CN109517026B - L-phenylalanine modified maslinic acid and synthetic method and application thereof - Google Patents
L-phenylalanine modified maslinic acid and synthetic method and application thereof Download PDFInfo
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Abstract
The invention discloses a series of L-phenylalanine modified maslinic acid and a synthesis method and application thereof. The L-phenylalanine modified maslinic acid is specifically 8 compounds, benzyl maslinic acid and Boc-L-phenylalanine react in a first organic solvent in the presence of dicyclohexylcarbodiimide and 4-dimethylaminopyridine, and the reactants are subjected to silica gel column chromatography after the solvent is recovered, so that a compound 1 and a compound 2 are obtained; and then the compound 1 and the compound 2 are respectively used as raw materials to react to obtain the other 6 compounds. The in vitro test results of the applicant show that the compounds have good proliferation inhibition activity on certain tumor cell strains, have good potential medicinal value and are expected to be used for preparing various antitumor medicaments.
Description
Technical Field
The invention relates to a maslinic acid derivative, in particular to L-phenylalanine modified maslinic acid and a synthesis method and application thereof.
Background
Maslinic acid is a pentacyclic triterpenic acid, exists in various natural plants, particularly hawthorn, red dates, loquat leaves and olive, and has high safety to human bodies. In recent years, maslinic acid has been found to have various biological activities such as anticancer, antioxidation, anti-AIDS, antibiosis, anti-diabetes and the like. However, no report about any activity of benzyl maslinic acid is found at present.
Amino acids, as basic building blocks of biologically functional macromolecular proteins, play an important role in the life process. Tumor cells are metabolized vigorously and require a large amount of nutrients to maintain the cells in a state of high-speed propagation, wherein amino acids are essential nutrients. L-phenylalanine belongs to essential amino acids for human body, which cannot be synthesized by human body and needs to be taken from food. At present, no reports related to the synthesis of L-phenylalanine modified maslinic acid derivatives and the application of the L-phenylalanine modified maslinic acid derivatives in tumor treatment are found.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a series of L-phenylalanine modified maslinic acids with novel structures, and a synthetic method and application thereof.
The invention relates to L-phenylalanine modified maslinic acid shown in the following formulas 1-8:
the invention also provides a synthesis method of the L-phenylalanine modified maslinic acid, which comprises the following steps:
the synthesis method of the compound 1 and the compound 2 mainly comprises the following steps: dissolving benzyl maslinic acid in a first organic solvent, adding Boc-L-phenylalanine, Dicyclohexylcarbodiimide (DCC) and 4-Dimethylaminopyridine (DMAP), reacting under heating or non-heating conditions, filtering the obtained material after the reaction is finished, collecting filtrate, recovering the solvent, performing silica gel column chromatography on the obtained residue, eluting with an eluent, and detecting and combining fractions by thin layer chromatography to respectively obtain a compound 1 and a compound 2;
the synthesis method of the compound 3 mainly comprises the following steps: dissolving the compound 1 in a second organic solvent, adding ammonium formate and a palladium-carbon catalyst, reacting under a heating condition, cooling to normal temperature after the reaction is finished, filtering, collecting filtrate, recovering the solvent, separating the residue with a third organic solvent, collecting a water phase, extracting with an extracting agent, collecting an organic phase, washing, drying and spin-drying to obtain a crude compound 3;
the synthesis method of the compound 4 mainly comprises the following steps: dissolving a compound 4 in a first organic solvent, adding a saturated hydrogen chloride ethyl acetate solution, reacting under heating or non-heating conditions, recovering the solvent after the reaction is finished, dispersing obtained residues in a composition of ethyl acetate and water, adjusting the pH value of the system to be alkaline, separating liquid, collecting an organic phase, washing, drying and spin-drying to obtain a crude compound 4;
the synthesis method of the compound 5 mainly comprises the following steps: dissolving the compound 1 in a first organic solvent, adding a saturated hydrogen chloride ethyl acetate solution, reacting under a heating condition or a non-heating condition, and recovering the solvent after the reaction is finished to obtain a crude compound 5;
the synthesis method of the compound 6 mainly comprises the following steps: taking the compound 2 as an initial raw material, and synthesizing according to the synthesis method of the compound 3 to obtain a crude product of the compound 6;
the synthesis method of the compound 7 mainly comprises the following steps: taking the compound 6 as an initial raw material, and synthesizing according to the synthesis method of the compound 5 to obtain a crude product of a compound 7;
the synthesis method of the compound 8 mainly comprises the following steps: taking the compound 2 as an initial raw material, and synthesizing according to the synthesis method of the compound 5 to obtain a crude product of a compound 8;
wherein the content of the first and second substances,
the first organic solvent is one or the combination of more than two of dichloromethane, chloroform, methanol, tetrahydrofuran and ethyl acetate;
the eluent is a mixed solvent consisting of petroleum ether and ethyl acetate;
the second organic solvent is one or the combination of more than two of methanol, ethanol and tetrahydrofuran;
the third organic solvent is a combination of water and one selected from dichloromethane, chloroform and ethyl acetate;
the extractant is dichloromethane, chloroform or ethyl acetate.
