Curcumin glycosyl derivative as well as preparation method and medical application thereof

Abstract

The invention discloses a curcumin glycosyl derivative as well as a preparation method and medical application thereof. The structure of the curcumin glycosyl derivative is shown in the specification. According to the invention, D-glucose is utilized to modify a drug through ester bond connection, then the drug is targeted to GLUT1, and the drug is transferred to penetrate through a blood brain barrier to reach brain tissues, so that the targeting property of the drug is remarkably improved. In addition, through F atom modification, the bond energy and reactivity of F atoms are lower than those of H atoms, the molecular weight of F atoms is smaller than that of other closed groups, and the influence on the molecular size is small. The compound can block sites which are prone to oxidative metabolism, so that the metabolic stability of the compound is improved, and the action time of the medicine in vivo is prolonged. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of curcumin glycosyl derivatives, and specifically relates to a curcumin glycosyl derivative and a preparation method and medical use thereof. Background Art

[0002] Curcumin is a natural product isolated from the rhizome extract of turmeric, a plant of the genus Curcuma in the ginger family. Studies have shown that it has multiple activities such as anti-inflammatory, antioxidant and anti-proliferative effects, and its anti-tumor effect is a hot topic for scientific researchers. At present, numerous in vivo and in vitro experiments have proved that curcumin has a wide range of anti-tumor activities and has low toxic side effects on normal cells. These results show that curcumin has great development potential in tumor treatment.

[0003] Previous studies have shown that the curcumin derivative DMC-BH has a certain therapeutic effect on brain glioma (Aging (Albany NY). 2020, 12 (23): 23795-23807; Aging (Albany NY). 2020, 12 (14): 14718-14735.), but its ability to penetrate the blood-brain barrier is not strong enough, and its plasma stability is poor and it is easily metabolized. Improving the metabolic stability of DMC-BH and enhancing its ability to penetrate the blood-brain barrier is expected to develop a curcumin derivative with better drugability and develop it into an effective clinical anti-glioma drug. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the purpose of the present invention is to provide a curcumin glycosyl derivative and its preparation method and medical use. The present invention obtains a curcumin derivative with good metabolic stability and GLUT1 transport activity by structural modification of curcumin, and provides a preparation method of the derivative and its application in the field of anti-tumor.

[0005] Technical solution: In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0006] A curcumin glycosyl derivative and a pharmaceutically acceptable salt thereof, wherein the structure of the curcumin glycosyl derivative is as follows:

[0007]

[0008] The present invention also provides a method for preparing the curcumin glycosyl derivative, comprising the following steps:

[0009]

[0010] (1) 4-Fluoro-3-formylbenzoic acid reacts with N,O-dimethylhydroxylamine hydrochloride to generate intermediate 1;

[0011] (2) Intermediate 1 reacts with tetrahydropyrrole to generate intermediate 2;

[0012] (3) Compound 2 generates intermediate 3 under the action of a reducing agent;

[0013] (4) Vanillin and acetyl bromide-α-D-glucose react under alkaline conditions to produce intermediate 4;

[0014] (5) Acetylacetone reacts with intermediate 4 to generate intermediate 5;

[0015] (6) reacting intermediate 3 with intermediate 5 to generate intermediate 6;

[0016] (7) reacting the intermediate 6 under acidic conditions to obtain the curcumin glycosyl derivative.

[0017] Preferably, the reaction formula is as follows:

[0018]

[0019] Preferably, in step (1), the reaction system further comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) and dichloromethane; the reaction is carried out under stirring at room temperature for 10-14 hours;

[0020] Preferably, the reaction is carried out in methanol, the reaction system further comprises sodium triacetoxyborohydride and glacial acetic acid, and the reaction is carried out at 60-70° C. for 10-14 hours.

[0021] Preferably, the reaction is carried out in anhydrous ether, the reducing agent is selected from lithium aluminum hydride, and the reaction is stirred under ice bath conditions for 1-3 hours;

[0022] Preferably, in step (4), the base of the alkaline condition is selected from anhydrous potassium carbonate, and the reaction is carried out in N,N-dimethylformamide; the reaction is carried out at 40-60° C. for 1-3 hours.

