CN103319365B

CN103319365B - Isotretinoin amido derivative, preparation method thereof and applications thereof

Info

Publication number: CN103319365B
Application number: CN201310219734.4A
Authority: CN
Inventors: 马鹏程; 张孟丽; 魏峻; 傅友军
Original assignee: INSTITUTE OF DERMATOLOGY CHINESE ACADEMY OF MEDICAL SCIENCES
Current assignee: INSTITUTE OF DERMATOLOGY CHINESE ACADEMY OF MEDICAL SCIENCES
Priority date: 2013-06-05
Filing date: 2013-06-05
Publication date: 2015-07-08
Anticipated expiration: 2033-06-05
Also published as: CN103319365A

Abstract

The invention discloses an isotretinoin amido alkyl benzoate derivative, a preparation method thereof and applications thereof. The derivative has stronger inhibition and differentiation regulating effects than isotretinoin on psoriasis, acne and epithelial cell tumors including, but not limited to skin squamous epithelial cell carcinoma, stomach cancer, lung cancer, and cervical cancer; the derivative has less influence on normal tissue cells, has a certain targeting effect on inhibiting proliferating cells, and has very less side effects than isotretinoin; and the derivative has wide promising prophylaxis and treatment applications such as cornification abnormality diseases and cell abnormal proliferation including tumors, psoriasis, acne, and other cornification abnormality dermatopathy.

Description

A kind of isotretinoin amide derivatives and its preparation method and application

Technical field

The present invention relates to biomedicine technical field, be specifically related to a kind of isotretinoin amide derivatives and preparation method thereof and pharmaceutical applications.

Background technology

Isotretinoin, also known as 13-cisRA (being called for short 13-cisRA), be the meta-bolites of vitamin A, belong to the one of retinoid, its molecular formula is C ₂₀h ₂₈o ₂, molecular weight is 300.44, and its structural formula is as follows:

Isotretinoin in impact epithelial propagation, differentiation etc. than all-trans-retinoic acid advantageously, can the generation of Tumor suppression and the proliferation and spreading of cancer cells, be widely used in Acne treatment, the control of the keratosa propagation such as psoriatic and prosoplasia disease and precancerous lesion and tumour.Retinoid medicine plays a very important role in the chemical prevention of the cancer of the brain, mammary cancer, uterus carcinoma, lung cancer, leukemia and other several skin carcinomas (Roos T C, JugertF K, Merk H F, et al.Retinoid metabolism in the skin.Pharmacol.ReV., 1998,50 (2): 315-333).Wherein isotretinoin is considered to the most effective retinoid medicine (the Niles RM.The use of retinoids in the prevention and treatment of skin cancer.Expert Opin Pharmacother preventing non-melanoma dermatoma (NMSC), 2002,3 (3): 299-303).

Regrettably, the isotretinoin of pharmacological dose has larger untoward reaction again simultaneously.The modal significant side effect of retinoid medicine to the irritant reaction of skin, mucous membrane, comprise erythema, furfur, cheilitis, mucocutaneous drying, dermatitis itch, acne increase the weight of and skin fragility increase etc., wherein cheilitis sees the patient of nearly all use isotretinoin.Isotretinoin also has teratogenecity and bone toxicity etc.These side effects greatly limit the application of isotretinoin medicine.

About the research report of the isotretinoin mechanism of action is very many, such as document " sucks isotretinoin for preventing the observation of curative effect of lung cancer " and reports white mouse lung tumor model after suction median dose isotretinoin, the expression of lung tissue retinoic acid receptor α (RAR α, β and γ) is caused to increase more than 3 times (Wang great Li etc., China's clinical tumor and rehabilitation, 2006,13 (3): 196-199).In G. cephalantha clone, system can raise RAR β and express (Bushue N after using isotretinoin, Wan YJ.Retinoid pathway and cancer therapeutics.Adv Drug Deliv Rev, 2010,62 (13): 1285-98).Oral cavity mucous membrane white spot patient system uses isotretinoin therapy after 3 months, RAR β expresses and is increased to 90% (Lotan R.Retinoids and their receptors in modulation ofdifferentiation from 40% before treatment, development, and prevention of head and neck cancers.Anticancer Res, 1996,16 (4C): 2415-9.), think that isotretinoin plays a role by directly or indirectly activating retinoic acid receptor α RAR thus.But, large quantifier elimination shows, the teratogenecity of retinoid medicine, bone toxicity and Mucocutaneous irritant reaction be different retinoic acid receptor α RAR hypotype exciting from retinoid medicine relevant (retinoic acid receptor α RAR has three kinds of hypotypes, i.e. RAR α, RAR β and RAR γ) just.Vitamin A acid teratogenesis ability and parallel (the Lee H Y of the exciting RAR ability of medicine are found, Dohu D F, Kim Y H.et al.All-trans-retinoic acid convertsE2F into a transcripitional suppressor and inhibits the growth of normal human bronchialepithelial cells through retinoic acid receptor-dependent signalingpathway.J.Clin.Invest, 1998,101 (5): 1012-1019), and think that vitamin A acid makes RAR α and β express and increases, and then there is (Zhang YP in induction deformity, Liu K, Wu YL.Modulation of retinoicreceptor alpha and beta and its links with beta-catenin and caspase-3 after maternalexposure to all-trans-tetinoic acid in KM mouse fetuses.Progress in biochemistry andbiophysics, 2007,34 (11): 1182-9.).The calcification of hyperostosis, tendon and ligament is that after oral retinoid medicine, modal bone is abnormal, research think these abnormal also with retinoic acid receptor α RAR by relevant (the DiGiovanna JJ.Isotretinoin effects on bone.J Am Acad Dermatol of retinoid drug activation, 2001,45 (5): S176-82.).Skin and mucous membrane irritation reaction is the common side reaction of retinoid medicine, main manifestations is erythema, furfur, drying, itch, burning sensation, sensation of pricking etc., severity and dosage are proportionate, the skin wound repair that this vitamin A acid causes is alpha mediated (hereinafter pretty by RAR, horse distance of travel of roc. the expression of Retinoic acid receptor mRNA in continuous topical Retinoids skin wound repair. Tropical China medical science, 2009,9 (5): 827-8).Therefore it has been generally acknowledged that, due to the mediation of retinoid receptor, the response to treatment of vitamin A acid is difficult to be separated with its side reaction.

Researchist wishes by carrying out structure of modification to isotretinoin, active to improve it further.Such as, Deng little Qiang etc. disclose synthesis (synthesis of 13-cis-isotretinoin derivative, sign and the Anticancer Activities of 7 isotretinoin ester derivatives.Synthetic chemistry, 13 (4): 327 ~ 330), wherein isotretinoin N3-ethyl-1-(tetrahydrochysene 2-furans)-5 FU 5 fluorouracil ester has certain anti-tumor activity (being respectively 85 μ g/mL to the ID50 of LCC, T9 cell, 120 μ g/mL).There is synthesis (the Y.Fulmer Shealy etal.Synthesis and Properties of Some 13-cis-and All-trans-retinamides.Journal ofpharmaceutical Sciences.1984 of bibliographical information isotretinoin fatty amide and fatty alkanol amide, 73 (6): 745-751), but all not mentioned activation capability to retinoic acid receptor α and side reaction research thereof, the acid amides that isotretinoin and aminobenzoic acid alkyl ester are formed is not related to yet, the latter may because the amino of aromatic amine affects owing to being subject to the inductive effect of electron-withdrawing substituent group (carbalkoxy) on the conjugative effect of aromatic ring and aromatic ring, cloud density in amino nitrogen atom is reduced, amino nucleophilicity reduces, acylation reaction is active in aliphatic amide, the acid amides not easily forming high yield pulp1 with isotretinoin is relevant.

Instant invention overcomes phenyl ring has electron-withdrawing group to replace aromatic amine when synthesizing amide because affecting by the inductive effect of electron-withdrawing substituent group in the conjugative effect of phenyl ring and aromatic ring; the technical barrier that its acylation reaction activity reduces greatly, has gone out to have no the isotretinoin amido benzoic acid alkyl derivative of bibliographical information with aminobenzoic acid alkyl ester and isotretinoin Reactive Synthesis.Higher fat-soluble owing to having, its ability entering cell significantly improves, thus improves antiproliferative effect and regulate the activity of cytodifferentiation.The more important thing is, the isotretinoin amide derivatives that the present invention synthesizes not only significantly is better than isotretinoin to the Carbazole alkaloid of abnormality proliferation and differentiation and regulating effect, and it is less to normal impact cell, optionally can suppress abnormal proliferative cell, but, there is not the hallmark cutaneous irritant reaction that equivalent volumetric molar concentration isotretinoin shows in the retinoic acid receptor α RAR of the reaction of not exciting mediation skin and mucous membrane irritation, teratogenesis and bone toxicity.This existing very strong anti-cell abnormality proliferation and differentiation effect, the invention of the compound of the retinoic acid receptor α RAR of not exciting induced toxicity reaction again, be an important breakthrough in isotretinoin research field, have broad application prospects comprising in the preventing and treating of the various kinds of cell abnormality proliferation of acne, tumour, psoriatic etc. and dyskeratosis disease.

Summary of the invention

According to a first aspect of the invention, a kind of isotretinoin amido benzoic acid alkyl derivative is provided, the acid amides of fat-solubility is formed by the carboxyl of isotretinoin and the amino of aminobenzoic acid alkyl ester, improve the ability that it enters cell, thus improve the activity of antiproliferative effect and adjustment cytodifferentiation, and improve the ability through skin and Lipid dissolution.Pharmaceutical research result shows, isotretinoin amido benzoic acid alkyl derivative provided by the invention is significantly better than isotretinoin to the suppression of the cell of abnormality proliferation and differentiation and regulating effect, the impact of normal tissue cell is less, optionally can suppress abnormal proliferative cell, do not activate the retinoic acid receptor α RAR of induced toxicity reaction, untoward reaction is little, does not produce isotretinoin hallmark cutaneous irritant reaction under clinical dosage to skin.

A kind of isotretinoin amido benzoic acid alkyl derivative, the compound for formula I structure:

Carbalkoxy wherein on phenyl ring can in the contraposition (p) of amino, ortho position (o) or a position (m); The alkyl R of isotretinoin amido benzoic acid alkyl ester is C _1-6alkyl, is preferably methyl, ethyl, n-propyl, sec.-propyl or normal-butyl.

Preferably, described isotretinoin amide derivatives is the compound of formula II-VIII structure, be followed successively by: p-(isotretinoin amide group) methyl benzoate, p-(isotretinoin amide group) ethyl benzoate, p-(isotretinoin amide group) propyl benzoate, p-(isotretinoin amide group) isopropyl benzoate, p-(isotretinoin amide group) n-butylbenzoate, m-(isotretinoin amide group) ethyl benzoate, o-(isotretinoin amide group) methyl benzoate, the structural formula of described compound is as follows:

According to a second aspect of the invention; the preparation method of isotretinoin amido benzoic acid alkyl derivative is provided; the method overcome phenyl ring has electron-withdrawing group (carbalkoxy) to replace aromatic amine when synthesizing amide because affecting by the inductive effect of electron-withdrawing substituent group in the conjugative effect of phenyl ring and aromatic ring; make the technical barrier that its acylation reaction activity reduces greatly; mild condition; technique is simple, and productive rate is higher.

Described isotretinoin amido benzoic acid alkyl derivative is formed by amide linkages by isotretinoin and aminobenzoic acid alkyl ester, comprises the following steps:

DMAP (DMAP) and tosic acid is added in reaction vessel, then solvent is added wherein, and then add isotretinoin, add aminobenzoic acid alkyl ester more subsequently, stir, then add N, N '-dicyclohexylcarbodiimide (DCC), 10 DEG C ~ 66 DEG C are stirred 3-10 hour, stopped reaction.Above-mentioned reaction is carried out under nitrogen protection.Above-mentioned reaction mixture is placed 12 hours, and filter, filtrate revolving steams to obtain crude product.Use column chromatography and obtain isotretinoin amido benzoic acid alkyl derivative.Above-mentioned all to carry out under lucifuge condition in steps.Wherein, the mol ratio of each raw material is: isotretinoin: aminobenzoic acid alkyl ester: DCC: DMAP: tosic acid=1: (1-1.5): (1.1-2): (0.1-0.15): (0.1-0.15); Preferably, the mol ratio of each raw material is: isotretinoin: aminobenzoic acid alkyl ester: DCC: DMAP: tosic acid=1: (1-1.3): (1.1-1.5): (0.1-0.13): (0.1-0.13); Solvent can be CH ₂cl ₂, tetrahydrofuran (THF), CHCl ₃deng; Temperature of reaction: 10 DEG C ~ 66 DEG C, preferred 10-40 DEG C; Reaction times 3-10 hour, preferred 4-8 hour; Column chromatography selects silica gel or neutral alumina dress post, and elutriant is ethyl acetate: sherwood oil 1: 10-1: 35 (V/V), preferred silicagel column, elutriant ethyl acetate: sherwood oil 1: 15 ~ 1: 25 (V/V).

As preferably, aminobenzoic acid alkyl ester described in preparation method is selected from the one in p-Methyl anthranilate, p-subcutin, p-propyl aminobenzoate, p-aminobenzoic isopropyl propionate, the positive butyl ester of p-benzaminic acid, m-subcutin, o-Methyl anthranilate, successively the compound of obtained formula II-VIII.

Because this isotretinoin amido benzoic acid alkyl derivative is with benzaminic acid alkyl group, so for bulk drug isotretinoin, greatly strengthen it fat-soluble, more easily enter cell, thus improve the activity of antiproliferative effect and adjustment cytodifferentiation.Equally, greatly strengthen because it is fat-soluble, enhance the dissolving to acne, make the sebum of hair follicle-sebiferous gland excessive secretion be able to drainage, be unfavorable for that anerobe grows, make medicine be easy to penetrate in pilosebaceous follicle to strengthen antibacterial, germicidal action.Again due to the introducing of benzaminic acid alkyl group, what considerably increase that compound is combined with retinoic acid receptor α RAR is sterically hindered, therefore isotretinoin amido benzoic acid alkyl derivative provided by the present invention does not activate the retinoic acid receptor α RAR albumen of induced toxicity reaction and crucial target gene thereof, untoward reaction is little, does not occur the significant vitamin A acid skin wound repair that isotretinoin shows under Clinical practice concentration.

According to a third aspect of the present invention, the invention provides the abnormal cutaneous keratinization that described isotretinoin amido benzoic acid alkyl derivative includes but not limited to psoriatic, acne in preparation treatment and the application including but not limited in cutaneous squamous cell carcinoma, cancer of the stomach, lung cancer, the cervical cancer medicine in interior epithelial cell tumour and other tumour.

Epithelial cell tumour originates from epithelial tumour, epithelial cell paraplasm and be divided into its principal character; Acne is sebocyte cell paraplasm, ductal epithelium abnormal differentiation, and then formation cutin embolism is principal character; Psoriatic be also with the epidermal hyper-proliferative of inflammation and prosoplasia for principal character.Wherein the high expression level of cytodifferentiation genes involved KRT1 and IVL etc. is the key character of epithelial cell tumour precancerous lesion and pathology, is also the important clinical index of epithelial cell tumour, acne ductal epithelial cell and psoriatic lesion cellular abnormality differentiation.

The present invention is by relevant measuring, research has been done to the activity of the anti-abnormality proliferation of isotretinoin amido benzoic acid alkyl derivative in treatment epithelial cell tumour, acne and psoriatic and epithelial differentiation and principle, and with the activity of isotretinoin and compare (see embodiment 8) Normocellular impact.Experiment proves, isotretinoin amido benzoic acid alkyl derivative of the present invention has very strong restraining effect, its IC to the squamous cell cancer cell (SCL-1 cell) of epithelial origin, gastric carcinoma cells (SGC-7901 cell), human cervical carcinoma cell (HELA cell), people's epidermis abnormal proliferative cell (A431 cell) ₅₀tens of to hundreds of times (embodiment 8) lower than isotretinoin.Meanwhile, the isotretinoin amido benzoic acid alkyl derivative of 10 μMs significantly can reduce the protein expression (the equal < 0.01 of P value, embodiment 9) of division guideline KRT1, IVL.Simultaneously from embodiment 8, isotretinoin amido benzoic acid alkyl derivative of the present invention to the inhibiting rate of the normal keratinocyte of people (HaCaT cell) significantly lower than the cell (p < 0.01) of abnormality proliferation.Experiment proves, isotretinoin amido benzoic acid alkyl derivative of the present invention is to acne epithelial cell, psoriatic lesion and include but not limited to that cutaneous squamous cell carcinoma, cancer of the stomach, lung cancer, cervical cancer have stronger suppression and differentiation regulating effect in interior epithelial cell tumour than isotretinoin, and the impact of normal tissue cell is less, the cell of abnormality proliferation and differentiation optionally can be suppressed.Wherein p-(isotretinoin amide group) phenylformic acid alkyl ester is better than m-(isotretinoin amide group) phenylformic acid alkyl ester and o-(isotretinoin amide group) phenylformic acid alkyl ester, and p-(isotretinoin amide group) ethyl benzoate is the most preferred.

The significant side reaction of isotretinoin medicine mucocutaneously occurs the irritant reaction such as erythema, furfur, also there will be the toxic reaction such as teratogenecity, bone toxicity, these toxic reactions are mediated by exciting retinoic acid receptor α RAR α, β or the γ of direct or indirect approach by isotretinoin.The present invention is by relevant measuring (see embodiment 10), after proving isotretinoin amido benzoic acid alkyl derivative of the present invention effect, RAR α, the RAR β of cell and the albumen relative expression quantity of RAR γ be no difference of science of statistics (p > 0.05) compared with not medication control group; And each albumen relative expression quantity is all significantly higher than isotretinoin amido benzoic acid alkyl derivative of the present invention and not medication control group (p < 0.05 or P < 0.01) after vitamin A acid effect.In addition, the present invention is also studied its mechanism for the compound of formula II I (being called for short ECPIRM), result is with after 10 μMs of vitamin A acid effects, and crucial target gene CYP26A1mRNA and TIG1mRNA of retinoid receptor transcriptional activation path expresses and be significantly higher than not medication control group (p < 0.01); And with after the ECPIRM effect of 10 μMs, CYP26A1mRNA with TIG1mRNA be no difference of science of statistics (the equal > 0.05 of p value) compared with the control group of not dosing.Experiment proves, the retinoic acid receptor α RAR that vitamin A acid is reacted by exciting induced toxicity and then activate its crucial target gene; And the retinoic acid receptor α RAR of the not exciting induced toxicity reaction of isotretinoin amido benzoic acid alkyl derivative of the present invention, activation is not had to the crucial target gene of retinoic acid receptor α RAR yet.Experimentation on animals of the present invention confirms, the retinoic acid receptor α RAR of not exciting induced toxicity reaction, toxic side effects just reduces.Use 0.05% isotretinoin gel namely to occur the skin wound repair such as erythema, furfur in 2 days, medication reaches peak in 5 ~ 6 days, disappears gradually subsequently, is continued until that medication is just disappeared for about 16 days completely.And each isotretinoin amido benzoic acid alkyl derivative gel 21 days of volumetric molar concentration such as to use continuously, all there is not skin wound repair (embodiment 11).Prove that the toxic side effect of each isotretinoin amido benzoic acid alkyl derivative of the present invention is much smaller than bulk drug isotretinoin.

According to a fourth aspect of the present invention, the invention provides isotretinoin amido benzoic acid alkyl derivative as the pharmaceutical composition of active constituents of medicine and preparation thereof.Isotretinoin amido benzoic acid alkyl derivative can separately as activeconstituents, various clinical suitable preparation is made after adding pharmaceutically acceptable customary adjuvant, also can as the first promoting agent, combine with the second promoting agent for identical indication, after adding auxiliary material, make compound preparation.Wherein the second promoting agent is selected from Dalacina, or Clindamycin Phosphate, or erythromycin, or paraxin, or tsiklomitsin, or metronidazole, or tinidazole, or secnidazole, or Ofloxacine USP 23, or Ciprofloxacin, or vitamin-E, or clobetasol propionate, or diflorasone, or Valisone, or beclometasone, or clobetasone butyrate, or Sch-11460, or furoic acid momisone, or fluocinonide, or fluticasone propionate etc.The form of preparation can be the external preparations such as the oral preparations such as coated tablet, conventional tablet, capsule, soft capsule, granule, or ointment, gelifying agent, creme, sprays, suspensoid, solution.The preparation of preparation can according to the existing preparation method of formulation art.Described isotretinoin amido benzoic acid alkyl derivative is the one in formula I structural compounds, preferred p-(isotretinoin amide group) ethyl benzoate (formula III).

A kind of prevention and therapy that is used for includes but not limited to acne, psoriatic abnormal cutaneous keratinization, or the combination of oral medication of epithelial cell tumour, the formula I wherein containing treatment significant quantity.

