CN103319371B - Preparation method and application of N-substituted hydroxamic acid compound - Google Patents

Abstract

The invention discloses a preparation method and application of an N-substituted hydroxamic acid compound. The histone compound with a 4-benzamido-N-hydroxy-N-benzoylphenyl ammonia structure, i.e., the N-substituted hydroxamic acid compound, is represented by formula 1 as described in the specification. The preparation method comprises the following steps: with methyl p-aminobenzoate, benzoyl chloride and nitrobenzene as raw materials, allowing methyl p-aminobenzoate and benzoyl chloride to undergo aminoacylation and subjecting a reaction product to hydrolysis and acylation so as to prepare 4-benzamido-benzoyl chloride or 4-(4-chlorphenyl-formamido)benzoyl chloride or 4-phenylacetamido-benzoyl chloride; and reducing nitrobenzene into N-hydroxy aniline under the catalysis of Raney nickel and reacting N-hydroxy aniline with 4-benzamido-benzoyl chloride so as to obtain the target compound.

Description

Its purposes of preparation method of N substituted isohydroxyloxime acid compounds

Technical field

The present invention relates to the Preparation method and use of N substituted isohydroxyloxime acid compounds.The present invention is exactly a kind of Antibiotic FR 901228 of 4-benzamido-N-hydroxy-N-phenyl benzene carbon amide structure, and this kind of use of a compound and preparation method.

Background technology

Tumorigenic reason has a lot, think that oncogenic formation is led in gene mutation in the past, and successfully find many antioncogenes, oncogene, but along with deepening continuously to tumor research, increasing example proposes new challenge to " Gene determinism ", such as when DNA base sequence does not change, the phenotype of organism there occurs change.For illustrating the reason that these phenomenons occur, also been proposed epigenetic modification in recent years and change hypothesis, this theory is mainly concerned with two aspects: one is the reversible modification for histone in cyto-chromatin ^[1-3], another is reversibly modified for methylating of gene DNA ^[4-5].DNA methylation normal mode changes, and causes cell carcinogenesis.The reversible modification of histone, except DNA sequence is as except genetic coding, apparent gene mechanism is the most basic regulate factors of gene expression and protein synthesis subsequently.In eukaryotic cell, in GO phase, DNA is closely filled by nucleosome, so that DNA can not close to transcriptional elements.Acetylation of histone reaction is a conclusive apparent gene process, can reinvent transcriptional elements and DNA close to ability, thus initiation gene expression.A large amount of evidences is had now to show; the Chromatin Protein reconstruction of surrounding DNA is the basic mechanism of gene regulation epigenetics; what these histones were reversible transcribes the acetylation modification modified and comprise lysine residue nitrogen-atoms; the modification that methylates of lysine residue and arginine residues; the phosphorylation of serine residue; the ubiquitination of lysine residue, ubiquitin-like modification etc. ^[6].These are modified in cellular gene expression and regulation process and play important effect, modify abnormal meeting trigger cell generation canceration.Wherein, the acetylation modification of histone is the most general.The acetylation modification of histone plays a role in gene expression regulation.Histone acetylation in vivo occurs on the conservative lysine amino of protein N end heredity, but the acetylation modification of histone H 3 and H4 is more extensive than H2A and H2B ^[7].The acetylizad important site of histone H 3 is Lys9 and Lysl4, and the acetylation sites of histone H 4 is Lys5, Lys8, Lys12 and Lys16 ^[8-10].

In the process of Mechanisms of Histone Acetylation Modification; that play a decisive role is acetyltransferase (histone aceyl transferase; and deacetylase (Histone deacetylase HAT); HDAC); the enzyme that these two effects are contrary; common regulate and control the degree of acetylation of histone, play regulator gene express effect.HDAC is the catalytic subunit of polyprotein matter complex, participates in histone and nonhistones protein deacetylation.HDAC mediates nucleosomal structure and changes and regulator gene expression, participates in cell cycle progression and differentiation, and with various diseases as the generation of cancer, acute myeloid leukaemia, virus and infection etc. is relevant with development.Generally, acetylation of histone level strengthens relevant with the enhancing of Gene Transcription in vitro, and Acetylation Level is too low relevant with gene expression inhibition ^[11-12].

By suppressing the activity of HDAC, make the excessive acetylation of histone, eliminate amino with positive charge, it is lax that the tight structure of nucleosome becomes because of electrical change, this lax structure is easy to the close of gene transcription factor and DNA molecular, thus the accurate translation of gene is facilitated, repressed antioncogene is made to recover to express, also can promote the expression of cell cycle inhibitors (P21WAF1/CIP1), make the stability of tumor-inhibiting factor P53 and active raising, improve genomic stability, reduce the probability that gene is undergone mutation, energy arresting cell cycle simultaneously, promote the differentiation of cell, the expression of VEGF (VEGF) and hypoxia inducible factor (HIF-1) can also be reduced, the effect such as inhibiting angiogenesis and cell death inducing.In addition, hdac inhibitor is to the therapeutical effect of nervous system disease and radiosensitizing effect ^[13-15].

HDAC is the promising anticancer target of a class nonkinase of current most.HDAC inhibitor is the New Policy of the treatment human cancer of empirical tests at present.Have been found that the Antibiotic FR 901228 of various structures type at present, comprised (1) short-chain fatty acid and salt thereof, as butanoic acid and isovaleric acid sodium; (2) hydroxamic acid, as SAHA (Vorinostat) and LBH-589 (Panobinostat); (3) Benzoylamide, as MS-275 (Entinostat) and MGCDOI03; (4) cyclic tetrapeptide, as trapoxin and FK-228 ^[16-17]; Ethyl ketone, trifluoromethyl ketone, 2-aminobenzene glue, mercaptan and acetyl derivatives thereof etc. are also had to go second extremely to change enzyme inhibitor as the histone of pharmacophore ^[18].At present, most compound is in clinical front or clinical research, only have Vorinostat and Romidepsin to be used for the treatment of T-cell lymphoma,cutaneous (CTCL) respectively at 2006 and 2009 by U.S. FDA approval listing, these all have wide development prospect predictive of this kind of novel antitumor drug.Above content can see with Publication about Document:

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[11] WSXu1, RBParmigiani1,PAMarks; Histone deacetylase inhibitors molecular mechanisms of action; Cell Biology Program,Memorial Sloan-Kettering Cancer Center,NewYork,NY,USA.

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[17] Roberto Rosato,Stefanie Hock, Paul Dent; LBH-589(Panobinostat) potentiates fludarabine anti-leukemic activity through a JNK- and XIAP-dependent mechanism. Leuk Res. Author manuscript; available in PMC 2013 April 1.

[18] PaulA.Marks,VictoriaM Richon,RichardA.Rifkind; Histone deacetylase inhibitors inducers of differentiation or apoptosis of transformed cells; JournaloftheNationalCancerInstitute,Vol.92,No.15,August2,2000.。

Summary of the invention

The invention provides the histone compound of 4-benzamido-N-hydroxy-N-phenyl benzene carbon amide structure, purposes of this compound and preparation method thereof is provided simultaneously.

The histone compound of 4-benzamido-N-hydroxy-N-phenyl benzene carbon amide structure of the present invention shows compound such as formula 1 i, the n in formula is 0 or 1; Substituent R 1 is H or 4-Cl;

R2 is H or 2-CH ₃or 3-CH ₃or 4-CH ₃or 2-Cl or 4-Cl.

