CN109438300B - Phenolic compound and preparation method thereof - Google Patents
Phenolic compound and preparation method thereof Download PDFInfo
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- CN109438300B CN109438300B CN201811454351.4A CN201811454351A CN109438300B CN 109438300 B CN109438300 B CN 109438300B CN 201811454351 A CN201811454351 A CN 201811454351A CN 109438300 B CN109438300 B CN 109438300B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C317/00—Sulfones; Sulfoxides
- C07C317/16—Sulfones; Sulfoxides having sulfone or sulfoxide groups and singly-bound oxygen atoms bound to the same carbon skeleton
- C07C317/22—Sulfones; Sulfoxides having sulfone or sulfoxide groups and singly-bound oxygen atoms bound to the same carbon skeleton with sulfone or sulfoxide groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
- C07C39/12—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic with no unsaturation outside the aromatic rings
- C07C39/15—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring polycyclic with no unsaturation outside the aromatic rings with all hydroxy groups on non-condensed rings, e.g. phenylphenol
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
- C07C65/01—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups
- C07C65/03—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing hydroxy or O-metal groups monocyclic and having all hydroxy or O-metal groups bound to the ring
Abstract
The invention provides a phenolic compound, and relates to the field of medicaments, wherein the chemical structure of the compound is shown as a formula I:
Description
Technical Field
The invention belongs to the field of chemical drugs, and particularly relates to a phenolic compound and a preparation method thereof.
Background
The tumor seriously harms the health of people, the tumor is harmed because the existing medicine is lack of effective medicine, and the toxic and side effects of part of the effective medicine are difficult to tolerate. People have undergone the times of operations, radiotherapy and chemotherapy aiming at tumors. At present, PD-1, PDL-1 inhibitors and CART-T (genetically engineered vaccine) are being used as cell therapy for immunotherapy of tumors. This new technology may revolutionize the treatment of cancer, but life is a fight process and whether tumors will escape immune surveillance is not negligible, so treatment of tumors is necessarily or largely a complex intervention. The micromolecule medicine is the main force of the anti-cancer medicine, reaches thousands of varieties on the market, has definite curative effect, but has the weak point that the single medicine and the single target point are obviously inverse regular behaviors for dealing with complex tumors, so the effect is easy to see, but the problem is far from being solved. The combination of different mechanisms of medicines is reasonable, and the effect of the medicine is superior to that of a single medicine probably. Following current thinking, it may be desirable to find new mechanisms for anti-tumor molecules. The invention is supported by Shenzhen scientific traumatism project (JCYJ20170412110504956) and the like, the related compounds are from Yi family lute beetles with anticancer application, and the antitumor of the related compounds and analogs is not reported, and the related compounds represent antitumor compounds of a new structural family.
Disclosure of Invention
The compound of the invention is 6-methyl-2- (methylsulfonyl) benzene-1,4-diol, 3,4 '-diethyl- [1, 1' -biphenyl ] -2,2 ', 5, 5' -tetraol and 2-ethyl-3, 6-dihydroxy methyl benzoate,
the invention provides a pharmaceutical composition of a compound, which comprises 6-methyl-2- (methylsulfonyl) bezene-1, 4-diol, 3,4 '-diethyl- [1, 1' -biphenyl ] -2,2 ', 5, 5' -tetraol and 2-ethyl-3, 6-dihydroxy methyl benzoate or pharmaceutically acceptable derivatives and salts thereof.
Furthermore, the pharmaceutical composition is a pharmaceutical preparation, and consists of 6-methyl-2- (methylsulfonyl) bezene-1, 4-diol, 3,4 '-diethyl- [1, 1' -biphenyl ] -2,2 ', 5, 5' -tetraol and 2-ethyl-3, 6-dihydroxy-benzoic acid methyl ester or pharmaceutically acceptable derivatives and salts thereof as active ingredients and pharmaceutically acceptable excipients.
Wherein the solid preparation comprises: tablets, capsules, pills, granules and the like; semisolid preparations include ointments, suppositories, and the like; the liquid formulation comprises: solutions, injections, sprays, and the like; the ophthalmic preparation comprises: eye drops, eye gels, etc.
