WO2015096640A1 - Thiazolyl-containing rapamycin derivative and application thereof - Google Patents

Abstract

The present invention relates to a rapamycin derivative and an optical isomer and pharmaceutically acceptable salts thereof. The present invention also relates to uses of the compound in preparation of drugs for treating and/or preventing cancers, and method of preparing the rapamycin derivative.

Description

Thiazolyl-containing rapamycin derivatives and applications thereof Technical field

The present invention belongs to the field of medical technology and relates to novel rapamycin derivatives, optically active substances thereof and pharmaceutically acceptable salts thereof, including a process for the preparation thereof and a pharmaceutical composition containing the same. The invention also relates to the use of the compounds and derivatives thereof for the preparation of a medicament for the treatment and/or prevention of cancer.

Background technique

Malignant tumors seriously endanger human health. There are more than 12 million new cases of cancer every year in the world, and there are about 1.3 million people who die from malignant tumors every year in China. The clinical application of existing anti-tumor drugs has inhibited the tumor to a certain extent. However, the treatment of malignant tumors that are most harmful to human life and health has not yet achieved satisfactory results. In the face of huge drug use population, it is urgent to develop new targeted anti-tumor drugs.

Rapamycin is a multifunctional drug produced by fermentation of Streptomyces hygroscopicus FC904, now known as sirolimus. Rapamycin is a macrolide compound that is used as an organ transplant anti-rejection drug in clinical practice and later as a drug-coated stent for the treatment of coronary artery restenosis. It is a mammalian intracellular serine/threonine. Protein kinase mTOR (mammalian target of rapamycin, mammalian target of rapamycin). mTOR is the central regulator of cell growth and proliferation. The mTOR pathway is dysregulated in various tumor cells. The mTOR inhibitor developed as a target can effectively inhibit tumor cell proliferation, induce tumor cell apoptosis and reverse cytotoxic drug resistance. Medicinal. Therefore, several chemical semi-synthetic derivatives of rapamycin are now approved by the FDA for late use. Cancer treatment; representative drugs are: Temsirolimus (CCI-779) developed by Wyeth, approved by the FDA for advanced kidney cancer; Everolimus developed by Novartis, approved by the FDA in 2009 For the treatment of advanced kidney cancer. Deferolimus was developed by Ariad and has no immunosuppressive activity. The drug was approved by the FDA in 2005 for rapid treatment of soft tissue and skeletal malignancies. In recent years, it has been found to have the effect of prolonging the lifespan of mammals, and has a very broad application prospect.

Related studies have shown that among the above sirolimus derivatives, the derivatives obtained by the chemical semi-synthesis method at the C40 position have outstanding antitumor activity and low immunosuppressive activity, and the present inventors have designed and synthesized based on the reference literature. A series of rapamycin derivatives, which were tested by in vitro immunosuppressive activity test and antitumor activity, showed no or low immunosuppressive activity and strong antitumor activity.

Summary of the invention

The present invention relates to a compound of the formula I, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof,

among them,

X is "single bond", or -O(CH ₂ )n-;

n is an integer of 2-4;

R ₁ is hydrogen, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 3 to 7 cycloalkyl groups, 2 to 10 C atoms or an alkynyl group having 2 to 10 C atoms;

R ₂ is hydrogen, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, carboxy, nitro, cyano, alkyl, alkoxy, N-alkylamino, N,N-dialkylamino, Alkylthio, alkylsulfinyl, alkylsulfonyl, alkoxymethyl, alkoxyethyl, alkyl acyl, carbamoyl, N-alkylcarbamoyl, N,N-dioxane a carbamoyl group, an aminosulfonyl group, an N-alkylaminosulfonyl group, an N,N-dialkylaminosulfonyl group or an alkylenedioxy group having a C atom number of 1-3, wherein the alkyl group C The number of atoms is 1-4, and the number of C atoms of the alkoxy group is 1-4.

a compound of formula I, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof,

among them,

X is "single bond" or -O(CH ₂ )n-;

n is an integer of 2-4;

R ₁ is hydrogen, an alkyl group having a C atom number of 1-3 or a C atom number of 3-7 cycloalkyl;

R ₂ is hydrogen, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, carboxy, nitro, cyano, alkyl, alkoxy, N-alkylamino, N,N-dialkylamino, Alkylthio, alkylsulfonyl, alkoxymethyl, alkoxyethyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, N-alkylamino a sulfonyl group, an N,N-dialkylaminosulfonyl group or an alkylenedioxy group having a C atom number of from 1 to 3, wherein the alkyl group has a C atom number of from 1 to 4, and the alkoxy group has C The number of atoms is 1-4.

a compound of formula I, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof,

among them,

X is "single bond" or -O(CH ₂ )n-;

n is an integer of 2-4;

R ₁ is an alkyl group having a C atom number of 1-3;

R ₂ is hydrogen, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, carboxy, nitro, cyano, alkyl, alkoxy, N-alkylamino, N,N-dialkylamino, Alkylthio, alkylsulfonyl, alkoxymethyl, alkoxyethyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, N-alkylamino a sulfonyl group, an N,N-dialkylaminosulfonyl group or an alkylenedioxy group having a C atom number of from 1 to 3, wherein the alkyl group has a C atom number of from 1 to 4, and the alkoxy group has C The number of atoms is 1-4.

a compound of formula I, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof,

among them,

X is "single bond";

R ₁ is an alkyl group having a C atom number of 1-3;