In the above-mentioned synthesis methods of the respective compounds, the molar ratios of the respective reaction raw materials are stoichiometric ratios thereof. In the synthesis method of the compound 1 and the compound 2, the raw material benzyl maslinic acid can be synthesized by referring to the existing literature (Carbohydrate Research,2016,424:42-53, European Journal of Medicinal Chemistry,2011,46: 5991-6001) or designing a synthetic route. The structure of the benzyl maslinic acid is shown as the following formula:
in the synthesis method of the compound 1 and the compound 2, dicyclohexylcarbodiimide is used as a condensing agent, and the addition amount of the dicyclohexylcarbodiimide is preferably 1-5 times of the molar amount of benzyl maslinic acid; the 4-dimethylamino pyridine is used as a catalyst, and the addition amount of the 4-dimethylamino pyridine is preferably 10-20% of the molar amount of benzyl maslinic acid. In the method for synthesizing the compound 3 or the compound 6, the palladium carbon catalyst is preferably 10% Pd/C, and in this case, the amount thereof is preferably 5 to 15% by mass of the compound 1 or the compound 2.
In the method for synthesizing the above compound, the first organic solvent or the second organic solvent is preferably used in an amount capable of dissolving the raw material to be reacted. For the third organic solvent, the prior art is referred to for its amount.
In particular, in the synthesis method of the compound 1 and the compound 2, the reaction is preferably carried out at 50 ℃ or lower, more preferably at normal temperature. Whether the reaction is complete can be followed by Thin Layer Chromatography (TLC). When the reaction is carried out under normal temperature conditions, the reaction time is generally controlled to be 8-24 h. In this synthesis method, the eluent preferably has a composition in which the volume ratio of petroleum ether to ethyl acetate is 20-5: 1, more preferably 20-8: 1.
Particularly in the synthesis method of the compound 3 and the compound 6, the reaction is preferably carried out at 40-80 ℃, and the reaction time is usually controlled at 0.5-24h under the temperature condition. In this synthesis method, the third organic solvent preferably has a composition in which the volume ratio of water to methylene chloride, chloroform or ethyl acetate is 1: 1 to 3.
Particularly in the synthesis method of the compound 4, the reaction is preferably carried out at 60 ℃ or lower, and the reaction time is generally controlled to be 1 to 24 hours under the temperature condition. In the synthesis method, the existing conventional alkaline substance is used for adjusting the pH of the system to be alkaline, and preferably, the pH is adjusted by using an aqueous solution of the alkaline substance, such as one or a combination of more than two selected from a sodium carbonate aqueous solution, a sodium bicarbonate aqueous solution, a potassium carbonate aqueous solution and a potassium bicarbonate aqueous solution; more preferably, the adjustment is carried out by using a saturated aqueous sodium carbonate solution, a saturated aqueous sodium bicarbonate solution, a saturated aqueous potassium carbonate solution or a saturated aqueous potassium bicarbonate solution. Preferably, the pH of the system is adjusted to 9-10.