Formula 1 of the present invention shows compound ipreparation method show reaction such as formula 2; namely with methyl p-aminobenzoate, Benzenecarbonyl chloride. and Nitrobenzol or substituted-nitrobenzene for raw material; make methyl p-aminobenzoate and Benzenecarbonyl chloride. generation aminoacylation; product is through hydrolysis; acidylate obtains 4-benzamido Benzenecarbonyl chloride.; by Nitrobenzol under raney ni catalysis, be reduced into N-hydroxyanilines, with 4-

Benzamido Benzenecarbonyl chloride. is obtained by reacting target compound i a.

Or formula 1 of the present invention shows compound ipreparation method show reaction such as formula 3; namely with Nitrobenzol or substituted-nitrobenzene, methyl p-aminobenzoate, 4-chlorobenzoyl chloride for raw material; methyl p-aminobenzoate and 4-chlorobenzoyl chloride generation aminoacylation; product is through hydrolysis; acidylate obtains 4-(4-chloro-benzoyl amino) Benzenecarbonyl chloride.; Nitrobenzol, under raney ni catalysis, is reduced into

N-hydroxyanilines, with 4-(4-chloro-benzoyl amino) Benzenecarbonyl chloride. is obtained by reacting target compound i b.

Or formula 1 of the present invention shows compound ipreparation method show reaction such as formula 4, namely with Nitrobenzol or substituted-nitrobenzene, methyl p-aminobenzoate, phenyllacetyl chloride for raw material, methyl p-aminobenzoate and phenyllacetyl chloride generation aminoacylation, product is through hydrolysis, and acidylate obtains

4-phenylacetyl amido Benzenecarbonyl chloride., Nitrobenzol, under raney ni catalysis, is reduced into N-hydroxyanilines, is obtained by reacting target compound with 4-benzamido phenyllacetyl chloride i c.

The preferred preparation method of compound of the present invention is:

Nitrobenzol or substituted-nitrobenzene 20mol are dissolved in dehydrated alcohol/1, in the mixed solvent (40ml) of 2-dichloroethanes (1:1), under ice-water bath condition, add a little Raney's nickel, stir, drip the hydrazine hydrate (60mol) of three times of equivalents in batches, a large amount of bubble is had to release, TLC monitoring is complete to raw material reaction, cross and filter Raney's nickel, distilling under reduced pressure to remaining a little solution, with adding suitable quantity of water and dichloromethane extraction, organic facies anhydrous magnesium sulfate drying, then distilling under reduced pressure is to grease or yellow solid nitrogen substituted-phenyl azanol.

Weigh PABA methyl ester 3g(20mmol) be dissolved in 40ml anhydrous tetrahydro furan, add sodium bicarbonate 30mmol, drip the anhydrous tetrahydrofuran solution that 20ml is dissolved with 20mmol Benzenecarbonyl chloride. or phenyllacetyl chloride or parachlorobenzoyl chloride, stirring at room temperature 4 hours, filter, distilling under reduced pressure obtains white solid 4-benzamido essence of Niobe or 4-phenyl acetamide yl benzoic acid methyl ester or 4-(4-chloro-benzoyl amino) essence of Niobe.

15mmol 4-benzamido essence of Niobe or 4-phenyl acetamide yl benzoic acid methyl ester or 4-(4-chloro-benzoyl amino is dissolved with 50ml oxolane) essence of Niobe, add the sodium hydroxide solution 30ml of 1mol/L, reflux 5 hours, decompression steams partial solvent, slowly pour 100ml into and be dissolved with 30mmol salt aqueous acid, filtration drying obtains compound 4-Benzoylamide yl benzoic acid or 4-phenyl acetamide yl benzoic acid or 4-(4-chloro-benzoyl amino) benzoic acid.

Getting compound 4-Benzoylamide yl benzoic acid or 4-phenyl acetamide yl benzoic acid or 4-(4-chloro-benzoyl amino) benzoic acid 15mmol is dissolved into 40ml anhydrous tetrahydro furan, add 4 DMF, oxalyl chloride 18mmol is dripped under ice-water bath condition, stirring at room temperature 4 hours, remove solvent under reduced pressure, with n-hexane, obtain white compound 4-benzamido Benzenecarbonyl chloride. or 4-phenylacetyl amido Benzenecarbonyl chloride. or 4-(4-chloro-benzoyl amino) Benzenecarbonyl chloride..

Nitrogen substituted-phenyl azanol 5mmol is dissolved into 40ml anhydrous tetrahydro furan, add 10mmol sodium bicarbonate, cryosel bath cools to-15 DEG C below and drips dissolving 5mmol 4-benzamido Benzenecarbonyl chloride .s or 4-phenylacetyl amido Benzenecarbonyl chloride. or 4-(4-chloro-benzoyl amino) the anhydrous tetrahydrofuran solution 40ml of Benzenecarbonyl chloride., stir 2 hours, TLC detection reaction is complete, pour crystallization in 150ml water into and obtain white solid, be that the mixed solvent of 3:1 does mobile phase with petrol ether/ethyl acetate, use column chromatography separating-purifying, obtain compound 4-benzamido-N-hydroxy-N-phenyl benzene carbon amide and analog thereof.

Formula 1 shows compound i can prepare the application in Therapeutic cancer medicine, or in preparation treatmentcervical cancer application in medicine, or in preparation treatmentthe esophageal carcinoma application in medicine, or in preparation treatmentlung carcinoma cell application in medicine, or in preparation treatmenthepatoma carcinoma cell application in medicine, or in preparation treatmentskin flbroblast disease before people application in medicine.

Having refer to acetylation of histone level above strengthens relevant with the enhancing of Gene Transcription in vitro, and Acetylation Level is too low relevant with gene expression inhibition.Can obtain from the X-ray structure of HDAC1 and HDAC2, they have the cavity of 11 degree of depth jointly, have individual active zinc ions in the innermost end of tube chamber.Tellable be hydroxamic acid compound is the good chelating agen of zinc ion; can reduce by the zygotic induction histone deacetylase activity stable with zinc ion; degree of acetylation strengthens; what promote gene translates expression; repressed antioncogene is made to recover to express; the generation of cell death inducing, plays antineoplastic action.But hydroxamic acid compound is easily hydrolyzed and unstable, advantage of the present invention is the hydrogen atom on design phenyl ring N substituted isohydroxyloxime acid, synthesize a series of N substituted isohydroxyloxime acid compounds, increased the stability of compound, reach the object of the anti-tumor activity that improve compound.And therefrom filter out two little compounds of the effective toxic and side effects of antitumor action.

SAHA(vorinostat) have inhibition of histone deacetylation enzymatic activity, research shows that dimethyl sulfoxide (DMSO) can the metabolism of elicit virus transformant and differentiation be because DMSO has the effect of polar amide base.Have the words effect meeting of two polarity acyl groups stronger in compound, this is owing to providing more acceptor site to chelating effect.Thus synthesized hexamethylenebisacetamide (HMBA), but find in the experiment of HMBA that it has the effect of good inducible metabolism and differentiation, (30 – 100mM suppress MELC cell proliferation) (Reuben etal., 1976), but find in clinical trial that it can cause myelodysplastic syndrome and acute myeloid leukemia, the derivant of a series of two hydroxamic acid HMBA has been synthesized according to the structure activity relationship of HMBA, the therefrom growth retardation of SAHA Induction Transformation cell and cell death two orders of magnitude higher than HMBA, and the valid density of SAHA drops to μM.Find that it can induce various transformant apoptosis or death in the inside and outside experiment of body of SAHA, and even there is no toxicity to normal cytotoxicity is very little, obtained FDA listing approval at U.S. SAHA in 2006, therefore the present invention at the experiment in vivo and vitro SAHA of 18 N substituted isohydroxyloxime acid compounds of synthesis as positive control.