[0023] Preferably, in step (5), the reaction method comprises the following: dissolving acetylacetone and boron trioxide in ethyl acetate, heating to 80-90°C and reflux, reacting for 0.5-1.5h, then cooling to 45-55°C, adding intermediate 4 and tri-n-butyl borate, dissolving n-butylamine in ethyl acetate and slowly adding dropwise, maintaining the temperature at 50±2°C, reacting for 1.5-2.5h, and then adding hydrochloric acid solution to adjust the pH.

[0024] Preferably, in step (6), the reaction method comprises the following: dissolving the intermediate 5 and boron trioxide in ethyl acetate, heating to 80-90°C and reflux, reacting for 0.5-1.5h, then cooling to 45-55°C, adding 4-fluoro-3-(pyrrolidin-1-ylmethyl)benzaldehyde and tri-n-butyl borate, dissolving n-butylamine in ethyl acetate and slowly adding dropwise, maintaining the temperature at 50±2°C, reacting for 1.5-2.5h, and then adding hydrochloric acid solution to adjust the pH.

[0025] Preferably, in step (7), the acidic condition is selected from 2N HCl solution, the reaction is carried out in dichloromethane, and the reaction is carried out at 55-65° C. for 1.5-2.5 hours; after the reaction is completed, the crude product is recrystallized with dichloromethane and petroleum ether to obtain the pure curcumin glycosyl derivative.

[0026] The present invention also provides a pharmaceutical composition, which contains the curcumin glycosyl derivative and / or its pharmaceutically acceptable salt.

[0027] Finally, the present invention provides the curcumin glycosyl derivative and / or its pharmaceutically acceptable salt, and the use of the pharmaceutical composition in preparing drugs for treating tumors.

[0028] Furthermore, the tumor is lung cancer or brain glioma.

[0029] Beneficial effects: The present invention uses D-glucose to modify the drug through an ester bond connection, and then targets it to GLUT1, and transports it through the blood-brain barrier to reach the brain tissue, significantly improving the targeting of the drug. In addition, through F atom modification, because its bond energy and reactivity are lower than H atoms, and its molecular weight is smaller than other blocking groups, it has less effect on the molecular size. It can block sites prone to oxidative metabolism, thereby improving the metabolic stability of the compound and prolonging the drug's action time in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a schematic diagram of the design principle of the curcumin glycosyl derivatives of the present invention. DETAILED DESCRIPTION

[0031] The present invention is further described by the following examples. These examples are purely illustrative and are only used to specifically describe the present invention and should not be construed as limiting the present invention. The invention is further described below in conjunction with the accompanying drawings and examples:

[0032] Example 1 Preparation of Intermediate 1

[0033]

[0034] 4-Fluoro-3-formylbenzoic acid (1.0 g), N,O-dimethylhydroxylamine hydrochloride (0.87 g), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.7 g), and 4-dimethylaminopyridine (1.09 g) were dissolved in 30 ml of dichloromethane in sequence, stirred at room temperature, and reacted for 12 h. The reaction was completed when the starting material disappeared after TLC detection. The solvent was spin-dried, extracted with EA, and the organic layer was purified by HPLC. 2 O and saturated brine three times, dried over anhydrous sodium sulfate, allowed to stand, filtered, and the solvent was distilled off under reduced pressure. The mixture was separated by silica gel column chromatography to obtain 0.94 g (yield 75%) of colorless oily liquid (Intermediate 1). 1 H NMR (300 MHz, DMSO-d 6 )δ10.23(s,1H),8.08(dd,J=6.8,2.3Hz,1H),8.00(ddd,J=8.7,5.1,2.3Hz,1H),7.51(dd,J=10.5,8.6Hz,1H),3.55(s,3H),3.29(s,3H).ESI-MS m / z:212.1[M+H] + .