Particularly in the synthesis method of the compound 5, the compound 7 and the compound 8, the reaction is preferably carried out at a temperature of less than or equal to 60 ℃, and the reaction time is generally controlled to be 1 to 24 hours under the temperature condition.
The above-mentioned synthesis of the compounds 3-8 gives crude products of the compounds 3-8, which can be purified by conventional purification methods to increase the purity of each compound. Preferably, in the purification of the crude compound 3, the crude compound 4 and the crude compound 6, silica gel column chromatography is used for purification. When the crude compound 3 or the crude compound 6 is subjected to silica gel column chromatography, the eluent used for elution is a mixed solvent composed of petroleum ether and ethyl acetate, wherein the volume ratio of the petroleum ether to the ethyl acetate is preferably 20-1: 1, and more preferably 15-3: 1. When the crude compound 4 is subjected to silica gel column chromatography, the eluent used for elution is a mixed solvent composed of petroleum ether and ethyl acetate, wherein the volume ratio of the petroleum ether to the ethyl acetate is preferably 60-10: 1, and more preferably 60-20: 1. When the crude compounds 5, 7 and 8 are purified, the crude compounds are preferably dispersed in dichloromethane, chloroform or ethyl acetate, the solvent is evaporated after the mixture is fully stirred, and the residue is subjected to the above operation for 1 to3 times to obtain the purified compounds 5, 7 and 8.
The invention further comprises the application of any compound of the compounds 1-8 in preparing antitumor drugs.
The invention further includes a pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of compounds 1-8 above. The dosage form of the pharmaceutical composition may be a pharmaceutically acceptable dosage form, such as a liquid dosage form, a solid dosage form, or a semi-solid dosage form.
Compared with the prior art, the invention provides a series of L-phenylalanine modified maslinic acid with novel structure and a synthesis method thereof, and in-vitro test results of the applicant show that the maslinic acid has good proliferation inhibition activity on certain tumor cell strains and good potential medicinal value, and is expected to be used for preparing various antitumor medicaments.
Detailed Description
The present invention will be better understood from the following detailed description of specific examples, which should not be construed as limiting the scope of the present invention.
Example 1: synthesis of Compounds 1 and 2
Dissolving benzyl maslinic acid (3.00g,5.33mmol) in CH2Cl2To (50mL) was added Boc-L-phenylalanine (3.50g,13.33mmol), DCC (3.00g,13.33mmol) and DMAP (0.16g,1.33mmol) in this order, and the mixture was stirred at room temperature for 12 hours, filtered, and added CH2Cl2Washing, distilling the filtrate under reduced pressure to remove solvent to obtain residue, and separating the residue by silica gel column chromatography (V)Petroleum ether∶VEthyl acetatePooled fractions were identified by thin layer chromatography at 20: 1 to 8: 1 to give compound 1(1.20g, 27.8%, white solid) and compound 2(2.90g, 51.5%, white solid), respectively.
Compound 1
Yield：1.20g，27.8％，white solid；Rf＝0.412(Petroleum ether：EtOAc＝5∶1).m.p.96-98℃.1H NMR(400MHz，CDCl3)6(ppm)：0.58，0.84，0.90，0.93，0.96，1.03，1.11(7s，each 3H，7×CH3)，1.41(s，9H，3×CH3 Boc)，0.71-2.05(m，20H)，2.91(d，1H，J＝10.3Hz，H-18)，3.05(m，2H，CH2Ar)，3.14(d，1H，J＝9.8Hz，H-3)，4.40(q，1H，J＝6.9Hz，CHCOO)，4.87-5.02(m，2H，H-2，CONH)，5.07(m，2H，OCH2Ar)，5.29(s，1H，H-12)，7.40-7.13(m，10H，Ar-H).13CNMR(100MHz，CDCl3)δ(ppm)：14.3，16.5，16.9，17.0，18.4，22.9，23.2，23.8，26.1，27.7，28.5，28.8，29.6，29.9，30.3，30.4，30.5，30.9，31.6，31.7，32.1，32.5，32.7，33.3，34.1，38.2，38.5，39.5，39.8，41.5，41.9，43.4，46.1，47.7，55.3，55.4，66.2，74.9，80.4，80.5，122.3，127.3，128.2，128.2，128.6，128.8，129.6，136.3，136.6，144.1，155.6，172.4，177.5.HRMS(ESI)m/z：calcd for C51H71NO7Cl[M+C1]-844.4919，found 844.4877.
