CN111647036B - Ocotillol esterified derivatives, preparation method thereof and application thereof in preparing anti-inflammatory drugs - Google Patents
Ocotillol esterified derivatives, preparation method thereof and application thereof in preparing anti-inflammatory drugs Download PDFInfo
- Publication number
- CN111647036B CN111647036B CN202010367674.0A CN202010367674A CN111647036B CN 111647036 B CN111647036 B CN 111647036B CN 202010367674 A CN202010367674 A CN 202010367674A CN 111647036 B CN111647036 B CN 111647036B
- Authority
- CN
- China
- Prior art keywords
- formula
- epoxy
- dammarane
- hydroxy
- boc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J17/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, having an oxygen-containing hetero ring not condensed with the cyclopenta(a)hydrophenanthrene skeleton
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
Abstract
The invention provides an Ocotillol type esterified derivative shown in a formula (I-R) or a formula (I-S) and an Ocotillol type esterified derivative shown in a formula (II-R) or a formula (II-S), wherein a compound shown in the formula (II-R) or the formula (II-S) is prepared by removing a protecting group from a compound shown in the formula (I-R) or the formula (I-S). The invention also provides a preparation method of the compound shown in the formula (I-R), the formula (I-S), the formula (II-R) or the formula (II-S) and pharmaceutically acceptable salts thereof, and application of the compound in preparation of anti-inflammatory drugs or anti-inflammatory drug compositions. The Ocotillol esterified derivative provided by the invention is obviously superior to the prior clinical medicine hydrocortisone sodium succinate in inhibiting the generation of inflammation signal molecules NO. And the biological friendliness is high, the safety of the medicine is good, and meanwhile, the long half-life period is shown in the in-vitro metabolic stability evaluation, so that the safety and the stability of the patent medicine can be improved.
Description
Technical Field
The invention relates to an Ocotillol esterified derivative, a preparation method thereof and application thereof in preparing anti-inflammatory drugs, belonging to the technical field of preparation and application of new compounds.
Background art:
inflammation is an important and common physiological role, and is thought to be a tissue protective immune response that occurs against harmful stimuli (such as damaged cells, irritants, and bacteria) that regulates the inflammatory process by turning signals on, off, and on. However, unbalanced inflammation may induce cell and tissue damage in different diseases, such as atherosclerosis, hypertension, diabetes, cancer and neurodegenerative diseases, which seriously threaten human health.
Nitric Oxide (NO) is an important pro-inflammatory mediator, NO activates COX-2, stimulates the production of inflammatory prostaglandins, promotes acute inflammatory reactions such as edema, and NO also has a significant effect on chronic inflammatory processes. Excessive NO production may exacerbate tissue damage, and selective iNOS inhibitors may have therapeutic effects on inflammation. At present, clinically used anti-inflammatory drugs comprise non-steroidal anti-inflammatory drugs, steroidal anti-inflammatory drugs and the like, and have obvious serious adverse reactions such as osteoporosis and the like. Therefore, there is still a great need for anti-inflammatory agents with high efficacy and low side effects.
Ginseng has a history of thousands of years in China, has various efficacies of invigorating primordial qi, recovering pulse, relieving depletion, benefiting intelligence, soothing nerves and the like, and has a wide pharmacological action by taking ginsenoside as a main active ingredient. Especially, in the aspect of anti-inflammatory activity, the ginsenoside Rb1, Re, Rg1, Rg3, Rh1 and the like have obvious treatment effect on the aspects of resisting neuritis, skin tissue inflammation, colitis and the like, and show better anti-inflammatory activity. The Ocotillol type ginsenoside is a tetracyclic triterpene saponin with a furan ring on a side chain, is possibly a key main body for exerting pharmacological activity, has better bioavailability and in vivo stability, and has great medicinal research and development values. The Ocotillol type sapogenin derivative has tumor drug resistance reversing activity (patent application publication No. CN109021058A), and a certain type of derivative is found to have an inhibiting effect on proinflammatory factors (patent application publication No. CN110642913A1), but hydroxyl in a synthesized compound is easier to be subjected to oxidative metabolism, so that the half-life period of the compound is shortened, and the stability is poor. Meanwhile, at present, no research on toxic and side effects of the compounds on normal cell physiological tissues and the like exists, the toxicity of active substances influences the safety of medicaments, and the activity is rootless wood in case of breaking the safety of medicaments. Therefore, it becomes more important to prolong the half-life of the drug while ensuring the medicinal value and to increase the safety and stability of the drug.
Disclosure of Invention
The invention aims to provide an Ocotillol esterified derivative, a preparation method and application thereof; another object of the present invention is to provide an anti-inflammatory agent and its use in a medicament or pharmaceutical composition for the treatment and prevention of diseases associated with acute lung injury, sepsis, etc.
The invention is realized by the following technical scheme:
an esterified derivative of the Ocotillol type, the structural formula of which is shown as formula (I-R) or formula (I-S), or a pharmaceutically acceptable salt of a compound shown as formula (I-R) or formula (I-S):
in the formula (I-R) or the formula (I-S), R, S represents chirality of carbon atom at position 24, respectively;
in the formula (I-R) or the formula (I-S), R1Is hydrogen, C1-C4 alkyl, phenyl, benzyl, p-hydroxybenzyl, -RaCOORb、-RaORb、-RaNHRcor-RaSRd；R3Is H or C1-C4 alkyl; or R1、R3Connecting with N to form a ring to form N-tetrahydropyrrole;
R1in, RaIs C1-C4 alkylene; rbH, Bn or tBu; rcH, Boc or Fmoc; rdIs H or methyl;
preferably R1Is hydrogen, isopropyl, benzyl, - (CH)2)2-COOH、-CH2-COOH、-(CH2)2-COO-tBu or-CH2-COO-tBu；
R3Preferably H.
The Bn is benzyl, and tBu is tert-butyl; boc is tert-butyloxycarbonyl; fmoc is fluorenyl-methoxycarbonyl.
The invention also provides an Ocotillol esterified derivative, the structure of which is shown as formula (II-R) or formula (II-S), or the pharmaceutically acceptable salt of the compound shown as formula (II-R) or formula (II-S):
in the formula (II-R) or the formula (II-S), R, S represents chirality of carbon atom at position 24, respectively;
in the formula (I-R) or the formula (I-S), R1Is hydrogen, C1-C4 alkyl, phenyl, benzyl, p-hydroxybenzyl, -RaCOORb、-RaORb、-RaNHRcor-RaSRd；R2Is tert-butyloxycarbonyl (Boc) and fluoreneOxycarbonyl (Fmoc) or benzyl (Bn); r3Is H or C1-C4 alkyl; or R1、R3Connecting with N to form a ring to form N-tetrahydropyrrole;
R1in, RaIs C1-C4 alkylene; rbH, Bn or tBu; rcH, Boc or Fmoc; rdIs H or methyl;
preferably R1Is hydrogen, isopropyl, benzyl, - (CH)2)2-COOH、-CH2-COOH、-(CH2)2-COO-tBu or-CH2-COO-tBu；
Preferably R2Is Boc; preferably R3Is H.
R in formula (II-R) or formula (II-S)2For the protecting group, the formula (I-R) or the formula (I-S) is prepared by deprotecting the formula (II-R) or the formula (II-S), respectively.