In above-mentioned combination of oral medication, the shared in the composition weight percentage of effective constituent formula I is 2% ~ 60% (wt), preferably 30% ~ 50%.

Various clinical suitable preparation is made after can adding pharmaceutically acceptable customary adjuvant in above-mentioned combination of oral medication, each preparation unit (as 1,1,1 bag, 1 bag) can contain above-mentioned isotretinoin amide derivatives 5mg ~ 300mg in preparation, preferably 25mg ~ 100mg.

In above-mentioned combination of oral medication, effective constituent formula I can as the first promoting agent, combine with the second promoting agent, various clinical suitable compound preparation is made after adding pharmaceutically acceptable customary adjuvant again, can above-mentioned formula I 5mg ~ 300mg be contained in each preparation unit (as 1,1) preparation, be preferably 25mg ~ 100mg.

The form of the preparation of combination of oral medication can be coated tablet, conventional tablet, capsule, soft capsule, granule etc.

Be used for the treatment of the externally-applied medicinal composition including but not limited to acne or psoriatic abnormal cutaneous keratinization, the formula I wherein containing treatment significant quantity.

In above-mentioned externally-applied medicinal composition, effective constituent formula I in the composition shared weight percentage is 0.01% ~ 5% (wt), preferably 0.025% ~ 0.25% (wt).

Various clinical suitable external preparation is made after can adding pharmaceutically acceptable conventional substrate carrier in above-mentioned externally-applied medicinal composition.Effective constituent formula I shared weight percentage in composite preparation is 0.01% ~ 5% (wt), preferably 0.025% ~ 0.25% (wt).

In above-mentioned externally-applied medicinal composition, effective constituent formula I can as the first promoting agent, combine with the second promoting agent, various clinical suitable externally used compound preparation is made after adding pharmaceutically acceptable customary adjuvant again, effective constituent formula I shared weight percentage in composite preparation is 0.01% ~ 5% (wt), preferably 0.025% ~ 0.25% (wt).

The conventional antimicrobial drug Dalacina that the second promoting agent in above-mentioned externally used compound preparation can infect for anti-acne, or Clindamycin Phosphate, or erythromycin, or paraxin, or tsiklomitsin, or metronidazole, or tinidazole, or secnidazole, or Ofloxacine USP 23, or Ciprofloxacin.Antimicrobial drug in the composition shared weight percentage is 0.25% ~ 5% (wt), preferably 0.5% ~ 3% (wt).This compound preparation can be used as a kind of externally used compound pharmaceutical composition being used for the treatment of infectious acne.

The second promoting agent in above-mentioned externally used compound preparation can be the psoriatic conventional anti-inflammatory agent clobetasol propionate for the treatment of, or diflorasone, or Valisone, or beclometasone, or clobetasone butyrate, or Sch-11460, or furoic acid momisone, or fluocinonide, or fluticasone propionate.Anti-inflammatory agent in the composition shared weight percentage is 0.01% ~ 0.5% (wt), preferably 0.025% ~ 0.15% (wt).This compound preparation can be used as one and is used for the treatment of psoriatic externally used compound pharmaceutical composition.

The form of the preparation of above-mentioned externally-applied medicinal composition can be the external preparations such as ointment, gelifying agent, creme, sprays, suspensoid, solution, can above-mentioned isotretinoin amido benzoic acid alkyl derivative 0.01% ~ 5% (wt) be contained in preparation, be preferably 0.025% ~ 0.25% (wt).

Compared with prior art, the present invention has the following advantages:

(1) the invention provides new isotretinoin amide derivatives, this area faced by include but not limited to the treatment of acne, psoriatic abnormal cutaneous keratinization and epithelial cell tumour time add new medication and select.

(2) instant invention overcomes phenyl ring has electron-withdrawing group to replace aromatic amine when synthesizing amide because affecting by the inductive effect of electron-withdrawing substituent group in the conjugative effect of phenyl ring and aromatic ring; make the technical barrier that its acylation reaction activity reduces greatly; with aminobenzoic acid alkyl ester and isotretinoin for raw material, synthesized isotretinoin amido benzoic acid alkyl derivative high yield pulp1.

(3) isotretinoin amido benzoic acid alkyl derivative of the present invention to acne epithelium, psoriatic lesion and include but not limited to cutaneous squamous cell carcinoma, cancer of the stomach, lung cancer, cervical cancer interior epithelial cell tumour than isotretinoin have stronger Inhibit proliferaton and differentiation regulating effect, and the impact of normal tissue cell is less, to the suppression of abnormal proliferative cell, there is certain targeting.

(4) the retinoic acid receptor α RAR of isotretinoin amido benzoic acid alkyl derivative of the present invention not exciting induced toxicity reaction and crucial target gene thereof, do not produce skin wound repair under clinical dosage.This existing very strong and there is the anti-cell abnormality proliferation of certain targeting and differentiation effect, do not activate again the retinoic acid receptor α RAR of induced toxicity reaction, do not have the hallmark cutaneous of isotretinoin medicine to stimulate the invention of the compound of side reaction, be an important breakthrough in isotretinoin research field.Have broad application prospects comprising in the preventing and treating of the abnormal cell proliferation of tumour, acne, psoriatic etc. and point voltinism disease.

Accompanying drawing illustrates: enumerate part illustration for formula III

Fig. 1 is embodiment 2 ¹h-NMR schemes (in figure, FI-2-61 is formula III compound sample presentation number)

Fig. 2 is embodiment 2 ¹³h-NMR schemes (in figure, FI-2-61 is formula III compound sample presentation number)

Fig. 3 is the effect of embodiment 8 pairs of SCL-1 cell proliferation

Fig. 4 is the effect of embodiment 8 pairs of HELA cell proliferation

Fig. 5 is the effect of embodiment 8 pairs of SGC-7901 cell proliferation

Fig. 6 is the effect of embodiment 8 pairs of A431 cell proliferation

Fig. 7 is the effect of embodiment 8 pairs of HaCaT cell proliferation

Fig. 8 is the impact that embodiment 10 is expressed retinoid receptor RAR

Embodiment

Below in conjunction with embodiment, the present invention is described further, to help the present invention of those skilled in the art's comprehend, but not in this, as limitation of the present invention.

The synthesis of embodiment 1:p-(isotretinoin amide group) methyl benzoate

In 250ml three-necked bottle, add DMAP (DMAP) (0.1g, 0.8mmol), tosic acid (0.14g, 0.8mmol), adds CH ₂cl ₂110ml, then add isotretinoin (2.4g, 8mmol), p-Methyl anthranilate (1.21g, 8.0mmol), in stirred at ambient temperature 10min, then add DCC (1.82g, 8.8mmol), 10 DEG C of stir abouts 4 hours, stopped reaction.Above-mentioned reaction is in nitrogen protection and carry out under lucifuge condition.Above-mentioned reaction mixture is placed about 12 hours at 0 ~ 5 DEG C, take out and filter, filtrate revolving steams to obtain crude product, use column chromatography (200-300 order silica gel, elutriant are ethyl acetate: sherwood oil 1: 15 (V/V) ~ 1: 10 (V/V)), gradient elution, products therefrom uses ethyl alcohol recrystallization again, obtains reaction product (3.02g).The molecular mass of reaction product is 433.60, proton nmr spectra ( ¹h-NMR) δ (ppm): 10.30 (s, 1H, NH), 7.94 (d, 1H, 12-H), 7.90 (d, 2H, 3 ', 5 '-H), 7.78 (d, 2H, 2 ', 6 '-H), 7.00 (dd, 1H, 11-H), 6.28,6.23 (2H, 10-H, 7-H), 6.18 (d, 1H, 8-H), 5.91 (s, 1H, 14-H), 3.87 (s, 3H ,-OC h3), 2.07 (s, 3H, 20-H), 2.02 (m, 2H, 4-H), 1.98 (s, 3H, 19-H), 1.70 (s, 3H, 18-H), 1.58 ~ 1.60 (m, 2H, 3-H), 1.45 ~ 1.46 (m, 2H, 2-H), 1.02 (s, 6H, 16,17-H).Show that the reaction product obtained is p-(isotretinoin amide group) methyl benzoate (formula II).

The synthesis of embodiment 2:p-(isotretinoin amide group) ethyl benzoate

In 250ml three-necked bottle, add DMAP (DMAP) (0.1g, 0.8mmol), tosic acid (0.14g, 0.8mmol), adds CH ₂cl ₂110ml, then add isotretinoin (2.4g, 8mmol), p-subcutin (1.4g, 8.4mmol), be about 15min in stirred at ambient temperature, then add DCC (1.87g, 9.1mmol),, 40 DEG C of stirring and refluxing about 3 hours, stopped reaction.Above-mentioned reaction is in nitrogen protection and carry out under lucifuge condition.Above-mentioned reaction mixture is placed about 12 hours at 0 ~ 5 DEG C, take out and filter, filtrate revolving steams to obtain crude product, use column chromatography (200-300 order silica gel, elutriant are ethyl acetate: sherwood oil 1: 20 (V/V)), products therefrom uses ethyl alcohol recrystallization again, obtains reaction product (3.58g).The molecular mass of reaction product is 447.62, proton nmr spectra ( ¹h-NMR) δ (ppm): 10.28 (s, N-H), 7.94 (d, 1H, 12-H), 7.90 (d, H, 3 '-H, 5 '-H), 7.78 (d, 2H, 2 '-H, 6 '-H), 7.01 (dd, 1H, 11-H), 6.29,6.25 (2H, 10-H, 7-H), 6.21 (d, 1H, 8-H), 5.91 (s, 1H, 14-H), 4.28 (m, 2H ,-OC h ₂cH ₃), 2.08 (s, 3H, 20-H), 2.01 (m, 2H, 4-H), 1.99 (s, 3H, 19-H), 1.70 (s, 3H, 18-H), 1.58 (m, 2H, 3-H), 1.45 (m, 2H, 2-H), 1.31 (t, 3H ,-OCH ₂c h ₃), 1.02 (s, 6H, 16-3H, 17-H), (Fig. 1).The carbon-13 nmr spectra of reaction product ( ¹³c-NMR) δ (ppm) 165.254 (7 '-C), 164.504 (15-C), 148.049 (13-C), 143.804 (1 '-C), 139.047 (9-C), 137.188 (6-C), 136.996 (7-C), 131.441 (11-C), 130.465 (10-C), 130.062 (3 '-C, , 5 '-C), 129.806 (12-C), 129.366 (5-C), 127.622 (8-C), 123.833 (4-C '), 119.888 (14-C), 118.257 (2 '-C, 6 '-C), 60.239 (-O ch ₂cH ₃), 39.162 (2-C), 33.768 (1-C), 32.550 (4-C), 28.718 (16-C, 17-C), 21.418 (18-C), 20.640 (20-C), 18.646 (3-C), 14.121 (-OCH ₂ ch ₃), 12.520 (19-C), (Fig. 2).Show that the reaction product obtained is p-(isotretinoin amide group) ethyl benzoate (formula II I).