Yield：2.90g，51.5％，white solid；Rf＝0.562(Petroleum ether：EtOAc＝5∶1).m.p.90-92℃.1H NMR(400MHz，CDCl3)δ(ppm)：0.58，0.91，0.93，1.02，1.12(5s，each 3H，5×CH3)，0.85(s，6H，2×CH3)，1.35，1.36(2s，each 9H，6×CH3 Boc)，0.72-2.12(m，20H)，2.85-2.92(m，1H，H-18)，2.96(m，2H，CH2Ar)，3.05-3.36(m，2H，CH2Ar)，4.27-4.62(m，2H，H-3，CHCOO)，4.81(t，1H，J＝9.7Hz，CHCOO)，4.98-5.24(m，5H，H-2，2×CONH，OCH2Ar)，5.30(s，1H，H-12)，7.12-7.41(m，15H，Ar-H).13C NMR(100MHz，CDCl3)δ(ppm)：16.4，16.5，16.9，17.6，17.7，18.3，21.0，23.1，23.6，23.7，26.0，27.6，28.4，30.8，32.5，32.6，33.2，34.0，37.4，37.7，38.2，38.3，39.4，39.5，39.7，41.5，41.8，43.7，45.9，46.8，47.6，54.7，54.9，55.0，66.0，71.2，71.6，79.8，81.4，122.1，126.8，126.9，128.0，128.1，128.5，129.3，129.4，129.5，136.5，143.9，144.0，155.7，171.8，172.2，177.5.HRMS(ESI)m/z：calcd for C65H88N2O10Na[M+Na]+1079.6337，found1079.6328.
Example 2: synthesis of Compound 3
1(0.50g,0.62mmol) was dissolved in methanol (30mL), and ammonium formate (0.39g,6.20mmol) and 10% Pd/C (0.05g) were added in this order to react at 55 ℃ for 1 h. Cooling to room temperature, filtering, washing the filter cake with methanol, evaporating the filtrate to remove the solvent to obtain residue, and purifying with hydrogen peroxide2O and CH2Cl2Compositional Mixed solvent (60mL, H2O and CH2Cl2In a volume ratio of 1: 1) is shaken up and separated, and the aqueous phase is treated with CH2Cl2(2X 30mL) extraction; mixing organic phases, washing with water and saturated sodium chloride water respectively, drying with anhydrous sodium sulfate, filtering, and evaporating to remove solvent to obtain residue; the residue is chromatographed on a silica gel column (V)Petroleum ether∶VEthyl acetate15: 1 to 3: 1) to give compound 3(0.40g, 89.5%, white solid).
Yield:0.40g,89.5％,white solid；Rf＝0.432(Petroleum ether∶EtOAc＝3∶1).m.p.157-159℃.1H NMR(400MHz,CDCl3)δ(ppm):0.73,0.83,0.91,0.93,0.99,1.04,1.12(7s,each 3H,7×CH3),1.41(s,9H,3×CH3 Boc),0.75–2.06(m,20H),2.83(d,1H,J＝11.6Hz,H-18),3.06(m,2H,CH2Ar),3.15(d,1H,J＝9.6Hz,H-3),4.41(q,1H,J＝7.0Hz,CHCOO),4.96(m,1H,H-2),5.02(d,1H,J＝7.0Hz,CONH),5.28(s,1H,H-12)，7.37–7.07(m,5H,Ar-H).13C NMR(100MHz,CDCl3)δ(ppm):14.3,16.5,16.9,17.3,18.4,23.0,23.7,23.8,25.1,25.8,26.2,27.8,28.5,28.8,29.9,30.4,30.9,31.6,32.6,33.3,34.0,38.2,38.5,39.5,39.8,41.1,41.8,43.4,46.0,46.7,47.7,55.3,55.4,74.8,80.4,80.6,122.4,127.3,128.8,129.6,136.3,144.0,155.6,172.5,184.0.HRMS(ESI)m/z:calcd for C44H65NO7Na[M+Na]+742.4659,found 742.4657.