Further, it is preferable that the esterified derivative of the Ocotillol type represented by the formula (I-R) or the formula (I-S) is one of the following:
(20S,24R) -epoxy-25-hydroxy-3 beta-O- (N-Boc-phenylalanyl) -dammarane-12-one, (20S,24R) -epoxy-25-hydroxy-3 beta-O- (N-Boc-valyl) -dammarane-12-one, (20S,24S) -epoxy-25-hydroxy-3 beta-O- (N-Boc-glycyl) -dammarane-12-one, (20S,24S) -epoxy-25-hydroxy-3 beta-O- (N-Boc-phenylalanyl) -dammarane-12-one, (20S,24S) -epoxy-25-hydroxy-3 beta-O- (N-Boc-valinyl) -dammarane-12-one Acyl ester group) -dammarane-12 ketone.
Preferably, the esterified derivative of the Ocotillol type represented by the formula (II-R) or the formula (II-S) is one of the following:
is (20S,24S) -epoxy-25-hydroxy-3 beta-O-glutamyl ester-dammarane-12-ketone, (20S,24S) -epoxy-25-hydroxy-3 beta-O-aspartyl ester-dammarane-12-ketone, (20S,24R) -epoxy-25-hydroxy-3 beta-O-phenylalanyl ester-dammarane-12-ketone, (20S,24R) -epoxy-25-hydroxy-3 β -O-valyloxy-dammaran-12-one, (20S,24R) -epoxy-25-hydroxy-3 β -O-aspartyl-dammaran-12-one.
The invention also provides a preparation method of the Ocotillol esterified derivative with the general formula (I-R), (I-S) and (II-R), (II-S), which comprises the following steps: protopanaxadiol (PDD) shown in a formula (III) is used as a raw material, and is subjected to double epoxidation and intramolecular nucleophilic attack reaction with m-chloroperoxybenzoic acid (m-CPBA) to prepare (20S,24R) -epoxy dammarane-3 beta, 12 beta, 25-triol shown in a formula (IV-R) after separation and purification and (20S,24S) -epoxy dammarane-3 beta, 12 beta, 25-triol shown in a formula (IV-S), the compound shown in the formula (IV-R) or the formula (IV-S) is oxidized by a dessimutan reagent (DMP) to oxidize 3-position hydroxyl and 12-position hydroxyl to generate ketone, and the (20S,24R) -epoxy-25-hydroxyl-dammarane-3, 12-diketone shown in the formula (V-R) or the (20S) shown in the formula (V-S) is respectively and correspondingly prepared, the compound shown in the formula (V-R) or the formula (V-S) is selectively reduced by sodium borohydride to reduce the carbonyl group at the 3-position into hydroxyl group, respectively and correspondingly prepare (20S,24R) -epoxy-25-hydroxyl-3 beta-hydroxyl-dammarane-12-ketone shown in the formula (VI-R) or (20S,24) -epoxy-25-hydroxyl-3 beta-hydroxyl-dammarane-12-ketone shown in the formula (VI-S), the compound shown in the formula (VI-R) or the formula (VI-S) and the amino acid derivative shown in the formula (VII) are subjected to esterification reaction, respectively and correspondingly prepare the protection group-containing Ocotillol type esterified derivative shown in the formula (II-R) or the formula (II-S) which is prepared .
Carrying out deprotection reaction on the protection group-containing Ocotillol esterified derivative shown as the formula (II-R) or the formula (II-S) to respectively and correspondingly prepare the Ocotillol esterified derivative shown as the formula (I-R) or the formula (I-S).
The reaction formula is shown as follows:
in the invention, because chiral isomers exist, R or S configuration raw materials respectively and correspondingly prepare R or S configuration products, and for convenience of description, the preparation methods of the two isomers are written together in an OR mode.
Further, the method comprises the steps of:
(1) raw material Protopanaxadiol (PDD) is used as a raw material, and is subjected to feeding reaction with m-chloroperoxybenzoic acid (m-CPBA) according to the mass ratio of 1: 1-2, and (20S,24R) -epoxy dammarane-3 beta, 12 beta, 25-triol shown in formula (IV-R) and (20S,24S) -epoxy dammarane-3 beta, 12 beta, 25-triol shown in formula (IV-S) are respectively prepared through separation and purification;
the reaction is carried out in an aprotic solvent, preferably dichloromethane;
the reaction temperature is-20 ℃ to 80 ℃; preferably-10 ℃ to 30 ℃, more preferably room temperature.
The reaction time is 3-5 hours.
The separation and purification adopts a column chromatography mode after washing;
(2) feeding a compound dessimutan reagent (DMP reagent) shown in a formula (IV-R) or a formula (IV-S) according to a mass ratio of 1: 2-3 for reaction, and respectively and correspondingly preparing (20S,24R) -epoxy-25-hydroxy-dammarane-3, 12-diketone shown in a formula (V-R) or (20S,24S) -epoxy-25-hydroxy-dammarane-3, 12-diketone shown in a formula (V-S);
the reaction is carried out in an aprotic solvent, preferably dichloromethane;
in the reaction, a dessimantine reagent (DMP) is used as an oxidant, tert-butyl alcohol is added as a cosolvent, and sodium bicarbonate is added as a pH regulator
Furthermore, the volume usage amount of the tertiary butanol is generally 0.2-1 mL/mmol based on the amount of the DMP reagent.
Further, the mass ratio of the DMP reagent to the sodium bicarbonate is 1: 1.2-3
The reaction temperature is-20 ℃ to 50 ℃; preferably, the reaction temperature is-5 ℃ to 30 ℃.
The reaction time is 4-8 hours.
More preferably, the initial reaction temperature is controlled below 0 ℃, generally from-5 ℃ to 0 ℃, and the reaction is carried out for 1 to 2 hours at the temperature of from-5 ℃ to 0 ℃ and then for 3 to 5 hours at room temperature;
after the reaction is completed, the reaction system is subjected to post-treatment to obtain (20S,24R) -epoxy-25-hydroxy-dammarane-3, 12-diketone shown in a formula (V-R) or (20S,24S) -epoxy-25-hydroxy-dammarane-3, 12-diketone shown in a formula (V-S).
The post-treatment steps are generally: adding saturated sodium bicarbonate and sodium sulfite aqueous solution into a reaction system to adjust the pH value, extracting with ethyl acetate, washing an organic phase with the saturated sodium bicarbonate aqueous solution and saturated salt water respectively, drying, concentrating, separating by silica gel column chromatography, and eluting to obtain (20S,24R) -epoxy-25-hydroxy-dammarane-3, 12-diketone shown in a formula (V-R) or (20S,24S) -epoxy-25-hydroxy-dammarane-3, 12-diketone shown in a formula (V-S).
(3) (20S,24R) -epoxy-25-hydroxy-dammarane-3, 12-dione shown in a formula (V-R) or (20S,24S) -epoxy-25-hydroxy-dammarane-3, 12-dione shown in a formula (V-S) reacts with sodium borohydride according to the mass ratio of 1: 2-3 to respectively and correspondingly prepare (20S,24R) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-ketone shown in a formula (VI-R) or (20S,24S) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-ketone shown in a formula (VI-S);
the reaction is carried out in an aprotic solvent, preferably isopropanol
The reaction temperature is room temperature;
the reaction time is 20-30 hours.
After the reaction is completed, the post-treatment mode of the reaction solution is as follows: quenching reaction with water, extracting with dichloromethane, drying organic phase, concentrating, and separating by column chromatography to obtain (20S,24R) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-ketone shown in formula (VI-R) or (20S,24S) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-ketone shown in formula (VI-S).