[0035] Example 2 Preparation of Intermediate 2

[0036]

[0037] The intermediate 1 (0.7 g) was dissolved in methanol, and tetrahydropyrrole (0.35 g), sodium triacetoxyborohydride (0.84 g) and glacial acetic acid (0.02 ml) were added. The mixture was reacted at 65 °C for 12 h. The reaction was completed after the disappearance of the starting material by TLC. The solvent was dried by spin drying, and the organic layer was extracted with EA. 2 O and saturated brine for 3 times, dried over anhydrous sodium sulfate, allowed to stand, filtered, and the solvent was distilled off under reduced pressure. The mixture was separated by silica gel column chromatography to obtain 0.57 g (yield 65%) of a yellow oily liquid (Intermediate 2). 1 H NMR (300 MHz, DMSO-d 6 )δ7.67(dd,J=7.2,2.3Hz,1H),7.56(ddd,J=8.5,5.1,2.3Hz,1H),7.23(dd,J=9.9,8.5Hz,1H ),3.68-3.62(m,2H),3.53(s,3H),3.25(s,3H),2.48-2.43(m,4H),1.72-1.66(m,4H).ESI-MS m / z:267.1[M+H] + .

[0038] Example 3 Preparation of Intermediate 3

[0039]

[0040] Weigh (0.24 g) lithium aluminum hydride and dissolve it in 10 ml anhydrous ether, and stir at room temperature for 30 min; dissolve intermediate 2 (1.5 g) in 20 ml anhydrous ether solution, and slowly add it dropwise to the anhydrous ether solution of lithium aluminum hydride at -40 ° C. After the addition is completed, move the reaction to an ice bath and stir for 2 h. The reaction is completed when the raw material disappears by TLC detection; spin dry the solvent, extract with EA, and separate the organic layer with H 2 O and saturated brine for three times, dried over anhydrous sodium sulfate, allowed to stand, filtered, and the solvent was distilled off under reduced pressure. The mixture was separated by silica gel column chromatography to obtain 0.793 g (yield 68%) of a yellow oily liquid (intermediate 3). 1 H NMR (300 MHz, DMSO-d 6 )δ9.99(s,1H),7.99(dd,J=7.2,2.2Hz,1H),7.89(ddd,J=7.8,5.2,2.2Hz,1H),7.41(dd,J=9.8,8 .4Hz,1H),3.69(d,J=1.6Hz,2H),2.49(tdd,J=6.7,4.0,1.7Hz,4H),1.70(h,J=3.2Hz,4H).ESI-MS m / z:208.1[M+H] + .

[0041] Example 4 Preparation of Intermediate 4

[0042]

[0043] Vanillin (1.0 g), acetyl bromide-α-D-glucose (3.24 g), and potassium carbonate (2.72 g) were dissolved in 50 mL of N,N-dimethylformamide, and the temperature was raised to 50 °C. The reaction was completed after TLC detection of the disappearance of the raw material. The organic layer was extracted with EA and the reaction mixture was purified by HPLC. 2 O and saturated brine for three times, dried over anhydrous sodium sulfate, allowed to stand, filtered, and the solvent was distilled off under reduced pressure. The mixture was separated by silica gel column chromatography to obtain 2.5 g (yield 80%) of a white solid (intermediate 4). 1 H NMR (300 MHz, DMSO-d 6)δ9.85(s,1H),7.51(dd,J=7.6,1.6Hz,1H),7.39(d,J=1.6Hz,1H),7.15(d,J= 7.5Hz,1H),5.30(t,J=6.9Hz,1H),5.26(t,J=6.9Hz,1H),5.16(t,J=6.8Hz,1H) ,4.93(d,J=6.8Hz,1H),4.28(dd,J=12.2,6.9Hz,1H),4.15(dd,J=12.2,7.0Hz ,1H),4.09(q,J=6.9Hz,1H),3.83(s,2H),2.06(dd,J=6.8,1.5Hz,12H).ESI-MS m / z:483.1[M+H] + .

