CN1108297C - Viformyl cumarins compound, process for preparing same and pharmaceutical composition contg. same - Google Patents

Abstract

The present invention discloses a viformyl cumarins compound denoted in the formula I, and salt thereof which is adacceptable by materia medica. In the formula, R1 is H, alkyl, aralkyl, haloalkyl, etc.; R<2> is H, alkyl, haloalkyl, alkoxy, ester base, halogen, OH, phenyl or halogeno- alkyl, etc.; R<3> is alkyl of H, OH, CHC2OH, halogen and C1 to C18, halogeno- alkyl, ester base, alkoxy, OR, CH2OR, etc.; R<4> is H, halogen, alkyl, halogeno- alkyl, alkoxy, ester base, OH, etc.; R<5> is H, alkyl, halogeno- alkyl, alkoxy, halogen, ester base, etc.; R<6> is H, alkyl, halogeno- alkyl, alkoxy, halogen, ester base, OH, etc. The present invention also relates to a method for preparing the compound and a medicine composition containing the compound.

Description

Retinoylcoumarin compound, its preparation method and pharmaceutical composition containing the compound

Technical field

The invention relates to a retinoyl coumarin compound, a preparation method of the compound and a pharmaceutical composition containing the compound, especially retinoyl coumarin for treating diseases such as tumors or precancerous changes and skin diseases A class of compounds, a preparation method of the compound and a pharmaceutical composition containing the compound.

Background technique

Retinoids play an important role in the growth and differentiation of epithelial cells and in blocking or reversing precancerous lesions. Xu Shiping designed and synthesized a series of retinoic acid derivatives for improving the performance of retinoids (Acta Pharmacologica Sinica 16 (9): 678-685 (1981)), wherein retinoid (RI) and retinoic acid (RII) are in It has good anti-carcinogenic effects and low toxicity, and is suitable for cancer chemoprevention and treatment of precancerous lesions. Cai Haiying et al. found that these compounds had a significant inhibitory effect on the stomach cancer in mice induced by ethyl sarcosinate nitrosamine (Acta Pharmaceutica Sinica 16(9):648-652(1981)). Wang Ruizhen and others found that these retinoid compounds have significant activity in inhibiting esophageal cancer cells (Chinese Journal of Tumor 5(4): 243-248(1983)). Du Congzhi et al. compared the toxicity of retinoids such as retinoic acid (RI), retinoic acid (RII), retinoic acid, and Kangshanling and Baofuling, and found that RI and RII were less toxic, which indicated that they were suitable for Application in the treatment of precancerous lesions (Acta Pharmacologica Sinica 17: 333-337 (1982)). Song Zhengping found that RI and RII induce differentiation (Acta Pharmacologica Sinica 19: 576-581 (1984)). Han Rui et al. reported that RII has good effects in treating precancerous lesions, such as oral white spots, vulvar white lesions, cervical and gastric mucosal dysplasia, and preleukemia (In Vivio 4:153-160 (1990)). Lin Peizhong found that RI in the high-risk area of esophageal cancer in Lin County, blockade treatment of esophageal precancerous lesions in a large population, reduced the incidence of esophageal cancer by 43.2% in five years (Proc.CAMS & PUMC5(3): 121-129 (1990)). There are also a large number of reports on retinoid research abroad. Isotretinoin (Baofuling) and aromatic retinoic acid (Kangshanling) have been listed as dermatological drugs. Gander et al., US Patent No. 4,126,693, also treat precancerous lesions with the above agents. Most retinoid drugs have considerable toxic and side effects when patients take them. Those with little toxic and side effects often have too low activity. Therefore, it is necessary to continuously develop new retinoid compounds with high activity, good effect and low toxic and side effects, so as to provide better drugs for clinical practice. Smith et al., J.Clin.Oncol.10 (5): 839-864 (1992) reviewed the application of retinoid compounds in the treatment of cancer, especially retinoic acid (RA) on human promyelocytic leukemia (APL) therapeutic effect. Such as Blazsek et al., Biomed.Pharmacother.45: 169-177 (1991); Degos et al., Biomed.Pharmather 46: 210-209 (1992); Castaigne et al., Blood74 (9): 1704-1709 (1990) Chomienne et al., Blood76 (9): 1710-1717 (1990): Lo Coco et al., Blood77 (8): 1657-1659 (1991), all proved that retinoids can treat APL by differentiation therapy. Kizaki et al., Cancer Res. 40:3413-3425 (1980), report the role of retinoic acid esters, amines and amides in cancer chemoprevention. Shealy et al., U.S. Patent No. 5,124,083, demonstrating differentiation-inducing and cancer chemopreventive effects of retinoic acid derivatives, and published in J. Clin. Invest. 21:574-579 (1991) and Loev et al., U.S. Patent No. 4,532,042, Both demonstrate the differentiation-inducing effect of retinoids. Gander, US Patent No. 4,323,581, demonstrates the role of 4-HPR in the treatment of breast cancer precancerous lesions. Nair et al., Carcinogenesis 12(1):65-69 (1991), demonstrated the anticancer effect of coumarins. Ishizuka et al., US Patent No. 5,096,924, demonstrate that substituted coumarins have anticancer effects. Marshall, et al., J. Biol. Resp. Mod. 8:62 (1989), report that coumarin enhances immune function. For example, the monocytes of cancer patients are significantly increased, thus increasing the anti-cancer ability. Preuss-Ueberschar et al., Drug Res. 34:1305-1313 (1984), demonstrated that coumarin is not teratogenic. These documents are cited as references of the present invention.