(4) Feeding (20S,24R) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-ketone shown in a formula (VI-R) or (20S,24S) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-ketone shown in a formula (VI-S) and an amino acid derivative shown in a formula (VII) according to the mass ratio of 1: 1-2, carrying out esterification reaction, and respectively and correspondingly preparing the protection group-containing Ocotillol type esterified derivative shown in a formula (II-R) or a formula (II-S);
the reaction solvent is an aprotic solvent, preferably dichloromethane;
a condensing agent and a dehydration catalyst are also added in the reaction; the condensing agent is 4-Dimethylaminopyridine (DMAP); the dehydration catalyst is ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI);
the amount of the EDCI substance is 3-4 times of that of (20S,24R) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-ketone shown in a formula (VI-R) or (20S,24S) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-ketone shown in a formula (VI-S);
the ratio of the amount of DMAP to the amount of (20S,24R) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-one represented by formula (VI-R) or (20S,24S) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-one represented by formula (VI-S) is 0.3-0.8: 1
The reaction temperature is-20 ℃ to 100 ℃; preferably-5 ℃ to 30 ℃.
More preferably, the initial reaction temperature is controlled below 0 ℃, generally from-5 ℃ to 0 ℃, and after the addition is finished at the temperature of from-5 ℃ to 0 ℃, the reaction is carried out for 20 to 30 hours at room temperature;
the reaction is carried out under the protection of argon or nitrogen
After the reaction is completed, the post-treatment mode of the reaction solution is as follows: quenching reaction with water, extracting with dichloromethane, drying organic phase, concentrating, and separating by column chromatography to obtain the protection group-containing Ocotillol esterified derivative shown as formula (II-R) or formula (II-S).
The protection group-containing Ocotillol esterified derivative shown as the formula (II-R) or the formula (II-S) can be subjected to deprotection reaction to prepare the Ocotillol esterified derivative shown as the formula (II-R) or the formula (II-S).
Further, R2In the case of Boc, the Boc protecting group is removed by treating with trifluoroacetic acid or hydrochloric acid;
R2in the case of Fmoc, the Fmoc protecting group is removed by treatment with diethylamine.
R2In the case of Bn, the Bn protecting group is removed by catalytic hydrogenation treatment with a palladium on carbon catalyst.
These are all deprotection methods well known to those skilled in the art.
The compound shown in the formula (I-R), the formula (I-S), the formula (II-R) or the formula (II-S) provided by the invention has anti-inflammatory activity, and the compound shown in the formula (I-R), the formula (I-S), the formula (II-R) or the formula (II-S) and pharmaceutically acceptable salts thereof can be used for preparing anti-inflammatory medicaments or anti-inflammatory pharmaceutical compositions, and further can be used for preparing medicaments or pharmaceutical compositions for treating and preventing diseases related to acute lung injury, sepsis and the like.
Further, preferred are (20S,24R) -epoxy-25-hydroxy-3 β -O- (N-Boc-phenylalanyl) -dammarane-12-one, (20S,24R) -epoxy-25-hydroxy-3 β -O- (N-Boc-valyl) -dammarane-12-one, (20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-glycyl) -dammarane-12-one, (20S,24S) -epoxy-25-hydroxy-3 β -O-glutamyl-dammarane-12-one, (20S,24S) -epoxy-25-hydroxy-3 β -O-aspartyl-dammarane-12-one, and (20S,24S) -epoxy-25-hydroxy-3 β -O-aspartyl-dammarane-12-one -12-one, (20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-phenylalanyl) -dammarane-12-one, (20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-valyl) -dammarane-12-one, (20S,24R) -epoxy-25-hydroxy-3 β -O-phenylalanyl-dammarane-12-one, (20S,24R) -epoxy-25-hydroxy-3 β -O-valyl-dammarane-12-one, (20S,24R) -epoxy-25-hydroxy-3 β -O-aspartyl-dammarane-12-one Can be used for preparing anti-inflammatory drugs or anti-inflammatory drug compositions.
The pharmaceutically acceptable salts of the compounds of the present invention refer to conventional acid addition salts which have the same pharmaceutical efficacy as the compounds and are salts with suitable non-toxic organic or inorganic acids.
The compound or the pharmaceutically acceptable salt thereof can be added with pharmaceutically acceptable carriers to prepare common pharmaceutical preparations, such as tablets, capsules, powder, syrup, liquid, suspending agents and injection, and common pharmaceutical excipients such as spices, sweeteners, liquid or solid fillers or diluents can be added.
The clinical administration mode of the compound of the invention can adopt oral administration, injection and other modes.
The clinical dosage of the compound of the invention is 0.01mg to 1000 mg/day, and the dosage can be deviated from the range according to the severity of the disease condition or different dosage forms.
Compared with the prior art, the invention has the following advantages:
the compound shown in the formula (I-R), the formula (I-S), the formula (II-R) or the formula (II-S) and the pharmaceutically acceptable salt thereof have high bioavailability, better anti-inflammatory activity and better safety.
The data in the examples show that the protective group-containing Ocotillol esterified derivatives 7, 8, 9, 12 and 13 all have an inhibition rate of NO generation of more than 20% at a concentration of 20. mu.M, which is more than 2 times of the inhibition rate (10%) of positive comparative hydrocortisone sodium succinate, wherein the inhibition rate of the compound 8(20S,24R) -epoxy-25-hydroxy-3 beta-O- (N-Boc-valyl) -dammarane-12 ketone on NO generation at a concentration of 20. mu.M is more than 40%, and show strong inhibition effect of LPS induced NO release. And the compounds 7, 8, 9, 12 and 13 show good safety and no cell toxicity, and the cell survival rate in an in vitro toxicity test reaches over 90 percent, and exceeds or is equivalent to the survival rate (90 percent) compared with a positive medicament. Therefore, compared with the existing clinical medicines, the compounds 7, 8, 9, 12 and 13 have better effect of inhibiting NO release induced by LPS, and have good safety and lower toxicity.
The compounds 10, 11 and 14 are protecting group-removed Ocotillol esterified derivatives, the inhibition rate of the compounds on NO generation is more than 26 percent under the concentration of 20 mu M, the anti-inflammatory activity is prominent, the compounds have low toxicity on cells, and the cell survival rate is more than 70 percent.
Therefore, the Ocotillol esterified derivative provided by the invention is obviously superior to the prior clinical medicine hydrocortisone sodium succinate in inhibiting the generation of inflammation signal molecules NO. And the biological friendliness is high, the safety of the drug is good, and meanwhile, the long half-life period is shown in the in-vitro metabolic stability evaluation, so that the active utilization time of the drug can be effectively prolonged. Therefore, the compound of the invention can improve the safety and stability of the patent medicine.
Drawings
FIG. 1 is a bar graph showing the NO inhibition ratio of the synthesized Ocotillol esterified derivative of the present invention.
FIG. 2 is a bar graph showing in vitro cytotoxicity of esterified derivatives of the Ocotillol type synthesized by the present invention.
Detailed Description
The technical solutions of the present invention are further described below by using specific examples, but the scope of the present invention is not limited thereto.
Example 1: (20S,24R) -epoxy-25-hydroxy-3-one-dammaran-12-one
20S-Protopanaxadiol (1.000g, 2.17mmol) was dissolved in dichloromethane (21.7mL), m-CPBA (549mg, 3.18mmol) was added, and the mixture was stirred at room temperature for 3 h. After diluting with chloroform, washing with water, washing with saturated brine, drying over anhydrous sodium sulfate, filtering, concentrating, and column chromatography, a white solid compound 1[ (20S,24R) -epoxydammara -3 β,12 β, 25-triol ] (530mg, 1.11mmol, yield 53%) and a white solid compound 2[ (20S,24S) -epoxydammara -3 β,12 β, 25-triol ] (419mg, 0.88mmol, yield 41%) were obtained.