R ₂ is hydrogen, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, carboxy, nitro, cyano, alkyl, alkoxy, N-alkylamino, N,N-dialkylamino, Alkylthio, alkylsulfonyl, alkoxymethyl, alkoxyethyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, N-alkylamino a sulfonyl group, an N,N-dialkylaminosulfonyl group or an alkylenedioxy group having a C atom number of from 1 to 3, wherein the alkyl group has a C atom number of from 1 to 4, and the alkoxy group has C The number of atoms is 1-4.

a compound of formula I, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof,

among them,

X is -O(CH ₂ )n-;

n is 2;

R ₁ is an alkyl group having a C atom number of 1-3;

R ₂ is hydrogen, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, carboxy, nitro, cyano, alkyl, alkoxy, N-alkylamino, N,N-dialkylamino, Alkylthio, alkylsulfonyl, alkoxymethyl, alkoxyethyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, N-alkylamino a sulfonyl group, an N,N-dialkylaminosulfonyl group or an alkylenedioxy group having a C atom number of from 1 to 3, wherein the alkyl group has a C atom number of from 1 to 4, and the alkoxy group has C The number of atoms is 1-4.

a compound of formula I, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof,

40-N-(6-chloro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T1)

40-N-(5-chloro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T2)

40-N-(3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T3)

40-N-(4-fluoro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T4)

40-N-(6-fluoro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T5)

40-O-(2-(3-methylbenzothiazole-2-imino)ethyl)oxyrapamycin (RAP-T6)

Wherein R ₁ is preferably a methyl group.

A pharmaceutical composition comprising a compound of the formula I according to the invention, a derivative thereof, an optical isomer thereof and a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable excipient.

Use of a compound of the formula I of the present invention, a derivative thereof, an optical isomer thereof and a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment and/or prevention of various cancers.

The above use, wherein the cancer is a solid tumor or a non-solid tumor.

Wherein the cancer is breast, lung, colon, rectum, stomach, prostate, bladder, pancreas Or ovarian cancer.

Wherein the cancer is cervical cancer, renal cancer, leukemia, melanoma, nasopharyngeal cancer or gastric cancer.

The preparation method of the compound of the formula I of the present invention, the amino compound A-2 is formed from the compound A-1, and the compound A-2 and the quaternary ammonium salt compound A-3 are subjected to an organic base catalytic reaction to obtain a formula I. compound of;

Wherein R ₁ , R ₂ , X and n are as defined above, respectively.

The derivatives of the invention may exist in stereoisomeric forms which may be enantiomers or diastereomers. The invention relates both to enantiomers or diastereomers, as well as to their respective mixtures, which, like diastereomers, can be separated into stereoisomeric individual components according to methods known per se.

Furthermore, derivatives of the compounds of the invention may be prodrugs. In accordance with the present invention, the prodrug itself may have weaker activity or even no activity, but after administration, it is converted to the corresponding biologically active form under physiological conditions (e.g., by metabolism, solvolysis, or otherwise).

Furthermore, the invention also includes esters of the compounds of the invention.

The term "halogen" as used herein, unless otherwise indicated, means a fluorine, chlorine, bromine or iodine atom; "alkyl" means a straight or branched substituted or unsubstituted alkyl group; "alkylene" means Straight chain or branch a substituted or unsubstituted alkylene group; "cycloalkyl" means a substituted or unsubstituted cycloalkyl group; and a heteroaryl group means a substituted or unsubstituted heteroaryl group which includes one or more selected from O The hetero atom of N and S may be monocyclic or polycyclic, and the cyclic system is aromatic, and examples thereof include imidazolyl, pyridyl, pyrimidinyl, (1, 2, 3)- and (1, 2,4)-triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, oxazolyl, pyrazolyl, pyrrolyl, thiazolyl, benzimidazolyl, pyridoimidazole A benzothiophenyl group, a benzothiazolyl group, a fluorenyl group, a quinolyl group, a pyridopyrimidinyl group or the like.

The present invention includes a pharmaceutical composition comprising a rapamycin compound of the formula I, an optical isomer thereof, and a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier that can be used in the pharmaceutical field. The derivatives of the present invention can be used in combination with other active ingredients as long as they do not cause other adverse effects such as allergic reactions.

The pharmaceutical compositions of the present invention may be formulated in several dosage forms containing some of the excipients commonly used in the pharmaceutical arts; for example, oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as Injectable solutions or suspensions, or injectable dry powders, ready to use by injecting water before injection; topical preparations (such as ointments or solutions).

The carrier used in the pharmaceutical composition of the present invention is a common type available in the pharmaceutical field, including: a binder for an oral preparation, a lubricant, a disintegrant, a solubilizer, a diluent, a stabilizer, a suspending agent, and a non-pigment. , flavoring agents, etc.; preservatives, solubilizers, stabilizers, etc. for injectable preparations; bases, diluents, lubricants, preservatives, etc. for topical preparations. The pharmaceutical preparations can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically), and if certain drugs are unstable under gastric conditions, they can be formulated into enteric coated tablets.

We have found that the compound of the present invention has an activity of inhibiting tumor cell growth in vitro, and therefore, it can be used as a medicament for preparing a cancer for treating and/or preventing cancer. Especially for the treatment of breast, lung, colon, rectum, stomach, prostate, bladder, pancreas and ovarian cancer. The compounds of the invention are also expected to be useful in the treatment of other cell proliferative disorders such as psoriasis, benign prostatic hypertrophy, atherosclerosis and restenosis. It is further contemplated that the rapamycins of the invention will have activity against leukemia, malignant lymphoma, and solid tumors such as cancer and sarcoma in tissues such as liver, kidney, prostate, and pancreas.