The invention also discloses a preparation method of the phenolic compound, which comprises the following steps:
50kg of Japanese blaps is crushed, extracted by cold soaking with 70% ethanol for 3 times, each time for 48 hours, stirring occasionally, filtered, and decompressed to recover ethanol to obtain 4kg of extract, which is suspended in acid water, adjusted pH to 1-2, and extracted with ethyl acetate for 4 times in equal volume to obtain 1kg of non-alkaloid part. Adjusting pH of the residual acid water layer with alkali to neutral or weakly alkaline, and extracting with ethyl acetate for 4 times in equal volume to obtain alkaloid fraction 300 g. The non-alkaloid fraction (1kg) was purified by MCI gel CHP 20P column chromatography with methanol/water (10:90,20:80, 3)0:70,40:60,50:50,60:40,70:30,80:20,90:10,100:0) gradient elution, and TLC detection to combine the same Fractions to obtain 6 Fractions A-F. And Fr.D (48g) is separated by Sephadex LH-20 column chromatography, eluted with methanol, and combined to obtain 4 fractions, D1-D4. D2(10g) was fractionated by silica gel column chromatography and eluted with chloroform/methanol gradient (30:1,20:1,15:1,10:1,7:1,5:1,3:1) to give 7 fractions, D2.1-D2.7. Fr.D2.2(3g) was fractionated by RP-18 column chromatography and eluted with a methanol/water gradient (2:8,3:7,4:6,5:5,6:4,7:3,1:0) to give 5 fractions, D2.2.1-D2.2.5. Fr.D2.2.2(120mg) was purified by semi-preparative HPLC (methanol/water 25:75) to give 6-methyl-2- (methylsulfonyl) bezene-1, 4-diol (1) (3mg, t;, t;)R30.3min, flow rate: 3 mL/min). Fr.D2.4(1g) was subjected to repeated RP-18 column chromatography (methanol/water 30:70) to give methyl 2-ethyl-3, 6-dihydroxybenzoate (3) (300 mg). Fr.E (75g) was separated by Sephadex LH-20 column chromatography, eluted with methanol and combined to give 4 fractions, E1-E4. Fr.E2(12g) was fractionated by MCI gel CHP 20P column chromatography and eluted with a methanol/water gradient (3:7,4:6,5:5,6:4,7:3,8:2,1:0) to give 5 fractions, E2.1-E2.5. Fr. E2.1(3.5g) was separated by Sephadex LH-20 column chromatography (methanol elution) to give 3 fractions, E2.1.1-E2.1.3. Fr.E2.1.2(160mg) was purified by semi-preparative RP-18HPLC (methanol/water 40:60) to give 3,4 '-diethyl- [1, 1' -biphenyl]-2,2′,5,5′-tetraol(2)(3mg，tR26.4min, flow rate: 3 mL/min).
Further, the compound is obtained by extracting from Japanese blaps beetles or artificially synthesizing.
Further, the new compound is applied to the aspect of resisting tumors.
The invention has the beneficial effects that:
1. the invention discloses a method for extracting and separating the compounds 6-methyl-2- (methylsulfonyl) bezene-1, 4-diol, 3,4 '-diethyl- [1, 1' -biphenyl ] -2,2 ', 5, 5' -tetraol and 2-ethyl-3, 6-dihydroxy methyl benzoate from Japanese blaps beetles for the first time.
2. The compounds 6-methyl-2- (methylsulfonyl) bezene-1, 4-diol and 3,4 '-diethyl- [1, 1' -biphenyl ] -2,2 ', 5, 5' -tetraol are novel compounds which are separated and identified from Japanese blaps beetles for the first time.
3. The compound 6-methyl-2- (methylsulfonyl) bezene-1, 4-diol, 3,4 '-diethyl- [1, 1' -biphenyl ] -2,2 ', 5, 5' -tetraol and 2-ethyl-3, 6-dihydroxy methyl benzoate have the function of anti-tumor activity.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.