Dissolving the compound 1(20S,24R) -epoxy dammarane-3 beta, 12 beta, 25-triol (727mg, 1.52mmol) and sodium bicarbonate (769mg, 9.15mmol) in anhydrous dichloromethane (15mL), adding DMP (1617mg, 3.81mmol), adding 2mL tert-butyl alcohol to accelerate the dissolution of the DMP, and removing the ice bath after reacting for 1 hour at normal temperature. After 4 hours of reaction, saturated aqueous sodium bicarbonate and sodium sulfite are added into the reaction system to adjust the pH and stir for half an hour, the mixture is extracted by ethyl acetate, the organic phase is washed by saturated aqueous sodium bicarbonate and saturated brine respectively and then is combined into a conical flask, and the mixture is dried by anhydrous sodium sulfate, filtered and concentrated. Column chromatography (petroleum ether: ethyl acetate ═ 4:1 → 2:1) elution gave compound 3(20S,24R) -epoxy-25-hydroxy-dammarane-3, 12-dione (672mg, 1.42mmol, 93% yield)
Example 2: (20S,24S) -epoxy-25-hydroxy-dammarane-3, 12-dione
The compound 2(20S,24S) -epoxydammar-3 beta, 12 beta, 25-triol (605mg, 1.27mmol) and sodium bicarbonate (639mg, 7.61mmol) were dissolved in anhydrous dichloromethane (13mL), DMP (1345mg, 3.17mmol) was added, 2mL of tert-butanol was added to accelerate the dissolution of DMP, and after 1 hour of reaction, the ice bath was removed and the reaction was carried out at room temperature. After 4 hours of reaction, saturated sodium bicarbonate and sodium sulfite aqueous solution are added into the reaction system to adjust the pH value and stirred for half an hour, the mixture is extracted by ethyl acetate, the organic phase is washed by the saturated sodium bicarbonate aqueous solution and the saturated saline solution respectively and then is combined into a conical flask, and the mixture is dried by anhydrous sodium sulfate, filtered and concentrated. Column chromatography (petroleum ether: ethyl acetate ═ 3:1 → 1:1) elution gave compound 4(20S,24S) -epoxy-25-hydroxy-dammarane-3, 12-dione (562mg, 1.18mmol, 93% yield)
Example 3: (20S,24R) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-one
Dissolving compound 3(214mg, 0.45mmol), sodium borohydride (34mg, 0.91mmol) in isopropanol (6.5mL), stirring at room temperature for 24h, quenching with water, extracting with dichloromethane, drying the organic phase over anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography to obtain compound 5(20S,24R) -epoxy-25-hydroxy-3 β -hydroxy-dammaran-12-one (180mg, 0.38mmol, 83.7% yield)
Example 4: (20S,24S) -epoxy-25-hydroxy-3 beta-hydroxy-dammarane-12-one
Dissolving compound 4(444mg, 0.93mmol), sodium borohydride (71mg, 1.87mmol) in isopropanol (9.3mL), stirring at room temperature for 24h, quenching with water, extracting with dichloromethane, drying the organic phase over anhydrous sodium sulfate, filtering, concentrating, and performing column chromatography to obtain compound 6(20S,24S) -epoxy-25-hydroxy-3 β -hydroxy-dammaran-12-one (377mg, 0.79mmol, 85% yield)
Example 5: (20S,24R) -epoxy-25-hydroxy-3 beta-O- (N-Boc-phenylalanyl) -dammarane-12-one
Compound 5(25mg, 0.05mmol), N-Boc-2-phenylalanine (17mg, 0.06 mmol 1) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 30mg, 0.16mmol) were dissolved in anhydrous dichloromethane (0.5mL), 4-dimethylaminopyridine (DMAP, 2mg, 0.02mmol) was added under argon protection in an ice bath, slowly warmed to room temperature and stirred for 24h, quenched with water, extracted with dichloromethane, the combined organic phases were dried over anhydrous sodium sulfate, concentrated, and subjected to column chromatography to give compound 7(20S,24R) -epoxy-25-hydroxy-3 β -O- (N-Boc-phenylalanyl) -dammarane-12 one (26mg, 0.04mmol, 72% yield).
Compound 71H NMR(400MHz,CHCl3)δ7.29(t,J＝1.6Hz,1H),7.26(d,J＝4.6Hz,1H),7.22(dd,J＝6.2,3.9Hz,1H),7.16(t,J＝1.8Hz,1H),7.15(d,J＝1.4Hz,1H),4.88(d,J＝8.4Hz,1H),4.55(d,J＝7.7Hz,1H),4.46(dd,J＝11.3,4.5Hz,1H),3.68(dd,J＝8.7,6.1Hz,1H),3.11(dd,J＝14.1,6.0Hz,1H),3.00(dd,J＝14.0,6.6Hz,1H),2.87(d,J＝9.5Hz,1H),2.55(td,J＝10.7,4.5Hz,1H),2.18(d,J＝7.4Hz,2H),1.96-1.38(m,18H),1.38(s,9H),1.19(s,3H),1.17(s,3H),1.09(s,3H),1.08(s,3H),0.92(s,3H),0.80(s,3H),0.77(s,3H),0.74(s,3H)
Example 6: (20S,24R) -epoxy-25-hydroxy-3 beta-O- (N-Boc-valoylester) -dammarane-12-one
Dissolving compound 5(33mg, 0.07mmol), N-Boc-2-valine (27mg, 0.08 mmol 1) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 40mg, 0.21mmol) in anhydrous dichloromethane (0.6mL), adding 4-dimethylaminopyridine (DMAP, 3mg,0.03mmol) under argon protection in an ice bath, slowly raising the temperature to room temperature and stirring for 24h, quenching the reaction with water, extracting with dichloromethane, drying the combined organic phases over anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain compound 8(20S,24R) -epoxy-25-hydroxy-3 β -O- (N-Boc-valyl) -dammarane-12 one (28mg, 0.04mmol, 57% yield)
Compound 81H NMR(400MHz,CHCl3)δ4.98(d,J＝9.2Hz,1H),4.49(dd,J＝11.3,4.9Hz,1H),4.19(dd,J＝9.2,4.1Hz,1H),3.67(dd,J＝8.7,6.1Hz,1H),2.87(d,J＝9.5Hz,1H),2.54(td,J＝10.7,4.5Hz,1H),2.18(d,J＝8.0Hz,2H),1.86-1.46(m,19H),1.42(s,9H),1.23(s,3H),1.18(d,J＝3.9Hz,6H),1.08(d,J＝1.6Hz,6H),0.96(d,J＝6.9Hz,3H),0.93(s,3H),0.87(s,3H),0.85(s,3H),0.74(s,3H).
Example 7: (20S,24S) -epoxy-25-hydroxy-3 beta-O- (N-Boc-glycylcarbonyl) -dammarane-12-one
Dissolving compound 6(25mg, 0.05mmol), N-Boc-2-glycine (11mg, 0.06 mmol 1) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 30mg, 0.16mmol) in anhydrous dichloromethane (0.5mL), adding 4-dimethylaminopyridine (DMAP, 3mg, 0.02mmol) under argon protection in an ice bath, slowly raising the temperature to room temperature and stirring for 24h, quenching the reaction with water, extracting with dichloromethane, drying the combined organic phases over anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain compound 9(20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-glycyl) -dammarane-12-one (20mg, 0.03mmol, 60% yield)
Compound 91H NMR(400MHz,CHCl3)δ4.99(d,J＝4.3Hz,1H),4.53(dd,J＝11.1,5.3Hz,1H),3.88(d,J＝5.2Hz,2H),3.69(dd,J＝9.7,5.5Hz,1H),2.94(d,J＝9.4Hz,1H),2.52(td,J＝9.9,4.3Hz,1H),2.19(d,J＝10.7Hz,2H),1.95-1.44(m,18H),1.43(s,9H),1.18(s,3H),1.17(s,3H),1.08(s,3H),1.02(s,3H),0.95(s,3H),0.86(s,3H),0.85(s,3H),0.73(s,3H).