[0044] Example 5 Preparation of Intermediate 5

[0045]

[0046] Acetylacetone (5 g) and boron trioxide (2.5 g) were dissolved in 60 ml of ethyl acetate, heated to 85 ° C and refluxed, reacted for 1 h, then cooled to 50 ° C, compound 4 (4.0 g) and tri-n-butyl borate (4.8 g) were added, n-butylamine (1.0 g) was dissolved in 10 ml of ethyl acetate and slowly added dropwise, the temperature was maintained at 50 ° C, and the reaction was completed after the raw material disappeared by TLC detection; 1 mol / L hydrochloric acid solution was added to adjust the pH, EA was extracted, and the organic layer was washed with H 2 O and saturated brine three times, dried over anhydrous sodium sulfate, allowed to stand, filtered, and the solvent was distilled off under reduced pressure. The mixture was separated by silica gel column chromatography to obtain 2.43 g (yield 52%) of a light yellow oily solid (Intermediate 5). 1 H NMR (300 MHz, DMSO-d 6 )δ7.51-7.44(m,1H),7.18-7.09(m,3H),6.66(d,J=15.0Hz,1H),5.31(t,J=6.9Hz,1H),5.26(t,J=6.9Hz,1H),5.19(t,J=6.9Hz,1H),4.9 3(d,J=6.8Hz,1H),4.23(dd,J=7.0,1.8Hz,2H),4.07(q,J=7.0Hz,1H),3.89(s,2H),3.76(s,2H),2.21(s,2H),2.10-2.02(m,11H).ESI-MS m / z:565.2[M+H] + .

[0047] Example 6 Preparation of Intermediate 6

[0048]

[0049] Compound 5 (0.5 g) and boron trioxide (0.075 g) were dissolved in 15 ml of ethyl acetate, heated to 85 ° C and refluxed, reacted for 1 h, then cooled to 50 ° C, intermediate 3 (0.22 g) and tri-n-butyl borate (0.2 g) were added, n-butylamine (0.07 g) was dissolved in 5 ml of ethyl acetate and slowly added dropwise, the temperature was maintained at 50 ° C, and the reaction was carried out for 12 h. The reaction was completed when the raw material disappeared by TLC detection; 1 mol / L hydrochloric acid solution was added to adjust the pH, EA was extracted, and the organic layer was washed with H 2 O and saturated brine three times, dried over anhydrous sodium sulfate, allowed to stand, filtered, and the solvent was distilled off under reduced pressure. The mixture was separated by silica gel column chromatography to obtain 0.226 g (yield 34%) of a yellow solid (Intermediate 6). 1 HNMR (300 MHz, DMSO-d 6 )δ7.57-7.43(m,4H),7.25(t,J=7.9Hz,1H),7.18-7.10(m,3H),6.65(d,J=15.1Hz,2H),5. 31-5.23(m,2H),5.19-5.12(m,1H),4.97-4.90(m,1H),4.27-4.15(m,2H),4.07(q,J=6.9H z,1H),3.89(s,2H),3.70(dd,J=12.3,1.0Hz,1H),3.65(dd,J=12.4,0.9Hz,1H),3.48(s,2 H),2.84(dddd,J=7.6,4.9,2.8,0.8Hz,4H),2.10-2.03(m,11H),1.78-1.66(m,4H).ESI-MS m / z:754.3[M+H] + .

[0050] Example 7 Preparation of Compound DMC-GF

[0051]

[0052] Compound 6 (0.1 g) was dissolved in 10 ml of dichloromethane, and 10 ml of 2N HCl aqueous solution was added. The mixture was reacted at 60°C for 2 h. TLC showed that the starting material disappeared and the reaction was complete. The solvent was dried and recrystallized from dichloromethane and petroleum ether to obtain 0.06 g of a yellow solid (compound DMC-GF) (yield 77%). 1 H NMR (300 MHz, DMSO-d 6)δ7.58-7.49(m,3H),7.49(dd,J=4.9,0.7Hz,1H),7.22-7.13(m,3H),7.15-7.09(m,1 H),6.66(d,J=15.1Hz,2H),5.12(d,J=6.9Hz,1H),4.82(d,J=10.1Hz,1H),4.78(d,J=9 .7Hz,1H),4.73(d,J=10.1Hz,1H),4.47(t,J=7.5Hz,1H),3.91-3.55(m,10H),3.48(s ,2H),3.40(dtd,J=9.7,6.9,1.8Hz,1H),2.89-2.79(m,4H),1.80-1.66(m,4H).ESI-MS m / z:586.2[M+H] + ..