Example 3: synthesis of Compound 3
Example 2 was repeated except that: THF is used to replace methanol, and the mixed solvent for liquid separation is changed from H2O and EtOAc are 1: 2; the reaction is carried out at 10 ℃, and the reactionThe time is controlled to be 10h, and the rest is unchanged. Finally, a white solid (0.34g, 75.8%) was obtained.
The obtained product is characterized by nuclear magnetic hydrogen spectrum, and is determined to be a compound 3.
Example 4: synthesis of Compound 4
Dissolve 3(0.30g,0.42mmol) in EtOAc (2mL), add saturated ethyl hydrogen chloride solution (12mmol,2mL) and stir at room temperature for 2 h; the reaction mass was evaporated under reduced pressure to remove the solvent and EtOAc (10mL) was added to evaporate the solvent again (repeated twice) to give a white solid; to the resulting white solid was added EtOAc (30mL) and water (10mL), the pH was adjusted to 9 with saturated sodium bicarbonate solution, the layers were separated, and the aqueous layer was extracted with EtOAc (2 × 30 mL); mixing the organic phases, washing with water, washing with saturated salt water, drying with anhydrous sodium sulfate, filtering, and evaporating to remove solvent to obtain residue; the residue was separated by column chromatography on silica gel (V)Methylene dichloride∶VMethanol60: 1 to 20: 1) to give compound 4(0.23g, 90.2%, white solid).
Yield:0.23g,90.2％,white solid；Rf＝0.323(CH2Cl2∶MeOH＝10∶1).m.p.206-208℃.1H NMR(400MHz,DMSO-d6)δ(ppm):0.70,0.73,0.92,0.95,1.10,(5s,each 3H,5×CH3),0.88(2s,each 3H,2×CH3),0.75–2.01(m,20H),2.63–2.81(m,2H,H-18,CHAr),2.85(dd,1H,J＝6.5,12.5Hz,CHAr),3.02(d,1H,J＝10.5Hz,H-3),3.56(t,1H,J＝6.5Hz,CHCOO),4.72(td,1H,J＝4.4,10.5Hz,H-2),4.82(s,1H,3-OH),5.19(s,1H,H-12),6.98–7.35(m,5H,Ar-H).13CNMR(100MHz,DMSO-d6)δ(ppm):16.1,16.8,17.0,18.0,22.6,23.0,23.4,25.7,27.2,28.5,30.4,32.1,32.9,33.3,37.7,40.6,40.8,41.4,43.0,45.4,45.7,46.8,54.3,55.6,72.5,78.0,121.3,126.2,128.0,128.1,129.2,129.3,138.0,143.9,174.8,178.6.HRMS(ESI)m/z:calcd for C39H58NO5[M+H]+620.4315,found620.4309.
Example 5: synthesis of Compound 4
Example 4 was repeated except that: the EtOAc was replaced with chloroform, the reaction was carried out at 60 ℃ for 1h, and the pH was adjusted to 10 with saturated potassium carbonate solution, and the remainder was unchanged. Finally, a white solid (0.21g, 82.0%) was obtained.
The obtained product is characterized by nuclear magnetic hydrogen spectrum, and is determined to be compound 4.
Example 6: synthesis of Compound 5
Dissolving compound 1(0.50g,0.62mmol) in EtOAc (4mL), adding saturated ethyl hydrogen chloride solution (18mmol,3mL), stirring at room temperature for 2h, evaporating the solvent under reduced pressure from the reaction mass, adding EtOAc (10mL) to the residue, stirring well, and evaporating the solvent again (repeating twice) to obtain a white solid; to the resulting white solid was added EtOAc (3mL), stirred at room temperature for 1h, filtered, the filter cake was washed with EtOAc and collected to give compound 5(0.43g, 91.2%, white solid).