Select HDAC-2 (3MAX) enzyme to simulate with target compound I c (1-6) to this few compound the present invention to dock, the data (table 1) of analog result are seen [Ic (4) > Ic (6) > SAHA>Ic (4) > Ic (3) > Ic (2) > Ic (5)] from big to small, when can find the substituent group para-orientation of N substituted isohydroxyloxime acid and the degree of fitting better than positive control SAHA [Ic (4) > Ic (6) > SAHA] of HDAC-2 enzyme, it is good that further methyl substituted replaces degree of fitting than chlorine, between the substituent group methyl neighbour in other site, the replacement of position is higher than the degree of fitting of chlorine.According to the principle of compound inhibition of histone deacetylation, the present invention SAHA does positive control, detection compound is to the suppression of histone deacetylase activity, the present invention obtains each compound and SAHA to the result data (table 3) of inhibition of enzyme activity by experiment, therefrom find that the compound activity of para-orientation is the highest, as Compound I a (4), Ia (6), Ib (4), Ib (6), Ic (4), Ic (6) has good inhibit activities, and activity is all less than SAHA higher than the IC50 of positive control drug SAHA(six compounds).Consistent with Computer simulation results, but ortho position chloro replacement inhibit activities is higher than methyl substituted activity.In the experiment of further cell in vitro poison, because the sensitivity of each cell to compound is different, again because mtt assay exists the error of 0-20%, can not generally must compare the result of all cells is unified, but the enzyme in the inhibition of enzyme activity that the present invention does extracts from hela cell, so result is consistent, and can compare with hela Cyto toxic experiment showed, IC50(table 4 from hela Cyto toxic experiment showed) find the compound [Ia (4) of para-orientation, Ia (6), Ib (4), Ib (6), Ic (4), Ic (6)] IC50 and the IC50 of positive control close, this can be described as consistent with experimental result above.Compound I a of the present invention (5), I b (1), also have good cytotoxicity (IC to HeLa cell ₅₀<200 μM) be better than SAHA(IC ₅₀=267 μMs); Compound is to the known Compound I a of cell toxicant result (4) of lung cell A549, Ib (6), Ic (3), Ic (4) has stronger cytotoxicity to lung carcinoma cell, wherein I a (4), the activity of I c (4) is the strongest, is obviously better than SAHA.To Compound I a (6), Ib (4) in the increment inhibit activities experimental result of the esophageal carcinoma, the active best (IC of Ic (6) ₅₀<200 μM), be better than SAHA(IC ₅₀=287 μMs); At compound in the cellulotoxic experiment of hepatoma carcinoma cell (HepG2), Compound I a (1), Ib (1), I c (1) activity and SAHA(IC ₅₀=342 μMs) substantially suitable, the Compound I a (4) of para-orientation, Ib (4), Ic (4), Ia (6), Ib (6), the activity of Ic (6) is better than SAHA; Target compound in the cytotoxicity of people's normal fiber cell, Compound I a (5), Ib (6), Ic (1), Ic (2), the IC of Ic (3), Ic (5) ₅₀<600 μM of inhibitory action (IC stronger than SAHA ₅₀=676 μMs), illustrate that compound has more selective inhibitory than SAHA to tumor cell.By screening, the present invention obtains Compound I a (1), Ib (1), Ib (4), and Ic (4), Ic (6) are better than SAHA or suitable, to normal fibrocellular effect lower than SAHA to tumor cell effect.So select these five compounds little to normal cytotoxicity to carry out interior animal experiment.At median lethal dose(LD 50) (LD ₅₀) measurement result in (table 7) LD of compound can be found out ₅₀value is far longer than positive control drug SAHA, illustrates that the toxicity of SCREENED COMPOUND is much smaller than SAHA, wherein compounds ib (4), the LD of Ic (6) ₅₀value is that the twice of SAHA value is many, shows good toleration in vivo.The present invention selects compounds ib (4), and Ic (6) carries out anti-tumor in vivo experiment further.From experimental result (table 7), little on each internal organs impact of mice under compound test dose, impact is had on the body weight increase of mice, positive control drug SAHA has impact comparatively large when high dose on Mouse Weight, and along with the increasing of dosage, Mouse Weight increases suppression ratio and becomes large, and target compound Ib (4), Ic (6) then shows the impact of body weight increase less, better to the inhibit activities of HeLa cell, all higher than positive control SAHA.Compounds ib (4), Ic (6) is also the N substituted isohydroxyloxime acid compounds of design para-orientation, LD ₅₀measurement result and ED ₅₀measurement result is consistent with related experiment result.

The present invention on the working foundation of forefathers according to the mechanism of action of histone deacetylase inhibitor and enzyme, 18 N substituted isohydroxyloxime acid compounds are designed and synthesized, and computer simulation is passed through to its activity, show that the compound inhibition of histone deacetylase of para-orientation is preferential, pass through Enzyme assay again, the experiment in vitro such as Cytotoxicity tests, LD ₅₀and ED ₅₀the experiment in vivo measured, not only demonstrates the result of computer simulation, and filters out two than the little compounds ib (4) of SAHA enzyme inhibition activity high toxic and side effects and Ic (6).

Detailed description of the invention

One, the preparation of compound

The preparation of embodiment 1 N-hydroxy-N-phenyl-4-benzamido-Benzoylamide I a (1)

Weigh PABA methyl ester 3g(20mmol) be dissolved in 40ml anhydrous tetrahydro furan; add sodium bicarbonate 30mmol, drip the anhydrous tetrahydrofuran solution that 20ml is dissolved with 20mmol Benzenecarbonyl chloride., stirring at room temperature 4 hours; filter, distilling under reduced pressure obtains white solid 4-benzamido essence of Niobe; Dissolve with 50ml oxolane; 15mmol 4-benzamido essence of Niobe; add the sodium hydroxide solution 30ml of 1mol/L; reflux 5 hours; decompression steams partial solvent; slowly pour 100ml into and be dissolved with 30mmol salt aqueous acid, filtration drying obtains compound 4-Benzoylamide yl benzoic acid; Get compound 4-Benzoylamide yl benzoic acid 15mmol and be dissolved into 40ml anhydrous tetrahydro furan, add 4 DMF, under ice-water bath condition, drip oxalyl chloride 18mmol, stirring at room temperature 4 hours, remove solvent under reduced pressure, with n-hexane, obtain white compound 4-benzamido Benzenecarbonyl chloride.; Get pyridyl azanol 5mmol and be dissolved into 40ml anhydrous tetrahydro furan, add 10mmol sodium bicarbonate, cryosel bath cools to less than-15 DEG C and drips the anhydrous tetrahydrofuran solution 40ml dissolving 5mmol 4-benzamido Benzenecarbonyl chloride., stir 2 hours, TLC detection reaction is complete, and pouring crystallization in 150ml water into and obtain white solid, is that the mixed solvent of 3:1 does mobile phase with petrol ether/ethyl acetate, use column chromatography separating-purifying, obtain compound 4-benzamido-N-hydroxy-N-phenyl benzene carbon amide.Product is white solid, and structural formula is shown in formula 5, yield 45.6%,

mp 236.1-238.0?C .; ¹HNMR (400MHz DMSO-d ₆): δ 10.72(s, 1H), 10.47(s, 1H), 7.97(d, J = 7.6 Hz, 3H), 7.85(d, J = 8.4Hz, 2H), 7.69(d, J = 8.4Hz, 2H), 7.56(d, J = 7.2Hz, 4H), 7.41-7.34(m, 2H), 7.21(t, J = 7.2Hz, 1H).; ¹³CNMR (100MHz DMSO-d ₆): δ 167.4, 165.8, 142.3, 141.1, 134.7, 131.8, 130.1, 129.5, 129.4, 128.4, 127.7, 125.4, 122.2, 119.0.。

The preparation of embodiment 2 N-hydroxy-n-o-aminomethyl phenyl-4-benzamido-Benzoylamide Ia (2)

Preparation method: the pyridyl azanol N-(2-aminomethyl phenyl by embodiment 1) azanol substitute, other preparation methoies are with embodiment 1.Product is white solid, and structural formula is shown in formula 6,

Yield 37.6%, mp 236.2-238.4 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.47 (s, 1H), 9.84 (s; 1H), 8.06-7.95 (m, 4H); 7.87-7.81 (m; 2H), 7.64-7.52 (m, 4H); 7.36-7.9515 (m; 4H), 2.26 (s, 3H).; ¹³cNMR (100MHz DMSO-d ₆): δ 165.7,164.7,141.4,141.0,136.5,134.6,133.6,131.7,130.2,129.6,129.2,128.3,127.7,126.5,125.9,119.4,17.9.; HRMS m/z: calcd [M+H] 347.1390, found 347.1381..