Although some progress has been made in the research of retinoid compounds, some of them have been used in clinical practice, but they are still far from meeting the desired requirements. On the one hand, compounds with high activity have too many side effects; on the other hand, the activity of compounds with low toxicity is also reduced, and it is difficult to reach the level of practical application, especially retinoids. The abnormal effect brings a lot of inconvenience to women of childbearing age.

Contents of Invention

The object of the present invention is to provide a new retinoid compound and its pharmaceutically acceptable salt, which have considerable advantages over previous retinoid compounds, especially non-teratogenic, anti-tumor and differentiation-inducing effects.

Another object of the present invention is to provide a pharmaceutical composition containing the novel retinoid compound of the present invention and pharmaceutically acceptable salts thereof.

Another object of the present invention is to provide a pharmaceutical composition containing the retinoid compound of the present invention for mammals, especially for humans.

Another object of the present invention is to provide a method for synthesizing the retinoid compound of the present invention and/or the composition containing the compound.

Description of drawings

Figure 1 shows the effect of the compound of Example 13 of the present invention on DMBA/croton oil-induced mouse skin papilloma.

Figure 2 shows the effect of the compound of Example 13 of the present invention on DMBA/croton oil-induced mouse skin papilloma.

Figure 3 shows the results of the inhibitory effect of the compound of Example 13 of the present invention on mouse epidermal ornithine decarboxylase (ODC) induced by croton oil.

Detailed description of the invention

The present invention provides a retinoylcoumarin compound with biological activity of anti-cancer and cancer chemoprevention, the structure of the compound is shown in the following formula I:

Wherein, R ¹ is H, C ₁ -C ₁₈ , preferably C ₁ -C ₄ alkyl, aralkyl and haloalkyl, CXYR ⁷ , where X is H, N, NH, C, CH, O, Y is H, N, NH, C, CH, O, Y is O or N, R ⁷ is H, halogen, OH, C ₁ -C ₁₈ , preferably C ₁ -C ₄ alkyl or alkoxy, haloalkyl , unsubstituted, mono-substituted or multi-substituted phenyl, wherein the substituents on the benzene ring can be C ₁ -C ₄ alkyl, haloalkyl, alkoxy, OH, halogen, CO ₂ H, ester group, NO ₂ , CF ₃ , SO ₃ H, NR ⁸ R ⁹ , wherein R ⁸ and R ⁹ are the same or different, and are independently selected from H, alkyl or cycloalkyl and heterocycle, etc.;

R ² is H, OH, C ₁ ~C ₁₈ alkyl, haloalkyl, alkoxy, preferably C ₁ ~C ₄ alkyl or alkoxy, unsubstituted or substituted aryl or aralkyl, on the benzene ring The substituents are the same as above, CXYR ⁷ or OR, R is retinoyl, X, Y and R ⁷ are as defined above;

R ³ is H, OH, CH ₃ , OR or CH ₂ OR, CXYR ⁷ , wherein X, Y, R and R ⁷ are as defined above;

R ⁴ is H, halogen, preferably Cl and Br, C ₁ -C ₁₈ alkyl (preferably C ₁ -C ₄ alkyl or alkoxy), OH, OR or CXYR ⁷ , wherein X, Y, R and R ⁷ as defined above;

R ⁵ is H, C ₁ -C ₁₈ alkyl or haloalkyl, alkoxy, OR, or CXYR ⁷ , wherein R, X, Y and R ⁷ are as defined above;

R ⁶ is H, C ₁ =C ₁₈ alkyl or haloalkyl, alkoxy, OR, or CXYR ⁷ , wherein R, X, Y or R ⁷ are as defined above.

Among the compounds of the present invention, R ¹ is preferably H or COR ⁷ , wherein R ⁷ is CH ₃ , OC ₂ H ₅ , OH or NHR ¹⁰ , wherein R ¹⁰ is H or substituted phenyl, etc.

In the compounds of the invention, R ² is preferably H, OR, Cl, CH ₃ , Ph, C═NR ¹⁰ , CH═CHR ¹⁰ wherein R and R ₁₀ are as defined above.

In the compounds of the present invention, R ³ is preferably H, OR, CO ₂ H, CO ₂ Et, COC ₁₂ H ₂₅ , CXYR ⁷ or CH ₂ OR, wherein X, Y, R and R ⁷ are as defined above.

In the compounds of the present invention, R ⁴ is preferably H, C ₂ H ₅ , CHO, COCH ₃ , C ₄ H ₉ , PhCO, CO ₂ H, Cl, Br, CH═CHR ₁₀ , wherein R ¹⁰ is as defined above.

In the compounds of the present invention, R ⁵ is preferably H, CH ₃ , OR, CHO, CH═NR ¹⁰ , CH═CHR ¹⁰ , wherein R and R ¹⁰ are as defined above.

In the compounds of the present invention, R ⁶ is preferably H, CH ₃ , OR, COCH ₃ , CO ₂ H, BR, CHO, CH═NR ¹⁰ , CH═CHR ¹⁰ , wherein R and R ¹⁰ are as defined above.

Examples of some preferred combinations of R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ according to the present invention are as follows: R ¹ =COCH ₃ , R ² =R ⁴ =R ⁶ =H, R ³ =R ⁵ = OR, R = retinoyl; R ¹ = COCH ₃ , R ² = R ⁴ = R ⁶ = H, R ³ = CO ₂ H, R ⁵ = OR, R = retinoyl; R ¹ = COCH ₃ , R ² =R ³ =R ⁴ =H, R ⁵ =R ⁶ =OR, R = tretinoyl; R ¹ =COCH ₃ , R ² =R ⁵ =R ⁶ =H, R ³ =OR, R ⁴ = CO ₂ H, R = retinoyl; R ¹ = COCH ₃ , R ² = R ³ = R ⁴ = H, R ⁵ = OR, R ⁶ = CO ₂ H, R = retinoyl; R ¹ = COCH ₂ , R ² =R ⁴ =R ⁶ =H, R ³ =CO ₂ H, R ⁵ =OR, R = tretinoyl; R ¹ =COCH ₃ , R ² =R ³ =R ⁶ =H, R ⁴ =Cl, ^R5 =OR, R=retinoyl.

