CN105985399B - The preparation method and purposes of (20S, 24R/S)-epoxy -12 β, 25- hydroxyl-dammarane-3 beta-amine derivative - Google Patents

Abstract

The present invention relates to organic syntheses and medicinal chemistry art, and in particular to dammarane's derivative of a kind of structure novel, structure such as formula (I) and (II).The invention also discloses 12 β of (20S, 24R/S) epoxy, the preparation method of 25 hydroxyl dammarane, 3 β amine derivatives, the pharmaceutical composition containing them and its applications in preparing reverse multiple drug resistance of tumor drug.

Description

Preparation of (20S, 24R/S)-epoxy-12β, 25-hydroxy-dammarane-3β-amine derivatives Preparation method and use

technical field

The present invention relates to the fields of organic synthesis and medicinal chemistry, in particular to (20S, 24R/S)-epoxy-12β, 25-hydroxyl-dammarane-3β-amine derivatives, and the present invention also discloses these dammarane derivatives The preparation method of the compound, the pharmaceutical composition containing the compound and the application of the compound in the preparation of drugs for reversing tumor multidrug resistance.

technical background

The emergence of antibiotics and chemotherapeutic drugs has made great contributions to human health, but with the application of these drugs, pathogens and tumor cells are less sensitive to chemotherapeutic drugs, resulting in drug resistance, and the emergence of drug resistance has become serious threaten human health. It has been found that although the mechanisms of drug resistance are diverse, the common main reason is that some active transporters pump a series of molecules with different chemical structures out of target cells, such as antibiotics, antibacterial drugs, antimalarials, and tumor chemotherapy drugs. Overexpression of drug transporters (efflux pumps) in tumor and bacterial cells is an important factor in the development of multidrug resistance. These drug efflux pumps are potential targets for overcoming multidrug resistance, and inhibition of membrane transporters can effectively enhance chemotherapeutic drug sensitivity. Among them, the largest class of membrane transporters is the ABC transporter (ATP-binding cassettetransporter, ATP-binding cassette protein) superfamily, which is known to be associated with tumor multidrug resistance (multidrug resistance, MDR). Members include P-glycoprotein (P-glycoprotein, P-gp), multidrug resistance-associated protein (MRP) and breast cancer multidrug resistance protein (BCRP). Currently, a large number of compounds of natural or synthetic origin have been demonstrated to have membrane transporter inhibitory and multidrug-reversal activities.

It has been reported in the literature that ginsenogenin protopanaxadiol (PPD) and ocotilol-type triterpenes have the effect of P-glycoprotein inhibition and reversal of tumor multidrug resistance [Bioorg Med Chem, 2013, 21 (14): 4279-4287.Bioorg Med Chem, 2010, 18(8): 2964-2975.]. In the process of structural modification and transformation of protopanaxadiol, the present inventor obtained a novel ocotilol-type triterpene saponin, and further structural modification obtained (20S, 24R/S)-epoxy-12β, 25- Hydroxy-dammarane-3β-amine derivatives. Pharmacological tests have proved that these derivatives can strongly reverse the tumor multidrug resistance mediated by P-glycoprotein.

Contents of the invention

The present invention provides a compound represented by formula (I) or (II) or a pharmaceutically acceptable salt thereof. The present invention provides a series of preparation methods of compounds with general formula (I) or (II) structural characteristics. It also includes the application of the compound of general formula (I) or (II) or its salt in the preparation of drugs for reversing tumor multidrug resistance.

Wherein R represents hydrogen, (C1-C4) straight chain alkyl, acyl.

The present invention preferably following any compound or pharmaceutically acceptable salt thereof:

(20S, 24S)-epoxydammarane-12β, 25-diol-3β-amine;

(20S, 24R)-epoxydammarane-12β, 25-diol-3β-amine;

(20S, 24S)-epoxy-3β-methylaminodammarane-12β, 25-diol;

(20S, 24R)-epoxy-3β-methylaminodammarane-12β, 25-diol;

(20S,24S)-epoxy-3β-acetamidodammarane-12β,25-diol;

(20S,24R)-epoxy-3β-acetamidodammarane-12β,25-diol;

Compounds of general formula (I) and (II) can be prepared according to the following reaction schemes and descriptions:

Using protopanaxadiol as raw material, acetylation protects the 3 and 12 hydroxyl groups, carries out epoxidation and intramolecular affinity attack, and deacetylates to obtain (20S, 24R/S)-epoxydammarane-3β, 12β , 25-triol; 3-oxidation into ketone, into oxime, amination; alkylation and acylation to obtain formula (I) and (II) derivatives.