The preparation of embodiment 3 N-hydroxy-n-m-aminomethyl phenyl-4-benzamido-Benzoylamide Ia (3)

Preparation method: the pyridyl azanol N-(3-aminomethyl phenyl by embodiment 1) azanol substitute, other preparation methoies are with embodiment 1.Product is white solid, and structural formula is shown in formula 7,

Yield 44.3%, mp 233.9-235.1 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.67 (s, 1H), 10.46 (s, 1H); (7.97 d, J=7.2 Hz, 2H), 7.84 (d; J=8.4Hz, 2H), 7.69-7.53 (m, 5H); (7.40 s, 1H), 7.34-7.24 (m; 2H), 7.02 (d, J=7.2 Hz; 1H), 2.32 (s, 3H).; ¹³cNMR (100MHz DMSO-d ₆): δ 167.4,165.8,142.3,141.0,137.8,134.7,131.8,130.2,129.4,128.4,127.9,127.6,126.1,122.7,119.5,119.2,21.1.; HRMS m/z: calcd [M+H] 347.1390, found 347.1383..

The preparation of embodiment 4 N-hydroxy-n-p-aminomethyl phenyl-4-benzamido-Benzoylamide Ia (4)

Preparation method: the pyridyl azanol N-(4-aminomethyl phenyl by embodiment 1) azanol substitute, other preparation methoies are with embodiment 1, and product is white solid, and structural formula is shown in formula 8,

Yield 40.4%, mp 234.8-236.2 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.70 (s, 1H), 10.45 (s; 1H), 8.08-8.02 (m, 1H); (8.00-7.92 m, 2H), 7.86-7.78 (m; 2H), 7.66-7.51 (m, 4H); (7.41-7.33 m, 2H), 7.24-7.18 (m; 2H), 2.29 (s, 3H).; ¹³cNMR (100MHz DMSO-d ₆): δ 167.1,165.8,140.9,140.0,131.7,130.2,129.4,128.9,128.4,127.7,126.6,122.7,119.0,20.5.; HRMS m/z: calcd [M+H] 347.1390, found 347.1395..

The preparation of embodiment 5 N-hydroxy-n-o-chlorphenyl-4-benzamido-Benzoylamide Ia (5)

Preparation method: the pyridyl azanol N-(2-chlorphenyl by embodiment 1) azanol substitute, other preparation methoies are with embodiment 1, and product is white solid, and structural formula is shown in formula 9,

Yield 48.8%, mp 241.3-243.2 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.57 (s, 1H), 10.44 (s, 1H); (8.11-7.82 m, 6H), 7.64-7.55 (m; 3H), 7.45-7.40 (m, 1H); (7.30 t, J=7.6Hz, 1H); (7.15 d, J=7.6Hz, 1H); 7.02 (t, J=7.6Hz, 1H).; ¹³cNMR (100MHz DMSO-d ₆): δ 167.1,164.7,141.1,140.2,136.6,133.3,131.2,130.2,129.9,129.5,129.7,129.3,128.5,128.1,119.7,119.2..

The preparation of embodiment 6 N-hydroxy-n-p-chlorphenyl-4-benzamido-Benzoylamide Ia (6)

Preparation method: the pyridyl azanol N-(4-chlorphenyl by embodiment 1) azanol substitute, other preparation methoies are with embodiment 1, and product is white solid, and structural formula is shown in formula 10,

Yield 37.1%, mp 240.8-242.1 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.88 (s, 1H), 10.49 (s, 1H); 7.99-7.97 (m, 2H), 7.88-7.83 (m, 3H); (7.73-7.56 m, 6H), 7.47-7.41 (m, 2H).; ¹³cNMR (100MHz DMSO-d ₆): δ 167.6,165.8,141.2,141.1,134.7,131.7,129.7,129.5,129.0,128.6,128.4,127.7,123.1,119.0..

Embodiment 7 N-hydroxy-N-phenyl-4-(4-chloro-benzoyl amino) preparation of-Benzoylamide Ib (1)

Preparation method: the Benzenecarbonyl chloride. 4-chlorobenzoyl chloride in embodiment 1 is substituted, other preparation methoies are with embodiment 2, and product is white solid, and structural formula is shown in formula 11,

Yield 45.6%, mp 214.8-216.2 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.75 (s, 1H), 10.53 (s; 1H), 8.00 (d, J=8.0 Hz; 2H), 7.83 (d, J=8.4Hz; 2H), 7.70-7.55 (m, 6H); (7.40 t, J=8.0Hz, 2H); 7.20 (t, J=8.0Hz, 1H).; ¹³cNMR (100MHz DMSO-d ₆): δ 167.4,164.7,142.3,140.9,136.6,133.4,130.3,129.7,129.5,128.5,125.4,122.2,119.1.; IR (KBr): 3413.1,3304.1,2914.6,2856.7,1668.4,1600.1,1517.5..

Embodiment 8 N-hydroxy-n-o-aminomethyl phenyl-4-(4-chloro-benzoyl amino) preparation of-Benzoylamide Ib (2)

Preparation method: the Benzenecarbonyl chloride. 4-chlorobenzoyl chloride in embodiment 2 is substituted, other preparation methoies are with embodiment 3, and product is white solid, and structural formula is shown in formula 12,

Yield 32.5%, mp 221.2-223.0 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.64 (s, 1H), 10.52 (s, 1H), 8.03-7.17 (m, 12H), 2.32 (s, 3H).; ¹³cNMR (100MHz DMSO-d ₆): δ 169.4,164.7,141.4,140.8,136.6,133.4,129.8,129.7,129.1,128.5,126.5,119.2,17.6..

Embodiment 9 N-hydroxy-n-m-aminomethyl phenyl-4-(4-chloro-benzoyl amino)-Benzoylamide Ib(3) preparation

Preparation method: the Benzenecarbonyl chloride. 4-chlorobenzoyl chloride in embodiment 3 is substituted, other preparation methoies are with embodiment 3, and product is white solid, and structural formula is shown in formula 13,

Yield 43.5%, mp 224.4-226.1 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.68 (s, 1H), 10.52 (s, 1H); (8.00 d, J=8.4 Hz, 2H), 7.83 (d; J=8.4Hz, 2H), 7.65 (dd, J1=8.4Hz; J2=27.6Hz, 4H), 7.40-7.24 (m; 3H), 7.02 (d, J=7.2Hz; 1H), 2.32 (s, 3H).; ¹³cNMR (100MHz DMSO-d ₆): δ 167.4,164.7,142.2,140.8,137.8,136.6,133.4,130.3,129.7,129.4,128.5,128.3,126.1,122.7,119.5,119.1,21.1..