[0053] Experimental Examples Pharmacological and pharmacokinetic experimental results of the compounds of the present invention:

[0054] (I) Anti-tumor activity test

[0055] 1. Cell digestion, counting, and preparation of a concentration of 5×10 4 100 μL of cell suspension (5×10 cells / mL) was added to each well of a 96-well plate. 3 cells);

[0056] 2. Place the 96-well plate at 37°C, 5% CO 2 Culture in an incubator for 24 hours;

[0057] 3. Dilute the drug to the required concentration with complete culture medium, add 100 μL of the corresponding drug-containing culture medium to each well, and set up negative control group, solvent control group, and positive control group;

[0058] 4. Place the 96-well plate at 37°C, 5% CO 2 Culture in an incubator for 72 hours;

[0059] 5. Perform MTT staining on the 96-well plate, λ=490nm, and measure the OD value.

[0060] 1) Add 20 μL MTT (5 mg / mL) to each well and continue to culture in the incubator for 4 hours;

[0061] 2) Discard the culture medium, add 150 μL DMSO to each well to dissolve, and gently mix on a shaker for 10 minutes; read the OD value of each well with an enzyme-labeled instrument at λ=490 nm, and calculate the inhibition rate.

[0062] Cell inhibition rate % = 100% × (OD value of negative control group - OD value of compound group) / OD value of negative control group

[0063] 6. Experimental Results

[0064] Table 1. Antiproliferative activity (IC) of compound DMC-GF against different tumor cell lines 50 : Unit: μg / mL)

[0065]

[0066]

[0067] (II) Liver microsomal stability of compound DMC-GF

[0068] 1. Experimental materials: ITEM (VENDOR); mouse liver microsomes (RILD); BSA (Solarbio); TRIZMABASE (Sigma); MgCl 2 (Sigma); NADPH (Solarbio); Acetonitrile (Merck); Methanol

[0069] (Merck).

[0070] 2. Experimental equipment: Mass Spectrometer (Waters 529); UPLC (UPLC H-Class, Waters); Temperature (Controlling System KK25V61TI, Seimens); Microplate Reader

[0071] (INFINITE 200PRO,TECAN).

[0072] 3. Incubation system: Add liver microsomes to 0.1M TRIS buffer solution at pH 7.4 (final concentration 0.33 mg / mL), and add the co-incubation factor MgCl 2 (final concentration 5 mM) and test compound (final concentration 1 μM, solvent (0.01%

[0073] DMSO) and 0.005% fetal bovine serum (BSA)) were incubated at 37°C for 10 min. NADPH (final concentration 1

[0074] The reaction was started after 500 mM. The solution was aliquoted at 0, 7, 17, 30 and 60 min and methanol (4°C) was added to terminate the reaction. After centrifugation (4000 rpm, 5 min), the samples were analyzed for compound content by LC-MS / MS.

[0075] 4. Experimental Results

[0076] Table 2: Liver microsome stability results

[0077]

[0078] (III) Study on brain permeability of compound DMC-GF in ICR mice

[0079] 1. Experimental animals: Male ICR mice were allowed free access to food and water during feeding and administration, and fasted 12 hours before blood collection.

[0080] 2. Experimental groups: 15 male ICR mice were divided into 5 time points (15min, 45min, 2h, 6h, 12h), 3 mice at each time point, and the drugs were given IV at a dose of 1.0 mg / kg.

[0081] 3. Blood collection: After the mice were administered, plasma and brain tissue were collected at the corresponding time points of each group. After collection, the plasma samples were placed in an EP tube anticoagulated with EDTA-K2 and centrifuged at 8000rpm for 5 minutes at 4°C, and the plasma was transferred to -20°C as soon as possible for storage for testing. The brain tissue was stripped of surface blood vessels with filter paper and placed in a corresponding numbered ziplock bag, which was stored at -20°C for testing. Before testing, 4 times the volume of 20% MeOH was added for homogenization, and a certain volume of samples was taken for pretreatment before LC-MS / MS testing.

[0082] 4. Detection: The concentration of the compound in the collected plasma / brain homogenate samples was determined using the established LC-MS / MS detection method. The pharmacokinetic parameters were calculated using Winnonlin 5.2.