Example 8: (20S,24S) -epoxy-25-hydroxy-3 beta-O-glutaminyl-dammaran-12-one
Compound 6(25mg, 0.05mmol), N-Boc-5-otBu-glutamic acid (18mg, 0.06mmo1) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 30mg, 0.16mmol) were dissolved in anhydrous dichloromethane (0.5mL), 4-dimethylaminopyridine (DMAP, 2mg, 0.02mmol) was added under argon protection in an ice bath, slowly warmed to room temperature and stirred for 24h, quenched with water, extracted with dichloromethane, the combined organic phases dried over anhydrous sodium sulfate, concentrated and subjected to column chromatography to give (20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-5-otBu-glutamyl) -dammaran-12-one (28mg, 0.04mmol, 74% yield).
(20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-5-otBu-glutamyl) -dammarane-12-one (26mg, 0.04mmol) obtained above was dissolved in trifluoroacetic acid (0.3mL), stirred at room temperature for 1h, concentrated, and subjected to column chromatography (chloroform: methanol: 10/1-6/1) to give compound 10(20S,24S) -epoxy-25-hydroxy-3 β -O-glutamyl-dammarane-12-one (25mg, 0.03mmol, 98%)
Compound 101H NMR(400MHz,CHCl3)δ4.56(t,J＝7.0Hz,1H),4.37-4.19(m,2H),3.75-3.62(m,1H),2.95(d,J＝9.3Hz,1H),2.70-2.59(m,1H),2.56-2.26(m,3H),2.20(d,J＝6.6Hz,2H),1.90-1.53(m,18H),1.18(d,J＝6.7Hz,6H),1.10(s,3H),1.04(s,3H),0.95(s,3H),0.88(d,J＝3.0Hz,3H),0.85(d,J＝1.9Hz,3H),0.73(s,3H).
Example 9: (20S,24S) -epoxy-25-hydroxy-3 beta-O-aspartyl-dammaran-12-one
Compound 6(25mg, 0.05mmol), N-Boc-2-otBu-aspartic acid (17mg, 0.06mmo1) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 30mg, 0.16mmol) were dissolved in anhydrous dichloromethane (0.5mL), 4-dimethylaminopyridine (DMAP, 2mg, 0.02mmol) was added under argon protection in an ice bath, slowly warmed to room temperature and stirred for 24h, quenched with water, extracted with dichloromethane, the combined organic phases dried over anhydrous sodium sulfate, concentrated and subjected to column chromatography to give (20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-2-otBu-aspartyl) -dammaran-12-one (26mg, 0.04mmol, 72% yield).
(20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-2-otBu-aspartyl) -dammarane-12 one (26mg, 0.04mmol) obtained above was dissolved in trifluoroacetic acid (0.3mL), stirred at room temperature for 1 hour, concentrated, and subjected to column chromatography (chloroform: methanol: 10/1-6/1) to give compound 11(20S,24S) -epoxy-25-hydroxy-3 β -O-aspartyl-dammarane-12-one (25mg, 0.03mmol, 98%)
Compound 111H NMR(400MHz,CHCl3)δ4.61-4.50(m,1H),4.29(t,J＝6.7Hz,2H),3.72-3.65(m,1H),3.17-3.00(m,2H),2.94(d,J＝7.8Hz,1H),2.57-2.47(m,1H),2.17(d,J＝10.0Hz,2H),1.75-1.33(m,18H),1.17(d,J＝9.3Hz,6H),1.08(s,3H),1.03(s,3H),0.94(d,J＝3.2Hz,3H),0.85(d,J＝7.0Hz,6H),0.73(s,3H).
Example 10: (20S,24S) -epoxy-25-hydroxy-3 beta-O- (N-Boc-phenylalanyl) -dammarane-12-one
Dissolving compound 6(25mg, 0.05mmol), N-Boc-2-phenylalanine (17mg, 0.06 mmol 1) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 30mg, 0.16mmol) in anhydrous dichloromethane (0.5mL), adding 4-dimethylaminopyridine (DMAP, 2mg, 0.02mmol) under argon protection in an ice bath, slowly raising the temperature to room temperature and stirring for 24h, quenching the reaction with water, extracting with dichloromethane, drying the combined organic phases over anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain compound 12(20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-phenylalanyl) -dammarane-12 one (27mg, 0.04mmol, 73% yield)
Compound 121H NMR(400MHz,CHCl3)δ7.30(t,J＝1.6Hz,1H),7.27(d,J＝1.3Hz,1H),7.23(d,J＝7.1Hz,1H),7.17(dd,J＝6.8,1.5Hz,2H),4.89(d,J＝8.8Hz,1H),4.61-4.52(m,1H),4.48(dd,J＝11.3,4.5Hz,1H),3.71(dd,J＝9.9,5.6Hz,1H),3.12(dd,J＝13.6,5.9Hz,1H),3.01(dd,J＝14.0,7.0Hz,1H),2.95(d,J＝9.4Hz,1H),2.53(td,J＝10.2,4.2Hz,1H),2.20(d,J＝7.1Hz,2H),1.90-1.48(m,18H),1.39(s,9H),1.19(d,J＝3.3Hz,6H),1.09(s,3H),1.03(s,3H),0.94(s,3H),0.82(s,3H),0.78(s,3H),0.75(s,3H)
Example 11: (20S,24S) -epoxy-25-hydroxy-3 beta-O- (N-Boc-valoylester) -dammarane-12-one
Dissolving compound 6(25mg, 0.05mmol), N-Boc-valine (13mg, 0.06 mmol 1) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 30mg, 0.16mmol) in anhydrous dichloromethane (0.5mL), adding 4-dimethylaminopyridine (DMAP, 2mg, 0.02mmol) under argon protection in an ice bath, slowly raising to room temperature and stirring for 24h, quenching the reaction with water, extracting with dichloromethane, drying the combined organic phases over anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain compound 13(20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-valyl) -dammarane-12 one (26mg, 0.04mmol, 77% yield)
Compound 131H NMR(400MHz,CHCl3)δ4.98(d,J＝9.1Hz,1H),4.49(dd,J＝11.5,4.8Hz,1H),4.20(dd,J＝9.2,4.1Hz,1H),3.69(dd,J＝9.8,5.6Hz,1H),2.94(d,J＝9.4Hz,1H),2.51(td,J＝10.1,4.3Hz,1H),2.18(d,J＝6.8Hz,2H),1.96-1.43(m,19H),1.42(s,9H),1.23(s,3H),1.17(d,J＝6.9Hz,6H),1.07(s,3H),1.01(s,3H),0.96(d,J＝6.8Hz,3H),0.94(d,J＝3.5Hz,3H),0.87(s,3H),0.85(s,3H),0.73(s,3H).