According to the present invention, the chemical formula of retinoyl is as follows:

The present invention also relates to the preparation method of above-mentioned compound, this method comprises:

(1) A coumarin compound (III) substituted with a hydroxyl group by reacting a phenolic compound represented by the following formula (II) with a corresponding compound

Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined above, but at least one of them is OH or both substituents are OH;

其中，R¹、R²、R³、R⁴、R⁵和R⁶如上所定义，R为维甲酰基。(2) The obtained compound (III) is reacted with retinoic acid to obtain the compound (I) of the present invention:

Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined above, and R is retinoyl.

According to the method of the present invention, the substituted hydroxyl-containing coumarin compound (III) can be prepared by methods known to those skilled in the art.

The compound of the present invention is a kind of new retinoid compound with anti-cancer and cancer chemopreventive activity, and can be used for in vivo and/or in vitro treatment of cancer, precancerous lesions and various diseases of dermatology.

The present invention further relates to a pharmaceutical composition containing a therapeutically effective dose of the retinoid coumarin compounds of the present invention and pharmaceutically acceptable salts thereof.

The administration route of the compound of the present invention may be oral administration or parenteral administration or a combination of both. The dosage forms for non-oral administration can be injections, various ointments, film preparations, suppositories, dosage forms for embedding, liniments, and other dosage forms for non-oral administration. The dosage forms for oral administration can be, for example, tablets, granules, capsules, syrups, suspensions, and granules. The dosage form used can be selected according to the patient's symptoms, age, sex, therapeutic purpose, physical condition and the like.

When the compound of the present invention is used as the active ingredient to prepare the pharmaceutical composition of the above dosage form, various adjuvants known in the art, such as excipients, binders, lubricants, disintegrants, etc., can be added.

Excipients can be commonly used excipients in the pharmaceutical field, such as crystalline cellulose, starch, sucrose, lactose, glucose, mannitol, etc.; binders can be hydroxypropyl cellulose, polyvinylpyrrolidone, etc.; The agent can be, for example, magnesium stearate, talc, etc.; the disintegrant, for example, can be hydroxymethylcellulose calcium, etc.

The preparation of the pharmaceutical composition of the present invention can be prepared by conventional methods well known in the art.

The dosage of the compound of the present invention is preferably 0.001-100 mg/kg body weight, more preferably 0.1-50 mg/kg body weight, particularly preferably 1-10 mg/kg body weight.

Example

The present invention will be further described below by way of examples. However, the present invention is not limited to these examples.

Example 1 Synthesis of 3-acetyl-5 carboxyl-7-retinoyloxycoumarin

Add 0.6g (0.002 moles) of retinoic acid, 20ml of benzene and 0.12ml (0.0013 moles) of PCl ₃ into a 100ml round bottom bottle equipped with an electromagnetic stirring bar, a reflux condenser and a drying tube, and stir at room temperature for 1 hour under the protection of N ₂ . The reaction mixture was concentrated and added to absolute anhydrous diethyl ether treated with sodium metal for later use.

Install a condenser tube and an addition funnel in a 100ml three-neck flask and make it a dry system. Then add 0.5g (0.002 moles) of 3-acetyl-5-carboxy-7-hydroxycoumarin, 0.5ml of anhydrous pyridine and 20ml of absolute anhydrous ether, and add dropwise the anhydrous ether solution obtained in the above steps while stirring. After stirring for two hours, the resulting solid was collected by filtration, dried and purified with ethanol to give 0.5 g of the title compound.

The melting point is 158-161°C.

¹ H-NMR (CDCl ₃ ); δ1.03 (s, 6H, 1'-2CH ₃ ); 1.38-2.14 (m, 6H, alicyclic CH ₂ ); 1.71 (s, 3H, 5'-CH ₃ ) ; 2.02(s, 3H, 9'-CH ₃ ); 2.42(s, 3H, 13'-CH ₃ ); 2.70(s, 3H, 3-COCH ₃ ); 4.76(b, 1H, 5-CO ₂ H ); 5.93-6.41 and 6.97-7.26 (m, 6H, retinoid double-bonded hydrogen); 7.50 (d, 1H, 8-H); 7.85 (d, 1H, 6-H); 9.51 (s, 1H, 4 -H).

MS: (C ₃₂ H ₃₄ O ₇ =530 (M ⁺ ), 282 (retinoyl), 248 (coumarin), 233 (248-CH ₃ ).

Example 2 Synthesis of 3-acetyl-5-ethoxycarbonyl-7-retinoyloxycoumarin

Adopt the same preparation method, equipment and reaction conditions as Example 1 to carry out, the raw material adopts 0.56g (0.002 mole) 3-acetyl-5-ethoxycarbonyl 0.7g hydroxycoumarin and 0.6g (0.002 mole) retinoic acid, This gave 0.5 g of the title compound (purified by ethanol).

Melting point: 162-64°C.

¹ H-NMR (CDCl ₃ ); δ1.04 (s, 6H, 1'-2CH ₃ ); 1.44 (t, 3H, 5-ester-CH ₃ ); 1.14-2.12 (m, 6H, alicyclic CH ₂ ); 1.72(s, 3H, 5'-CH ₃ ); 2.03(s, 3H, 9'-CH ₃ ); 2.42(s, 3H, 13'-CH ₃ ); 2.70(s, 3H, 3-COCH ₃ ); 4.24(q, 2H, 5-ester group CH ₂ ); 5.88-6.48 and 6.98-7.25(m, 6H, retinoid double-bonded hydrogen); 7.36(d, 1H, 8-H); 7.73(d , 1H, 6-H); 9.47 (s, 1H, 4-H).