Embodiment 10 N-hydroxy-n-p-aminomethyl phenyl-4-(4-chloro-benzoyl amino) preparation of-Benzoylamide Ib (4)

Preparation method: the Benzenecarbonyl chloride. 4-chlorobenzoyl chloride in embodiment 4 is substituted, other preparation methoies are with embodiment 4, and product is white solid, and structural formula is shown in formula 14,

Yield 47.6%, mp 223.8-225.2 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.69 (s, 1H), 10.50 (s, 1H); (8.00 d, J=8.0 Hz, 2H), 7.81 (d; J=8.4Hz, 2H), 7.63 (t, J=8.4Hz; 4H), 7.40 (d, J=8.0Hz; 2H), 7.18 (d, J=8.0Hz; 2H), 2.30 (s, 3H).; ¹³cNMR (100MHz DMSO-d ₆): δ 167.1,164.7,140.7,140.0,136.6,134.9,133.4,130.4,129.7,129.4,128.9,128.5,122.7,119.1,20.5..

Embodiment 11 N-hydroxy-n-o-chlorphenyl-4-(4-chloro-benzoyl amino) preparation of-Benzoylamide Ib (5)

Preparation method: the Benzenecarbonyl chloride. 4-chlorobenzoyl chloride in embodiment 5 is substituted, other preparation methoies are with embodiment 5, and product is white solid, and structural formula is shown in formula 15,

Yield 36.4%, mp 231.6-233.4 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.64 (s, 1H), 10.54 (s, 1H), 8.03-7.90 (m, 3H), 7.80 (s, 2H), 7.63-7.53 (m, 5H), 7.41-7.35 (m, 2H).; ¹³cNMR (100MHz DMSO-d ₆): δ 167.1,164.7,141.1,140.2,136.6,133.3,131.2,130.2,129.9,129.5,129.7,129.3,128.5,128.1,119.7,119.2..

Embodiment 12 N-hydroxy-n-p-chlorphenyl-4-(4-chloro-benzoyl amino) preparation of-Benzoylamide Ib (6)

Preparation method: the Benzenecarbonyl chloride. 4-chlorobenzoyl chloride in embodiment 6 is substituted, other preparation methoies are with embodiment 6, and product is white solid, and structural formula is shown in formula 16,

Yield 42.1%, mp 233.1-235.2 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.74 (s, 1H), 10.35 (s; 1H), 8.10-7.45 (m, 9H); (7.42 d, J=8.8Hz, 1H); 7.35 (d; J=8.4Hz, 1H), 7.04 (d; J=8.8Hz, 1H).; ¹³cNMR (100MHz DMSO-d ₆): δ 167.7,164.8,146.8,144.1,141.1,136.9,133.2,130.3,130.0,129.8,129.1,128.6,123.3,116.1..

Embodiment 13 N-hydroxy-N-phenyl-4-(4-phenylacetyl amido) preparation of-Benzoylamide I c (1)

Preparation method: the Benzenecarbonyl chloride. phenyllacetyl chloride in embodiment 1 is substituted, other preparation methoies are with embodiment 1, and product is white solid, and structural formula is shown in formula 17,

Yield 43.4%, mp 236.4-238.1 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.49 (s, 1H), 10.13 (s; 1H), 7.94 (d, J=8.4 Hz; 2H), 7.75 (t, J=8.4Hz; 4H), 7.36-7.25 (m, 7H); (7.08 t, J=7.6,1H); 3.69 (s, 2H).; ¹³cNMR (100MHz DMSO-d ₆): δ 169.5,164.8,142.1,139.3,135.7,129.2,128.6,128.5,128.3,126.6,123.5,120.3,118.3,43.3.; IR (KBr): 3364.0,3161.4,2912.5,2854.4,1664.3,1612.2,1520.1.; HRMS m/z: calcd [M+H] 347.1390, found 347.1381..

Embodiment 14 N-hydroxy-n-o-aminomethyl phenyl-4-(4-phenylacetyl amido) preparation of-Benzoylamide (I14)

Preparation method: the Benzenecarbonyl chloride. phenyllacetyl chloride in embodiment 2 is substituted, other preparation methoies are with embodiment 2, and product is white solid, and structural formula is shown in formula 18,

Yield 36.8%, mp 240.1-242.6 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.49 (s, 1H), 10.26 (s, 1H), 7.89-7.23 (m, 13H), 3.68 (s, 2H), 2.28 (s, 3H).; ¹³cNMR (100MHz DMSO-d ₆): δ 169.4,164.5,141.4,140.0,135.7,130.6,129.3,129.2,129.1,128.3,126.6,126.5,117.9,43.3,17.5.; IR (KBr): 3306.8,3192.6,3113.4,3030.1,2922.7,1665.7,1601.5,1530.8.; HRMS m/z: calcd [M+H] 361.1547, found 361.1553..

Embodiment 15 N-hydroxy-n-m-aminomethyl phenyl-4-(4-phenylacetyl amido) preparation of-Benzoylamide Ic (3)

Preparation method: the Benzenecarbonyl chloride. phenyllacetyl chloride in embodiment 3 is substituted, other preparation methoies are with embodiment 3, and product is white solid, and structural formula is shown in formula 19,

Yield 43.4%, mp 242.5-244.3 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.63 (s, 1H), 10.39 (s, 1H), 7.63-7.22 (m, 12H), 7.01 (d, J=7.2,1H), 3.67 (s, 2H), 2.30 (s, 3H).; ¹³cNMR (100MHz DMSO-d ₆): δ 169.5,167.3,142.3,141.0,137.8,135.8,129.8,129.6,129.1,128.3,128.26,126.6,126.1,122.7,119.5,117.8,43.3,21.1.; IR (KBr): 3349.6,3158.7,3063.0,3030.4,2919.0,1670.2,1607.3,1526.5.; HRMS m/z: calcd [M+H] 361.1547, found 361.1551..

Enforcement case 16 N-hydroxy-n-p-aminomethyl phenyl-4-(4-phenylacetyl amido) preparation of-Benzoylamide Ic (4)

Preparation method: the Benzenecarbonyl chloride. phenyllacetyl chloride in embodiment 4 is substituted, other preparation methoies are with embodiment 4, and product is white solid, and structural formula is shown in formula 20,

Yield 49.7%, mp 243.4-245.5 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.48 (s, 1H), 10.05 (s; 1H), 7.95-7.64 (m, 6H); (7.37-7.24 m, 5H), 7.14 (d; J=8.4; 2H), 3.70 (s, 2H); (2.27 s, 3H).; ¹³cNMR (100MHz DMSO-d ₆): δ 169.5,164.6,142.1,136.7,135.7,132.4,129.3,129.0,128.6,128.5,128,3,126.6,120.3,118.2,43.2,20.6.; IR (KBr): 3320.3,3298.3,3028.8,2916.5,2859.9,1668.9,1641.8,1515.5.; HRMS m/z: calcd [M+H] 361.1547, found 361.1551..

Case study on implementation 17 N-hydroxy-n-o-chlorphenyl-4-(4-phenylacetyl amido) preparation of-Benzoylamide Ic (5)

Preparation method: the Benzenecarbonyl chloride. phenyllacetyl chloride in embodiment 5 is substituted, other preparation methoies are with embodiment 5, and product is white solid, and structural formula is shown in formula 21,

Yield 40.3%, mp 255.1-257.3 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.64 (s, 1H), 10.54 (s, 1H), 7.83-7.30 (m, 13H), 3.73 (s, 2H).; ¹³cNMR (100MHz DMSO-d ₆): δ 169.3,167.2,142.3,141.5,135.3,130.7,128.5,128.1,128.0,127.3,126.3,123.2,116.9,43.7.; IR (KBr): 3326.7,3132.4,3030.0,2927.4,1670.0,1611.6,1525.9.; HRMS m/z: calcd [M+H] 381.1000, found 381.1003..