Example 12: (20S,24R) -epoxy-25-hydroxy-3 beta-O-phenylalanyl-dammarane-12-one
Dissolving compound 7(20S,24R) -epoxy-25-hydroxy-3 β -O- (N-Boc-phenylalanyl) -dammarane-12-one (26mg, 0.04mmol) in trifluoroacetic acid (0.3mL), stirring at room temperature for 1h, concentrating, and performing column chromatography (chloroform: methanol: 10/1-6/1) to give compound 14(20S,24R) -epoxy-25-hydroxy-3 β -O-phenylalanyl-dammarane-12-one (25mg, 0.04mmol, 98%)
Compound 141H NMR(400MHz,Chloroform-d)δ7.70-7.68(m,1H),7.53-7.50(m,1H),7.28(s,2H),6.98(s,1H),4.46(d,J＝5.8Hz,1H),4.32-4.16(m,1H),3.70(t,J＝7.0Hz,1H),3.37-3.17(m,2H),2.84(d,J＝9.5Hz,1H),2.60-2.50(m,1H),2.15(d,J＝7.9Hz,2H),1.85-1.34(m,18H),1.17(d,J＝6.1Hz,6H),1.09(s,3H),1.06(s,3H),0.89(s,3H),0.76-0.68(m,9H).
Example 13: (20S,24R) -epoxy-25-hydroxy-3 beta-O-valoylester-dammaran-12-one
Dissolving compound 8(20S,24R) -epoxy-25-hydroxy-3 β -O- (N-Boc-valyloxy) -dammaran-12-one (28mg, 0.04mmol) in trifluoroacetic acid (0.3mL), stirring at room temperature for 1h, concentrating, and performing column chromatography (chloroform: methanol 10/1-6/1) to give compound 15(20S,24R) -epoxy-25-hydroxy-3 β -O-valyloxy-dammaran-12-one (27mg, 0.04mmol, 98%)
Compound 151H NMR(400MHz,CHCl3)δ4.57(d,J＝7.3Hz,1H),4.29(t,J＝6.7Hz,1H),3.68(dd,J＝30.0,7.0Hz,1H),2.85(d,J＝9.7Hz,1H),2.55(td,J＝9.8,4.3Hz,1H),2.33(d,J＝11.2Hz,2H),2.18(d,J＝8.0Hz,1H),1.85-1.36(m,18H),1.24(s,6H),1.18(s,6H),1.10(s,3H),1.05(s,3H),0.94(s,3H),0.87(d,J＝6.6Hz,6H),0.75(s,3H).
Example 14: (20S,24R) -epoxy-25-hydroxy-3 beta-O-aspartyl-otBuaminoacyl-dammarane-12-one
Dissolving compound 5(25mg, 0.05mmol), N-Boc-2-otBu-aspartic acid (17mg, 0.06 mmol 1) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI, 30mg, 0.16mmol) in anhydrous dichloromethane (0.5mL), adding 4-dimethylaminopyridine (DMAP, 2mg, 0.02mmol) under argon protection in an ice bath, slowly raising to room temperature and stirring for 24h, quenching with water, extracting with dichloromethane, drying the combined organic phases over anhydrous sodium sulfate, concentrating, and performing column chromatography to obtain (20S,24R) -epoxy-25-hydroxy-3 β -O- (N-Boc-2-otBuaspartyl) -dammarane-12 one (26mg, 0.04mmol, 72% yield)
(20S,24R) -epoxy-25-hydroxy-3 β -O- (N-Boc-2-otBu-aspartyl) -dammarane-12 one (26mg, 0.04mmol) obtained above was dissolved in trifluoroacetic acid (0.3mL), stirred at room temperature for 1 hour, concentrated, and subjected to column chromatography (chloroform: methanol: 10/1-6/1) to give compound 16(20S,24R) -epoxy-25-hydroxy-3 β -O-aspartyl-dammarane-12-one (25mg, 0.03mmol, 98%)
Compound 161H NMR(400MHz,CHCl3).δ4.58-4.22(m,2H),3.79-3.66(m,1H),3.16-2.97(m,2H),2.86(d,J＝9.2Hz,1H),2.62-2.46(m,1H),2.16(d,J＝6.1Hz,2H),1.85-1.38(m,18H),1.18(s,3H),1.10(s,3H),1.06(s,3H),0.97(s,3H),0.92(s,3H),0.84(s,3H),0.79(s,3H),0.74(s,3H).
Example 15: detection of the inhibitory activity of the Ocotillol-type esterified derivatives on NO production:
NO is an important inflammatory mediator. The amount of NO produced in the body exceeds normal levels when certain pathological changes occur in the body. Thus, NO inhibitors have the potential and opportunity to find new treatments for inflammation-related disorders. To evaluate the anti-inflammatory effect of the Ocotillol-type esterified derivatives, Lipopolysaccharide (LPS) -induced NO release levels in RAW264.7 cells were measured using Griess reagent. RAW264.7 cells were plated at 1X106Individual cells/well were seeded in 96-well plates and cultured for 1 hour; then LPS (1ug/mL) is used for stimulating and molding, and after the cells are treated with 20 mu M of Ocotillol type esterified derivative and positive drug (hydrocortisone sodium succinate) for 24 hours, the amount of NO generated is determined by detecting the nitrite level by using Griess reagent (Beyotime, China); the absorbance of the sample at 540nm (OD540) was then measured in a microplate reader (spectra maxm 3); wherein the blank group is LPS-free and drug-treated group; the control group is a LPS-stimulated molding group but no compound treatment group;
NO inhibition ═ 100% for [ control (OD540) -compound (OD540) ]/[ control (OD540) -blank (OD540) ];
the NO inhibition results are shown in figure 1:
the legends illustrate the following:
(1) each value is from the mean of three parallel experiments ± SD (n ═ 3);
(2) the different letters between each set of data represent significant differences (p < 0.05);
compared with the control group, # P <0.05, # P < 0.01, # P < 0.001.
The results of example 15 show that:
the protective group-containing Ocotillol esterified derivatives 7, 8, 9, 12 and 13 have the inhibition rate of over 20 percent on NO generation at the concentration of 20 mu M and are over 2 times of the inhibition rate (10 percent) of positive comparative hydrocortisone sodium succinate, wherein the inhibition rate of the compound 8(20S,24R) -epoxy-25-hydroxy-3 beta-O- (N-Boc-valyl) -dammarane-12 ketone on NO generation at the concentration of 20 mu M is over 40 percent, and the compound shows stronger effect of inhibiting NO release induced by LPS.
The compounds 10, 11 and 14 are protecting group-removed Ocotillol esterified derivatives, the inhibition rate of the compounds on NO generation is more than 25 percent under the concentration of 20 mu M, the anti-inflammatory activity is obvious, and the inhibition rate is more than 2.5 times of the inhibition rate (10 percent) of positive contrast hydrocortisone sodium succinate.
The Ocotillol esterified derivative provided by the invention has good anti-inflammatory activity, and is obviously superior to the anti-inflammatory activity of a positive medicament (hydrocortisone sodium succinate); the Ocotillol esterified derivatives provided by the invention remarkably inhibit the increase of NO release induced by LPS, and the anti-inflammatory activity of inhibiting NO generation is more efficient than that of a clinical drug hydrocortisone sodium succinate.
Example 16: drug in vitro cytotoxicity assay
The experimental method comprises the following steps:
the MTT method, also known as MTT colorimetric method, is a method for detecting cell survival and growth. The principle is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-purple crystalline Formazan (Formazan) and deposit in cells, but the phenomenon does not occur in dead cells. Formazan crystals can be dissolved in DMSO, and absorbance is measured at a wavelength of 570nm by an enzyme linked immunosorbent assay (ELISA) detector, and the absorbance value is directly proportional to the number of living cells in a certain cell number range, thereby indirectly reflecting the number of the living cells.
1. Cell plating: taking adherent RAW cells which grow in logarithmic growth phase and are in good state, and digesting the adherent RAW cells into single cell suspension by pancreatin. After counting, the cells are diluted to 3-4 multiplied by 106one/mL and 100 uL/well in 96-well plate, at 37 degrees C, 5% CO2 incubator stationary culture.