Yield:0.43g,91.2％,white solid；Rf＝0.612(CH2Cl2∶MeOH＝10∶1).m.p.223-225℃.1H NMR(400MHz,DMSO-d6)δ(ppm):0.50,0.72,0.87,0.88,0.89,0.94,1.09(7s,each 3H,7×CH3),0.56–2.06(m,20H),2.83(dd,1H,J＝3.9,13.6Hz,H-18),3.00(dd,1H,J＝3.4,10.5Hz,H-3),3.08and 3.22(dd,each 1H,J＝7.0,13.9Hz,CH2Ar),4.20(t,1H,J＝7.0Hz,CHCOO),4.76(td,1H,J＝4.2,10.5Hz,H-2),4.99(d,1H,J＝4.2Hz,3-OH),5.04(d,2H,J＝7.3Hz,OCH2Ar),5.22(s,1H,H-12),7.15–7.42(m,10H,Ar-H),8.72(s,3H,NH2HCl).13C NMR(100MHz,DMSO-d6)δ(ppm):16.0,16.5,16.9,17.8,22.5,22.9,23.3,25.7,27.0,28.4,30.4,32.0,32.7,33.1,35.9,37.6,40.9,41.2,42.6,45.4,46.0,46.8,53.3,54.1,65.3,74.7,77.8,121.6,127.1,127.7,127.9,128.4,128.4,129.6,134.8,136.3,143.5,168.5,176.3.HRMS(ESI)m/z:calcd for C46H64NO5[M+H]+710.4784,found 710.4776.
Example 7: synthesis of Compound 5
Example 6 was repeated except that: the EtOAc was replaced by ethanol, and the reaction was carried out at 10 ℃ for 5h with the remainder being unchanged. Finally, a white solid (0.22g, 46.3%) was obtained.
The obtained product is characterized by nuclear magnetic hydrogen spectrum, and is determined to be the compound 5.
Example 8: synthesis of Compound 6
Dissolving compound 2(1.00g,0.95mmol) in methanol (50mL), adding ammonium formate (0.60g,9.46mmol) and 10% Pd/C (0.10g) in sequence, reacting at 55 deg.C for 1H, cooling the reaction product to room temperature, filtering, washing with methanol, collecting filtrate, evaporating solvent, collecting residue with hydrogen peroxide solution H2Mixed solvent of O and EtOAc (60mL, H)2Volume ratio of O and EtOAc 1: 1) shaking the separated liquid, extracting the aqueous phase with EtOAc (2X 30 mL); mixing organic phases, washing with water and saturated sodium chloride water respectively, drying with anhydrous sodium sulfate, filtering, and evaporating to remove solvent to obtain residue; the residue is separated by column chromatography (V)Petroleum ether∶VEthyl acetate15: 1 to 3: 1) to give compound 6(0.87g, 94.5%, white solid).
Yield:0.87g,89.5％,white solid；Rf＝0.441(Petroleum ether∶EtOAc＝3∶1).m.p.213-215℃.1H NMR(400MHz,CDCl3)δ(ppm):0.74,0.87,0.91,0.94,1.01,1.05,1.13(7s,each 3H,7×CH3),1.35(d,18H,6×CH3 Boc),0.76–2.07(m,20H),2.83(d,1H,J＝10.3Hz,H-18),2.94(m,2H,CH2Ar),3.11(m,2H,CH2Ar),4.36–4.61(m,2H,H-3,CHCOO),4.82(t,1H,J＝9.4Hz,CHCOO),5.18(m,4H,2×CONH,H-2,H-12),6.96–7.47(m,10H,Ar-H).13C NMR(100MHz,CDCl3)δ(ppm):16.6,16.6,17.3,17.8,18.3,23.0,23.7,23.8,26.1,27.8,28.5,28.5,29.9,30.4,30.9,31.6,32.6,33.3,34.0,37.4,37.8,38.3,38.4,39.5,39.6,39.8,41.1,41.8,43.7,46.0,46.7,47.7,54.8,55.1,71.3,79.9,81.5,122.4,127.0,128.6,128.7,129.3,129.5,129.6,136.8,136.9,137.2,144.0,155.8,172.0,172.3,184.0.HRMS(ESI)m/z:calcd for C58H82N2O10Na[M+Na]+989.5867,found989.5858.