MS: (C ₃₄ H ₃₈ O ₇ =558 (M ⁺ , near base peak), 283, 276, 261, 233, 231, 175 (base peak, 231-28-28).

Example 3 Preparation of 4-retinoyloxy-6-tert-butylcoumarin

Using the same preparation method, equipment and reaction conditions as in Example 1, 0.6 g of the title compound was obtained using 0.6 g of 4-hydroxy-6-tert-butylcoumarin and 1.0 g of retinoic acid as raw materials.

Melting point: 142-144°C.

¹ H NMR (CDCl ₃ ): δ1.05 (s, 6H, 1'-2CH ₃ ); 1.37 (s, 9H, t-butyl); 1.48, 1.62 and 2.04 (m, 6H, alicyclic CH ₂ ) ; 1.73(s, 3H, 5'-CH ₃ ); 2.05(s, 3H, 9'-CH ₃ ); 2.46(s, 3H, 13'-CH ₃ ); 6.06(s, 1H, 3-H) ; 6.16-6.52 and 7.16-7.26 (m, 6H, retinoid double-bonded hydrogen); 7.30 (d, 1H, Ar-H); 7.61 (m, 2H, Ar-H).

MS: 500 (M ⁺ ), 282, 203, 175, 161.

Example 4 Synthesis of 4-retinoyloxy-7-methylcoumarin

Using the same preparation method, equipment and reaction conditions as in Example 1, 0.4 g of 4-hydroxy-7-methyl-coumarin and 1.0 g of retinoic acid were used as raw materials to obtain 0.5 g of the title compound.

Melting point: 115-117°C.

¹ H NMR (CDCl ₃ ): δ1.03 (s, 6H, 1'-2CH ₃ ); 1.32-2.14 (m, 6H, alicyclic CH ₂ ); 1.731 (s, 3H, 5'-CH ₃ ); 2.30 (s, 3H, 9'-CH ₃ ); 2.42 (s, 6H, 7, 13'-2CH ₃ ); 5.92 (s, 1H, 3-H); 5.96-6.50 and 6.92-7.26 (m, 6H , retinoid double bond hydrogen); 7.10 (d, 1H, Ar-H); 7.48 (d, 1H, Ar-H).

MS: 458 (M ⁺ ), 430, 355, 321, 307, 282, 267, 175, 159, 147.

Embodiment 5 4, the synthesis of 8-dimethyl-7-retinoyloxycoumarin

Adopt the same preparation method, equipment and reaction conditions as in Example 1 to carry out. 0.5 g of the title compound was prepared from 0.57 g of 4,8-dimethyl-7-hydroxy-coumarin and 1.0 g of retinoic acid.

Melting point: 178-80°C.

¹ H NMR (CDCl ₃ ); δ1.04 (s, 6H, 1'-2CH ₃ ); 1.16-2.28 (m, 6H, alicyclic CH ₂ ); 1.73 (s, 3H, 5'-CH ₃ ); 2.01 (s, 3H, 9'-CH ₃ ); 2.28 (s, 3H, 13'-CH ₃ ); 2.41 (s, 6H, 4, 8-2CH ₃ ); 5.99 (s, 1H, 3-H) ; 6.01-6.56 and 6.86-7.28 (m, 6H, retinoid double-bonded hydrogen); 706 (d, 1H, Ar-H); 7.40 (d, 1H, Ar-H).

MS: 472 (M ⁺ ), 283, 190, 175, 161.

Example 6-45

Using the same preparation method, equipment and reaction conditions as in Example 1, and selecting corresponding raw materials, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ were respectively obtained as the compounds of the present invention shown in the following table.

Table 1 Example R ¹ R ² R ³ R ⁴ R ⁵ R ⁶ Melting point (°C) 12345678910111213141516171819202122 COCH ₃ COCH ₃ COOC ₂ H ₅ HHCOCH ₃ COCH ₃ HCOCH ₃ COCH ₃ COCH ₃ COCH ₃ COCH ₃ COCH ₃ COCH ₃ COOC ₂ H ₅ HCOOC ₂ H ₅ COOC ₂ H ₅ COOC ₂ H ₅ COOC ₂ H ₅ COOC ₂ H ₅ HHHORCH ₃ HHPhHHHHHHHHPhHHHHH COOHCO ₂ C ₂ H ₅ HHHCH ₃ HHHHORHORHHHORHHCOOHHOR HHHHHHHHHClCOOHHHC ₂ H ₅ n-HexHHHClHHH ORORORCH ₃ ORORORORORORHOROROROROROROROROROROR HHCOCH ₃ HCH ₃ HCH ₃ HCOOHHHORHHHCH ₃ HHHHORH 158-61162-64141115178179-81174-76113-16174-76167-69185-87136-38156-58149-50112-14122-24112-14174-7612415078-80138-40

(Continued from Table 1) Example R ¹ R ² R ³ R ⁴ R ⁵ R ⁶ Melting point (°C) 2324252627282930313233343536373839404142434445 HCOOC ₂ H ₅ COOH CONH ₂ CONH ₂ HHHHHHHHHHHHHHCO ₂ HHCOCH ₃ HHH ORHHHHORCH ₃ CH ₃ CH ₃ CH ₃ ORCH ₃ CH ₃ CH ₃ CH ₃ ORORHORHCH ₃ CH ₃ Ph HORHCOOHHHHHORHHHHHHCH ₃ HCO ₂ HHCO ₂ EtHHH n-HexC ₆ H ₅ COn-HexHClt-ButC ₂ H ₅ HHn-HexCH ₃ HClHBrHHHHHHHH ORORORORORHORORORORORORORORCH ₃ ORORHORORNHROR HHHHHHHHHHHHORHHHHCH ₃ ORHORCO ₂ HHCH ₃ 100106159191198142120154134114104106126161-62160-6213315323588122-38145239154

The anticancer activity of the compounds and/or compositions of the invention is determined using known in vivo assays. These methods are known, common tests. The compounds of the present invention have specific anticancer activity.

The following are examples of screening methods for retinoylcoumarin compounds in formula (I) of the present invention, but are not limited thereto.

The compound biological activity test of the present invention includes (not limited to these):

1) Inducing the differentiation of human promyelocytic leukemia cells;

2) Inhibition of cancer cells (MTT test).

3) Inhibit the formation of skin papillomas in mice (including the combination of anti-carcinogenic and anti-cancer-promoting experiments);

4) anti-skeletal effect;

5) Inhibition of cyclophosphamide-induced micronucleus formation in bone marrow polychromatic erythrocytes;

6) Inhibitory effect on mouse ear swelling induced by croton oil;

7) inhibition of the increased activity of croton oil-induced mouse epidermal ornithine decarboxylase (ODC);

8) Inhibitory effect on rat chondrosarcoma;

9) Teratogenicity experiments.

The therapeutic or prophylactic effect (reduction of morbidity) on animals or humans does not have to be complete or eradicated, but can give a statistically significant improvement (P ≥ 0.05) compared with the control compound, and can also be a severe Reduced or acute symptoms. The administration route can be oral and/or parenteral administration, such as subcutaneous, intravenous, intradermal, intramuscular injection, peritoneal, intranasal, intracranial, transdermal, oral spray inhalation and other routes, and each administration route can be used alternately or Use with each other.

Experimental Example 1 The Differentiation Induction Effect of Retinoylcoumarin Compounds on Human Promyelocytic Leukemia HL-60 Cells

Human promyelocytes (HL-60) were cultured and passaged in RPMI-1640 medium containing 10% calf serum, penicillin 100U/ml and streptomycin 100μg/ml in an incubator at 37°C and 5% _CO2 . Inoculate the cells growing logarithmically at a concentration of 1.2×10 ⁵ -1.4×10 ⁵ /ml into culture flasks containing 5 ml of culture solution, 3 flasks in each group. After 24 hours, the cell growth concentration reached 2.5×10 ⁵ ml. The experimental group was added with different concentrations of drugs dissolved in ethanol, and the control group was added with equal volume of ethanol (the final concentration of ethanol was not more than 0.01%. At this concentration, ethanol had no effect on cell growth. and differentiation), and the whole process of the experimental operation was carried out under dark conditions. Collect each 250 μl of administration and control group cells every day, add the same amount of (calcium-magnesium-free) phosphate buffer solution containing 0.2% NBT, each ml contains TPA600mg, 37 ℃, 5% CO _The incubator is incubated for three hours, and a certain amount of A small amount of cold phosphate buffer was used to stop the reaction. At 4°C, take a small amount of cells and count them. There are blue-purple granules deposited in the cells, which are NBT-positive cells. Check at least 200 cells for each sample, count the positive rate, and calculate the ED ₅₀ (see Table 2)

Table 2 The compounds of the present invention induce the differentiation experiment results of HL-60 cells compound Concentration (M) NBT(+)% ED ₅₀ (M) Example 6 Example 1 Example 7 Example 9 Example 10 Example 11 Example 12 Example 13 10 ^-4 77 3×10 ^-6 10 ^-5 7210 ^-6 4910 ^-7 810 -5 92 7 ^× 10 ^-8 10 ^-6 8410 ^-7 7310 ^-8 1510 ^-5 88 8×10 ^-7 10 ^-6 7710 ^{- 7} 1210 ^-5 89 2.8×10 ^-7 10 ^-6 6910 ^-7 4110 ^-4 96 6×10 ^-7 10 ^-5 8110 ^-6 7810 ^-7 3110 ^-5 98 5.9×10 ^-8 10 ^-6 6910 ^-7 5510 ^-8 3410 ^-5 96 6.5×10 ^-8 10 ^-6 8810 ^-7 5510 ^-8 316.4×10 -6 99 1.3×10 ^-8 6.4×10 ^-7 966.4×10 ^-8 826.4×10 ^-9 336.4× ^{10 - 10} 10

(Continued from Table 2) compound Concentration (M) NBT(+)% ED ₅₀ (M) Example 2 Example 16 Example 18 Example 19 Example 20 Example 22 Example 26 Example 27 10 ^-4 77 3×10 ^-6 10 ^-5 7210 ^-6 4910 ^-7 810 ^-5 93 2.6×10 ^-7 10 ^-6 8410 ^-7 4610 ^-8 1210 ^-5 84 1×10 ^-6 10 ^-6 5410 ^{- 7} 210 ^-5 44 1×10 ^-5 10 ^-6 810 ^-4 92 9×10 ^-6 10 ^-5 8410 ^-6 510 ^-5 94 2.5×10 ^-7 10 ^-6 8010 ^-7 4110 ^-8 1710 ^-5 97 8.0×10 ^-8 10 ^-6 9110 ^-7 5310 ^-8 3410 ^-5 88 5.5×10 ^-7 10 ^-6 8010 ^-7 17

Experimental example 2 The cytotoxic effect (MTT experiment) of compound of the present invention to HL-60 cell