The pharmaceutically acceptable salt of the compound of the present invention is characterized in that it refers to a conventional acid addition salt, which has the same pharmaceutical effect as the compound, and is formed with a suitable non-toxic organic acid or inorganic acid.

The invention also discloses a pharmaceutical composition, which contains the compound of the invention or a pharmaceutically acceptable salt thereof, which can be prepared by adding a pharmaceutically acceptable carrier to make common pharmaceutical preparations, such as tablets, capsules, powders, and syrups , liquids, suspensions, injections, commonly used pharmaceutical excipients such as spices, sweeteners, liquid or solid fillers or diluents can be added.

The clinical administration of the compound of the present invention can be oral administration, injection and the like.

The clinically used dose of the compound of the present invention is 0.01 mg-1000 mg/day, and may also deviate from this range according to the severity of the disease or different dosage forms.

The following are the pharmacological experiment results of some compounds of the present invention, and the compound codes used in the tests are shown in the examples.

laboratory apparatus:

microplate

Micro Adjustable Pipette

OPSYS microplate reader (DYNEX Technology, Inc., Chantilly, VA).

Liquid scintillation analyzer (Packard Instrument Company, Inc (Downers Grove, IL))

Reagent materials:

paclitaxel, vincristine, cisplatin, verapamil,

Dulbecco's modified Eagle's medium(DMEM),fetal bovine serum(FBS),DMSO

Cell line:

SW620, SW620/Ad300

HEK293/pcDNA3.1, HEK/ABCB1,

experimental method:

1. Cell digestion, counting, and making a cell suspension with a concentration of 5×10 ⁴ cells/mL, adding 100ul of cell suspension to each well of a 96-well plate (5×10 ³ cells per well);

2. Place the 96-well plate in a 37°C, 5% CO ₂ incubator for 24 hours;

3. Dilute the drug to the required concentration with the complete medium, add 100 μL of the corresponding drug-containing medium to each well, and set up a negative control group, a vehicle control group, and a positive control group at the same time;

4. Place the 96-well plate in a 37°C, 5% CO ₂ incubator for 72 hours;

5. The 96-well plate was stained with MTT, λ=570nm, and the OD value was measured.

1) Add 20 μL of MTT (5 mg/mL) to each well and continue culturing in the incubator for 4 hours;

2) Discard the medium, add 150 μL DMSO to each well to dissolve, shake gently for 10 minutes; λ=570nm,

Read the OD value of each well with a microplate reader, and calculate the inhibition rate and IC ₅₀ value. Drug resistance (Resistance Fold)

The IC ₅₀ value of the drug on normal sensitive cancer cells is divided by the IC ₅₀ value on MDR cells.

The experimental results are shown in Table 1 and Table 2:

Table 1. Reversion of P-glycoprotein-mediated multidrug resistance by ORA and OSA in P-glycoprotein overexpressing tumor cells.

Table 2. Reversion of ORA and OSA to P-glycoprotein-mediated multidrug resistance in transfected cells.

Detailed ways

The present invention will be further elaborated below in conjunction with specific examples, but the present invention is not limited to these examples.

Example 1

(20S, 24S)-epoxydammarane-12β, 25-diol-3β-amine (OSA)

Dissolve 20(S)-protopanaxadiol (500mg, 1.08mmol) in anhydrous pyridine (3mL), add DMAP (20mg, 0.16mmol), stir well and slowly add acetic anhydride (0.42mL, 4.43mmol) dropwise , stirred at room temperature for 12h. Dilute with ethyl acetate (20 mL), wash with 10% hydrochloric acid until acidic, wash with water, wash with saturated brine, dry over anhydrous sodium sulfate, filter, concentrate, and column chromatography (petroleum ether: ethyl acetate = 10:1) to give white Solid 1 (508 mg, 85%).

The above-obtained 1 (208 mg, 0.38 mmol) was dissolved in anhydrous dichloromethane (6 mL), and the dichloromethane of m-chloroperoxybenzoic acid (185 mg, 75%, 0.16 mmol) was slowly added dropwise under ice-salt bath precooling. Methane (5 mL) solution, half an hour after the dropwise addition was completed, the mixture was raised to room temperature and stirred for 2 h. Add isopropanol (0.1mL), continue to stir for one hour, add saturated sodium bicarbonate solution and stir for one hour, then extract by separation, the organic phase is washed successively with saturated sodium thiosulfate solution, water, saturated brine, anhydrous sulfuric acid It was dried over sodium, filtered, concentrated, and column chromatographed (petroleum ether: ethyl acetate = 8:1) to obtain 2 (132 mg, 62%) as a white solid.

The above obtained 2 (132mg, 0.24mmol) was dissolved in DMSO (8mL) and H ₂ O (2mL), potassium hydroxide (85mg, 1.52mmol) was added, and the reaction was stirred at 135°C for 2h. After the reaction solution was cooled to room temperature, an appropriate amount of water was added, and a large amount of white solid precipitated out, which was filtered by suction, dried, and column chromatography (petroleum ether: ethyl acetate = 2:1-1:1) to obtain white solid compound 3 [(20S ,24S)-epoxydammarane-3β,12β,25-triol] (50mg, 43.3%) and white compound 4[(20R,24S)-epoxydammarane-3β,12β,25-triol ].

Compound 3: ¹ H NMR (CDCl ₃ , 300MHz) δ3.87(dd, J=10.2Hz, 5.1Hz, 1H), 3.52(td, J=10.2Hz, 4.8Hz, 1H), 3.19(dd, J= 10.8Hz, 5.4Hz, 1H), 2.25(td, J=10.5Hz, 4.2Hz, 1H), 1.27(s, 3H), 1.23(s, 3H), 1.10(s, 3H), 1.01(s, 3H ), 0.97 (s, 3H), 0.91 (s, 3H), 0.88 (s, 3H), 0.78 (s, 3H); MS (ESI) m/z: 477.3 [M+H] ⁺ .

Compound 4: ¹ H NMR (CDCl ₃ , 300MHz) δ 3.84 (dd, J=8.4Hz, 6.6Hz, 1H), 3.52 (td, J=10.5Hz, 4.8Hz, 1H), 3.19 (dd, J= 10.8Hz, 5.1Hz, 1H), 2.19(td, J=10.8Hz, 3.6Hz, 1H), 1.28(s, 3H), 1.27(s, 3H), 1.10(s, 3H), 0.99(s, 3H ), 0.97 (s, 3H), 0.90 (s, 3H), 0.86 (s, 3H), 0.78 (s, 3H); MS (ESI) m/z: 477.3 [M+H] ⁺ .

Compound 3 (40 mg, 0.08 mmol) was dissolved in anhydrous dichloromethane (3 mL), PCC (pyridinium chlorochromate, 36 mg, 0.17 mmol) was added, and stirred at room temperature for 3 hours. The solvent was removed under reduced pressure, dissolved in ethyl acetate, extracted, the organic layer was washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography (petroleum ether:ethyl acetate=10:1) gave a white solid 5 (20S,24S)-epoxydammarane-12[beta],25-diol-3-one (23 mg, 58%). ¹ H NMR (CDCl ₃ , 500MHz) δ3.88(dd, J=10.5Hz, 5.0Hz, 1H), 3.55(td, J=10.0Hz, 4.5Hz, 1H), 2.49-2.54(m, 1H), 2.45(ddd, J=11.0Hz, 8.0Hz, 3.5Hz, 1H), 2.24-2.29(td, J=10.5Hz, 4.5Hz, 1H), 1.28(s, 3H), 1.23(s, 3H), 1.11 (s, 3H), 1.08(s, 3H), 1.053(s, 3H), 1.046(s, 3H), 0.96(s, 3H), 0.93(s, 3H); MS(ESI) m/z: 475.3 [M+H] ⁺ .

Compound 5 (80mg, 0.17mmol) was dissolved in anhydrous pyridine, hydroxylamine hydrochloride (28mg, 0.39mmol) was added at room temperature, and reacted at 60°C for three hours. Diluted with ethyl acetate, washed with 10% hydrochloric acid until acidic, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography (petroleum ether: ethyl acetate = 1:1) to give a white solid 7 ( 20S,24S)-epoxydammarane-12[beta],25-diol-3-ketoxime (70 mg, 85%). ¹ H NMR (CDCl ₃ , 300MHz) δ3.88(dd, J=10.2Hz, 4.9Hz, 1H), 3.52(td, J=10.4Hz, 5.5Hz, 1H), 2.93-3.03(m, 1H), 2.21-3.35(m, 2H), 1.96-2.09(m, 2H), 1.28(s, 3H), 1.24(s, 3H), 1.14(s, 3H), 1.10(s, 3H), 1.06(s, 3H), 1.04 (s, 3H), 0.98 (s, 3H), 0.90 (s, 3H); MS (ESI) m/z: 490.3 [M+H] ⁺ .

Dissolve compound 7 (310mg, 0.63mmol) and ammonium acetate (500mg, 6.3mmol) in methanol (50mL), add sodium cyanoborohydride (400mg, 6.3mmol) at room temperature, slowly drop trichloro Titanium chloride hydrochloric acid solution (2.6 mL, 15-20% in HCl), after the dropwise reaction was completed at room temperature for 5 hours. The reaction solution was adjusted to pH=10 with 2N NaOH solution. The aqueous layer was sequentially extracted with dichloromethane and ethyl acetate, the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and column chromatography (dichloromethane:methanol=20:1) gave white solid OSA (220mg, 71%) . ¹ H NMR (CDCl ₃ , 300MHz) δ3.87(dd, J=10.2Hz, 5.0Hz, 1H), 3.52(td, J=9.8Hz, 4.0Hz, 1H), 2.62(dd, J=10.2Hz, 4.9Hz, 1H), 2.24(td, J=9.8Hz, 5.8Hz, 1H), 1.27(s, 3H), 1.23(s, 3H), 1.10(s, 3H), 1.04(s, 3H), 1.01 (s, 3H), 0.91 (s, 6H), 0.88 (s, 3H), 0.85 (s, 3H); ¹³ C NMR (CDCl ₃ , 75 MHz) δ87.4, 87.1, 70.4, 70.1, 60.4, 56.4, 52.1, 50.2, 48.9, 48.8, 39.7, 38.8, 37.3, 37.1, 34.7, 32.2, 31.6, 29.7, 28.9, 28.5, 28.0, 25.1, 24.2, 18.4, 17.8, 16.0, 15.5; ESI-MSm/z476.4[ M+H] ⁺ ; HR-MS (ESI) m/z: calculated for calculated for C ₃₀ H ₅₄ NO ₃ [M+H] ⁺ : 476.4098, found: 476.4094.

Example 2

(20S, 24R)-epoxydammarane-12β, 25-diol-3β-amine (ORA)

Referring to the synthesis method of (20S, 24S)-epoxydammarane-12β, 25-diol-3-amine (OSA), a white solid ORA (120 mg, 73%) was obtained from compound 4 through compounds 6 and 8. ¹ H NMR (CDCl ₃ , 300MHz) δ3.84(dd, J=13.7Hz, 7.0Hz, 1H), 3.50(td, J=10.4Hz, 4.6Hz, 1H), 2.35(dd, J=11.5Hz, 4.4Hz, 1H), 2.18(td, J=10.9Hz, 3.5Hz, 1H), 1.28(s, 3H), 1.26(s, 3H), 1.09(s, 3H), 0.98(s, 3H), 0.94 (s, 3H), 0.90 (s, 3H), 0.83 (s, 6H), 0.74 (s, 3H). ¹³ C NMR (CDCl ₃ , 75MHz) δ86.5, 85.4, 80.0, 70.1, 59.7, 56.6, 52.0, 50.6, 49.4, 47.9, 39.6, 39.5, 38.1, 37.3, 34.8, 32.6, 31.2, 28.6, 28.3, 27.9, 27.6, 26.1, 25.0, 18.6, 18.1, 16.2, 15.5, 15.4; ESI-MS m/z476 .4[M+H] ⁺ ; HR-MS (ESI) m/z: calculated for C ₃₀ H ₅₄ NO ₃ [M+H] ⁺ : 476.4098, found: 476.4091.