The synthesis of embodiment 3:p-(isotretinoin amide group) propyl benzoate

In 250ml three-necked bottle, add DMAP (DMAP) (0.12g, 0.96mmol), tosic acid (0.17g, 0.96mmol), adds CH ₂cl ₂110ml, then add isotretinoin (2.4g, 8mmol), p-propyl aminobenzoate (1.72g, 9.6mmol), in stirred at ambient temperature 15min, then add DCC (2.15g, 10.4mmol), 30 DEG C of stir abouts 5 hours, stopped reaction.Above-mentioned reaction is in nitrogen protection and carry out under lucifuge condition.Above-mentioned reaction mixture is placed about 12 hours at 0 ~ 5 DEG C, take out and filter, filtrate revolving steams to obtain crude product, use column chromatography (200-300 order silica gel, elutriant are ethyl acetate: sherwood oil 1: 20 (V/V)), products therefrom uses ethyl alcohol recrystallization again, obtains reaction product (2.9g).The molecular mass of reaction product is 461.65, proton nmr spectra ( ¹h-NMR) δ (ppm): 10.32 (s, 1H, NH), 7.94 (d, 1H, 12-H), 7.90 (d, 2H, 3 ', 5 '-H), 7.78 (d, 2H, 2 ', 6 '-H), 7.01 (dd, 1H, 11-H), 6.28,6.24 (2H, 10-H, 7-H), 6.19 (d, 1H, 8-H), 5.90 (s, 1H, 14-H), 4.24 (t, 2H, OC h ₂cH ₂cH ₃), 2.07 (s, 3H, 20-H), 2.01 (m, 2H, 4-H), 1.98 (s, 3H, 19-H), 1.80 (m, 2H ,-OCH ₂c h ₂cH ₃), 1.70 (s, 3H, 18-H), 1.59 ~ 1.62 (m, 2H, 3-H), 1.47 (m, 2H, 2-H), 1.03 (s, 6H, 16,17-H), 0.97 (t, 3H ,-OCH ₂cH ₂c h ₃).Show that the reaction product obtained is p-(isotretinoin amide group) propyl benzoate (structural formula IV).

The synthesis of embodiment 4:p-(isotretinoin amide group) isopropyl benzoate

In 250ml three-necked bottle, add DMAP (DMAP) (0.13g, 1.04mmol), tosic acid (0.18g, 1.04mmol), adds CH ₂cl ₂110ml, add isotretinoin (2.4g again, 8mmol), p-aminobenzoic isopropyl propionate (1.86g, 10.4mmol), in stirred at ambient temperature 15min, then DCC (2.15g is added, 10.4mmol), 40 DEG C of (backflow) stir abouts 6 hours, stopped reaction.Above-mentioned reaction is in nitrogen protection and carry out under lucifuge condition.Above-mentioned reaction mixture is placed about 12 hours at 0 ~ 5 DEG C, take out and filter, filtrate revolving steams to obtain crude product, use column chromatography (200-300 order silica gel, elutriant are ethyl acetate: sherwood oil 1: 25 (V/V)), products therefrom uses ethyl alcohol recrystallization again, obtains reaction product (2.6g).The molecular mass of reaction product is 461.65, proton nmr spectra ( ¹h-NMR) δ (ppm): 1H NMR δ: 10.27 (s, 1H, NH), 7.97 (d, 1H, 12-H), 7.91 (d, 2H, 3 ', 5 '-H), 7.77 (d, 2H, 2 ', 6 '-H), 7.01 (dd, 1H, 11-H), 6.29,6.26 (2H, 10-H, 7-H), 6.21 (d, 1H, 8-H), 5.92 (s, 1H, 14-H), 4.30 (m ,-OC h(CH ₃) ₂), 2.08 (s, 3H, 20-H), 2.02 ~ 2.06 (m, 2H, 4-H), 2.00 (s, 3H, 19-H), 1.71 (s, 3H, 18-H), 1.59 ~ 1.63 (m, 2H, 3-H), 1.45 ~ 1.48 (m, 2H, 2-H), 1.36 (d, 6H ,-OCH (C h ₃) ₂), 1.02 (s, 6H, 16,17-H).Show that the reaction product obtained is p-(isotretinoin amide group) isopropyl benzoate (structural formula V).

The synthesis of embodiment 5:p-(isotretinoin amide group) n-butylbenzoate

In 250ml three-necked bottle, add DMAP (DMAP) (0.13g, 1.04mmoll), tosic acid (0.18g, 1.04mmol), adds CH ₂cl ₂110ml, add isotretinoin (2.4g again, 8mmol), p-butyl aminobenzoate (2.01g, 10.4mmol), in stirred at ambient temperature 20min, then DCC (2.15g is added, 10.4mmol), 40 DEG C of (backflow) stir abouts 5 hours, stopped reaction.Above-mentioned reaction is in nitrogen protection and carry out under lucifuge condition.Above-mentioned reaction mixture is placed about 12 hours at 0 ~ 5 DEG C, take out and filter, filtrate revolving steams to obtain crude product, use column chromatography (200-300 order silica gel, elutriant are ethyl acetate: sherwood oil 1: 20 (V/V)), products therefrom uses ethyl alcohol recrystallization again, obtains reaction product (3.01g).The molecular mass of reaction product is 475.68, proton nmr spectra ( ¹h-NMR) δ (ppm): 1H NMR δ: 10.29 (s, 1H, NH), 7.93 (d, 1H, 12-H), 7.90 (d, 2H, 3 ', 5 '-H), 7.76 (d, 2H, 2 ', 6 '-H), 7.00 (dd, 1H, 11-H), 6.27,6.23 (2H, 10-H, 7-H), 6.20 (d, 1H, 8-H), 5.89 (s, 1H, 14-H), 4.23 (t, 2H, OC h ₂cH ₂cH ₂cH ₃), 2.07 (s, 3H, 20-H), 2.03 (m, 2H, 4-H), 1.99 (s, 3H, 19-H), 1.76 (m, 2H ,-OCH ₂c h ₂cH ₂cH ₃), 1.70 (s, 3H, 18-H), 1.62 (m, 2H, 3-H), 1.47 (m, 2H, 2-H), 1.34 (m, 2H ,-OCH ₂cH ₂c h ₂cH ₃), 1.03 (s, 6H, 16,17-H), 0.97 (t, 3H ,-OCH ₂cH ₂cH ₂c h ₃).Show that the reaction product obtained is p-(isotretinoin amide group) n-butylbenzoate (structural formula VI).

The synthesis of embodiment 6:m-(isotretinoin amide group) ethyl benzoate

In 250ml three-necked bottle, add DMAP (DMAP) (0.14g, 1.12mmol), tosic acid (0.19g, 1.12mmol), adds CHCl ₃120ml, then add isotretinoin (2.4g, 8mmol), m-subcutin (1.85g, 11.2mmol), in stirred at ambient temperature 20min, then add DCC (2.48g, 12.0mmol), 50 DEG C of stir abouts 8 hours, stopped reaction.Above-mentioned reaction is in nitrogen protection and carry out under lucifuge condition.Above-mentioned reaction mixture is placed about 12 hours at 0 ~ 5 DEG C, take out and filter, filtrate revolving steams to obtain crude product, use column chromatography (200-300 order silica gel, elutriant are ethyl acetate: sherwood oil 1: 30 (V/V)), products therefrom uses ethyl alcohol recrystallization again, obtains reaction product (2.03g).The molecular mass of reaction product is 447.62, proton nmr spectra ( ¹h-NMR) δ (ppm): 10.32 (s, 1H, NH), 8.28 (d, 1H, 2 '-H), 7.98 (d, 1H, 12-H), 7.31 ~ 7.88 (m, 3H, 4 ', 5 ', 6 '-H), 6.88 (dd, 1H, 11-H), 6.34,6.29 (2H, 10-H, 7-H), 6.24 (d, 1H, 8-H), 5.99 (s, 1H, 14-H), 4.28 (q, 2H, OC h ₂cH ₃), 2.08 (s, 3H, 20-H), 2.02 (m, 2H, 4-H), 1.97 (s, 3H, 19-H), 1.70 (s, 3H, 18-H), 1.61 ~ 1.63 (m, 2H, 3-H), 1.47 ~ 1.48 (m, 2H, 2-H), 1.31 (t, 3H ,-OCH ₂c h ₃), 1.02 (s, 6H, 16,17-H).Show that the reaction product obtained is m-(isotretinoin amide group) ethyl benzoate (structural formula VII).

The synthesis of embodiment 7:o-(isotretinoin amide group) methyl benzoate

DMAP (DMAP) (0.15g is added in 250ml three-necked bottle, 1.2mmol), tosic acid (0.21g, 1.2mmol), add tetrahydrofuran (THF) 130ml, add isotretinoin (2.4g again, 8mmol), o-Methyl anthranilate (1.81g, 12.0mmol), in stirred at ambient temperature 20min, then DCC (3.3g is added, 16.0mmol), 66 DEG C of (backflow) stir abouts 10 hours, stopped reaction.Above-mentioned reaction is in nitrogen protection and carry out under lucifuge condition.Above-mentioned reaction mixture is placed about 12 hours at 0 ~ 5 DEG C, take out and filter, filtrate revolving steams to obtain crude product, use column chromatography (200-300 order silica gel, elutriant are ethyl acetate: sherwood oil 1: 35 (V/V)), products therefrom uses ethyl alcohol recrystallization again, obtains reaction product (1.75g).The molecular mass of reaction product is 433.60, proton nmr spectra ( ¹h-NMR) δ (ppm): 10.24 (s, 1H, NH), 7.96 (d, 1H, 12-H), 7.10 ~ 7.91 (m, 4H, 3 ', 4 ', 5 ', 6 '-H), 7.05 (dd, 1H, 11-H), 6.32,6.27 (2H, 10-H, 7-H), 6.23 (d, 1H, 8-H), 5.95 (s, 1H, 14-H), 3.90 (s, 3H ,-OC h3), 2.09 (s, 3H, 20-H), 2.03 (m, 2H, 4-H), 1.96 (s, 3H, 19-H), 1.71 (s, 3H, 18-H), 1.60 (m, 2H, 3-H), 1.46 ~ 1.47 (m, 2H, 2-H), 1.03 (s, 6H, 16,17-H).Show that the reaction product obtained is o-(isotretinoin amide group) methyl benzoate (structural formula VIII).