Example 9: synthesis of Compound 6
Example 8 was repeated except that: by CH2Cl2And THF in a volume ratio of 1: 1 instead of EtOAc, and the reaction is carried out at 30 ℃ for 1h, and the rest is unchanged. Finally, a white solid (0.47g, 51.2%) was obtained.
The obtained product was characterized by nuclear magnetic hydrogen spectrum and was identified as compound 6.
Example 10: synthesis of Compound 7
Dissolving compound 6(0.50g,0.52mmol) in EtOAc (5mL), adding saturated ethyl hydrogen chloride solution (24mmol,4mL), stirring at room temperature for 2h, evaporating the solvent from the reaction mass, adding EtOAc (10mL) to the residue, stirring well, and evaporating the solvent again (repeating twice) to obtain a white solid; to the resulting white solid was added EtOAc (3mL), stirred at room temperature for 1h, filtered, the filter cake was washed with EtOAc and collected to give compound 7(0.40g, 91.0%, white solid).
Yield:0.40g,91.0％,white solid；Rf＝0.512(CH2Cl2∶MeOH＝10∶1).m.p.291-293℃.1H NMR(400MHz,DMSO-d6)δ(ppm):0.50,0.60,0.66,0.85,1.10(5s,each 3H,5×CH3),0.88(s,6H,2×CH3),0.69–1.99(m,20H),2.75(d,1H,J＝11.6Hz,H-18),3.03and 3.12(dd,each 1H,J＝7.9,14.0Hz,CH2Ar),3.21–3.32(m,2H,CH2Ar),4.34(s,1H,H-3),4.56(m,2H,2×CHCOO),4.80(s,1H,H-2),5.20(s,1H,H-12),7.29(m,10H,Ar-H),8.93(s,6H,2×NH2HCl),12.05(s,1H,COOH).13C NMR(100MHz,DMSO-d6)δ(ppm):15.8,16.6,16.8,22.5,23.3,25.6,27.6,30.4,31.8,32.8,35.8,37.2,38.6,38.7,40.7,41.3,45.4,46.4,52.6,53.4,66.8,71.1,74.5,81.3,120.9,127.1,128.3,128.5,129.3,129.6,134.5,134.7,144.0,168.6,169.0,173.2,178.4.HRMS(ESI)m/z:calcd for C48H67N2O6[M+H]+767.4999,found 767.4997.
Example 11: synthesis of Compound 8
Dissolving compound 2(1.50g,1.42mmol) in EtOAc (6mL), adding saturated ethyl hydrogen chloride solution (36mmol,6mL), stirring at room temperature for 2h, evaporating the solvent from the reaction mass, adding EtOAc (10mL) to the residue, stirring thoroughly, and evaporating the solvent again (repeating twice) to obtain a white solid; to the resulting white solid was added EtOAc (5mL), stirred at room temperature for 1h, filtered, the filter cake was washed with EtOAc and collected to give compound 8(1.21g, 91.7%, white solid).
Yield:1.21g,91.7％,white solid；Rf＝0.510(CH2Cl2∶MeOH＝15∶1).m.p.279-281℃.1H NMR(400MHz,DMSO-d6)δ(ppm):0.48,0.53,0.62,0.83,0.89,0.90,1.10(7s,each 3H,7×CH3),0.71–2.07(m,20H),2.84(dd,1H,J＝3.6,13.5Hz,H-18),3.03and 3.13(dd,each 1H,J＝7.8,14.2Hz,CH2Ar),3.21and3.23(dd,each 1H,J＝6.0,11.7Hz,CH2Ar),4.38(t,1H,J＝7.9Hz,CHCOO),4.63(m,2H,H-3,CHCOO),4.84(td,1H,J＝4.7,10.9Hz,H-2),5.04(m,2H,OCH2Ar),5.24(s,1H,H-12),7.43–7.14(m,15H,Ar-H),8.83(s,6H,2×NH2HCl).13C NMR(100MHz,DMSO-d6)δ(ppm):15.8,16.4,16.9,17.5,22.5,22.9,23.3,25.6,27.0,27.6,30.4,31.8,32.0,32.7,33.2,35.9,37.2,38.7,38.8,41.0,41.3,42.4,45.4,46.0,46.4,52.6,53.4,65.3,71.2,81.4,121.5,127.2,127.2,127.7,127.8,128.3,128.4,,128.6,129.3,129.6,134.4,134.6,136.3,143.6,168.7,169.1,176.2.HRMS(ESI)m/z:calcd for C55H73N2O6[M+H]+857.5469,found857.5457.