Case study on implementation 18 N-hydroxy-n-p-chlorphenyl-4-(4-phenylacetyl amido) preparation of-Benzoylamide Ic (6)

Preparation method: the Benzenecarbonyl chloride. phenyllacetyl chloride in embodiment 6 is substituted, other preparation methoies are with embodiment 6, and product is white solid, and structural formula is shown in formula 22,

Yield 46.2%, mp 256.4-257.9 C.; ¹hNMR (400MHz DMSO-d ₆): δ 10.83 (s, 1H), 10.48 (s, 1H), 7.67-7.25 (m, 13H), 3.69 (s, 2H).; ¹³cNMR (100MHz DMSO-d ₆): δ 169.5,167.6,141.3,141.2,135.8,129.7,129.3,129.1,128.9,128.3,126.6,123.2,117.9,43.3.; IR (KBr): 3333.4,3137.4,3032.0,2922.0,1671.0,1612.6,1527.9.; HRMS m/z: calcd [M+H] 381.1000, found 381.1006..

The preparation method of the nitrogen substituted-phenyl azanol used in above-described embodiment is as follows: be dissolved in dehydrated alcohol/1 by Nitrobenzol or for Nitrobenzol A 20mol, in the mixed solvent (40ml) of 2-dichloroethanes (1:1), under ice-water bath condition, add a little Raney's nickel, stir, drip the hydrazine hydrate (60mol) of three times of equivalents in batches, a large amount of bubble is had to release, TLC monitoring is complete to raw material reaction, cross and filter Raney's nickel, distilling under reduced pressure is to remaining a little solution, with adding suitable quantity of water and dichloromethane extraction, organic facies anhydrous magnesium sulfate drying, then distilling under reduced pressure is to grease or yellow solid nitrogen substituted-phenyl azanol B, its reaction scheme is such as formula 23, this method is based on existing technology, (such as SarahKr ü ger synthesizes 8-(the intermediate aryl azanol of acetylaminohydroxyphenylarsonic acid 2-deoxy-guanine just in this way) although the method preparing phenylhydroxylamine has several, but be also the modal method preparing phenylhydroxylamine, just can complete at room temperature ice bath, the productive rate that takes a short time is high, see SarahKr ü ger, ChrisMeier, Synthesis of Site-Specific Damaged DNA Strands by 8-(Acetylarylamino)-2deoxyguanosine Adducts and Effectson Various DNA Polymerases, Eur.J.Org.Chem. 2013,1158 – 1169.

Two Computer-Aided Drug Designs

Computer-Aided Drug Design is the important means of new drug development, by simulating and calculate the interaction of receptor and part, carries out the Optimum and design of lead compound, is widely used in the exploitation of new drug.At present, obtained the crystal structure of four kinds of histon deacetylase (HDAC) hypotypes, these crystal structures have been widely used in the design of hdac inhibitor.Select HDAC-2 (3MAX) enzyme to simulate with target compound I c (1-6) in the present invention to dock, result display Compound I c (1), I c (4), I c (6) is better with HDAC-2 matching, wherein the degree of fitting of I c (1) is substantially close with SAHA, the fitting degree of I c (4) is better than SAHA, the results are shown in Table 1.

Three, the compounds of this invention antitumor activity evaluation

1 histon deacetylase (HDAC) Inhibition test

1.1 experiment material

96 well culture plates, HDAC enzyme inhibitor medicine screening reagent box (Biovision:K340-100), comprises the HDAC enzyme extracted in HeLa nucleus, wherein containing abundant HDAC-1, HDAC-2 enzyme; Histone Phe-Gly-Lys-Ala-Val-Leu-Lys-Lys oc-Lys (the Ac)-AMC of lysine acetylation; Developer Lysine Developer; Buffer, SAHA(Hubei Yi Kangyuan Chemical Co., Ltd.)

1.2 experimental principle

The HDAC enzyme extracted can make the acetyl group on substrate histone Phe-Gly-Lys-Ala-Val-Leu-Lys-Lys oc-Lys (Ac)-AMC slough; product is after developer Lysine Developer process; light absorption value is detected at 450nm place by microplate reader; because acetylizad histone oligopeptide can not react with developer; do not absorb at 450nm place, therefore the size of light absorption value has just reacted the height of enzymatic activity.

determination of activity

Experiment is using SAHA as positive control drug, and arrange solvent control group, blank negative control group and compound group, it is 0.8 μ g/ml that each medicine group arranges 4 Concentraton gradient, 3.1 μ g/ml, 12.5 μ g/ml, 50 μ g/ml, and each concentration arranges three secondary orifices.Weigh each compound and each 0.25mg of SAHA with analytical balance, dissolve with 0.1mlDMSO, be diluted to 10ml with buffer, compound concentration 100 μ g/ml solution, gets 2ml solution, is diluted to 8ml, obtain concentration 25 μ g/ml solution, prepare the solution of each 2 times of experimental concentrations successively.

Add buffer successively in 96 well culture plates, HeLa cell extraction enzyme, substrate protein oligopeptide and compound solution, respectively group addition is as table 2, mix homogeneously.Add 50 μ l compound solutions, mixing, cultivate 30 minutes for 37 DEG C.Each hole adds developer 10 μ l, cultivates 30 minutes for 37 DEG C.Under 450nm wavelength, light absorption value is measured by microplate reader.Calculate each concentration hole meansigma methods, calculate suppression ratio, with the half-inhibition concentration IC of each compound of Spss 10.0 computed in software ₅₀, suppression ratio=(blank group absorption value-experimental group absorption value)/white group absorption value * 100% result of calculation is as table 3.

From experimental data, major part target compound has inhibitory action to HeLa cell extraction enzyme, half-inhibition concentration is in micromole's level, as Compound I a (2) when on azanol phenyl ring, ortho position replaces, Ib (2), Ic (2) activity is lower, ortho position chlorine replaces active active higher than ortho methyl group replacement, during compound para-orientation, activity is the highest, as Compound I a (4), Ia (6), Ic (4), Ic (6) has good inhibit activities, and activity is due to positive control drug SAHA.

cell in vitro Inhibition test

2.1 experiment material

HeLa cell (human cervical carcinoma), Ec109 cell (human esophagus cancer), A549(lung small cell lung cancer cell), skin flbroblast before HFF(people) and Hep G2(human liver cancer cell) (all cells is obtained by preclinical medicine institute of Lanzhou University experimental center), DMEM high glucose medium, superfine hyclone, DMSO, PBS, tetramethyl azo azoles salt (MTT), trypsin, sterilizing filter.

experimental principle

Experiment adopts mtt assay, and MTT can be reduced to hepatic formazan by the succinum dehydrogenase in living cells mitochondrion, and is deposited in cell, and dead cell does not then have this function.By dimethyl sulfoxide (DMSO) Rong Xie formazan, measure its absorption value at 570nm wavelength place light by microplate reader.Due within the scope of certain cell quantity, growing amount and the living cells quantity of formazan are directly proportional, and the absorption value therefore measured can reflect the situation of cell proliferation indirectly, can be used for understanding the ability of Drug inhibition or killing tumor cell.

determination of activity

The concentration of experimental compound is 400,200,100,50 μ g/ml, weighing target compound 4mg is dissolved in 50 μ L DMSO, 10ml is diluted to complete culture solution, be made into the solution of 400 μ g/ml, the solution getting 5ml concentration 400 μ g/ml adds 5ml complete culture solution, the solution of compound concentration 200 μ g/ml, prepares each strength solution successively, uses 0.22 μm of membrane filtration degerming.