Embodiment 8: the inhibition of cell proliferation test of isotretinoin amido benzoic acid alkyl derivative

Epithelial cell tumour originates from epithelial tumour, epithelial cell paraplasm and be divided into its principal character; Acne is also ductal epithelium paraplasm and differentiation, and then formation cutin embolism is principal character; Psoriatic is also for principal character with epidermal hyper-proliferative and prosoplasia.The present invention is with the anti-abnormal proliferative cell of isotretinoin amido benzoic acid alkyl derivative (epithelium abnormal proliferative cell (SCL-1 cell), human gastric adenocarcinoma (SGC-7901 cell), people's adenocarcinoma of the uterine cervix cell (HELA cell), people's epidermis abnormal proliferative cell (A431 cell)) and the propagation of the normal keratinocyte of people (HaCaT cell) be example, to isotretinoin amido benzoic acid alkyl derivative Acne treatment, the activity of epithelial cell tumour and psoriatic principle and anti-abnormality proliferation and differentiation has done research, and with the activity of the isotretinoin of medicine in contrast and compare research to Normocellular impact.

One, method:

Take a certain amount of isotretinoin amido benzoic acid alkyl derivative, dissolve with DMSO respectively, concentration is 0.1M, is stored in-80 DEG C, be diluted to the concentration set by test with the DMEM substratum containing 10% foetal calf serum before experiment, wherein the final concentration of DMSO is no more than 0.1%.More than operate equal lucifuge.

Growth of Cells, in the DMEM substratum containing 10%FBS, 100U/ml penicillin and 0.1mg/mL Streptomycin sulphate, is placed in 5%CO ₂, 37 DEG C of constant-temperature enclosed formula CO ₂incubator (relative humidity 95%) interior subculture, growing state observed by inverted microscope.Within 2-3 days, go down to posterity once.The cell of logarithmic phase is inoculated in 96 orifice plates, at 37 DEG C and 5%CO ₂under hatch 24h, abandoning supernatant, every hole adds the substratum 200 μ l of the isotretinoin amido benzoic acid alkyl derivative containing setting concentration, establishes 5 concentration altogether, and each concentration establishes 3 multiple holes, control group adds the substratum 200 μ L containing 0.2%DMSO, blank well does not add cell, only adds the substratum containing 0.2%DMSO, cultivates after 48 hours, every hole adds the MTT solution (5mg/ml) of 20 μ L, continues at 37 DEG C and 5%CO ₂under hatch 4h after, under microplate reader 490nm wavelength, measure each hole absorbance.Cell proliferation rate=(treating each concentration mean absorbance values of sieve thing-blank group mean absorbance values)/(control group mean absorbance values-blank group mean absorbance values)] × 100%.Described cell is the one in SCL-1 cell, SGC-7901 cell, HELA cell, A431 cell, HaCaT cell.In SCL-1 cell, HELA cell and HaCaT cell experiment, trial drug isotretinoin amido benzoic acid alkyl derivative concentration is in the medium respectively 5 μMs, 10 μMs, 20 μMs, 40 μMs, 80 μMs; In SGC-7901 cell and A431 cell experiment, trial drug isotretinoin amido benzoic acid alkyl derivative concentration is in the medium respectively 12.5 μMs, 25 μMs, 50 μMs, 75 μMs, 100 μMs.IC ₅₀use SPSS18.0 computed in software.Data processing adopts SPSS18.0 software, and all experimental datas represent with Mean ± SD, adopts independent samples t test.

Two, result:

Isotretinoin amido benzoic acid alkyl derivative of the present invention all has very strong restraining effect to SCL-1 cell, SGC-7901 cell, HELA cell, A431 cell.To SCL-1 cell, the drug level (IC50) needed for half T suppression cell of formula III compound is 37.3 μMs, the IC50 of the compound of formula II compound and formula IV ~ formula VI is between 37.9 μMs ~ 43.5 μMs, the IC50 of formula VII and formula VIII compound is respectively 49.5 μMs and 54.3 μMs, and isotretinoin is to the IC50 > 1000 μMs of SCL-1 cell; To SGC-7901 cell, the IC50 of formula II and formula III compound is respectively 41.8 μMs and 42.4 μMs, the IC50 of formula IV ~ formula VI compound is between 43.1 μMs ~ 48.1 μMs, and isotretinoin is to the IC50 > 1000 μMs of SGC-7901 cell; To HELA cell, the IC50 of formula III compound is 40.2 μMs, and the IC50 of formula II and formula IV ~ formula VI compound is between 40.9 μMs ~ 51.3 μMs, and isotretinoin is to the IC50 > 1000 μMs of HELA cell; To A431 cell, the IC50 of formula III compound is 49.7 μMs, the IC50 of formula II and formula IV ~ formula VI compound is between 49.9 μMs ~ 53.5 μMs, the IC50 of formula VII and formula VIII compound is respectively 55.3 μMs and 57.6 μMs, and isotretinoin is to the IC50 > 1000 μMs of A431 cell.The IC50 of isotretinoin amido benzoic acid alkyl derivative is generally tens of to hundreds of times lower than isotretinoin.Along with the increase of compound concentration, its restraining effect is stronger, >=40 μMs time, is significantly better than former medicine isotretinoin (the equal < 0.01 of p value) to the restraining effect of above-mentioned cell.Comparatively speaking, p-(isotretinoin amide group) phenylformic acid alkyl ester is better than m-(isotretinoin amide group) phenylformic acid alkyl ester and o-(isotretinoin amide group) phenylformic acid alkyl ester, and formula III compound is preferred.To the normal keratinocyte of people (HaCaT cell), the inhibiting rate of formula II ~ formula VIII compound is significantly lower than the cell (p < 0.01) of abnormality proliferation, and its IC50 is between 73.0 μMs ~ 77.5 μMs.Experiment proves, isotretinoin amido benzoic acid alkyl derivative of the present invention is to acne epithelium, psoriatic lesion and include but not limited to that cutaneous squamous cell carcinoma, cancer of the stomach, lung cancer, cervical cancer have stronger suppression and differentiation regulating effect in interior epithelial cell tumour than former medicine isotretinoin, and the impact of normal tissue cell is less, can Selective depression abnormal proliferative cell.(Fig. 3, Fig. 4, Fig. 5, Fig. 6 and Fig. 7 are the part illustration enumerated).

Embodiment 9: isotretinoin amido benzoic acid alkyl derivative is tested the adjustment of cytodifferentiation

Epithelial cell tumour originates from epithelial tumour, epithelial cell paraplasm and be divided into its principal character; Acne is also ductal epithelium paraplasm and differentiation, and then formation cutin embolism is principal character; Psoriatic is also for principal character with epidermal hyper-proliferative and prosoplasia.The high expression level of cytodifferentiation genes involved Keratin 1 (KRT1) and involucrin (IVL) is the key character of epithelial cell tumour precancerous lesion and pathology, is also the important clinical index of acne epithelium, epithelial cell tumour and psoriatic lesion cellular abnormality differentiation.The present invention for model, evaluates the impact that isotretinoin amido benzoic acid alkyl derivative breaks up acne epithelium, epithelial cell tumour and psoriatic lesion cellular abnormality with the epithelial cell SCL-1 cell of abnormality proliferation and differentiation.

One, method:

Isotretinoin amido benzoic acid alkyl derivative is on the impact of KRT1 and IVL level: the model cell (SCL-1) selecting logarithmic phase, is modulated into cell suspension, with every hole about 5 × 10 with the DMEM substratum containing 10% foetal calf serum ⁵the density of individual cell is seeded in 10cm plate, at 37 DEG C and 5%CO ₂lower overnight incubation, abandoning supernatant, adds the substratum 8mL containing 10 μMs of isotretinoin amido benzoic acid alkyl derivatives in every plate, control group adds the substratum 8mL containing DMSO (final concentration of DMSO is 0.05%).After effect 24h, with 0.25% trypsinase-EDTA collecting cell and in 4 DEG C, the centrifugal 5min of 1200rpm, gets supernatant and washs twice with the PBS of precooling, centrifugal, obtains cell precipitation.Extract cell whole protein and carry out protein quantification with Bradford method, adopting Western Blotting method to measure the expression amount of Keratin 1 (KRT1) and involucrin (IVL) in each group.Data processing adopts SPSS 18.0 software, and all experimental datas represent with Mean ± SD, adopts independent samples t test.

Two, result:

Result shows, isotretinoin amido benzoic acid alkyl derivative of the present invention all significantly can reduce the protein expression of early differentiation genes involved KRT1 and IVL.For KRT1 albumen, the relative expression quantity of formula III compound group is 0.38 ± 0.06, the difference (P > 0.05) of formula II and formula IV ~ VI compound group and formula III compound group not statistically significant, all remarkable in not dosing group (the equal < 0.01 of 0.99 ± 0.35, P value); For IVL albumen, the relative expression quantity of formula III group is 0.18 ± 0.06, the difference (P > 0.05) of formula II and formula IV ~ VI compound group and formula III compound group not statistically significant, all remarkable in not dosing group (the equal < 0.01 of 0.39 ± 0.10, P value).

Illustrate that isotretinoin amido benzoic acid alkyl derivative of the present invention is to acne ductal epithelium, psoriatic lesion and include but not limited to that cutaneous squamous cell carcinoma, cancer of the stomach, lung cancer, cervical cancer have stronger adjustment active at interior epithelial cell tumour cell abnormal differentiation.

Embodiment 10: the impact experiment that isotretinoin amido benzoic acid alkyl derivative is expressed retinoid receptor RAR

The significant side reaction of isotretinoin medicine mucocutaneously occurs the irritant reaction such as erythema, furfur, also there will be the toxic reaction such as teratogenecity, bone toxicity, these toxic reactions are mediated by exciting retinoic acid receptor α RAR α, β or the γ of direct or indirect approach by isotretinoin.The present invention tests the impact that retinoid receptor RAR expresses with regard to isotretinoin amido benzoic acid alkyl derivative.And for formula III compound (being called for short ECPIRM), carried out mechanism test with regard to the impact of isotretinoin amido benzoic acid alkyl derivative on crucial target gene CYP26A1 and TIG1 of retinoid receptor transcriptional activation path.