Cells in the logarithmic growth phase were seeded in 96-well plates at 10 ⁴ ml. The next day, discard the medium and replace it with a medium containing different concentrations of drugs and corresponding solvent controls. Set up 3-5 parallel wells for each group. After culturing at 37°C for 4-5 days, discard the supernatant and add 0.2ml of freshly prepared 0.2ml to each well. mg/ml MTT serum-free medium, continue to culture for 4 hours. Carefully discard the supernatant and add 200 μl DMSO to dissolve the MTT formazan precipitate, mix well with a micro-ultrasonic oscillator, measure the optical density value at 570 mm on the MR700 microplate reader, calculate the cell survival rate according to the following formula, and calculate the ED ₅₀ (See Table 3)

Table 3 MT experimental results of compounds of the present invention on HL-60 cells compound Concentration (M) Inhibition Rate% ED ₅₀ (M) Example 9 Example 11 Example 12 Example 15 Example 16 Example 22 Example 26 ^10-5 51.6±0.0 8.5× ^{10-6 10-6} 34.1±7.110-7 ^11.5 ±4.210-5 ^63.9 ±2.9 1× ^{10-7 10-6} 54.0± ^1.910-7 50.5± ^0.910-8 29.7± ^1.410-5 61.5±1.9 9.5× ^{10-7 10-6} 50.6± ^0.510-7 48.8± ^0.510-8 28.0±5.210-5 ^52.6 ±1.3 9.0× ^{10-6 10-6} 30.4±1.410-7 ^10.4 ± ^2.410-5 54.5±2.8 9×10 ^-7 10 ^-6 48.8±1.310 ^-7 34.6±0.010 ^-8 16.2”2.410 ^-5 61.2±2.4 1×10 ^-6 10 ^-6 49.9±2.510 ^-7 32.5±1.110 ^-8 21.8±3.110 ^-5 68.7 ±0.2 9×10 ^-7 10 ^-6 52.6±0.510 ^-7 43.8±1.310 ^-8 9.4±3.7

Experimental example 3 Inhibition of mouse skin papilloma experiment (combination of anti-carcinogenic and anti-cancer-promoting experiments)

Kunming mice are both male and female, with a body weight of 18-22g. Three days before the start of the experiment, the back hair was removed with a depilatory agent, and they were randomly divided into groups (see Table 4), with 20 mice in each group, half male and half male. The control group received DMBA acetone solution (150 μg/200 μl) on the first, seventh, and 14th day of the experiment, 200 μl each time, twice a week until the end of the experiment. The experiment lasted for 12 weeks. During this period, the number of papillomas and the number of tumor-bearing mice were recorded every weekend. The whole species was orally administered with different doses; the compound of Example 14 (10 mg/Kg, 20 mg/Kg) and the control drug RII (40 mg/Kg). The method of smearing DMBA and croton oil and the method of tumor observation and counting in the treatment group were the same as those in the control group.

Table 4 The treatment plan of the compound of Example 13 on the combination experiment of carcinogenesis and cancer promotion group Initial stage (4 weeks) Cancer preservation stage (8 weeks) 123456 DMBA Croton oil DMBA+Example 13 compound Croton oil+Example 13 compound (10mg/Kg/2 days) (10mg/Kg/2 days) DMBA+Example 13 compound Croton oil+Example 13 compound (20mg/Kg/2 days ) (20mg/Kg/2 days) DMBA+Example 13 compound Croton oil (20mg/Kg/2 days) DMBA Croton oil+Example 13 compound (20mg/Kg/2 days) DMBA+RII Croton oil+RII (40mg/Kg/2 days) Kg/2 days) (40mg/Kg/2 days)

Experimental results

Skin papillomas occurred in the control group from the fifth week, and the incidence of tumors and the average number of tumors in the mice were 6.75/mouse. Oral administration of Example 14 compound groups (10mg/Kg, 20mg/Kg) can prolong the incubation period of mouse skin papillomas, reduce the incidence of tumors, and significantly reduce the average number of tumors in mice, showing a certain dose-effect relationship . Its effect is better than similar drugs RII (see Figures 1 and 2).

Experimental example 4 mouse ear swelling test

The ICR mice were randomly divided into control groups and administration groups with different doses. The compound obtained in Example 14 was orally administered to mice in advance, once a day, for three consecutive days. Half an hour after the third administration, 25 μl of croton oil solution (containing 2% croton oil, 20% ethanol, 73% ether and 5% water) was applied to both sides of the mouse's right ear. Six hours later, the mice were killed by dislocation, and the ears were perforated with a keratome. The difference between the weight of the left and right ears was measured as the degree of swelling. The results are shown in Table 5.

Table 5 The effect of the compound of Example 13 on croton oil-induced mouse ear swelling (n=10) group Dose (mg/Kg) Mouse ear swelling (mg) Inhibition rate Comparative example 13 compound 16.5±1.450 14.2±2.8 13.9*100 12.3±3.0 31.5**

*p<0.05

**p<0.01

Example 5 Inhibitory effect on the increased activity of ornithine decarboxylase (ODC) in the back epidermis of mice induced by croton oil

Male ICR mice weighing 18-22 g were randomly divided into a blank control group, a positive control group and an administration group. The compound obtained in Example 13 was orally administered to mice in advance, at a dose of 50 mg/Kg body weight, once a day for 2 consecutive days. One hour after the second administration, 200 µl of 1% croton oil in acetone was applied to the back of the depilated mouse. After 5 hours, the animal was sacrificed, and the back skin was cut off, first immersed in ice-cold physiological saline, then immersed in 52°C hot water for 30 seconds, and finally immersed in ice-cold saline. Use a scalpel to scrape off the dermis, cut the epidermis into pieces, add an appropriate amount of extract, use a homogenizer to homogenize at high speed in an ice bath, centrifuge at 30,000×g for 30 minutes, and measure the protein content and enzyme activity of the supernatant Determination. The enzyme activity unit is expressed in nmol CO ₂ /30min/mg protein. The experimental results are shown in Figure 3.