Example 3

(20S, 24S)-epoxy-3β-methylaminodammarane-12β, 25-diol

(20S,24S)-epoxydammarane-12β,25-diol-3-amine (OSA, 200 mg, 0.42 mmol) and anhydrous potassium carbonate (60 mg, 0.42 mmol) were added to anhydrous THF (8 mL) , then iodomethane (60 mg, 0.42 mmol) was added. The reaction was carried out at room temperature under N ₂ protection for 5 hours. Diluted with ethyl acetate, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to give a pale yellow product (155 mg, 75%). ¹ H NMR (CDCl ₃ , 300MHz) δ3.86(dd, J=10.2Hz, 5.0Hz, 1H), 3.51(td, J=9.8Hz, 4.0Hz, 1H), 3.16(s, 3H), 2.54( m, 1H), 2.22(td, J=9.8Hz, 5.8Hz, 1H), 1.26(s, 3H), 1.21(s, 3H), 1.10(s, 3H), 1.02(s, 3H), 1.00( s, 3H), 0.89 (s, 6H), 0.88 (s, 3H), 0.85 (s, 3H); ESI-MS m/z 490.4 [M+H] ⁺ .

Example 4

(20S,24R)-epoxy-3β-methylamino-dammarane-12β,25-diol

Referring to the synthesis method in Example 3, a white solid (120 mg, 73%) was obtained from (20S, 24R)-epoxydammarane-12β, 25-diol-3β-amine (ORA). ¹ H NMR (CDCl ₃ , 300MHz) δ 3.85 (dd, J=13.7Hz, 7.0Hz, 1H), 3.49 (td, J=10.4Hz, 4.6Hz, 1H), 3.15(s, 3H), 2.33( m, 1H), 2.16(td, J=10.9Hz, 3.5Hz, 1H), 1.27(s, 3H), 1.25(s, 3H), 1.09(s, 3H), 0.99(s, 3H), 0.95( s, 3H), 0.90 (s, 3H), 0.84 (s, 6H), 0.75 (s, 3H); ESI-MS m/z 490.4 [M+H] ⁺ .

Example 5

(20S,24S)-epoxy-3β-acetamidodammarane-12β,25-diol

Dissolve (20S,24S)-epoxydammarane-12β,25-diol-3-amine (OSA, 200 mg, 0.42 mmol) and a catalytic amount of DMAP in anhydrous dichloromethane (8 mL), then add Acetic anhydride (0.06 mL, 0.65 mmol). React at room temperature for 5 hours. Diluted with dichloromethane, washed with water, washed with saturated brine, dried over anhydrous sodium sulfate, and column chromatographed to give a white solid (155 mg, 75%). ¹ HNMR (CDCl ₃ , 300MHz) δ3.88(dd, J=10.2Hz, 5.0Hz, 1H), 3.53(td, J=9.8Hz, 4.0Hz, 1H), 3.41(m, 1H), 2.23(td , J=9.8Hz, 5.8Hz, 1H), 2.00(s, 3H), 1.27(s, 3H), 1.23(s, 3H), 1.11(s, 3H), 1.02(s, 3H), 1.01(s , 3H), 0.90 (s, 6H), 0.88 (s, 3H), 0.85 (s, 3H); ESI-MS m/z 518.4 [M+H] ⁺ .

Example 6

(20S,24R)-epoxy-3β-acetamidodammarane-12β,25-diol

Referring to the synthesis method of Example 5, a white solid (100 mg, 85%) was obtained from (20S, 24R)-epoxydammarane-12β, 25-diol-3β-amine (ORA). ¹ H NMR (CDCl ₃ , 300MHz) δ 3.84 (dd, J=13.7Hz, 7.0Hz, 1H), 3.48(td, J=10.4Hz, 4.6Hz, 1H), 3.45(m, 1H), 2.14( td, J=10.9Hz, 3.5Hz, 1H), 1.99(s, 3H), 1.25(s, 6H), 1.07(s, 3H), 0.98(s, 3H), 0.93(s, 3H), 0.88( s, 3H), 0.82 (s, 6H), 0.73 (s, 3H); ESI-MS m/z 518.4 [M+H] ⁺ .

Claims (2)

1. The application of the following structural compounds or pharmaceutically acceptable salts thereof in the preparation of drugs for reversing tumor multidrug resistance: where R represents hydrogen, The name of compound I is: (20S, 24S)-epoxydammarane-12β, 25-diol-3β-amine, The name of compound II is: (20S, 24R)-epoxydammarane-12β, 25-diol-3β-amine. 2. The application of the compound as claimed in claim 1 in the preparation of drugs for reversing tumor multidrug resistance.