One, method:

Select the model cell (SCL-1) of logarithmic phase, be modulated into cell suspension, with every hole about 5 × 10 with the DMEM substratum containing 10% foetal calf serum ⁵the density of individual cell is seeded in 10cm plate, at 37 DEG C and 5%CO ₂lower overnight incubation, abandoning supernatant, every plate adds the substratum 8mL of medicine containing 10 μMs or 10 μMs of all-trans-retinoic acids (ATRA) respectively, and Matrix controls group adds the substratum 8mL containing DMSO (final concentration of DMSO is 0.05%).After effect 24h, with 0.25% trypsinase-EDTA collecting cell and in 4 DEG C, the centrifugal 5min of 1200rpm, goes supernatant to wash twice with the PBS of precooling, centrifugal, obtains each group of cell precipitation.Extract each group of Nuclear extract respectively, adopt Bradford method carry out nucleoprotein quantitatively after, adopt Western Blotting method to measure the relative expression quantity (the expression gray-scale value of β-actin is internal reference) of retinoid receptor albumen in each group (RAR α, RAR β, RAR γ).IC ₅₀use SPSS18.0 computed in software.Data processing adopts SPSS18.0 software, and all experimental datas represent with Mean ± SD, adopts independent samples t test.

(2) detection experiment of target gene CYP26A1 and TIG1 of retinoid receptor transcriptional activation path

Select the model cell (SCL-1) of logarithmic phase, be modulated into cell suspension, with every hole about 5 × 10 with the DMEM substratum containing 10% foetal calf serum ⁴the density of individual cell is seeded in 6 well culture plates, at 37 DEG C and 5%CO ₂lower overnight incubation, abandoning supernatant, every hole adds the substratum 2mL containing 10 μMs of medicines, control group adds the substratum 2mL containing DMSO (final concentration of DMSO is 0.05%), after effect 24h, carry out the extraction of RNA and measure concentration and the purity of RNA, then carrying out the synthesis of cDNA.The primer of pre-synthesis CYP26A1 and TIG1, adopts real-time fluorescence quantitative PCR (Real-time PCR) technology, using house-keeping gene (GAPDH) as internal reference, measures goal gene CYP26A1 and TIG1mRNA relative content.Data processing adopts SPSS18.0 software, and all experimental datas represent with Mean ± SD, adopts independent samples t test.

Two, result:

1, after each isotretinoin amido benzoic acid alkyl derivative effect of 10 μMs, retinoic acid receptor α RAR α in the cell of formula III compound group, the albumen relative expression quantity of RAR β and RAR γ is respectively 0.42 ± 0.06, 0.28 ± 0.10 and 0.45 ± 0.15, formula II, retinoic acid receptor α RAR α in the cell of formula IV ~ VI compound group, the albumen relative expression quantity of RAR β and RAR γ and formula III compound group no significant difference (p > 0.05), each group (is respectively 0.43 ± 0.11 with not dosing control group, 0.27 ± 0.08 and 0.45 ± 0.10) more equal no significant difference (p > 0.05).And vitamin A acid significantly can induce the high expression level (0.83 ± 0.15,0.62 ± 0.05 and 0.96 ± 0.33) of RAR α, RAR β and RAR γ albumen, be significantly higher than each isotretinoin amido benzoic acid alkyl derivative group and not dosing control group (P < 0.05 or P < 0.01).(illustration is shown in Fig. 8)

The significant side reaction of isotretinoin medicine mucocutaneously occurs the irritant reaction such as erythema, furfur, also there will be the toxic reaction such as teratogenecity, bone toxicity, these toxic reactions are mediated by exciting retinoic acid receptor α RAR α, β or the γ of direct or indirect approach by isotretinoin.This experimental result shows, the not exciting retinoic acid receptor α RAR α of isotretinoin amido benzoic acid alkyl derivative, RAR β, RAR γ, and this untoward reaction for this kind of medicine of reduction very has using value.

2, the formula III compound effects of 10 μMs is after model cell 24h, and in cell, the mrna expression amount of CYP26A1 is 0.49 ± 0.05, with not dosing control group (1.00 ± 0.35) no significant difference (P > 0.05); In cell, the mrna expression amount of TIG1 is 0.58 ± 0.22, with not dosing control group (1.00 ± 0.43) no significant difference (P > 0.05).And after the vitamin A acid of 10 μMs acts on model cell 24h, the mrna expression amount of CYP26A1 is 25.49 ± 1.37, is 25 times (p < 0.01) of not dosing control group; In cell, the mrna expression amount of TIG1 is 3.88 ± 0.11, is nearly 4 times (p < 0.01) of not dosing control group (1.00 ± 0.43).

Experiment proves, the retinoic acid receptor α RAR that vitamin A acid is reacted by exciting induced toxicity and then activate its crucial target gene; And the retinoic acid receptor α RAR of the not exciting induced toxicity reaction of isotretinoin amido benzoic acid alkyl derivative of the present invention, activation is not had to the crucial target gene of retinoic acid receptor α RAR yet.

Embodiment 11: isotretinoin amido benzoic acid alkyl derivative is tested the untoward reaction of mouse skin

The significant side reaction of isotretinoin medicine mucocutaneously occurs the irritant reaction such as erythema, furfur, also there will be the toxic reaction such as teratogenecity, bone toxicity, these toxic reactions are mediated by exciting retinoic acid receptor α RAR α, β or the γ of direct or indirect approach by isotretinoin.This experiment, for hallmark cutaneous irritant reaction, observes isotretinoin amido benzoic acid alkyl derivative to the irritant reaction of mouse skin, and with etc. the isotretinoin of volumetric molar concentration contrast.

One, method:

The BALB/c mouse in 6 week age (18 ~ 20g) is divided into groups, often organizes male and female half and half, sub-cage rearing.Mark one piece of 1.5cm × 1cm size area with skin mark pen at every nearly head of mouse back and caudad, two is interregional every about 1.5cm.Isotretinoin is made 0.05% gel, formula II of the present invention and III compound make 0.075% gel respectively, structural formula IV, V, VI compound makes 0.08% gel respectively, take the above-mentioned preparation of about 0.030g and gel matrix (1% carbomer gel containing 15% ethanol and 1% dimethyl sulfoxide (DMSO)) respectively, be applied to mark zone (the nearly head zone painting medicine of above-mentioned mouse respectively, caudad region is coated with matrix), medication every day 1 time, continuous use 21d.Observe mouse skin change and record local skin reaction.Skin reaction grading system: 0 is divided into medication local without visible reaction; 0.5 is divided into erythema of maying be seen indistinctly, furfur; 1 is divided into slight erythema, furfur; 2 are divided into moderate erythema, furfur; 3 are divided into severe erythema, furfur.The different administration time of local skin on average stimulates the local excitation score value sum/observation number of animals on the score value=all observation animal same day.

Two, result:

Namely there is the skin wound repair such as erythema, furfur in 2 days in external application 0.05% isotretinoin gel, medication reaches peak in 5 ~ 6 days, disappears gradually subsequently, is continued until that medication 16 talent is disappeared completely.And each isotretinoin amido benzoic acid alkyl derivative gel 21 days of volumetric molar concentration such as to use continuously, all there is not skin wound repair (table 4).Compare without considerable change before all external-use substrate positions and medication.The results are shown in Table 1.Prove the security of isotretinoin amido benzoic acid alkyl derivative much larger than etc. the isotretinoin of volumetric molar concentration.

Table 1. is group skin reaction appraisal result respectively

Embodiment 12: isotretinoin amido benzoic acid alkyl ester coating tablet

Core formulation:

Label preparation technology:

P-(isotretinoin amide group) ethyl benzoate, Magnesium Stearate are crossed 120 mesh sieves respectively, for subsequent use; The p-(isotretinoin amide group) ethyl benzoate, starch, dextrin and the tartrate that take prescription proportional quantity mix, ethanolic soln with 50% is as wetting agent softwood, 30 mesh sieves are granulated, 50 DEG C ~ 60 DEG C dryings 2 ~ 4 hours, the whole grain of 30 mesh sieve, adds the Magnesium Stearate of recipe quantity, mixing, compressing tablet, to obtain final product.All operations process is carried out in dim light environment.Dressing preparation technology:

The ethanolic soln of preparation 80%, puts stirring on slurry shape agitator and makes into a vortex, slowly add in 80% ethanolic soln, stir 45min by the coating material Opadry of recipe quantity; Put by label in coating pan, adjustment rotating speed is to per minute 30 turns, and drum hot blast, makes temperature maintain about 50 DEG C, spray into coating liquid, after heavily increasing about 3%, stops heating, Free-rolling about 30 minutes to sheet, to label cooling.

Above-mentioned p-(isotretinoin amide group) ethyl benzoate can use in p-(isotretinoin amide group) methyl benzoate, p-(isotretinoin amide group) propyl benzoate, p-(isotretinoin amide group) isopropyl benzoate, p-(isotretinoin amide group) n-butylbenzoate, m-(isotretinoin amide group) ethyl benzoate, o-(isotretinoin amide group) methyl benzoate any one substitute.

Embodiment 13: isotretinoin amido benzoic acid alkyl ester ordinary tablet

Prescription:

P-(isotretinoin amide group) ethyl benzoate, Magnesium Stearate are crossed 120 mesh sieves respectively, for subsequent use; The p-(isotretinoin amide group) ethyl benzoate, starch, dextrin and the tartrate that take prescription proportional quantity mix, ethanolic soln with 50% is as wetting agent softwood, 30 mesh sieves are granulated, 50 DEG C ~ 60 DEG C dryings 2 ~ 4 hours, the whole grain of 30 mesh sieve, adds the Magnesium Stearate of recipe quantity, mixing, compressing tablet, to obtain final product.All operations process is carried out in dim light environment.

Embodiment 14: isotretinoin amido benzoic acid alkyl ester capsule

Prescription:

Preparation technology:

P-(isotretinoin amide group) ethyl benzoate, Magnesium Stearate are crossed 120 mesh sieves respectively, for subsequent use; The p-(isotretinoin amide group) ethyl benzoate, starch, dextrin and the tartrate that take prescription proportional quantity mix, be that wetting agent is by above-mentioned mixing fine powders softwood with 50% ethanol, cross 30 mesh sieves to granulate, 50 DEG C ~ 60 DEG C dryings 2 ~ 4 hours, the whole grain of 30 mesh sieve, add the Magnesium Stearate of recipe quantity, mixing, filling capsule.All operations process is carried out in dim light environment.