2. Cell administration: after the cells were plated for 2h and adhered to the wall, 20 μ M of different compounds and corresponding solvents were added for control culture, and 3 parallel wells were placed in each group. And (4) after the medicine is added, placing the 96-hole plate in an incubator, and performing static culture for 24 hours.
3. MTT detection: after culturing the cells for 24 hours by administering the corresponding drug, MTT solution at a concentration of 5mg/mL was added to 20uL per well, and the cells were incubated at 37 ℃ in an incubator for 3 hours, and the medium containing MTT was discarded. Under a dark environment, 150uLDMSO is added into each hole to dissolve formazan, after shaking and mixing evenly, the absorbance value of each hole is measured at 570nm, macrophage groups added with DMSO to be used as a control group, and hydrocortisone sodium succinate to be used as a positive control group, and the survival rate of cells is calculated and obtained.
Cell survival (%). The mean of administration group/mean of blank group X100%
The results are shown in FIG. 2.
The Ocotillol esterified derivative provided by the invention has no obvious toxicity to cells, and the cell survival rate basically reaches the standard of lower toxicity or higher.
The cell survival rates of the compounds 7, 8, 9, 12 and 13 in-vitro toxicity experiments reach over 90 percent, are equal to or exceed the survival rate (90 percent) compared with a positive medicament, and show good safety and cell non-toxicity. Therefore, compared with the existing clinical medicines, the compounds 7, 8, 9, 12 and 13 have better effect of inhibiting NO release induced by LPS, and have good safety and lower toxicity.
Compounds 10, 11, 14 showed low toxicity to cells, with cell survival rates above 70%.
The applicant speculates that the protecting group on the amino group reduces the toxicity of the compound to cells, increases the lipid solubility of the compound, and makes the compound have higher membrane permeability, thereby having better anti-inflammatory activity.
Example 17: metabolic stability evaluation
The total volume of each incubation system was 200uL according to the literature, the system included 188 uL of 0.1M PBS buffer (pH 7.4) and 12uL of NADPH generating system. The experiment was divided into 3 groups (n ═ 3), and the experimental group, the positive control group, and the negative control group were sequentially included. The experimental group was an incubation system containing compound 8/compound a (20S,24R) -epoxy-3 β -O- (Boc-glycylglycidylcarbonyl) -dammarane-12 β, 25-diol (CN109776647A example 1 compound)/compound b (20S,24R) -epoxy-12 β, 25-dihydroxy-3 β -O-alanylcylato-dammarane (CN110642913A example 1 compound); the positive control group is an incubation system containing a positive control hydrocortisone to determine the activity of the reaction system; the negative control group contained no NADPH in the incubation system to determine the stability of Compound 8/Compound a/Compound b in the reaction system. After pre-incubation in 37 ℃ water bath for 3min, NADPH is added to start reaction, 0,5,10,20,40 and 60min are set, and 400 mu L of glacial methanol containing an internal standard is added to stop reaction after 6 sampling points are reacted.
Taking the concentration of the compound to be detected at the incubation time point of 0min as 100%, converting the concentrations of other incubation time points into the residual percentage, performing linear regression on the incubation time by using the natural logarithm of the residual percentage of each time point, calculating the slope k, and obtaining the slope k according to the formula T1/2The in vitro half-life can be calculated as-0.693/k.
Compound 8T1/2＝7.14
Compound aT1/2＝6.34
Compound aT1/2＝6.19
The hydroxyl at the 12-position in the structural formulas of the compounds a and b is easy to be oxidized and metabolized, so that the half life of the compounds is reduced, and the active action time of the compounds is correspondingly reduced. The Ocotillol esterified derivative provided by the application converts 12-site hydroxyl into carbonyl which is not easy to oxidize, increases the half-life period of the compound, and is beneficial to improving the active action time of a medicament.
Claims (10)
1. An esterified derivative of the Ocotillol type, represented by formula (I-R) or formula (I-S), or a pharmaceutically acceptable salt of a compound represented by formula (I-R) or formula (I-S):
in the formula (I-R) or the formula (I-S), R, S represents chirality of carbon atom at position 24, respectively;
in the formula (I-R) or the formula (I-S), R1Is hydrogen, C1-C4 alkyl, phenyl, benzyl, p-hydroxybenzyl, -RaCOORb、-RaORb、-RaNHRcor-RaSRd；
R1In, RaIs C1-C4 alkylene; rbH, Bn or tBu; rcH, Boc or Fmoc; rdIs H or methyl;
R3is H or C1-C4 alkyl.
2. An esterified derivative of the Ocotillol type, which is represented by formula (II-R) or formula (II-S), or a pharmaceutically acceptable salt of a compound represented by formula (II-R) or formula (II-S):
in the formula (II-R) or the formula (II-S), R, S represents chirality of carbon atom at position 24, respectively;
in the formula (II-R) or the formula (II-S), R1Is hydrogen, C1-C4 alkyl, phenyl, benzyl, p-hydroxybenzyl, -RaCOORb、-RaORb、-RaNHRcor-RaSRd；R2Is tert-butyloxycarbonyl, fluorenylmethyloxycarbonyl or benzyl; r3Is H or C1-C4 alkyl;
R1in, RaIs C1-C4 alkylene; rbH, Bn or tBu; rcH, Boc or Fmoc; rdIs H or methyl.
3. The process for the preparation of an esterified derivative of the Ocotillol type represented by the formula (I-R) or the formula (I-S) according to claim 1, which comprises deprotecting an esterified derivative of the Ocotillol type represented by the formula (II-R) or the formula (II-S) to produce an esterified derivative of the Ocotillol type represented by the formula (I-R) or the formula (I-S);
in the formula (II-R) or the formula (II-S), R, S represents chirality of carbon atom at position 24, respectively;
in the formula (II-R) or the formula (II-S), R1Is hydrogen, C1-C4 alkyl, phenyl, benzyl, p-hydroxybenzyl, -RaCOORb、-RaORb、-RaNHRcor-RaSRd；R2Is tert-butyloxycarbonyl, fluorenylmethyloxycarbonyl or benzyl; r3Is H or C1-C4 alkyl;
R1in, RaIs C1-C4 alkylene; rbH, Bn or tBu; rcH, Boc or Fmoc; rdIs H or methyl.
4. Esterified derivative of the Ocotillol type of formula (I-R) or (I-S) according to claim 1, characterized in that R is1Is hydrogen, isopropyl, benzyl, - (CH)2)2-COOH、-CH2-COOH、-(CH2)2-COO-tBu or-CH2-COO-tBu；R3Is H.
5. Esterified derivative of the Ocotillol type of formula (I-R) or (I-S) according to claim 4, characterized in that it is one of the following:
(20S,24S) -epoxy-25-hydroxy-3 beta-O-glutamyl-dammarane-12-one, (20S,24S) -epoxy-25-hydroxy-3 beta-O-aspartyl-dammarane-12-one, (20S,24R) -epoxy-25-hydroxy-3 beta-O-phenylalanyl-dammarane-12-one, (20S,24R) -epoxy-25-hydroxy-3 β -O-valyloxy-dammaran-12-one, (20S,24R) -epoxy-25-hydroxy-3 β -O-aspartyl-dammaran-12-one.
6. Esterified derivative of the Ocotillol type of formula (II-R) or (II-S) according to claim 2, characterized in that R is1Is hydrogen, isopropyl, benzyl, - (CH)2)2-COOH、-CH2-COOH、-(CH2)2-COO-tBu or-CH2-COO-tBu；R2Is Boc; r3Is H.