Experimental Example 6 Inhibition of transplanted rat chondrosarcoma

Under sterile conditions, take the well-grown rat chondrosarcoma, cut it into pieces, dilute it 1:3, and inoculate it in the armpit of 60-80g Wistar rats with a 16-gauge needle, 0.4ml/only. On the next day, they were randomly divided into groups and administered orally, once every other day, for a total of 18 administrations. The rats were sacrificed on the 37th day after inoculation, and the tumors and body weight were weighed after the tumors were peeled off. The results were evaluated by tumor inhibition rate. The experimental results are shown in Table 6:

Table 6 Inhibition of Example 11 Compound and Retinoic Acid on Transplanted Rat Chondrosarcoma group Dose Body Weight Tumor Weight Inhibition Rate (%) Comparative example 11 compound retinoic acid 245.7 15.345 262.3 2.68** 82.510 262.3 0.39** 97.520 244.0 0.03** 99.820 222.0 0.00** 100.0

**p<0.01

Example 7 Inhibitory effect of CTX on the formation of micronuclei in polyphagocytic erythrocytes in mouse bone marrow

50 male ICR mice, 18-22 g, were randomly divided into 4 groups; the control group without any treatment; the cyclophosphamide mutagenesis group and two administration groups. The administration group administered different doses of the compound obtained in Example 13 to the mice in advance, once a day, for three consecutive days. Two hours after the third administration, cyclophosphamide 100 mg/Kg was injected intraperitoneally except for the control group. At 24 hours, 48 hours and 71 hours after the injection of CTX, 5 mice in each group were killed, and femoral bone marrow smears were taken , Wright-Giemsa staining, the number of micronuclei formed in polyphagocytic erythrocytes (PCE) in bone marrow was counted under a microscope, and 1000 polyphagocytic erythrocytes were counted for each operation.

The compound obtained in Example 13 orally given to mice in different doses in advance can significantly reduce the formation rate of micronuclei induced by CTX and has a certain dose-effect relationship (see Table 7). Inhibition of micronuclei formation in erythrocytes (n=5) group Dose CTX bone marrow micronucleus formation 1000 PCE Control group CTX embodiment 13 compound+CTX mg/kg mg/kg 24h 48h 72h4.0±1.6100 25.6±2.9 33.2±5.9 23.6±4.050 100 12.9±2.0 17.0±3.2 11.4±2.3**100 100 8.6±2.6 11.8±2.9 7.2±1.9**

CTX = cyclophosphamide

PCE＝ploychromadic erythrocytes

**p<0.01

Experimental example 8 Ames experiment

According to the Salmonella reverse mutation experiment of Ames, the used bacterial strains are TA97 and TA98. The experiment adopts the plate incorporation method, and the detection system contains 0.1ml bacterial liquid and 80 μl of the compound obtained in Example 14 with different concentrations. In the anti-mutagenic experiment, also Add 20 μl positive mutagen, or control solvent DMSO, 0.5ml PBS (pH7.4) and 0.2ml soft agar. The glass plate was incubated upside down in a 37°C incubator for 48 hours, and then the number of reverted colonies of the bacteria was counted and the results of the mutagenesis test are shown in Tables 8, 9 and 10.

Table 8 The effect of the compound of Example 13 on 4-NQO-induced back mutation of Salmonella TA97 variant (n=3) group Concentration 4NQO Number of reverse mutations/dish Inhibition rate DMSO erythrobimycin Example 13 compound μl/dish μl/dish X±SD ％80μl/dish 0 111±161 552±9350 1 429±62 27.9100 1 302±35** 56.7200 1 265±7** 65.1

**p<0.01

Table 9 The effect of the compound of Example 13 on the back mutation of Salmonella TA97 variant induced by erythrobimycin (n=3) group Concentration 4NQO Number of reverse mutations/dish Inhibition rate DMSO erythrobimycin Example 13 compound μl/dish μl/dish X±SD ％80μl/dish 0 24±25 107±1350 5 67±9** 48.2100 5 52±6** 66.3200 5 43±4** 77.1300 5 38±8** 83.1

**p<0.01

Table 10 Mutagenic experiment of the compound of Example 13 on Salmonella variant TA97TA98 (n=3) Concentration Number of mutations/dish Group DMSO Example 13 compound μl/dish TA97 TA98μl/dish 111± 24±2200 101±32300 21±4

Experiments show that the compound of Example 13 has anti-syringogenic effect, but it has no shock-inducing effect at a larger dose.

Experimental example 9 The teratogenic experiment of the compound of embodiment 13 and the compound of embodiment 12

Choose healthy, sexually mature mice, weighing 29±2g, and house them together at a sex ratio of 2:1 (or 1:1). The vaginal plugs of the female rats were checked every morning, and the day when sperm was found was defined as day 0 of conception (D ₀ ). Pregnant mice were randomly assigned to each experimental group and control group. Pregnant mice were reared in separate cages in groups, and male mice were put into female cages to mate every night, and the mating was stopped when there were 15-20 mice in each group. Two doses of 40 mg/Kg and 20 mg/Kg were given to each pregnant mouse by intragastric administration from the 7th day of pregnancy, and the control drug was RA once a day for 10 consecutive days. The normal control group was given the same amount of normal saline. Pregnant mice were weighed every three days from D ₀ , and the dosage was adjusted according to the body weight change. The positive control group was given retinoic acid 10mg/Kg. Before delivery (D18), the female mice were killed by spinal dislocation, the uterus was opened by laparotomy, and the live fetuses were taken out, and the number of absorbed fetuses was recorded. Observe the shape of the fetus and weigh the litter. The fetal mice were randomly divided into two parts, one part was fixed with Bouins' solution, and the viscera were sectioned in due course. The other part was fixed with 95% ethanol, stained with alizarin red, decolorized and transparent to check the bone development. The experimental results are shown in Table 11 and Table 12. The teratogenicity experiment of the compound of table 11 embodiment 13 and 12 (on the influence of absorbing fetus) (n=10) group Dose fetal absorption (%) fetal body weight Compared with Tween80 embodiment 13 embodiment 12 retinoic acid mg/Kg Absorption number/Implantation number Average weight of total fetal mice (gX±SD)0/135 135 1.2±0.20.3% 0/104 104 1.2±0.240 0/107 107 1.1±0.220 0/104 104 1.1±0.240 0/119 119 1.1±0.220 0/113 113 1.1±0.210 73/99 (73.7%)26* 0.7±0.1**

*p<0.001 Table 12 Effects of Compounds of Examples 13 and 12 on Fetal Rat Bone and Viscera (n=10) Poor ossification of occipital sternum in fetal rats (%) Enlargement of lateral ventricles in fetal rats (%) Group Control Tween80 Example 13 Example 12 Retinoic Acid Occipital ossification difference/sternalization difference/lateral ventriculomegaly lateral ventriculomegaly test number test number/test number/total number of fetuses 0/79(0) 0/79(0) 0/54( 0) 0/10(0)0/69(0) 0/69(0) 0/34(0) 0/10(0)40 15/81(18.1) 20/81(24.7) 0/39(0 ) 0/10(0)20 22/61(33.3) 14/66(21.2) 0/38(0) 0/10(0)40 3/77(3.9) 2/77(2.6) 0/43(0 ) 0/10(0)20 0/75(0) 0/75(0) 0/38(0) 0/10(0)10 16/16(100)* 16/16(100)* 1/3 (33.3)*0/10(0)

*p<0.001

Claims (14)

1. A retinoid coumarin compound represented by the following formula (I) and a pharmaceutically acceptable salt thereof: In the formula, R ¹ is H or COR ⁷ , wherein R ⁷ is OH, CH ₃ , C ₁ -C ₄ alkoxy, or NH ₂ ; R ² is H, C ₁ -C ₄ alkyl, phenyl or OR, wherein R = tretinoyl; R ³ is H, CH ₃ , COR ⁸ or OR, wherein R ⁸ is OH, C ₁ -C ₈ alkoxy, and R is as defined above; R ⁴ is H, halogen, C ₁ -C ₁₈ alkyl, COR ⁹ , wherein R ⁹ =OH or phenyl; R ⁵ is H, CH ₃ or OR, wherein R is as defined above; R ⁶ is H, CH ₃ , COOH, COCH ³ or OR, wherein R is as defined above; And, in the above cases, any compound R ² , R ³ , R ⁵ or R ⁶ has at least one retinoyloxy group. 2. The compound of claim 1, wherein R ¹ is H or COR ⁷ , wherein R ⁷ is CH ₃ , OC ₂ H ₅ , OH or NH ₂ . 3. The compound of claim 1, wherein R ² is H, CH ₃ , phenyl or OR, wherein R is retinoyl. 4. The compound according to claim 1, characterized in that R ⁴ is H, CH ₃ , C ₂ H ₅ , tC ₄ H ₉ , nC ₆ H ₁₃ , C ₆ H ₅ CO, CO ₂ H, Cl, Br . 5. The compound of claim 1, wherein R ⁶ is H, CH ₃ , COOH or OR, wherein R is retinoyl. 6. The compound according to claim 1, characterized in that R ¹ =COCH ₃ , R ² =R ⁴ =R ⁶ =H, R ³ =R ⁵ =OR, R = tretinoyl. 7. The compound according to claim 1, characterized in that R ¹ =COCH ₃ , R ² =R ⁴ =R ⁶ =H, R ³ =CO ₂ H, R ⁵ =OR, R = tretinoyl. 8. The compound according to claim 1, characterized in that R ¹ =COCH ₃ , R ² =R ³ =R ⁴ =H, R ⁵ =R ⁶ =OR, R = tretinoyl. 9. The compound according to claim 1, characterized in that R ¹ =COCH ₃ , R ² =R ⁵ =R ⁶ =H, R ³ =OR, R ⁴ =CO ₂ H, R = retinoyl. 10. The compound according to claim 1, characterized in that R ¹ =COCH ₃ , R ² =R ³ =R ⁴ =H, R ⁵ =OR, R ⁶ =CO ₂ H, wherein R = retinoyl. 11. The compound according to claim 1, characterized in that R ¹ =CONH ₂ , R ² =R ⁴ =R ⁶ =H, R ³ =CO ₂ H, R ⁵ =OR, R = tretinoyl. 12. The compound according to claim 1, characterized in that R ¹ =COOC ₂ H ₅ , R ² =R ³ =R ⁶ =H, R ⁴ =Cl, R ⁵ =OR, R = tretinoyl. 13. The method for preparing the compound as claimed in claim 1, characterized in that: (1) Reaction of a phenolic compound represented by the following formula (II) with a corresponding compound to obtain a coumarin compound (III) substituted and containing a hydroxyl group:

Wherein, at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ is OH or both substituents are OH; (2) The obtained compound (III) is reacted with retinoic acid to obtain the compound (I) of the present invention:

Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are as defined in claim 1, and at least one of R ² , R ³ , R ⁵ and R ⁶ is retinoyloxy. 14. A pharmaceutical composition comprising a therapeutically effective dose of the retinoid compound as claimed in claim 1 and a pharmaceutically acceptable salt thereof.

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