect to hormone independent form human prostata cancer PC-3 cell under used dosage, and inhibition rate of tumor growth reaches 63%, with positive control cyclome phosphamide therapeutic equivalence.On the other hand, endoxan is a cell toxicity medicament, and is the same with other cytotoxic drug, has effect soon, acts on strong characteristics, but lacks selectivity.Can also find out that from our experiment under used dosage, toxic action has appearred in endoxan.It shows with control group and compares, endoxan treated animal weight loss 10.9%.In contrast, JC-5411 has good selectivity, under active drug dosage, obviously suppress the generation of prostate cancer, but tangible toxic reaction does not appear in animal.The weight of animals is compared with control group after the medication, does not have any difference.This is consistent with the initial design objective of medicine.Therefore, we think that JC-5411 has good application prospects.

Table 3:JC-5411 is to inoculation people hormone independent form prostate cancer PC-3 nude mice antitumous effect (X ± SD)

Group	The front/rear number of animals of administration (only)	Dosage (mg/kg)	Knurl heavy (g)	Body weight (g)		Tumour inhibiting rate (%)
							Before the treatment	After the treatment
				Model group	9/9				0	2.14±0.267	20.76±1.3	21.89±1.7	-
Positive drug a	9/9	60 a	0.616±0.016 *			20.54±1.2	18.30±1.2 *	71.2
				JC-5411 b	9/9				100 b	0.784±0.154 *	19.87±1.2	20.93±1.4	63.4

A: the 5th day and administration (i.p.) on the 15th; B: administration every day (p.o); Compare with model control group, ^*P＜0.01.

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Claims (15)

1. A natural and synthetic isothiocyanate compound, JC-5411, is characterized in that the chemical structure of the isothiocyanate compound is:

2. The in vivo metabolite of JC-5411, PEITC-NAC, has the following chemical structure characteristics:

3. The compound according to claim 1 or 2, characterized in that the isothiocyanate compound is a dual inhibitor of DNA methylation and histone deacetylase, which can restart the abnormally closed gene expression. 4. The use of natural and synthetic isothiocyanate compounds as claimed in claim 1 or 2 in the preparation of drugs for inducing cancer cell apoptosis, inhibiting cancer cell growth, and treating and preventing malignant tumors. 5. The use of natural and synthetic isothiocyanate compounds according to claim 4, characterized in that said malignant tumors include leukemia and solid tumors. 6. The application of natural and synthetic isothiocyanate compounds according to claim 5, characterized in that the leukemia is acute lymphoblastic leukemia, acute myeloid leukemia, promyelocytic leukemia, erythrocytic leukemia, chronic Lymphocytic leukemia, chronic myelogenous leukemia, T-cell leukemia, B-cell leukemia. 7. The application of natural and synthetic isothiocyanate compounds according to claim 5, characterized in that the solid tumor is liver cancer, lung cancer, oral cavity tumor, ear cancer, nasopharyngeal cancer, tongue cancer, esophageal cancer , gastric cancer, colon cancer, pancreatic cancer, breast cancer, cervical cancer, ovarian cancer, prostate cancer, penile cancer, melanoma and brain tumor, etc. 8. The natural and synthetic isothiocyanate compounds according to claim 1 or 2, characterized in that the isothiocyanate compounds are produced by all applicable preparation methods in the pharmaceutical industry: artificially synthesized, or Obtained by extraction from natural materials. 9. The natural and synthetic isothiocyanate compounds according to claim 1 or 2, characterized in that, when the isothiocyanate compounds are used for the treatment and prevention of various tumors, they can be made into All applicable dosage forms. 10. The natural and synthetic isothiocyanate compounds according to claim 9, characterized in that the dosage forms include: small needle injection, medium needle injection, large needle injection, powder injection, emulsion for injection, Tablets, pills, capsules, ointments, creams, patches, liniments, powders, sprays, implants, drops, suppositories, ointments, confectionary. 11. The natural and synthetic isothiocyanate compounds according to claim 1 or 2, characterized in that, when the isothiocyanate compounds are used for the treatment and prevention of various tumors, they can be administered in various ways. Medicine: oral administration, injection administration, implant administration, intracavitary administration, sublingual administration, anal administration, transdermal administration, internal and external application. 12. The isothiocyanate compound according to claim 11, wherein said injection administration can be: intravenous injection, intramuscular injection, subcutaneous injection, intracavitary injection. 13. The natural and synthetic isothiocyanate compounds according to claim 1 or 2, characterized in that, when the isothiocyanate compounds are used for the treatment and prevention of various tumors, they can be used as monotherapy, It can also be used in combination. 13. The natural and synthetic isothiocyanate compounds according to claim 1 or 2, characterized in that the combined drug includes: combined use with radiation therapy, combined use with Chinese herbal medicine, combined use with surgery, and combined use with biological conditions Agents are used in combination with gene therapy. 14. The natural and synthetic isothiocyanate compounds according to claim 1 or 2, characterized in that they have no adverse effects on normal human cells under effective drug dosage.

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