7. An esterified derivative of the Ocotillol type represented by the formula (II-R) or the formula (II-S) as claimed in claim 6, wherein said derivative is (20S,24R) -epoxy-25-hydroxy-3 β -O- (N-Boc-phenylalanyl) -dammarane-12-one, (20S,24R) -epoxy-25-hydroxy-3 β -O- (N-Boc-valyloxy) -dammarane-12-one, (20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-glycyl) -dammarane-12-one, (20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-phenylalanyl) -dammarane-12-one, or (20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-phenylalanyl) -dammarane -12 ketones, (20S,24S) -epoxy-25-hydroxy-3 β -O- (N-Boc-valyloxy) -dammaran-12 ketones.
8. A process for the preparation of esterified derivatives of the octillol type, represented by formula (II-R) or (II-S), according to claim 2, characterized in that it comprises: protopanoxadiol shown in formula (III) is used as a raw material, and is subjected to double-building epoxidation and intramolecular nucleophilic attack reaction with m-chloroperoxybenzoic acid, separation and purification are carried out to prepare (20S,24R) -epoxy dammarane-3 beta, 12 beta, 25-triol shown in formula (IV-R) and (20S,24S) -epoxy dammarane-3 beta, 12 beta, 25-triol shown in formula (IV-S), the compound shown in formula (IV-R) or formula (IV-S) is oxidized by a desimadine reagent, 3-position hydroxyl and 12-position hydroxyl are oxidized to generate ketone, and (20S,24R) -epoxy-25-hydroxyl-dammarane-3, 12-diketone shown in formula (V-R) or (20S) shown in formula (V-S) are respectively and correspondingly prepared, the compound shown in the formula (V-R) or the formula (V-S) is selectively reduced by sodium borohydride to reduce the carbonyl group at the 3-position into hydroxyl group, respectively and correspondingly prepare (20S,24R) -epoxy-25-hydroxyl-3 beta-hydroxyl-dammarane-12-ketone shown in the formula (VI-R) or (20S,24) -epoxy-25-hydroxyl-3 beta-hydroxyl-dammarane-12-ketone shown in the formula (VI-S), the compound shown in the formula (VI-R) or the formula (VI-S) and the amino acid derivative shown in the formula (VII) are subjected to esterification reaction, respectively and correspondingly prepare the protection group-containing Ocotillol type esterified derivative shown in the formula (II-R) or the formula (II-S) which is prepared (ii) a The reaction formula is as follows:
9. use of an esterified derivative of the Ocotillol type of formula (I-R) or (I-S) according to claim 1 and of its pharmaceutically acceptable salts for the preparation of anti-inflammatory drugs.
10. Use of an esterified derivative of the octillol type of formula (II-R) or (II-S) according to claim 2 and pharmaceutically acceptable salts thereof for the preparation of anti-inflammatory drugs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010367674.0A CN111647036B (en) | 2020-04-30 | 2020-04-30 | Ocotillol esterified derivatives, preparation method thereof and application thereof in preparing anti-inflammatory drugs |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010367674.0A CN111647036B (en) | 2020-04-30 | 2020-04-30 | Ocotillol esterified derivatives, preparation method thereof and application thereof in preparing anti-inflammatory drugs |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111647036A CN111647036A (en) | 2020-09-11 |
| CN111647036B true CN111647036B (en) | 2022-05-13 |
Family
ID=72346056
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010367674.0A Active CN111647036B (en) | 2020-04-30 | 2020-04-30 | Ocotillol esterified derivatives, preparation method thereof and application thereof in preparing anti-inflammatory drugs |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111647036B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113214339A (en) * | 2021-03-20 | 2021-08-06 | 延边大学 | Panaxadiol derivatives, preparation method and medical application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102875628A (en) * | 2012-10-30 | 2013-01-16 | 中国药科大学 | (20S,24S)-ocotillol ginsenoside derivatives with antibacterial activity, and preparation method and application thereof |
| CN102924556A (en) * | 2012-11-05 | 2013-02-13 | 烟台大学 | (20S, 24R)-ocotillol type ginsenoside derivative having antibacterial activity and preparation method and application thereof |
| WO2019128608A1 (en) * | 2017-12-26 | 2019-07-04 | 厦门本素药业有限公司 | Blank mixed micelle, preparation method therefor and application thereof |
-
2020
- 2020-04-30 CN CN202010367674.0A patent/CN111647036B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102875628A (en) * | 2012-10-30 | 2013-01-16 | 中国药科大学 | (20S,24S)-ocotillol ginsenoside derivatives with antibacterial activity, and preparation method and application thereof |
| CN102924556A (en) * | 2012-11-05 | 2013-02-13 | 烟台大学 | (20S, 24R)-ocotillol type ginsenoside derivative having antibacterial activity and preparation method and application thereof |
| WO2019128608A1 (en) * | 2017-12-26 | 2019-07-04 | 厦门本素药业有限公司 | Blank mixed micelle, preparation method therefor and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111647036A (en) | 2020-09-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2762522B2 (en) | Angiogenesis inhibitor | |
| CA2304770C (en) | A use of steroidal saponins for the prophylaxis or treatment of dementia, and novel steroidal saponin compounds | |
| US8835134B2 (en) | Cycloastragenol monoglucoside, preparation, pharmaceutical composition and application thereof | |
| WO2022257995A1 (en) | Cryptotanshinone derivative and preparation method therefor and application thereof in lowering lipid and resisting obesity | |
| CN111647036B (en) | Ocotillol esterified derivatives, preparation method thereof and application thereof in preparing anti-inflammatory drugs | |
| CN108992453B (en) | New application of ocotillol type sapogenin derivative in tumor drug resistance reversal | |
| CN110964078B (en) | Hederagenin compound H-X with anti-lung cancer effect and preparation method and application thereof | |
| CN111471080B (en) | ocotillol type ginsengenin A-ring amino thiazole ring derivative and preparation method thereof | |
| CN109776647B (en) | Pyxinol esterified derivative with anti-inflammatory activity and preparation method and application thereof | |
| TW201335177A (en) | Sterol derivatives and preparation method and use thereof | |
| CN106967143A (en) | Novel pyxinol derivatives of structure and its production and use | |
| CN108659089B (en) | Sterol compound with antioxidant effect and application thereof in preparation of medicines | |
| CN111333694A (en) | Application of hederagenin derivative in medicine for resisting myocardial anoxia reoxygenation injury | |
| KR100564383B1 (en) | Process for preparing ginsenoside derivatives | |
| CN104224796B (en) | Application of oleanane triterpene ester derivative in preparation for anti-neurodegeneration medicine | |
| KR100547253B1 (en) | Treating and prevention of cancer with new ginsenoside derivatives | |
| EP2789604B1 (en) | Daidzein derivative, pharmaceutically acceptable salt and preparation method thereof, and pharmaceutical composition containing same | |
| JPH0678231B2 (en) | Blood viscosity reducing agent | |
| CN113730419B (en) | 20S, 24R-epoxy-dammarane-3 beta, 12 beta, 25-triol derivative and pharmaceutical composition thereof | |
| CN116715706B (en) | Chemical modification method for prolonging in-vivo half-life of theanine | |
| CN111825608A (en) | Tetrahydroquinoline and tetrahydroisoquinoline compounds and application thereof | |
| CN115181154B (en) | Pyxinol amide derivative, preparation method thereof and application thereof in pharmaceutical field | |
| CN104788528B (en) | Rosy clouds grass Triterpenoids sapogenins compound, the pharmaceutical composition containing this compound and application thereof | |
| CN107056877A (en) | A kind of steroid compound and application thereof | |
| CN103239459A (en) | Use of sterol derivative in preparation of medicines for preventing and/or treating and/or adjunctively treating cancers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |