CN105399757A - Acid-sensitive camptothecin-site 20 norcantharidate derivative and antineoplastic application thereof - Google Patents

Abstract

The invention provides a camptothecin-site 20 norcantharidate derivative with a structural formula as shown in I which is described in the specification, and a preparation method and antineoplastic application thereof. Results of activity test prove that the camptothecin-site 20 norcantharidate derivative as shown in I is an appropriate candidate antineoplastic drug, especially a candidate drug for resisting liver cancers, stomach cancers and colorectal cancers. Compared with positive contrast drugs, i.e., camptothecin and cantharidin, the camptothecin-site 20 norcantharidate derivative has improved water solubility and stability; and the camptothecin-site 20 norcantharidate derivative is sensitive to acid and can be easily hydrolyzed. Moreover, the preparation method for the camptothecin-site 20 norcantharidate derivative employs easily available raw materials, has high yield and is easy to operate and implement.

Description

Acid Sensitive Camptothecin-20 Demethylcantharidinate Derivatives and Its Antitumor Application

technical field

The invention belongs to the field of new drug design and synthesis, and specifically relates to a novel acid-sensitive camptothecin-20-position norcantharidinate derivative and its antitumor application.

Background technique

Campylodendron is endemic to my country and is mainly distributed in the Yangtze River Basin and several southwestern provinces such as Guizhou, Sichuan, and Guangxi. Guizhou is one of the main producing areas of camptophylla, which grows in almost all counties of the province, and many counties have successfully carried out artificial cultivation. Camptothecin (CPT for short) was first extracted from camptothecin by American chemist Wall et al. Camptothecin contains a five-membered ring parallel structure, wherein the AB ring is a quinoline ring, the C ring is a pyrrole ring, the D ring is a conjugated pyridine ring, and the E ring is a chiral center with an S configuration (C20 ) of α-hydroxyl six-membered lactone. The ABCD ring is a huge conjugated system with a planar structure.

Due to the unique anti-tumor mechanism of camptothecin, pharmacists have developed and synthesized a large number of camptothecin derivatives, and several new camptothecin derivatives have successfully entered the clinical research stage or entered the market.

Cantharidin (C ₁₀ H ₁₂ O ₄ ) is an anti-cancer substance extracted from Cantharidin, a folk traditional Chinese medicine in China. It has a special inhibitory effect on ascites liver cancer or primary liver cancer. But cantharidin is in clinical application, and its toxic and side effects are bigger, can not use for a long time. Norcantharidin with two less methyl groups greatly reduces its toxic and side effects, and can better inhibit cancer; it also has a unique effect of stimulating the bone marrow growth of white blood cells.

In order to find more effective and more toxic anticancer drug candidates, we designed and fully synthesized the following drug molecules, linking camptothecin and norcantharidin through the 20-position ester bond to obtain a new type of drug with a unique structure Camptothecin-20-norcantharidinate derivatives.

Contents of the invention

On the one hand, the present invention provides a novel acid-sensitive camptothecin-20-position demethylcantharidinate derivative; its structural formula is shown in formula I,

On the other hand, the present invention provides the synthetic method of camptothecin-20-position norcantharidinate derivative I as described above, comprising the following steps: 1), side chain 5-ene-norcantharidin monoacid benzyl Ester 3 (compound 3) reacts with camptothecin in an organic solvent under the action of a coupling agent and an organic base catalyst to obtain a coupling product 4 (compound 4), 2), compound 4 is catalytically hydrogenated to obtain camptothecin shown in formula I Base-20 norcantharidinate derivatives, the synthetic route is as follows:

The synthetic method of camptothecin-20 position norcantharidinate derivative I, comprises the following steps: 2), compound 4 catalytic hydrogenation obtains the camptothecin-20 position norcantharidate derivative shown in formula I, synthesizes See below for directions:

In a preferred embodiment, the coupling agent described in the above step 1) is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (abbreviated EDCI), bicyclic Hexylcarbodiimide (abbreviated as DCC) and the like; the organic base is selected from triethylamine, diisopropylamine, 4-dimethylaminopyridine (abbreviated as DMAP) and the like.

In a preferred embodiment, the catalyst used in the catalytic hydrogenation reaction described in the above step 2) is selected from palladium carbon and platinum carbon including Pd/C and Pd(OH) ₂ /C; preferably Pd/C; The solvent of step 2) is selected from methanol, ethanol and other alcohol solvents, preferably methanol.

In yet another aspect, the present invention also provides a side chain compound 3 and a preparation method thereof for synthesizing camptothecin-20-position norcantharidinate derivatives shown in formula I, comprising the steps of: a), taking furan as a raw material, and Maleic anhydride (compound 1) reacts in an organic solvent to obtain 5-ene-norcantharidin 2 (compound 2), b), 5-ene-norcantharidin 2 reacts with benzyl alcohol in an organic solvent to obtain Side chain 5-ene-norcantharidin monoacid benzyl ester 3 (compound 3), the synthetic route is as follows:

In a preferred embodiment, the organic solvent used in step a) is an ether solvent or a halogenated hydrocarbon, such as diethyl ether, propyl ether, tetrahydrofuran, dichloromethane or chloroform.

In a preferred embodiment, step a) can be carried out at room temperature, or can be properly heated; room temperature is preferred.

In a preferred embodiment, the organic solvent used in step b) is selected from ether solvents or halogenated hydrocarbons, such as diethyl ether, propyl ether, tetrahydrofuran, dichloromethane or chloroform.

In the above synthesis and preparation methods, the organic solvent can also be selected from N,N-dimethylformamide (abbreviated as DMF), dimethyl sulfoxide (abbreviated as DMSO), dichloro Choose from methane (abbreviated as DCM), chloroform, acetonitrile, tetrahydrofuran or diethyl ether. The reaction temperature can be appropriately selected depending on the type of reaction. The reaction time can be obtained by tracking the reaction status through monitoring means such as thin-layer chromatography TLC, high-performance liquid chromatography HPLC or LC-MS liquid chromatography-mass spectrometry.

The activity test proves that the camptothecin-20 demethylcantharidinate derivatives shown in formula I designed and synthesized by the present invention have good antitumor effects; especially for liver cancer, gastric cancer and colon cancer; in vitro inhibition rate As high as 64%-72%, which is equivalent to the inhibitory activity of positive control drugs camptothecin and cantharidin; it is expected to be applied clinically as cantharidin antitumor drugs.

Therefore, the fourth aspect of the present invention provides the use of camptothecin-20-position norcantharidinate derivatives shown in formula I for the preparation of antitumor drugs; preferably, for the preparation of anti-liver cancer drugs and anti-gastric cancer drugs and the use of anti-colon cancer drugs.

The benefits of the present invention are: the present invention provides camptothecin-20 demethylcantharidinate derivatives shown in formula I, which is a suitable anti-tumor drug candidate, especially as anti-liver cancer, anti-gastric cancer and Drug candidates against colon cancer. With respect to positive contrast drug camptothecin and cantharidin, compound I molecular structure of the present invention has introduced carboxyl, improved water solubility, stability; In addition, due to introducing the similar structure of norcantharidin with oxygen bridging ring, it is for Acids are sensitive and prone to hydrolysis, which enhances pharmacokinetic potency. In addition, the synthesis method of the camptothecin norcantharidin ester derivative of the present invention has easy-to-obtain raw materials, high yield of the synthesis route, and is very easy to operate and implement.

detailed description

The following will further illustrate the present invention through specific examples, but it is not intended to limit the protection scope of the present invention. Without departing from the concept of the present invention, those skilled in the art may make improvements or combinations to the parameters or conditions of the claims, and these improvements or combinations shall also be regarded as the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

The furan and maleic anhydride used in the present invention come from Shanghai Sinopharm Group; the solvent used comes from Zunyi Shuangju Chemical Co., Ltd. Unless otherwise specified, all reagents used were chemically pure.

The synthetic method of aforementioned side chain 5-ene-norcantharidin monoacid benzyl ester 3 of the present invention,

Specifically include the following steps:

1) Take maleic anhydride, grind it finely, add organic solvent to dissolve, add furan dropwise after it is completely dissolved, react at 35°C-45°C for 24 hours, and filter with suction to obtain the white solid product 5-ene-norcantharidin 2. Dry for later use;

2) Suspending the 5-ene-norcantharidin 2 obtained in step a) in benzyl alcohol; then adding triethylamine dropwise to the suspension, stirring at room temperature; rotary evaporation to remove the solvent. The resulting residue was dissolved in dichloromethane, extracted once with 1N (N represents the unit concentration of mol/L in this specification) hydrochloric acid, the organic phase was extracted once with saturated brine, and dried with anhydrous MgSO to obtain ₅ -ene - benzyl norcantharidin monoate 3 as a white solid.

In the above reaction, chromatography and liquid mass spectrometry can be used to monitor the progress of the reaction. In chromatography, thin-layer chromatography can be applied, and gas chromatography or liquid chromatography such as HPLC can be used instead.

The preparation of embodiment 1 , 5-ene-norcantharidin 2:

Take out a certain amount of maleic anhydride 1 from the reagent bottle, place it in a dry grinding body and grind it finely, then weigh 12.021g of the finely ground maleic anhydride with an electronic balance, put it in a dry three-necked flask, and stopper Put on the stopper, add diethyl ether and stir, and when the amount of diethyl ether is 90mL, the maleic anhydride is completely dissolved. After the maleic anhydride was completely dissolved, 13 mL of furan was slowly added through the dropping funnel for 13 minutes. The temperature was controlled to start the reaction at 38°C. After reacting for 1 hour, white solids appeared in the solution, and the longer the time, the more white solids there were. After reacting for 24 hours, it was suction-filtered to obtain Compound 2 as a white solid, namely 5,6-didehydronorcantharidin. The dry weight is 17.459g, and the yield is 85.75%. Melting point: 122-123°C, ratio shift R _f : 0.52 (developing solvent is petroleum ether: ethyl acetate = 3:1); ¹ HNMR (CDCl ₃ ): δ3.18(s, 2H), 5.47(s, 2H), 6.58 (s, 2H).

The organic solvent used to dissolve maleic anhydride in the step a) of the above-mentioned embodiment 1 can also be N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, chloroform, acetonitrile, Any substitution in tetrahydrofuran; the reaction temperature can be between 35°C and 45°C.

Embodiment 2 , the preparation of 5-ene-norcantharidin monoacid benzyl ester 3:

Weigh 5 g of raw material 2 and dissolve it in 40 ml of dichloromethane to form a suspension. Then, 4.7ml of benzyl alcohol and 366mg of DMAP were added dropwise to the suspension. Stirred at room temperature for 3 days, the solvent was removed by rotary evaporation to obtain the crude product of 5,6-didehydronorcantharidin monomethyl ester 2. Dissolve the obtained crude product in 25ml of dichloromethane, wash once with 7ml of 1N hydrochloric acid, leave the organic phase, then wash once with 10ml saturated brine, leave the organic phase, add an appropriate amount of anhydrous _MgSO4 and dry for 15-30 minutes, suction filtration, the filter cake was rinsed 2-3 times with dichloromethane, the filtrate was left, and the filtrate was rotary evaporated to obtain a white solid (4.08g) of 5-ene-norcantharidin monoacid benzyl ester 3, the yield 50%. ¹ HNMR (DMSO-d ₆ ): δ12.43(s, 1H), 7.30-7.35(m, 5H), 7.38(m, 5H), 6.43(d, J=4Hz, 2H), 4.92-5.10(m ,4H); ¹³ CNMR (DMSO-d ₆ ): δ173.07, 171.93, 137.15, 136.97, 128.79, 128.40, 80.49, 80.16, 66.31, 47.14, 46.41.

Example 3. Preparation of camptothecin (2-benzyloxycarbonyl-5-ene) norcantharidinate 4

Add camptothecin (60mg, 0.17mmol) and 10ml methylene chloride in the reaction flask, then add 5-ene-norcantharidin monoacid benzyl ester (3,93.2mg, 0.34mmol, 2equ.), EDCI ( 161.3 mg, 0.84 mmol) and DMAP (13.4 mg, 0.11 mmol). The reaction solution was heated to reflux for 24 hours. Add dichloromethane (30ml) to dilute the reaction solution, and wash with water, saturated sodium carbonate solution and saturated brine successively. The organic phase was dried with anhydrous _MgSO4 . After the solvent was spin-dried, the residue was purified by column chromatography to obtain a coupling product (compound 4, 73.5 mg) as a yellow solid, with a yield of 71.6%. R _f = 0.44 (DCM/MeOH = 95/5). ¹ H NMR (CDCl ₃ ): δ8.39 (s, 1H), 8.20 (d, J = 8Hz, 1H), 7.94 (d, J = 8Hz, 1H ),7.84(d,J=8Hz,1H),7.81(s,1H),7.67(t,J=8Hz,1H),7.38(m,5H),7.18(s,1H),6.99(d,J =4Hz,1H),5.71(d,J=8Hz,1H),5.42(d,J=8Hz,1H),5.27(d,J=12Hz,1H),2.18-2.34(m,2H),1.01( t,J＝8Hz,3H),0.87-0.88(m,4H) ^.13 CNMR(CDCl ₃ ):δ165.84,163.27,162.53,156.27,151.16,147.82,145.41,144.22,134.38,133.94,131.22,130.61, .

Example 4. Preparation of camptothecin-20 demethylcantharidinate derivatives

The compound 4 (500mg) obtained in Example 3 was dissolved in methanol (10mL), and 10% palladium carbon (50mg) was added; the reaction was stirred, and the reaction was stopped after the disappearance of the raw material was detected by TLC. The catalyst was filtered off, and the filtrate was spin-dried to obtain the target compound I as a yellow solid (100%). R _f = 0.29 (DCM/MeOH = 10/1). ¹ HNMR (DMSO-d ₆ ): δ8.67 (s, 1H), 8.17 (d, J = 8Hz, 1H), 8.11 (d, J = 8Hz ,1H),7.87(t,J=8Hz,1H),7.71(t,J=8Hz,1H),7.13(s,1H),5.49(t,J=16Hz,2H),5.27(t,J= 20Hz, 2H), 2.69-2.85(m, 2H), 2.45-2.51(m, 4H), 2.16(d, J=4Hz, 2H), 1.23(s, 2H), 0.92(t, J=8Hz, 3H ). ¹³ CNMR(DMSO-d ₆ ):δ171.81,167.64,156.95,152.79,148.29,146.33,145.71,131.97,130.83,130.18,129.41,128.95,128.37,128.13,119.33,95.56,76.28,66.73,50.62,30.82 ,29.08,7.99.

Example 5, Activity Test of Novel Camptothecin Derivatives

Cell lines and solvents

Human liver cancer cells HEPG2;

Human gastric cancer cell BGC803;

Human colon cancer cells SW480;

Cells were cultured in RPMI1640 medium containing 10% fetal bovine serum;

Solvent: dimethyl sulfoxide (abbreviated as DMSO).

CCK-8 staining method to detect cell anti-tumor activity embodiment

The cells whose ratio of live tumor cells to be tested reaches more than 90% are selected for the experiment. The cell proliferation inhibition test used the EnoGeneCell ^™ CountingKit-8 (abbreviated as CCK-8) cell viability detection kit. Cells were digested, counted, and made into a cell suspension with a concentration of 1×105/mL, and 100 μL of cell suspension was added to each well of a 96-well plate (1×104 cells per well); the 96-well plate was placed at 37°C for 5 %CO2Cultivate in the incubator for 24 hours; add 100 μ L of corresponding drug-containing medium to each well, and the effect concentration is 50 μ Mol/L (ie: micromol/liter), and set up a negative control group, a vehicle control group, and a positive control group ( Cantharidin and camptothecin were selected as positive controls respectively), and each group had 5 multiple wells; the 96-well plate was placed at 37° C., and 5% CO2 was cultivated for 72 hours in an incubator; Incubate in the box for 4 hours, measure the absorbance value at 450nm (abbreviated as OD value) with a microplate reader, and calculate the inhibition rate of each compound on human liver cancer cell HEPG2, human gastric cancer cell BGC803 and colon cancer cell SW480. The experimental results are detailed in Table 1-3.

Table 1: Inhibitory rate of in vitro proliferation activity of human hepatoma cell HEPG2

group Inhibition rate(%) camptothecin 72.46 Cantharidin 78.58 Compound I 72.30

Table 2: Inhibitory rate of in vitro proliferation activity of human gastric cancer cell BGC803

group Inhibition rate(%) camptothecin 63.00 Cantharidin 70.74 Compound I 69.77

Table 3: Inhibitory rate to the in vitro proliferation activity of human colon cancer cell SW480

group Inhibition rate(%) camptothecin 76.77 Cantharidin 70.97 Compound I 64.39

It can be seen from the experimental results in Table 1-3 that the compound I of the present invention has good antitumor activity in vitro, and its 50 μMol/L concentration is as high as 64% in vitro for human liver cancer cell HEPG2, human gastric cancer cell BGC803 and colon cancer cell SW480. 72%, comparable to the inhibitory activity of positive control drugs camptothecin and cantharidin.

In addition, compared with the positive control drugs camptothecin and cantharidin, the molecular structure of compound I of the present invention has introduced carboxyl groups, which has improved water solubility and stability; in addition, due to the introduction of norcantharidin similar structures with oxygen bridge rings, It is acid sensitive and prone to hydrolysis, which enhances pharmacokinetic potency. Therefore, Compound I is a suitable antitumor drug candidate, especially as a candidate drug against liver cancer, gastric cancer and colon cancer.

Claims (6)

1. camptothecin-20 demethylcantharidinate derivatives, its structural formula is as shown in formula I, 2. the synthetic method of camptothecin-20 position demethylcantharidinate derivative I according to claim 1, comprising: 1), the side chain compound 3 and camptothecin react in an organic solvent under the action of a coupling agent and an organic base catalyst to obtain compound 4, 2), compound 4 is reduced by a hydrogenation catalyst to obtain a derivative of formula I, and the synthetic route is as follows : 3. synthetic method according to claim 2, is characterized in that, step 1) described coupling agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or Dicyclohexylcarbodiimide; the organic base is selected from triethylamine, diisopropylamine or 4-dimethylaminopyridine. 4. synthetic method according to claim 2, is characterized in that, step 2) described hydrogenation catalyst is selected from palladium carbon or platinum carbon; The solvent of step 2) is selected from alcohol solvent. 5. The camptothecin-20-position norcantharidate derivative I according to claim 1 is used for the preparation of antitumor drugs. 6. The use according to claim 5, characterized in that the tumor comprises liver cancer, gastric cancer or colon cancer.