The derivative according to the present invention can be used as an active ingredient for the preparation of a medicament for treating and/or preventing various cancers, and the present invention also provides a method for treating or preventing the above-mentioned diseases, comprising administering a therapeutically effective amount to a patient suffering from or susceptible to the disease. A derivative according to the invention. The clinical dose of the rapamycin compound of Formula I for use in a patient must depend on the subject being treated, the particular route of administration, the severity of the condition being treated, and the optimal dosage will be determined by the physician treating the particular patient.

The active compounds of the invention may be used as the sole anticancer drug or may be used in combination with one or more other antineoplastic agents. Combination therapy is achieved by administering the individual therapeutic components simultaneously, sequentially or separately.

The examples and preparations provided below further illustrate and exemplify the compounds of the invention and methods for their preparation. It is to be understood that the scope of the following examples and preparations are not intended to limit the scope of the invention in any way.

Scheme A below describes the preparation of the compounds of formula I of the present invention, all of which are prepared by the methods described in these schematics, by methods well known to those of ordinary skill in the art of organic chemistry, or are commercially available. All of the final compounds of the present invention are prepared by the methods described in these schematics or by methods analogous thereto, which are well known to those of ordinary skill in the art of organic chemistry. All the variable factors applied in these schematic diagrams can be as follows Righteousness.

Compound A-1 is reacted in steps 2-4 to form amino compound A-2. The compound A-2 and the quaternary ammonium salt compound A-3 are subjected to an organic base-catalyzed reaction to obtain a derivative of the formula I.

According to the compound of the formula I according to the invention, in Scheme A, n, R ₁ , R ₂ and X are as defined in the Summary of the Invention.

Wherein, when X is "-", A-2 is prepared according to the method described in the first step: using A-1 as a raw material, reacting with trifluoromethanesulfonic anhydride to obtain B-1, and then reacting with sodium azide to obtain B- 2, finally restored to A-2.

When X is O(CH ₂ )n, A-2 is prepared according to the method described in Step 2: A-1 is used as a raw material, and etherification, azide and reduction are carried out to obtain A-2.

The preparation method of the invention is simple, and the prepared compounds all have significant antitumor activity.

In the above route, the compounds of the starting materials A-1, A-3 and C-1 can be prepared by a method well known to those skilled in the art of organic chemistry or are commercially available. The preparation method of the invention is simple, and the prepared compound has good antitumor activity.

detailed description

The examples are intended to illustrate and not to limit the scope of the invention. The nuclear magnetic resonance spectrum of the compound prepared in the present invention was measured by Bruker ARX-300, and the mass spectrum was measured by Agilent 1100 LC/MSD; the reagents used were either analytically pure or chemically pure.

Table 1. Structural formulae of Examples 1-6

Example X R ₁ R ₂ 1 -* CH ₃ 6-Cl 2 -* CH ₃ 5-Cl 3 -* CH ₃ H 4 -* CH ₃ 4-F 5 -* CH ₃ 6-F 6 OCH ₂ CH ₂ CH ₃ 6-Cl

Where - indicates a single bond

Example 1: Preparation of 40-N-(6-chloro-3-methylbenzothiazole-2-ylidene)-aminorapamycin (RAP-T1)

Step A: Preparation of C-40 triflate rapamycin

Rapamycin (11 mmol, 10 g) was added to 50 mL of dichloromethane solution. After the addition was completed, it was cooled to 0 ° C, 2,6-lutidine (52 mmol, 5.6 g) was added, and then trifluoromethane was added dropwise. Anhydride (17.7 mmol, 5 g) was incubated for 2 hours. The reaction was completed. After adding 1 mL of water, stirring was continued for 10 minutes. The reaction mixture was poured into water (80 mL), extracted with dichloromethane, and the combined extracts were washed with water and dried over anhydrous sodium sulfate. The oil was triturated with 9.3 mg of triflate rapamycin in a yield of 79.5%, MS: 1068.5 (M+Na).

Step B: Preparation of C-40 azide rapamycin

The triflate rapamycin (8.6 mmol 9.0 g) was gradually added to 130 mL of acetone solution, and an aqueous solution of sodium azide (34.4 mmol, 2.24 g, 5 mL of H ₂ O) was added at room temperature. After completion, the reaction was carried out at room temperature for 16 hours. After the completion of the reaction, the reaction mixture was poured into a large amount of water, extracted with ethyl acetate, evaporated, evaporated, evaporated, evaporated, MS: 961 (M+Na).

Step C: Preparation of C-40 aminorapamycin

Azide rapamycin (3.2 mmol, 3.0 g) was added to a tetrahydrofuran solution, and triphenylphosphine (12.8 mmol, 3.35 g) was added at room temperature. After the addition was completed, the mixture was stirred and heated to 50 ° C for 2 hours. 1 mL of distilled water was added dropwise, and the reaction was continued for 6 hours. After the completion of the reaction, the reaction mixture was cooled to EtOAc.

Step D: Preparation of 40-N-(6-chloro-3-methylbenzothiazol-2-ylidene)-amino rapamycin

C-40 aminorapamycin (0.44 mmol, 0.4 g) and A-3 (1.17 mmol 0.4 g) were added to acetonitrile at room temperature, and 3.6 g of triethylamine (2 mmol, 0.7 g) was added to the reaction mixture. The reaction was stirred at room temperature for 3 h. After completion of the reaction, 200 mL of H ₂ O was added, and the mixture was extracted with ethyl acetate. EtOAc was evaporated. m/z: 1093.6 (M+1); ¹ H NMR (500 MHz, DMSO) δ 7.61 (s, 1H), 7.27 (d, J = 7.9 Hz, 1H), 7.04 (d, J = 8.5 Hz, 1H) ), 6.44 (s, 1H), 6.43-6.38 (m, 1H), 6.22 (m, 1H), 6.20-6.14 (m, 1H), 6.13-6.07 (m, 1H), 5.46 (dd, J = 15.3) , 9.1 Hz, 1H), 5.24 (s, 1H), 5.10 (d, J = 9.7 Hz, 1H), 5.09 (m, 1H), 5.06-5.00 (m, 1H), 4.96 (d, J = 4.7 Hz , 1H), 4.06-3.96 (m, 2H), 3.90 (d, J = 4.1 Hz, 1H), 3.68-3.59 (m, 1H), 3.50-3.40 (m, 2H), 3.36 (s, 3H), 3.21(s,3H), 3.16(s,3H), 3.06(s,3H), 2.77(d,J=16.8Hz,1H), 2.47-2.29(m,2H),2.19(s,1H),2.15 -1.97 (m, 2H), 1.93-1.78 (m, 2H), 1.75 (s, 3H), 1.66 (s, 3H), 1.64-1.00 (m, 14H), 0.97 (d, J = 6.3 Hz, 3H ), 0.89 (d, J = 6.4 Hz, 3H), 0.83 (d, J = 6.3 Hz, 3H), 0.80 (d, J = 6.5 Hz, 3H), 0.73 (d, J = 6.4 Hz, 3H); ¹³ C NMR (126 MHz, DMSO) δ 210.64, 207.50,199.01, 169.15,166 .98,151.31,139.72,139.29,137.83,137.19,132.27,130.42,127.00,126.95,126.09,125.20,124.26,123.37,121.77,109.73,98.99,85.70,82.32,81.18,75.68,73.59,66.19,61.16,56.99 , 55.45, 55.40, 50.59, 45.19, 43.43, 35.20, 34.78, 33.07, 32.94, 32.80, 30.17, 30.10, 29.57, 27.52, 26.39, 26.22, 24.46, 21.61, 20.32, 15.54, 15.52, 14.65, 13.53, 13.19, 10.47 .

The compounds of Examples 2-5 were prepared according to the procedure of Example 1 by selecting the appropriate starting materials and reagents. When referring to a specific reaction material, it should be understood that the technology is well versed in this field. Personnel can select the appropriate materials and reagents according to the needs of the examples.

Example 2:

40-N-(5-chloro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T2)

MS (ESI) m/z: 1093.6 (M+H) ⁺ ; ¹³ C NMR (126 MHz, DMSO) δ 210.64, 207.50,199.01,169.15,166.98,151.65,142.04,139.30,137.83,137.19,132.29,131.04, 130.43, 127.00, 126.95, 125.19, 123.24, 120.33, 120.17, 108.78, 99.00, 85.71, 82.33, 81.18, 75.67, 73.59, 66.19, 61.23, 56.99, 55.46, 55.41, 50.59, 45.19, 43.44, 40.07, 39.64, 39.54, 39.20, 39.00, 35.21, 34.78, 33.06, 32.95, 32.81, 30.14, 30.10, 29.56, 27.52, 26.39, 26.22, 24.47, 21.61, 20.33, 15.54, 15.53, 14.66, 13.53, 13.20, 10.47; ¹ H NMR (500 MHz, DMSO) δ 7.47 (d, J = 8.2 Hz, 1H), 7.15 (s, 1H), 7.00 (d, J = 8.2 Hz, 1H), 6.43 (s, 1H), 6.42 (dd, J = 14.5, 11.1 Hz, 1H), 6.26-6.20 (m, 1H), 6.21-6.13 (m, 1H), 6.11 (dd, J = 10.8, 5.5 Hz, 1H), 5.46 (dd, J = 14.8, 9.6 Hz, 1H) ), 5.23 (s, 1H), 5.10 (d, J = 10.2 Hz, 1H), 5.06-5.00 (m, 1H), 4.95 (d, J = 5.2 Hz, 1H), 4.06 - 3.96 (m, 2H) , 3.90 (d, J = 4.8 Hz, 1H), 3.63 (d, J = 10.3 Hz, 1H), 3.44 (dd, J = 12.0, 2.1 Hz, 2H) , 3.37 (s, 3H), 3.20 (s, 3H), 3.15 (s, 3H), 3.06 (s, 3H), 2.77 (dd, J = 17.6, 2.0 Hz, 1H), 2.48-2.36 (m, 2H) ), 2.23-2.16 (m, 1H), 2.07-1.99 (m, 1H), 1.90 - 1.78 (m, 2H), 1.74 (s, 3H), 1.65 (s, 3H), 1.64-1.46 (m, 4H) ), 1.46-1.20 (m, 8H), 1.13 - 0.99 (m, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.89 (d, J = 6.5 Hz, 3H), 0.83 (d, J = 6.4 Hz, 3H), 0.80 (d, J = 6.7 Hz, 3H), 0.73 (d, J = 6.6 Hz, 3H).

Example 3:

40-N-(3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T3)

MS (ESI) m / z: 1060.5 (M + H) +; 1 H NMR (400MHz, DMSO) δ7.46 (d, J = 7.6Hz, 1H), 7.24 (t, J = 7.8Hz, 1H), 7.04 (d, J = 7.9 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 6.47 (s, 1H), 6.45-6.35 (m, 1H), 6.25 (m, 1H), 6.23-6.16 (m, 1H), 6.13 (m, 1H), 5.46 (dd, J = 14.7, 9.7 Hz, 1H), 5.27 (d, J = 4.5 Hz, 1H), 5.09 (d, J = 10.1 Hz, 1H) , 5.05-5.00 (m, 1H), 4.96 (d, J = 5.1 Hz, 1H), 4.02 (m, 2H), 3.92 (d, J = 4.7 Hz, 1H), 3.63 (d, J = 12.2 Hz, 1H), 3.52-3.39 (m, 2H), 3.37 (s, 3H), 3.31-3.24 (m, 3H), 3.21 (s, 3H), 3.15 (s, 3H), 3.06 (s, 3H), 2.78 (d, J = 16.9 Hz, 1H), 2.48-2.34 (m, 2H), 2.26 (m, 3H), 2.14 - 2.01 (m, 2H), 1.92-1.80 (m, 2H), 1.75 (s, 3H) ), 1.66 (s, 3H), 1.63-1.21 (m, 10H), 0.96 (d, J = 6.4 Hz, 3H), 0.88 (d, J = 6.4 Hz, 3H), 0.83 (d, J = 6.6 Hz) , 3H), 0.80 (d, J = 7.0 Hz, 3H), 0.73 (d, J = 6.6 Hz, 3H); ¹³ C NMR (101 MHz, DMSO) δ 211.18, 207.93, 199.54, 169.65, 167.50, 152.32, 141.17, 139.81, 138.35, 137.73, 132.79, 130.94, 127.52, 127.47, 126. 82,125.72,122.61,121.83,121.14,109.22,103.51,99.51,86.23,82.84,81.73,76.18,74.10,66.71,61.59,60.21,57.51,55.97,55.87,51.10,45.70,43.94,35.73,35.30,33.61, 33.45, 33.38, 30.68, 30.43, 30.08, 28.07, 26.90, 26.73, 24.95, 22.13, 21.22, 16.04, 15.15, 14.55, 14.06, 13.69. Example 4:

40-N-(4-fluoro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T4)

MS (ESI) m/z: 1077.5 (M+H) ⁺ ; ¹ H NMR (400 MHz, DMSO) δ 7.31 (m, J = 6.9 Hz, 1H), 7.11 (dd, J = 12.1, 8.7 Hz, 1H ), 7.01-6.90 (m, 1H), 6.46 (s, 1H), 6.41 (dd, J = 14.2, 11.4 Hz, 1H), 6.23 (m, 1H), 6.17 (d, J = 13.4 Hz, 1H) , 6.11(m,1H), 5.46(dd,J=14.6,9.7Hz,1H), 5.26(s,1H),5.09(d,J=10.0Hz,1H),5.05-4.98(m,1H), 4.95 (m, 1H), 4.08-3.97 (m, 2H), 3.93 (m, 1H), 3.66-3.59 (m, 1H), 3.29 (m, 3H), 3.20 (s, 3H), 3.18 (s, 3H), 3.15 (s, 3H), 3.05 (s, 3H), 2.76 (d, J = 16.0 Hz, 1H), 2.41 (dd, J = 24.6, 8.9 Hz, 1H), 2.29-1.94 (m, 3H) ), 1.85 (dd, J = 21.1, 9.0 Hz, 2H), 1.75 (s, 3H), 1.64 (s, 3H), 1.62-0.99 (m, 16H), 0.96 (d, J = 6.4 Hz, 3H) , 0.87 (d, J = 6.5Hz , 3H), 0.83 (d, J = 7.0Hz, 3H), 0.80 (d, J = 7.2Hz, 3H), 0.73 (d, J = 6.5Hz, 3H); 13 C NMR (101 MHz, DMSO) δ 211.08, 208.03, 199.49, 169.68, 167.48, 139.78, 138.35, 137.68, 132.78, 130.94, 128.67, 128.57, 127.53, 127.46, 125.60, 124.51, 124.49, 121.70,119.04, 114.60, 99.51, 86.14, 82.81, 81.67, 76.20, 74.06, 66.70, 61.87, 57.46, 55.96, 55.91, 51.14, 45.70, 43.96, 35.71, 35.28, 33.72, 33.55, 33.45, 30.77, 30.58, 30.10, 27.99, 26.91, 26.74, 24.98, 22.11, 20.85, 16.07, 16.03, 15.19, 13.95, 13.79, 10.97.

Example 5:

40-N-(6-fluoro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T5)

MS (ESI) m/z: 1077.6 (M+H) ⁺ ; ¹ H NMR (400 MHz, DMSO) δ 7.46 (dd, J = 8.3, 1.9 Hz, 1H), 7.08 (t, J = 7.9 Hz, 1H) ), 7.03 (d, J = 4.5 Hz, 1H), 6.46 (s, 1H), 6.42 (m, 1H), 6.24 (d, 1H), 6.17 (d, J = 13.7 Hz, 1H), 6.10 (m) , 1H), 5.45 (dd, J = 14.6, 9.7 Hz, 1H), 5.27 (s, 1H), 5.08 (d, J = 10.1 Hz, 1H), 5.05 - 4.98 (m, 1H), 4.95 (d, J = 5.2 Hz, 1H), 4.03 (m, 1H), 4.01 (d, J = 4.2 Hz, 1H), 3.91 (d, J = 4.7 Hz, 1H), 3.62 (d, J = 12.1 Hz, 1H) , 3.43 (m, 2H), 3.35 (s, 3H), 3.20 (s, 3H), 3.15 (s, 3H), 3.05 (s, 3H), 2.77 (d, J = 16.1 Hz, 1H), 2.48- 2.31 (m, 2H), 2.25 - 1.94 (m, 3H), 1.85 (m, 2H), 1.74 (s, 3H), 1.65 (s, 3H), 1.63-1.46 (m, 5H), 1.44-1.00 ( m, 10H), 0.96 (d, J = 6.4 Hz, 3H), 0.87 (d, J = 6.5 Hz,), 0.82 (d, J = 6.5 Hz, 3H), 0.79 (d, J = 6.9 Hz, 3H) ), 0.73 (d, J = 6.6 Hz, 3H); ¹³ C NMR (101 MHz, DMSO) δ 211.16, 208.02, 199.54, 169.66, 167.50, 158.58, 156.23, 152.08, 139.81, 138.35, 137.82, 137.72, 132.78, 130.95 , 127.52, 127.47, 125.70, 123.30 , 110.36, 109.56, 99.51, 86.21, 82.83, 81.70, 76.19, 74.09, 66.70, 61.56, 57.49, 55.97, 55.89, 51.10, 45.70, 43.95, 35.72, 35.29, 33.58, 33.44, 33.34, 30.73, 30.66, 30.08, 28.03 , 26.90, 26.73, 24.98, 22.12, 20.84, 16.06, 16.03, 15.14, 14.03, 13.71, 10.98.

Example 6: Preparation of 40-O-(2-(3-methylbenzothiazole-2-imino)ethyl)oxyrapamycin (RAP-T6)

Step A: Preparation of 40-O-(2-bromoethyl)oxyrapamycin

Rapamycin (3.3 mmol, 3 g) was added to 60 mL of toluene solution, 12 g of 2-bromoethyl trifluoromethanesulfonate and 4 mL of diisopropylethylamine (DIPEA) were added, and the temperature was raised to 60 ° C. After the completion of the reaction, the reaction mixture was poured into 100 mL of water, and washed with water, diluted with hydrochloric acid and saturated sodium hydrogen carbonate and brine, and dried over anhydrous sodium sulfate. The oil was evaporated to dryness (yield: 41 g,yield: 41.7%, MS: 1044.6 (M+Na).

Step B: Preparation of 40-O-(2-azidoethyl)oxyrapamycin

40-O-(2-bromoethyl)oxyrapamycin (1.17 mmol, 1.2 g) was added to 30 mL of N,N-dimethylformamide (DMF) solution, and sodium azide (4.6 mmol) was added. 0.3 g) and potassium iodide (0.1 g) were heated to 60 ° C for 2 hours. After the completion of the reaction, the reaction mixture was poured into 100 mL of water, and ethyl acetate was added, and the mixture was washed with water and brine, and then evaporated. By Column chromatography gave 0.62 g of a yellow solid, yield 53.7%, MS: 1005.6 (M+Na).

Step C: Preparation of 40-O-(2-aminoethyl)oxyrapamycin

40-O-(2-azidoethyl)oxyrapamycin (0.2 mmol, 0.21 g) was gradually added to tetrahydrofuran solution, and triphenylphosphine (0.8 mmol, 0.21 g) was added in portions at room temperature. After the completion of the addition, the mixture was heated to 60 ° C for 2 hours, and 1 mL of distilled water was added dropwise to continue the reaction 36. After completion of the reaction, the reaction mixture was cooled to EtOAc EtOAc.

Step D: 40-O-(2-(3-methylbenzothiazole-2-imino)ethyl)oxyrapamycin (RAP-T6)

40-O-(2-aminoethyl)oxyrapamycin (300 mmol, 0.16 g,), A-34.1 g (300 mmol) was added to acetonitrile at room temperature, and triethylamine 3.6 was added to the reaction mixture. g (900 mmol), stirred at room temperature for 3 h. After completion of the reaction, 200 mL of H ₂ O was added, and the mixture was extracted with EtOAc. MS (ESI) m/z: 1128.6 (M+H) ⁺ ; ¹ H NMR (400 MHz, DMSO) δ 7.66 (s, 1H), 7.29 (d, J = 8.5 Hz, 1H), 7.08 (d, J) = 8.6 Hz, 1H), 6.47 (s, 1H), 6.45-6.34 (m, 1H), 6.23 (m, 1H), 6.17 (m, 1H), 6.14-6.06 (m, 1H), 5.46 (dd, J=14.6, 9.6 Hz, 1H), 5.29 (s, 1H), 5.09 (d, J = 10.0 Hz, 1H), 5.01-4.96 (m, 1H), 4.94 (d, J = 5.4 Hz, 1H), 4.05-3.98 (m, 2H), 3.94 (d, J = 4.4 Hz, 1H), 3.79-3.57 (m, 4H), 3.49-3.38 (m, 2H), 3.34 (s, 3H), 3.33 (s, 3H), 3.30-3.19 (m, 2H), 3.15 (s, 3H), 3.05 (s, 3H), 2.86-2.67 (m, 2H), 2.45-2.32 (m, 2H), 2.20 (m, 1H) , 2.14-1.79 (m, 4H), 1.74 (s, 3H), 1.63 (s, 3H), 1.61 - 0.99 (m, 12H), 0.97 (d, J = 6.4 Hz, 3H), 0.87 (d, J) = 6.4 Hz, 3H), 0.82 (d, J = 6.2 Hz, 3H), 0.77 (d, J = 6.6 Hz, 3H), 0.73 (d, J = 6.6 Hz, 3H).

¹³ C NMR (101 MHz, DMSO) δ 211.03, 208.02, 199.44, 169.68, 167.49, 155.02, 148.32, 140.34, 140.28, 139.81, 138.33, 137.67, 132.83, 130.91, 127.50, 126.75, 125.50, 124.88, 123.75, 122.36, 110.50,99.58,86.05,83.09,82.93,82.76,76.22,74.07,69.94,66.70,57.70,57.45,55.95,55.20,51.21,45.69,43.98,38.76,36.64,35.69,35.30,33.85,32.77,31.40,30.54, 30.31, 29.45, 26.93, 26.71, 24.96, 22.15, 20.86, 16.04, 15.19, 13.95, 13.84, 10.96.

Pharmacological study of the product of the invention

In vitro immunosuppressive activity test

The in vitro immunosuppressive activity was measured by lymphocyte transformation assay (LTT):

(1) The spleen of clean-grade Kunming mice (20-22 g) was aseptically placed in a dish containing PBS buffer, and the spleen was gently squeezed through a 100-mesh screen with a glass syringe handle. After the pipette is fully blown, the cell liquid is aspirated into a 15 ml centrifuge tube, centrifuged for 5 minutes (1000 r/min), and the precipitate is washed twice with 1640 medium, centrifuged for 5 minutes (1000 r/min), and the precipitate is adjusted with 1640 medium. The cell concentration was 1 x 107 cells/ml.

(2) Concanavalin A (ConA)-induced lymphocyte transformation in mice, weigh the powder to be tested, dissolved in absolute ethanol as the sample mother liquor, and then use 1640 culture solution to prepare test solutions of various concentrations, add 50 μl The sample test solution was added to a 96-well cell culture plate, and 100 μl of the above mouse spleen lymphocyte suspension was added thereto, and 50 μl of ConA (20 μg/ml) was separately added. Among them, 100 μl of the cell suspension and 100 μl of the 1640 medium were used as a blank control, and 100 μl of the cell suspension and 100 μl of ConA were added as a ConA control. After the addition was completed, the 96-well cell culture plate was cultured at 37 ° C for 72 hours in a 5% CO 2 incubator. Add 4 μl of MTT (5 mg/ml) to each well 4 hours before termination of culture, continue to culture for 4 hours, centrifuge for 10 minutes (4000 r/min), discard the supernatant, and add 150 μl of dimethyl sulfoxide (DMSO) per well. The plate reader measures the light absorption value (OD ₅₇₀ ) of the sample and calculates the inhibition rate of the sample on the lymphocyte reaction I:

The immunosuppressive activity of the compounds is shown in Table 2.

Table 2 Example compound in vitro immunosuppressive activity

Using rapamycin as a control drug, the results of LTT assay showed that the immunological activity of the new derivatives of rapamycin from Example 1 to Example 5 was significantly lower than that of the positive control drug rapamycin. Example 1 and Examples 2 It has no immunosuppressive activity at different concentration gradients. Example 3 has no immunosuppressive activity at low concentrations, while high concentration is significantly lower than rapamycin activity. The immunological activities of Example 4 and Example 5 were weaker than rapamycin.

Since the example compounds exhibited low or no immunosuppressive activity, we performed an in vitro antitumor activity screening according to the rapamycin derivative of the above formula I of the present invention.

In vitro antitumor activity test

(1) cervical cancer cells CasKi, renal cancer cells SK-NEP-1, human leukemia cells HL-60, human malignant melanoma cells A-375, human nasopharyngeal carcinoma cells CNE2 and human gastric cancer cells 7901 cells The strain was resuscitated and passaged 2-3 times to stabilize cell viability for in vitro cell viability assays. The suspended cells do not need to be digested, the adherent cells trypsin (0.25%) is digested, the serum-containing cell culture solution is added to terminate the digestion, and the cell liquid is transferred to a centrifuge tube by a pipette, centrifuged at 1500 r/min for 3 min, and gently discarded. After the supernatant was added, 5 mL of the culture solution was added, and the cells were mixed by blowing, counted, 5000-10000 cells/well of adherent cells, 20,000 cells/well of suspension cells, added to a 96-well plate, cultured at 37 ° C, 5% CO 2 , and cultured for 24 cells. The test drug was added after an hour.

(2) Dissolve the test sample as a mother liquid with dimethyl sulfoxide, and then dilute the test sample with the cell culture solution: take 10 ul of the sample mother liquid, add 990 ul of the cell culture solution, and dilute the sample to the test concentration. The sample dilutions were added to the cell culture medium which had been cultured in a 96-well plate for 24 hours, and each well was added to 3 wells, and a blank control (no drug treatment) was placed. The 96-well plate was further cultured at 37 ° C, 5% CO 2 for 72 h, and 20 μL of MTT (tetrazole) (5 mg/mL) was added to each well. After 4 h in the incubator, the suspension cells were centrifuged at 4000 r/min for 10 min. Discard the supernatant, add 150 μL of dimethyl sulfoxide, fully dissolve the viable cells and the MTT reaction product formazan, and place the results in a microplate reader. The IC ₅₀ value of the drug can be determined by the Bliss method. The IC ₅₀ (μmol/L) of the strain is shown in Table 3.

Table 3 Example compound in vitro antitumor activity

Note: - indicates untested activity.

It is clear from the above test results that the compound of the formula I to be protected by the present invention has no immunosuppressive activity or low immunosuppressive activity and exhibits superior anticancer activity, and the compound of the example is applied to most cancerous strains. The antitumor activity is stronger than rapamycin. Its Example 2 has no immunological activity, but the antitumor activity against SK-NEP-1 is about 30 times higher than that of rapamycin. Therefore, the compound of the present invention has a good potential application prospect, and it is more likely to develop no or A novel rapamycin candidate compound with low immunosuppressive activity but strong antitumor activity.

As described above, the compounds of the present invention have strong anticancer activity against a wide range of cancers including cervical cancer, renal cancer, leukemia, melanoma, nasopharyngeal cancer or gastric cancer, in the treatment of solid tumors and non-solid tumors. Both have good application prospects.

Claims (10)

a compound of formula I, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof,

among them, X is "single bond", or -O(CH ₂ )n-; n is an integer of 2-4; R ₁ is hydrogen, an alkyl group having 1 to 10 carbon atoms, an alkenyl group having 3 to 7 cycloalkyl groups, 2 to 10 C atoms or an alkynyl group having 2 to 10 C atoms; R ₂ is hydrogen, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, carboxy, nitro, cyano, alkyl, alkoxy, N-alkylamino, N,N-dialkylamino, Alkylthio, alkylsulfinyl, alkylsulfonyl, alkoxymethyl, alkoxyethyl, alkyl acyl, carbamoyl, N-alkylcarbamoyl, N,N-dioxane a carbamoyl group, an aminosulfonyl group, an N-alkylaminosulfonyl group, an N,N-dialkylaminosulfonyl group or an alkylenedioxy group having a C atom number of 1-3, wherein the alkyl group C The number of atoms is 1-4, and the number of C atoms of the alkoxy group is 1-4. A compound of the formula I according to claim 1, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof, among them, X is "single bond" or -O(CH ₂ )n-; n is an integer of 2-4; R ₁ is hydrogen, an alkyl group having a C atom number of 1-3 or a C atom number of 3-7 cycloalkyl; R ₂ is hydrogen, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, carboxy, nitro, cyano, alkyl, alkoxy, N-alkylamino, N,N-dialkylamino, Alkylthio, alkylsulfonyl, alkoxymethyl, alkoxyethyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, N-alkylamino a sulfonyl group, an N,N-dialkylaminosulfonyl group or an alkylenedioxy group having a C atom number of from 1 to 3, wherein the alkyl group has a C atom number of from 1 to 4, and the alkoxy group has C The number of atoms is 1-4. A compound of the formula I according to claim 2, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof, among them, X is "single bond" or -O(CH ₂ )n-; n is an integer of 2-4; R ₁ is an alkyl group having a C atom number of 1-3; R ₂ is hydrogen, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, carboxy, nitro, cyano, alkyl, alkoxy, N-alkylamino, N,N-dialkylamino, Alkylthio, alkylsulfonyl, alkoxymethyl, alkoxyethyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, N-alkylamino a sulfonyl group, an N,N-dialkylaminosulfonyl group or an alkylenedioxy group having a C atom number of from 1 to 3, wherein the alkyl group has a C atom number of from 1 to 4, and the alkoxy group has C The number of atoms is 1-4. A compound of the formula I according to claim 3, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof, among them, X is "single bond"; R ₁ is an alkyl group having a C atom number of 1-3; R ₂ is hydrogen, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, carboxy, nitro, cyano, alkyl, alkoxy, N-alkylamino, N,N-dialkylamino, Alkylthio, alkylsulfonyl, alkoxymethyl, alkoxyethyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, N-alkylamino a sulfonyl group, an N,N-dialkylaminosulfonyl group or an alkylenedioxy group having a C atom number of from 1 to 3, wherein the alkyl group has a C atom number of from 1 to 4, and the alkoxy group has C The number of atoms is 1-4. A compound of the formula I according to claim 3, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof, among them, X is -O(CH ₂ )n-; n is 2; R ₁ is an alkyl group having a C atom number of 1-3; R ₂ is hydrogen, halogen, trifluoromethyl, trifluoromethoxy, hydroxy, carboxy, nitro, cyano, alkyl, alkoxy, N-alkylamino, N,N-dialkylamino, Alkylthio, alkylsulfonyl, alkoxymethyl, alkoxyethyl, carbamoyl, N-alkylcarbamoyl, N,N-dialkylcarbamoyl, N-alkylamino a sulfonyl group, an N,N-dialkylaminosulfonyl group or an alkylenedioxy group having a C atom number of from 1 to 3, wherein the alkyl group has a C atom number of from 1 to 4, and the alkoxy group has C The number of atoms is 1-4. A compound of the formula I according to claim 1, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof, 40-N-(6-chloro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T1) 40-N-(5-chloro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T2) 40-N-(3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T3) 40-N-(4-fluoro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T4) 40-N-(6-fluoro-3-methylbenzothiazol-2-ylidene)-amino rapamycin (RAP-T5) 40-O-(2-(3-methylbenzothiazol-2-imino)ethyl)oxyrapamycin (RAP-T6). A pharmaceutical composition comprising the compound of the formula I according to any one of claims 1 to 6, a derivative thereof, an optical isomer thereof, and a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable composition Acceptable excipients. Use of a compound of the formula I, a derivative thereof, an optical isomer thereof and a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 6, for the preparation of a medicament for the treatment and/or prevention of cancer. The use of claim 8 wherein the cancer is a solid tumor or a non-solid tumor. A process for the preparation of a compound of formula I according to any one of claims 1 to 6, wherein amino compound A-2 is formed from compound A-1, and compound A-2 and quaternary ammonium salt compound A-3 are subjected to an organic base-catalyzed reaction. Obtaining a compound of the formula I;

Wherein R ₁ , R ₂ , X and n are as defined above, respectively.