Embodiment 15: isotretinoin amido benzoic acid alkyl ester granule

Prescription:

Preparation technology:

P-(isotretinoin amide group) ethyl benzoate, Microcrystalline Cellulose, cane sugar powder, sodium starch glycolate, lactose, aspartame are crossed 100 mesh sieves respectively, and orange essence, sodium lauryl sulphate cross 80 mesh sieves respectively, for subsequent use; Take p-(isotretinoin amide group) ethyl benzoate of prescription proportional quantity, Microcrystalline Cellulose, cane sugar powder, sodium starch glycolate, lactose, aspartame mix by equal increments method, with 30% ethanol softwood of 3% polyvidone, 20 mesh sieves are granulated, 50 DEG C ~ 60 DEG C dryings 3 ~ 4 hours, the whole grain of 18 mesh sieve, then add orange essence, the sodium lauryl sulphate of recipe quantity, mixing, pack is sealed.All operations process is carried out in dim light environment.

Embodiment 16: isotretinoin amido benzoic acid alkyl ester soft capsule

Liquid prescription:

Preparation technology:

Liquid configures: at room temperature p-(isotretinoin amide group) ethyl benzoate and auxiliary material are configured to liquid;

The configuration of soft capsule shell material: by gelatin, glycerine, water mixing, dissolve, make soft capsule shell feed liquid;

Prepare soft capsule: use dropping method, every content weight is 500mg, the amount comprising p-(isotretinoin amide group) ethyl benzoate in controlling every is 25mg, by soft capsule shell feed liquid, liquid is sealed spherical soft capsule, then at 35 ~ 37 DEG C, air seasoning, to remove the moisture in soft capsule shell, to obtain final product.All operations process is carried out in dim light environment.

Note: above liquid formula and soft capsule shell formula are preparations separately, do not have to be related between the two consumption, generally according to the thickness producing concrete situation control soft capsule shell.

Embodiment 17

The compound containing formula I structure prepared according to embodiment 12, embodiment 13, embodiment 14, embodiment 15, embodiment 16 is as the coated tablet of activeconstituents, or conventional tablet, or capsule, or soft capsule, or granule etc. may be used for the control of cutaneous squamous cell carcinoma, cancer of the stomach, lung cancer, cervical cancer, also for the control of other epithelial cell tumours, also for psoriatic treatment, also for the treatment of acne, also for the treatment of other abnormal cutaneous keratinization.Quantity is 0.1mg ~ 5mg/kg every day, and can divide and take for 1 time ~ 3 times, concrete dosage can adjust the assessment of the state of an illness according to clinician.

Embodiment 18: isotretinoin amido benzoic acid alkyl ester cream formulation

Prescription

Preparation technology: getting p-(isotretinoin amide group) ethyl benzoate, to add dimethyl sulfoxide (DMSO) hydrotropy for subsequent use.

Oil phase: get white vaseline, stearyl alcohol, Liquid Paraffin, glyceryl monostearate, PC (to phenyl methylcarbamate between chlorine) puts heat fused in water-bath, maintains the temperature at 70 ~ 80 DEG C.

Aqueous phase: separately get Betacylcodextrin, sodium lauryl sulphate, glycerine, ethyl p-hydroxybenzoate, distilled water heating maintain the temperature at 70 ~ 80 DEG C.

Emulsification: slowly joined in aqueous phase by oil phase, limit edged stirs, and makes emulsification, adds above-mentioned p-for subsequent use (isotretinoin amide group) ethyl benzoate solution and vitamin-E respectively, continue to be stirred to condensation, to obtain final product below 60 DEG C.All operations process is carried out in dim light environment.

The compound containing formula I structure prepared according to above-mentioned each prescription may be used for psoriatic treatment as 0.01% emulsifiable paste of activeconstituents or 0.075% emulsifiable paste, 0.125 emulsifiable paste or 5% cream formulation, also for the treatment of acne, also for the treatment of other abnormal cutaneous keratinization.Clinical usage is outer for skin damage place, every day 1 time ~ 2 times.

Embodiment 19: isotretinoin amido benzoic acid alkyl ester gel preparation

Prescription:

Preparation technology:

Get after p-(isotretinoin amide group) ethyl benzoate adds dimethyl sulfoxide (DMSO) hydrotropy, then it is for subsequent use to add propylene glycol.Carbomer is swelling in distilled water, spill in liquid level (about 600g), gel substrate is become after overnight, leave standstill final vacuum deaeration, above-mentioned p-for subsequent use (isotretinoin amide group) ethyl benzoate solution and rest materials are mixed, join gradually in slurry, mixing, to obtain final product.All operations process is carried out in dim light environment.

The compound containing formula I structure prepared according to above-mentioned each prescription may be used for psoriatic treatment, also for the treatment of acne, also for the treatment of other abnormal cutaneous keratinization as each gel preparation of activeconstituents.Clinical usage is outer for skin damage place, every day 1 time ~ 2 times.

Embodiment 20: isotretinoin amido benzoic acid alkyl ester solution preparation

Prescription:

Preparation technology:

Get p-(isotretinoin amide group) ethyl benzoate and add dimethyl sulfoxide (DMSO), ethanol, N, after dinethylformamide, hydrotropy, add again 2,6 ditertiary butyl p cresol, butylated hydroxy anisole, propylene glycol, ethylparoben miscible after, adding distil water is to 1000g.

Above-mentioned solution directly can be applied in skin damage place, also can be placed in the bottle of band shower nozzle, as spray in skin damage place.

The compound containing formula I structure prepared according to above-mentioned each prescription may be used for psoriatic treatment, also for the treatment of acne, also for the treatment of other abnormal cutaneous keratinization as each solution of activeconstituents or sprays or suspensoid etc.Clinical usage is outer for skin damage place, every day 1 time ~ 2 times.

Embodiment 21: treat psoriatic isotretinoin amido benzoic acid alkyl ester compound external-use preparation

(1) compound cream preparation

Prescription

Oil phase: get Liquid Paraffin, white vaseline, hexadecanol, stearyl alcohol, glyceryl monostearate, Arlacel-60, puts heat fused in water-bath, maintains the temperature at 70 ~ 80 DEG C.

Aqueous phase: separately get peregal O-20, propylene glycol, phenylcarbinol, distilled water heating maintain the temperature at 70 ~ 80 DEG C.

Emulsification: slowly joined in aqueous phase by oil phase, limit edged stirs, and makes emulsification, adds above-mentioned p-for subsequent use (isotretinoin amide group) ethyl benzoate solution and Sch-11460, continue to be stirred to condensation, to obtain final product below 60 DEG C.All operations process is carried out in dim light environment.

Sch-11460 0.5g ~ 5g in above-mentioned prescription, Triamcinolone Acetonide 0.1g ~ 0.5g can be used, or clobetasol propionate 0.1g ~ 0.5g, or diflorasone 0.5g ~ 2.5g, or Valisone 1g ~ 5g, or beclometasone 0.1g ~ 0.5g, or clobetasone butyrate 0.5g3g, or furoic acid momisone 0.5g ~ 3g, or fluocinonide 0.1g ~ 1g, or fluticasone propionate 0.1g ~ 1.5g substitutes.Can be made into all size compound cream preparation.

(2) compound gel preparation

Prescription:

Preparation technology:

Get after p-(isotretinoin amide group) ethyl benzoate adds dimethyl sulfoxide (DMSO) hydrotropy, then it is for subsequent use to add hexylene glycol.Carbomer is swelling in distilled water, spill in liquid level (about 600g), gel substrate is become after overnight, leave standstill final vacuum deaeration, above-mentioned p-for subsequent use (isotretinoin amide group) ethyl benzoate solution and rest materials are mixed, join gradually in slurry, mixing, to obtain final product.All operations process is carried out in dim light environment.

Triamcinolone Acetonide 0.1g ~ 0.5g in above-mentioned prescription, Sch-11460 0.5g ~ 5g can be used, or clobetasol propionate 0.1g ~ 0.5g, or diflorasone 0.5g ~ 2.5g, or Valisone 1g ~ 5g, or beclometasone 0.1g ~ 0.5g, or clobetasone butyrate 0.5g3g, or furoic acid momisone 0.5g ~ 3g, or fluocinonide 0.1g ~ 1g, or fluticasone propionate 0.1g ~ 1.5g substitutes.

The compound containing formula I structure prepared according to above-mentioned preparation method as the compound external-use ointment of activeconstituents and anti-inflammatory agent composition or gelifying agent or solution or, sprays or suspensoid etc. may be used for psoriatic treatment.Clinical usage is outer for skin damage place, every day 1 time ~ 2 times.

Embodiment 22: the isotretinoin amido benzoic acid alkyl ester compound external-use preparation for the treatment of infectious acne

(1) compound cream preparation

Prescription

Aqueous phase: separately get sodium lauryl sulphate, glycerine, ethyl p-hydroxybenzoate, distilled water heating maintain the temperature at 70 ~ 80 DEG C.

Emulsification: slowly joined in aqueous phase by oil phase, limit edged stirs, and makes emulsification, adds above-mentioned p-for subsequent use (isotretinoin amide group) ethyl benzoate solution and Clindamycin Phosphate, continue to be stirred to condensation, to obtain final product below 60 DEG C.All operations process is carried out in dim light environment.

Clindamycin Phosphate consumption in above-mentioned prescription can be 5g ~ 15g; Clindamycin Phosphate 10g in above-mentioned prescription can use Dalacina 5g ~ 15g, preferred 10g, or erythromycin 10g ~ 40g, preferred 20g, or paraxin 2.5g ~ 10g, preferred 5g, or tsiklomitsin 10g ~ 30g, preferred 20g, or metronidazole 10g ~ 50g, preferred 20g, or tinidazole 5g ~ 30g, preferred 15g, or secnidazole 10g ~ 50g, preferred 20g, or Ofloxacine USP 23 2.5g ~ 20g, preferred 5g, or Ciprofloxacin 2.5g ~ 20g, preferred 5g substitutes.

(2) compound gel preparation

Prescription:

Preparation technology:

Erythromycin consumption in above-mentioned prescription can be 10g ~ 40g; Erythromycin 20g in above-mentioned prescription can use Clindamycin Phosphate 5g ~ 15g, preferred 10g, Dalacina 5g ~ 15g, preferred 10g, or paraxin 2.5g ~ 10g, preferred 5g, or tsiklomitsin 10g ~ 30g, preferred 20g, or metronidazole 10g ~ 50g, preferred 20g, or tinidazole 5g ~ 30g, preferred 15g, or secnidazole 10g ~ 50g, preferred 20g, or Ofloxacine USP 23 2.5g ~ 20g, preferred 5g, or Ciprofloxacin 2.5g ~ 20g, preferred 5g substitutes.

For the acne with infection or inflammation, the auxiliary conventional antibiotic adding anti-acne and infect on the basis with stronger epitheliosis suppression and differentiation regulating, can increase the curative effect to infectious acne.So the compound containing formula I structure prepared according to above-mentioned preparation method as the compound external-use ointment of activeconstituents and antibiotic composition or gelifying agent or solution or, sprays or suspensoid etc. can be used for the treatment of infectious acne.Clinical usage is outer for skin damage place, every day 1 time ~ 3 times.