EXAMPLE 1 isolation and Structure characterization of the Compounds of the invention
50kg of Japanese blaps is crushed, extracted by cold soaking with 70% ethanol for 3 times, each time for 48 hours, stirring occasionally, filtered, and decompressed to recover ethanol to obtain 4kg of extract, which is suspended in acid water, adjusted pH to 1-2, and extracted with ethyl acetate for 4 times in equal volume to obtain 1kg of non-alkaloid part. Adjusting pH of the residual acid water layer with alkali to neutral or weakly alkaline, and extracting with ethyl acetate for 4 times in equal volume to obtain alkaloid fraction 300 g. The non-alkaloid fraction (1kg) was subjected to MCI gel CHP 20P column chromatography, methanol/water (10:90,20:80,30:70,40:60,50:50,60:40,70:30,80:20,90:10,100:0) gradient elution, and the same Fractions were combined for TLC detection to give 6 Fractions A-F. And Fr.D (48g) is separated by Sephadex LH-20 column chromatography, eluted with methanol, and combined to obtain 4 fractions, D1-D4. D2(10g) was fractionated by silica gel column chromatography and eluted with chloroform/methanol gradient (30:1,20:1,15:1,10:1,7:1,5:1,3:1) to give 7 fractions, D2.1-D2.7. Fr.D2.2(3g) was fractionated by RP-18 column chromatography and eluted with a methanol/water gradient (2:8,3:7,4:6,5:5,6:4,7:3,1:0) to give 5 fractions, D2.2.1-D2.2.5. Fr.D2.2.2(120mg) was purified by semi-preparative HPLC (methanol/water 25:75) to give 6-methyl-2- (methylsulfonyl) bezene-1, 4-diol (3mg, t;)R30.3min, flow rate: 3 mL/min). Fr.D2.4(1g) was subjected to repeated RP-18 column chromatography (methanol/water 30:70) to give methyl 2-ethyl-3, 6-dihydroxybenzoate (300 mg). Fr.E (75g) was separated by Sephadex LH-20 column chromatography, eluted with methanol and combined to give 4 fractions, E1-E4. Fr.E2(12g) was fractionated by MCI gel CHP 20P column chromatography and eluted with a methanol/water gradient (3:7,4:6,5:5,6:4,7:3,8:2,1:0) to give 5 fractions, E2.1-E2.5. Fr. E2.1(3.5g) was separated by Sephadex LH-20 column chromatography (methanol elution) to give 3 fractions, E2.1.1-E2.1.3. Fr.e2.1.2(160mg) menstruation halfPreparation of RP-18HPLC (methanol/water 40:60) purification to obtain 3,4 '-diethyl- [1, 1' -biphenyl]-2,2′,5,5′-tetraol(3mg，tR26.4min, flow rate: 3 mL/min).
The compound structure was identified as follows:
the compound 6-methyl-2- (methylsulfonyl) bezene-1, 4-diol (1), light yellow solid, UV (MeOH). lamdamax(log)207(4.32),224(3.76),313(3.55)nm；ESIMS m/z 203[M+H]+；HRESIMS m/z 203.0377[M+H]+(calcd for C8H10O4S,203.0373),1H(600MHz)and13C NMR (150MHz) data, see Table 1.
Compound 3,4 '-diethyl- [1, 1' -biphenyl]-2,2 ', 5, 5' -tetraol (2), light yellow solid, uv (meoh) λmax(log)207(4.64),304(4.06)nm；ESIMS m/z 273[M–H]–,1H(600MHz)and13C NMR (150MHz) data, see Table 2.
The compound methyl 2-ethyl-3, 6-dihydroxybenzoate (3), a pale yellow solid,1H NMR(acetone-d6,500MHz)H10.36(1H,s),7.26(1H,s),6.92(1H,d,J＝8.8Hz),6.75(1H,d,J＝8.8Hz),3.97(3H,s),2.90(2H,q,J＝7.0Hz),1.19(3H,t,J＝7.0Hz)。
TABLE 1 preparation of Compound 11H and13c NMR data
TABLE 2 preparation of Compound 21H and13c NMR data
EXAMPLE 2 test method for antitumor Activity of Compounds (A549, K562 and Huh-7)
A method for detecting the in vitro anti-tumor activity (A549, K562 and Huh-7) of the compound by using a CCK-8 reagent. The method comprises the following specific steps:
1. the various cancer cell lines were plated in 384-well plates at 50. mu.L/well volume at an appropriate cell concentration and incubated overnight at 37 ℃.
2. Compound dilution the compound was first dissolved in DMSO to 10mM stock and diluted with a 3-fold gradient of DMSO to 10 concentration gradients to 1000 Xcompound series stock before being transferred to a compound transfer apparatus adapter 384PP plate for use.
3. The 1000 Xseries concentration of compounds were transferred to corresponding wells of 384-well cancer cells plates at 50nL per well using a compound transfer instrument Liquid Handler Echo520, and 50nL DMSO was added to blank wells. Gently mixing, and further culturing at 37 deg.C.
After 4.72 hours, CCK8 cell proliferation-toxicity test reagent was added thereto at 3. mu.L/well, and the mixture was incubated at 37 ℃ for 2 hours. Then the 450nm light absorption intensity is detected by a microplate reader.