The blank group of 96 well culture plates adds 100 μ L complete culture solutions, and other are organized every hole and add cell suspension 100 μ L(5000 cell), after cell inoculation, 37 DEG C of temperature, 90% relative humidity, containing 5%CO ₂, 95% air incubator preculture 12h.

Suck the culture fluid of each culture hole, blank group, groups of cells adds 100 μ L complete culture solutions, and positive controls adds each strength solution of the SAHA prepared, and experimental group adds the corresponding each concentration compound complete culture solution 100 μ L prepared.Put into incubator to continue to cultivate 36h.

Every hole adds the MTT solution of 10 μ L 5mg/ml, continues to cultivate 4h.Suck supernatant, every hole adds 150 μ L DMSO, and Shi formazan is dissolved.Measure 570nm wavelength place OD value by microplate reader, calculate the meansigma methods of each concentration, suppression ratio=(groups of cells OD value-experimental group OD value)/(groups of cells OD value-blank group OD value) * 100%, with the IC of Spss computed in software cell ₅₀value.Result, as following table 4, is suppressed to live to the increment of lung cell A549 by compound in table 4

The property known Compound I a of testing result (4), Ib (6), Ic (3), Ic (4) has stronger inhibit activities to lung carcinoma cell, wherein Ia (4), and the activity of Ic (4) is respectively IC the most by force ₅₀=155 μMs; IC ₅₀=106 μMs, be obviously better than control compound SAHA(IC ₅₀=204 μMs), and Compound I a (2), Ib (2), the adjacent methyl substituted compound of Ic (2) does not act on IC substantially ₅₀>2mM; Compound I a (5), Ib (1), Ic (6) has good inhibitory action IC to HeLa cell ₅₀<200 μM, is better than SAHA(IC ₅₀=267 μMs), Ia (4), Ib (3), the activity of Ib (4), Ic (4) is slightly better than SAHA; To Compound I a (6), Ib (4) in the increment inhibit activities experimental result of the esophageal carcinoma, the active preferably IC of Ic (6) ₅₀<200 μM, is better than SAHA(IC ₅₀=287 μMs), Ia (4), Ic (3), Ic (4) is active substantially suitable with SAHA; In the external increment Inhibition test of compound to hepatoma carcinoma cell (HepG2), Compound I a (2), Ib (2), Ic (2), Ia (5), Ib (5), Ic (5) do not have inhibit activities IC ₅₀>2mM, Compound I a (1), Ib (1), Ic (1) activity and SAHA(IC ₅₀=342 μMs) substantially suitable, the Compound I a (3) that a position replaces, the inhibit activities (IC of Ib (3), Ic (3) ₅₀>800 μM) be far weaker than SAHA, the Compound I a (4) of para-orientation, Ib (4), Ic (4), Ia (6), the activity of Ib (6), Ic (6) is higher than positive controls, and the compound that during para-position methyl substituted, active a little higher than chlorine replaces; The growth inhibition effect of target compound to people's normal fiber cell is tested, Compound I a (5), Ib (6), Ic (1), Ic (2), the IC of Ic (3), Ic (5) in the present invention's experiment ₅₀<600 μM of inhibitory action (IC stronger than SAHA ₅₀=676 μMs), the inhibitory action of all the other majority of compounds is weaker than SAHA, illustrates that compound has more selective inhibitory than SAHA to tumor cell.Obtain Compound I a (1), Ib (1), Ib (4) by screening, Ic (4), Ic (6) are better than SAHA or suitable, to normal fibrocellular effect lower than SAHA to tumor cell effect.Interior animal experiment is carried out with these five compounds.

four, the compounds of this invention anti-tumor in vivo activity rating

1 experiment material

Kunming mice, body weight is at 20 ~ 25 grams, and male and female dual-purpose, is purchased from Lanzhou University's animal experimental center.Experiment is raised in 20 ~ 25 DEG C of environment the last week, and relative humidity is 35 ~ 45%, and except experimental period, animal can freely be looked for food and drink water.Mouse cage, 1ml, 5ml syringe, olive oil, DMSO, Hela cell, normal saline, electronic balance, analytical balance, scalpel, formalin.

median lethal dose(LD 50) (LD ₅₀ ) mensuration

2.1 LD ₅₀ preliminary Determination

Getting Compound I a (1) 0.65g is dissolved in 0.5mlDMSO, adds 4.5ml olive oil, and even Emulsion is made in mixing, and joining concentration is 130mg/ml, is the solution of 10% containing DMSO.Getting 0.325g Compound I a (1) is dissolved in 0.5mlDMSO, adds 4.5ml olive oil, and even Emulsion is made in mixing, and joining concentration is 65mg/ml, is the solution of 10% containing DMSO.Get 12 kunming mices to be divided into 4 groups at random and often to organize 3, label of weighing, water is can't help in fasting, administration 400 after 12 hours, 800,1600,3200mg/kg, in observed and recorded mice 7, death condition is as table 5, and deterministic compound Ia (1) lethal range is between 800mg/kg to 3200mg/kg.

formal experiment

The lethal range of Compound I a (1) between 800mg/kg to 3200mg/kg, determine test dose maximum (Dm) for 3200mg/kg and minima (Dn) be 800mg/kg.Setup Experiments 5 dosage groups, dosage common ratio R=4 √ (Dm/Dn) of each group, experimental group dosage is Dn, Dn × R, Dn × R ², Dn × R ³, Dm, calculates R=1.414, and getting each group of dosage is 800,1130,1600,2260,3200.Get kunming mice 248, male and female half and half, random packet becomes 31 groups, and one group is matched group, all the other 30 groups is compound S AHA, Ia (1), Ib (1), Ib (4), Ic (4), Ic (6) experimental group, each compound arranges 5 groups, every mouse weights label.The preparation of experimental compound, according to the preparation of preliminary experiment Compound I a (1), is made into two strength solution 65mg/ml, 130mg/ml.Body weight according to mice calculates dosage and administration volume, and mice fasting, after 12 hours, draws amount of calculation compound solution, 0.5ml is diluted to olive oil, lumbar injection, administration fore-and-aft observing record Mouse Weight, takes food, occurs the situations such as symptom, records dead mouse information slip 6 in seven days.

Occur after some animals administration that action reduces, eyeball is dimmed, and health is twitched, and afterbody bleaches, and in experiment, mice solution plane that is dead and that put to death is observed, and the color of the internal organs such as liver, kidney, stomach, intestinal, feature and normal mouse internal organs are not obviously not extremely.The LD of SCREENED COMPOUND can be found out by table 7 data ₅₀value is far longer than positive control drug SAHA, illustrates that the toxicity of SCREENED COMPOUND is much smaller than SAHA, wherein compounds ib (4), the LD of Ic (6) ₅₀value is that the twice of SAHA value is many, shows good toleration in vivo.Select compounds ib (4), Ic (6) carries out anti-tumor in vivo experiment further.

compounds ib (4), Ib (6) anti-tumor in vivo activity rating

the foundation of 3.1 pharmacological models

(1) get the attached cell HeLa of cultivation, digestion, normal saline dilution, be inoculated into mouse peritoneal and pass three generations, extract ascites normal saline dilution to 10 ⁸individual/ml, for subsequent use.