[0083] 5. Experimental Results

[0084] Table 3. Contents in plasma / brain homogenate after administration of compound DMC-GF (ng / mL)

[0085]

[0086]

[0087] The above describes the implementation mode of the present invention in detail with reference to the accompanying drawings and specific embodiments. However, the present invention is not limited to the above implementation mode. Various changes can be made within the knowledge scope of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A curcumin glycosyl derivative and a pharmaceutically acceptable salt thereof, It is characterized in that The structure of the curcumin glycosyl derivative is shown below:

2. The method for preparing the curcumin glycosyl derivative according to claim 1, It is characterized in that The following steps are involved: (1) 4-Fluoro-3-formylbenzoic acid reacts with N,O-dimethylhydroxylamine hydrochloride to generate intermediate 1; (2) Intermediate 1 reacts with tetrahydropyrrole to generate intermediate 2; (3) Compound 2 generates intermediate 3 under the action of a reducing agent; (4) Vanillin and acetyl bromide-α-D-glucose react under alkaline conditions to produce intermediate 4; (5) Acetylacetone reacts with intermediate 4 to generate intermediate 5; (6) reacting intermediate 3 with intermediate 5 to generate intermediate 6; (7) reacting the intermediate 6 under acidic conditions to obtain the curcumin glycosyl derivative.

3. The method for preparing the curcumin glycosyl derivative according to claim 2, It is characterized in that In step (1), the reaction system further comprises 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) and dichloromethane; the reaction is carried out under stirring at room temperature for 10-14 hours; In step (2), the reaction is carried out in methanol, and the reaction system also includes sodium triacetoxyborohydride and glacial acetic acid, and the reaction is carried out at 60-70° C. for 10-14 hours.

4. The method for preparing the curcumin glycosyl derivative according to claim 2, It is characterized in that In step (3), the reaction is carried out in anhydrous ether, the reducing agent is selected from lithium aluminum hydride, and the reaction is stirred under ice bath conditions for 1-3 hours; In step (4), the base of the alkaline condition is selected from anhydrous potassium carbonate, and the reaction is carried out in N,N-dimethylformamide; the reaction is carried out at 40-60° C. for 1-3 hours.

5. The method for preparing the glycosyl derivative of curcumin according to claim 2, It is characterized in that In step (5), the reaction method comprises the following steps: dissolving acetylacetone and boron trioxide in ethyl acetate, heating to 80-90°C and reflux, reacting for 0.5-1.5h and then cooling to 45-55°C, adding intermediate 4 and tri-n-butyl borate, dissolving n-butylamine in ethyl acetate and slowly adding dropwise, maintaining the temperature at 50±2°C, reacting for 1.5-2.5h, and then adding hydrochloric acid solution to adjust the pH.

6. The method for preparing the curcumin glycosyl derivative according to claim 2, It is characterized in that In step (6), the reaction method comprises the following steps: dissolving the intermediate 5 and boron trioxide in ethyl acetate, heating to 80-90°C and reflux, reacting for 0.5-1.5h, then cooling to 45-55°C, adding 4-fluoro-3-(pyrrolidin-1-ylmethyl)benzaldehyde and tri-n-butyl borate, dissolving n-butylamine in ethyl acetate and slowly adding dropwise, maintaining the temperature at 50±2°C, reacting for 1.5-2.5h, and then adding hydrochloric acid solution to adjust the pH.

7. The method for preparing the glycosyl derivative of curcumin according to claim 2, It is characterized in that In step (7), the acidic condition is selected from 2N HCl solution, the reaction is carried out in dichloromethane, and the reaction is carried out at 55-65° C. for 1.5-2.5 hours; after the reaction is completed, the crude product is recrystallized with dichloromethane and petroleum ether to obtain the pure curcumin glycosyl derivative.

8. A pharmaceutical composition comprising the curcumin glycosyl derivative and / or a pharmaceutically acceptable salt thereof as claimed in claim 1.

9. Use of the curcumin glycosyl derivative and / or its pharmaceutically acceptable salt according to claim 1 and the pharmaceutical composition according to claim 8 in the preparation of a drug for treating tumors.

10. The use according to claim 9, It is characterized in that The tumor is lung cancer or brain glioma.

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