Above embodiment, only for common medicament specification, concrete according to the needs of preparation specification and technique, can to adjust each component content, makes the medicament of various required specification when implementing.

Claims

1. isotretinoin amido benzoic acid alkyl derivative, is characterized in that the structure with formula I:

Wherein R is C _1-6alkyl.

2. isotretinoin amido benzoic acid alkyl derivative as claimed in claim 1, is characterized in that R is the one in methyl, ethyl, n-propyl, sec.-propyl, normal-butyl.

3. isotretinoin amido benzoic acid alkyl derivative as claimed in claim 1 or 2, is characterized in that the carbalkoxy in formula I on phenyl ring is in the contraposition of amido, ortho position or a position.

4. isotretinoin amido benzoic acid alkyl derivative as claimed in claim 3, it is characterized in that described isotretinoin amido benzoic acid alkyl derivative is the one in the compound of formula II, formula III, formula IV, formula V, formula VI, formula VII, formula VIII structure, specific as follows

Formula II:p-(isotretinoin amide group) methyl benzoate

Formula III: p-(isotretinoin amide group) ethyl benzoate

Formula IV:p-(isotretinoin amide group) propyl benzoate

Formula V:p-(isotretinoin amide group) isopropyl benzoate

Formula VI:p-(isotretinoin amide group) n-butylbenzoate

Formula VII:m-(isotretinoin amide group) ethyl benzoate

Formula VIII:o-(isotretinoin amide group) methyl benzoate.

5. the preparation method of the isotretinoin amido benzoic acid alkyl derivative described in the arbitrary claim of claim 1-4, is characterized in that, is formed by connecting, comprises the following steps by isotretinoin and aminobenzoic acid alkyl ester by amido linkage:

(1) under nitrogen protection and lucifuge condition, in reaction vessel, DMAP and tosic acid is added;

(2) in reaction vessel, reaction solvent is added;

(3) in reaction vessel, isotretinoin is added;

(4) in reaction vessel, add aminobenzoic acid alkyl ester, stir;

(5) in reaction vessel, add N, N'-dicyclohexylcarbodiimide, stir lower reaction;

(6) above-mentioned reaction mixture is placed after stopping by reaction; Take out and filter, filtrate revolving steams to obtain crude product; Use column chromatography the isotretinoin amido benzoic acid alkyl derivative namely obtained shown in formula I.

6. method as claimed in claim 5, described aminobenzoic acid alkyl ester is p-aminobenzoic acid alkyl ester, o-aminobenzoic acid alkyl ester or m-aminobenzoic acid alkyl ester, and described alkyl is the one in methyl, ethyl, n-propyl, sec.-propyl, normal-butyl.

7. method as claimed in claim 5, wherein the mol ratio of each raw material is:

Isotretinoin: aminobenzoic acid alkyl ester: DCC:DMAP: tosic acid=1:(1-1.5): (1.1-2): (0.1-0.15): (0.1-0.15).

8. method as claimed in claim 5, wherein reaction solvent is CH ₂cl ₂, tetrahydrofuran (THF) or CHCl ₃.

9. method as claimed in claim 5, wherein column chromatography selects silica gel to fill post, and elutriant is ethyl acetate: the volume ratio of sherwood oil is the mixed solution of 1: 10-1: 35.

10. the method according to any one of claim 5-9, wherein step 5 temperature of reaction is 10-66 DEG C, time 3-10 hour.

11. methods as claimed in claim 10, wherein, the mol ratio of each raw material is:

Isotretinoin: aminobenzoic acid alkyl ester: DCC:DMAP: tosic acid=1:(1-1.3): (1.1-1.5): (0.1-0.13): (0.1-0.13); , column chromatography selects silica gel to fill post, and elutriant is ethyl acetate: the volume ratio of sherwood oil is 1: 15 ~ 1: 25; Step 5 temperature of reaction is 10-40 DEG C, time 4-8 hour.

The application of isotretinoin amido benzoic acid alkyl derivative in the medicine for the preparation of prevention and therapy disease shown in 12. formula I, described disease is selected from acne, psoriatic or epithelial cell tumour;

Wherein R is C _1-6alkyl; Carbalkoxy in formula I on phenyl ring is in the contraposition of amido, ortho position or a position.

13. application according to claim 12, wherein R is methyl, ethyl, n-propyl, sec.-propyl, normal-butyl.

Application described in 14. claims 12 or 13, epithelial cell tumour is wherein selected from cutaneous squamous cell carcinoma, cancer of the stomach, lung cancer or cervical cancer.

15. for the pharmaceutical composition of prevention and therapy acne, psoriatic or epithelial cell tumour, wherein containing the isotretinoin amido benzoic acid alkyl derivative shown in activeconstituents formula I;

Wherein R is C _1-6alkyl.

16. pharmaceutical compositions as claimed in claim 15, wherein isotretinoin amido benzoic acid alkyl derivative is as the first promoting agent, and described pharmaceutical composition is also containing the second promoting agent, and the second promoting agent is a kind of antimicrobial drug or antiphlogiston.

17. pharmaceutical compositions as claimed in claim 15, wherein R is methyl, ethyl, n-propyl, sec.-propyl, normal-butyl; Carbalkoxy in formula I on phenyl ring is in the contraposition of amido, ortho position or a position.

18. pharmaceutical compositions as claimed in claim 16, the second promoting agent antimicrobial drug of described pharmaceutical composition is selected from the one in Dalacina, Clindamycin Phosphate, erythromycin, paraxin, tsiklomitsin, metronidazole, tinidazole, secnidazole, Ofloxacine USP 23, Ciprofloxacin.

19. pharmaceutical compositions as claimed in claim 16, the second promoting agent antiphlogiston of described pharmaceutical composition is selected from the one in clobetasol propionate, diflorasone, Valisone, beclometasone, clobetasone butyrate, Sch-11460, furoic acid momisone, fluocinonide or fluticasone propionate.

20. pharmaceutical compositions as described in claim 15 or 16, wherein said composition is with coated tablet, conventional tablet, capsule, soft capsule, granule oral dosage form; The dosage form of ointment, gelifying agent, creme, sprays, suspensoid, solution exterior-applied formulation exists.

21. pharmaceutical compositions as claimed in claim 20, in oral dosage form activeconstituents formula I in the composition shared weight percentage be 2% ~ 60%, all the other are auxiliary material.

22. pharmaceutical compositions as claimed in claim 20, in oral dosage form, the content of activeconstituents formula I is every preparation unit 5mg ~ 300mg.

23. pharmaceutical compositions as claimed in claim 20, in exterior-applied formulation activeconstituents formula I in the composition shared weight percentage be 0.01% ~ 5%, all the other are auxiliary material.

24. pharmaceutical compositions as claimed in claim 15, the percentage composition 0.25% ~ 5% (wt) of the second activeconstituents antimicrobial drug in exterior-applied formulation, all the other are auxiliary material, are used for the treatment of the externally used compound pharmaceutical composition of infectious acne.

25. pharmaceutical compositions as claimed in claim 19, the percentage composition 0.01% ~ 0.5% (wt) of the second activeconstituents anti-inflammatory agent in exterior-applied formulation, all the other are auxiliary material, are used for the treatment of psoriatic externally used compound pharmaceutical composition.

26. pharmaceutical compositions as claimed in claim 23, in exterior-applied formulation activeconstituents formula I in the composition shared weight percentage be 0.025% ~ 0.25%.

27. pharmaceutical compositions as claimed in claim 24, the percentage composition 0.5% ~ 3% (wt) of the second activeconstituents antimicrobial drug in exterior-applied formulation.

28. pharmaceutical compositions as claimed in claim 25, the percentage composition 0.025% ~ 0.15% (wt) of the second activeconstituents anti-inflammatory agent in exterior-applied formulation.

29. pharmaceutical compositions as claimed in claim 20, in oral dosage form activeconstituents formula I in the composition shared weight percentage be 30% ~ 50%, all the other are auxiliary material.

30. pharmaceutical compositions as claimed in claim 20, in oral dosage form, the content of activeconstituents formula I is every preparation unit 25mg ~ 100mg.

31. pharmaceutical compositions as claimed in claim 20, in exterior-applied formulation activeconstituents formula I in the composition shared weight percentage be 0.025% ~ 0.25%, all the other are auxiliary material.

32. pharmaceutical compositions as claimed in claim 20, in exterior-applied formulation, the percentage composition of the second activeconstituents antimicrobial drug is 0.5% ~ 3% (wt), and all the other are auxiliary material, is used for the treatment of the externally used compound pharmaceutical composition of infectious acne.

33. pharmaceutical compositions as claimed in claim 20, in exterior-applied formulation, the percentage composition of the second activeconstituents anti-inflammatory agent is 0.025% ~ 0.15% (wt), and all the other are auxiliary material, is used for the treatment of psoriatic externally used compound pharmaceutical composition.

34. pharmaceutical compositions as claimed in claim 20, it is made into tablet or capsule, and auxiliary material is selected from starch, dextrin, tartrate and Magnesium Stearate; Wherein tablet is with dressing, and coating liquid is the ethanolic soln of Opadry; It is made into granule, and auxiliary material is selected from Microcrystalline Cellulose, cane sugar powder, sodium starch glycolate, lactose, aspartame, orange essence, 30% ethanol of sodium lauryl sulphate and 3% polyvidone; It is made into soft capsule, and liquid auxiliary material is soybean oil, and capsule shell auxiliary material is selected from gelatin, glycerine and distilled water; It is made into cream formulation, and auxiliary material is selected from vitamin-E, dimethyl sulfoxide (DMSO), beta-cyclodextrin, sodium lauryl sulphate, stearyl alcohol, white vaseline, Liquid Paraffin, ethyl p-hydroxybenzoate, glycerine, glyceryl monostearate, to chlorine m-cresol, hexadecanol, stearyl alcohol, glyceryl monostearate, paregal O-20, Arlacel-60, propylene glycol, phenylcarbinol and distilled water; It is made into gel preparation, and auxiliary material is selected from dimethyl sulfoxide (DMSO), propylene glycol, ethanol, carbomer, trolamine, ethylparoben, PEG400, hexylene glycol, Tween-80, phenylcarbinol and distilled water; It is made into pharmaceutical solutions, and auxiliary material is selected from dimethyl sulfoxide (DMSO), propylene glycol, ethanol, DMF, 2,6 ditertiary butyl p cresol, butylated hydroxy anisole, ethylparoben and distilled water.