(2) the male mice of 128 body weight at 19-23g is got, at the HeLa Cell sap (10 of the right fore oxter of every mice inoculation 0.1ml ⁷individual) take out ascites to the time controling of the complete all operations of tumor inoculation and complete within 60 min.The 4th day of normal raising, with vernier caliper measurement transplanted tumor diameter, selects the good animal of tumor growth and carries out grouping experiment.

the selection of dosage

As shown in Table 7, compounds ib (4), the LD of Ic (6) ₅₀value is greater than 1.5g/kg, and maximum tolerated dose is greater than 1g/kg, the LD of positive control drug SAHA ₅₀for 0.77g/kg, maximum tolerated dose 0.4g/kg.Get the LD of SAHA ₅₀value 1/5 that is 150 mg/kg are as maximum dosage, arranging five administration concentration gradients is 10,25,50,100,150mg/kg.

the random packet of mice

(1) weigh the body weight of Mice Inoculated, be divided into eight intervals, as 19-19.5,19.5-20,20-20.5 by body weight ... 22.5-23 put into eight cages respectively.

(2) get 16 mouse cages, numbering 1-16, generate eight groups of random digits of to ten six with software Spss11.0.

(3) mice in the cage of body weight between 19-19.5g is got at random, by first group of random digit, put into the mouse cage of reference numeral, if the mice between 19-19.5g is less than 16, then take off one group of mice (i.e. mice in the cage of body weight between 19.5-20g) at random and put into the mouse cage with random digit reference numeral, in each mouse cage, put into second mice according to second group of random digit, 128 mices are divided into 16 groups by Using such method, often organize eight.

the preparation of solution

Get compound S AHA respectively, Ib (4), Ic (6), 0.2g, dissolve with 1mlDMSO, be diluted to 10ml with olive oil, mix homogeneously, obtain concentration 20mg/ml, get 2ml solution dilution to 4ml, obtain the solution that concentration is 10mg/ml.Get 1ml and be diluted to 2ml, obtain the solution of concentration 5mg/ml.

the administration of mice and process

Numbered by mouse weights in every mouse cage, calculate the liquor capacity of dosage and administration, each compound establishes five administration gradients, the administration 10,25,50 respectively of mice 1-5 group, 100,150mg/kg, separately establishes one group of blank group, the DMSO olive oil solution 0.2ml of lumbar injection 10%, draws the solution of the corresponding concentration of amount of calculation, is diluted to 0.2ml with olive oil, intraperitoneal injection, once a day, continuous 7 days, the sign change of observed and recorded mice, weighs the body weight of mice.Put to death mice, carefully take off tumor block, liver, kidney, spleen is weighed, and calculates the meansigma methods often organized, calculates the tumour inhibiting rate of each experimental compound and each organ index respectively, organ index=Organ weight (mg)/Mouse Weight (g); Tumour inhibiting rate=(matched group average tumor weight-administration group average tumor weight) average tumor heavy * 100% of/matched group.Body weight suppression ratio=(matched group average weight-administration group average weight)/matched group average weight * 100%.Data processed result in table 8, with the median effective dose (ED of Spss11.0 computed in software experimental compound ₅₀), result of calculation is in table 9

Above-mentioned experiment shows, after the modeling of lotus tumor model mice HeLa solid tumor laboratory animal, compared with blank group, solid tumor negative control group animal ingestion, movable all compared with normals, come into play after 7d minimizing, be slow in action, feed also reduces, and during off-test, the tumor of negative control group mice HeLa solid tumor is great in 1g, simultaneously, positive controls and administration group are compared with negative control group, and the MST difference that mice HeLa solid tumor tumor weighs has statistical significance (P<0.01).Above result shows that the modeling of the lotus tumor model of this experiment is successful no matter from lotus tumor model modeling standard or the analysis of statistics aspect.

(2) antitumor activity of compound

From experimental result table 7, little on each internal organs impact of mice under compound test dose, impact is had on the body weight increase of mice, positive control drug SAHA has impact comparatively large when high dose on Mouse Weight, and along with the increasing of dosage, Mouse Weight increases suppression ratio and becomes large, and target compound Ib (4), Ic (6) then shows the impact of body weight increase less, better to the inhibit activities of HeLa cell, all higher than positive control SAHA.In the test of mouse entity tumor, 5 dosage of administration group and corresponding tumour inhibiting rate carry out linear regression, compounds ib (4) equation is y=15.339x-5.4876(R2=0.9934), Compound I c (6) equation is y=15.702x+0.1653(R2=0.9813); The equation of linear regression of positive controls SAHA is y=16.281x-10.992(R2=0.9969).There is good dose-effect relationship in the respective antitumor action of SAHA and Ib (4), Ic (6).In table 8, compounds ib (4), the median effective dose of Ic (6) is the value 77.78mg/kg that 71.29mg/kg and 51.91mg/kg is all less than contrast medicine SAHA, and the inhibit activities of Compound I c (6) is better than Ib (4), is the twice of SAHA.Related experiment shows that Formula 1 of the present invention can prepare the application in Therapeutic cancer medicine, particularly in preparation treatment cervical cancer medicine, in preparation treatment esophageal carcinoma medicine, application in preparation treatment lung carcinoma cell medicine, preparing in Hepatoma therapy cell drug, preparation treatment people before skin flbroblast disease medicine in application.

Claims (1)

1. show compound such as formula 1 i,

N in formula is 0 or 1; Substituent R ₁for H or 4-Cl; R ₂for H or 2-CH ₃or 3-CH ₃or 4-CH ₃or 2-Cl or 4-Cl.

2. as claim 1 compound ipreparation method; it is characterized in that reaction is shown such as formula 2; namely with the Nitrobenzol of methyl p-aminobenzoate, Benzenecarbonyl chloride. and Nitrobenzol or replacement for raw material; make methyl p-aminobenzoate and Benzenecarbonyl chloride. generation aminoacylation; product is through hydrolysis; acidylate obtains 4-benzamido Benzenecarbonyl chloride., by the Nitrobenzol of Nitrobenzol or replacement at raney ni catalysis

Under, be reduced into the N-hydroxyanilines of N-hydroxyanilines or replacement, be obtained by reacting target compound with 4-benzamido Benzenecarbonyl chloride. i a.

3. as claim 1 compound ipreparation method; it is characterized in that reaction is shown such as formula 3; namely with the Nitrobenzol of Nitrobenzol or replacement; methyl p-aminobenzoate; 4-chlorobenzoyl chloride is raw material, methyl p-aminobenzoate and 4-chlorobenzoyl chloride generation aminoacylation, and product is through hydrolysis; acidylate obtains 4-(4-chloro-benzoyl amino) Benzenecarbonyl chloride.

The Nitrobenzol of Nitrobenzol or replacement, under raney ni catalysis, is reduced into N-hydroxyanilines, with 4-(4-chloro-benzoyl amino) Benzenecarbonyl chloride. is obtained by reacting target compound i b.

4. as claim 1 compound ipreparation method, it is characterized in that reaction is shown such as formula 4, namely with the Nitrobenzol of Nitrobenzol or replacement, methyl p-aminobenzoate, phenyllacetyl chloride is raw material, and methyl p-aminobenzoate and phenyllacetyl chloride are sent out

）

Raw aminoacylation, product is through hydrolysis, and acidylate obtains 4-phenylacetyl amido Benzenecarbonyl chloride., and the Nitrobenzol of Nitrobenzol or replacement, under raney ni catalysis, is reduced into N-hydroxyanilines, is obtained by reacting target compound with 4-benzamido phenyllacetyl chloride i c.

5. compounds I as claimed in claim 1 is preparing the application in Therapeutic cancer medicine.

6. the application of compounds I as claimed in claim 1 in preparation treatment cervical cancer medicine.

7. the application of compounds I as claimed in claim 1 in preparation treatment esophageal carcinoma medicine.

8. the application of compounds I as claimed in claim 1 in preparation treatment lung carcinoma cell medicine.

9. compounds I as claimed in claim 1 is preparing the application in Hepatoma therapy cell drug.

10. the application in the medicine of compounds I as claimed in claim 1 skin flbroblast disease before preparation treatment people.