TABLE 3 IC of Compounds 1-3 against A549, K562 and Huh-750(μM)
The results show (Table 3), that the compounds 1-3 have proliferation inhibition effect on 3 tumor cell strains of A549, K562 and Huh-7, and show that the compounds have anti-tumor effect and potential.
Example 3
The compound in the example 1 is added with injection solvent according to the conventional method, fine filtered, encapsulated and sterilized to prepare injection.
Example 4
The compound in the example 1 is dissolved in sterile water for injection, filtered by a sterile funnel, subpackaged, frozen and dried at low temperature, and then aseptically sealed by melting to obtain the powder injection.
Example 5
The compound in the example 1 can be prepared into tablets or capsules by being matched with various pharmaceutical excipients according to a conventional method. Using the compound of example 1 as a pharmaceutically active ingredient, several conventional excipients as auxiliary ingredients for the preparation of combination pharmaceutical tablets or capsules, samples containing 10-300mg of the pharmaceutical ingredient per tablet or capsule were prepared according to conventional methods.
Claims (6)
1. The phenolic compound is characterized in that the phenolic compound is 1 phenolic compound with antitumor activity, is named as 3,4 '-diethyl- [1, 1' -biphenyl ] -2,2 ', 5, 5' -tetraol, and has a chemical structure shown as a formula I:
2. The compound according to claim 1, which is obtained by extraction from blaps japonica.
3. The use of a compound according to claim 1 for the preparation of an antineoplastic medicament.
4. The pharmaceutical composition of phenolic compounds of claim 1, wherein said pharmaceutical composition comprises 3,4 '-diethyl- [1, 1' -biphenyl ] -2,2 ', 5, 5' -tetraol or a pharmaceutically acceptable salt thereof.
5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition is a pharmaceutical formulation comprising 3,4 '-diethyl- [1, 1' -biphenyl ] -2,2 ', 5, 5' -tetraol or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable excipient.
6. A process for preparing the compound of claim 1 by:
50kg of Japanese beetle is crushed, is extracted by cold soaking with 70 percent ethanol for 3 times, each time is 48 hours, is stirred for 3 times each day, is filtered, is decompressed and recovered with ethanol, is 4kg of extract, is suspended in acid water, is adjusted in pH value to be 1-2, is extracted by ethyl acetate for 4 times with equal volume, is obtained non-alkaloid part 1kg, is adjusted in pH value to be neutral, is extracted by ethyl acetate for 4 times with equal volume, is obtained alkaloid part 300g, is non-alkaloid part 1kg, is subjected to MCI gel CHP 20P column chromatography, is subjected to methanol water washing/eluting system with 10:90,20:80,30:70,40:60,50:50,60:40,70:30,80:20,90:10,100:0, is detected by TLC and is combined with same flow part, is obtained 6 fraction sections of Fractions A-F, Fr. D48 g, is subjected to Sephadex LH-20 column chromatography separation, eluting with methanol, mixing to obtain 4 fractions, D1 ‒ D4, Fr. D210 g, fractionating with silica gel column chromatography, eluting with chloroform/methanol, eluting with solvent gradient of 30:1,20:1,15:1,10:1,7:1,5:1,3:1 to obtain 7 fractions, D2.1 ‒ D2.7, Fr. D2.23 g, fractionating with RP-18 column chromatography, eluting with methanol/water gradient of 2:8,3:7,4:6,5:5,6:4,7:3,1:0 to obtain 5 fractions, subjecting D2.2.1 ‒ D2.2.5, Fr. D2.41 g to repeated RP-18 column chromatography, eluting with methanol/water gradient of 30:70 to obtain 2-ethyl-3, 6-dihydroxy methyl benzoate 300mg, Fr. E75 g, separating with Sephadex-20, eluting with methanol, combining to obtain 4 parts, carrying out MCI column chromatography segmentation on E1 ‒ E4 and Fr. E212 g, eluting with methanol/water with gradient of 3:7,4:6,5:5,6:4,7:3,8:2 and 1:0 to obtain 5 components, carrying out Sephadex LH-20 column chromatography separation on E2.1 ‒ E2.5 and Fr. E2.13.5 g to obtain 3 parts, wherein E2.1.1 ‒ E2.1.3 and Fr. E2.1.2160 mg are subjected to semi-preparative HPLC purification with methanol/water of 40:60 to obtain 3,4 '-diethyl- [1, 1' -biphenyl ] -2,2 ', 5, 5' -tetraol and 3 mg.
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