Experimental example: the compounds 1 to 8 of the invention have proliferation inhibition activity experiments on various tumor cell strains:
1. cell lines and cell cultures
In the experiment, 3 human cell strains including human bladder cancer cell T24, human gastric cancer cell MGC-803 and human liver cancer cell HepG2 are selected.
All cell lines were cultured in RPMI-1640 medium containing 10% calf serum and 100U/mL streptomycin, and placed at 37 ℃ in a volume concentration of 5% CO2Culturing in an incubator.
2. Preparation of test Compounds
The purity of the used test drug is more than or equal to 95 percent, the DMSO stock solution is diluted by physiological buffer solution to be prepared into 200 mu mol/L final solution, wherein the concentration of the cosolvent DMSO is less than or equal to 1 percent, and the degree of inhibition of the compound on the growth of various tumor cells under the concentration is tested.
3. Cell growth inhibition assay (MTT method)
(1) Taking tumor cells in a logarithmic growth phase, digesting the tumor cells by trypsin, preparing a cell suspension with the concentration of 5000/mL by using a culture solution containing 10% calf serum, inoculating 190 mu L of the cell suspension in a 96-well culture plate in a non-well manner, and enabling the concentration of the cells to be detected to reach 1000-10000/well per well (filling the marginal well with sterile PBS);
(2)5％CO2incubating for 24h at 37 ℃ until a cell monolayer is paved on the bottom of each well, adding 10 mu L of medicine with a certain concentration gradient into each well, and arranging 4 compound wells in each concentration gradient;
(3)5％CO2incubating at 37 ℃ for 48h until the observation is carried out under an inverted microscope;
(4) add 10. mu.L of MTT solution (5mg/mL PBS, i.e., 0.5% MTT) to each well and continue culturing for 4 h;
(5) terminating the culture, carefully removing the culture solution in the wells, adding 150 μ L of DMSO into each well to sufficiently dissolve formazan precipitate, mixing uniformly with an oscillator, and measuring the optical density of each well with a microplate reader at a wavelength of 570nm and a reference wavelength of 450 nm;
(6) simultaneously, a zero setting hole (culture medium, MTT, DMSO) and a control hole (cells, a drug dissolving medium with the same concentration, a culture solution, MTT, DMSO) are arranged.
(7) The number of living cells was judged from the measured optical density values (OD values), and the larger the OD value, the stronger the cell activity. Using the formula:
tumor cell growth inhibition (%) [ (1-mean OD of experimental group)/(mean OD of control group) ] ×%;
IC50and (3) determination: by using the method, each compound is required to be provided with a concentration gradient which comprises a plurality of (generally 5-8) concentrations, each concentration is also required to be provided with 3-5 secondary holes, the inhibition rate of each different concentration is obtained through experiments, and then the IC of the compound is calculated in SPSS software50The results are shown in Table 1 below.
Table 1: IC of each compound on different cell lines50Value (μ M)a
| Compound (I) | T24 | MGC-803 | HepG2 |
| 1 | 38.52±8.31 | 46.97±49.76 | >200 |
| 2 | 14.25±9.54 | >200 | >200 |
| 3 | 6.63±0.30 | 0.94±0.48 | 0.27±0.19 |
| 4 | 1.50±0.14 | 1.30±0.10 | 1.64±0.30 |
| 5 | 8.96±0.86 | 6.64±0.55 | 19.46±6.44 |
| 6 | 0.56±1.14 | 0.17±0.06 | 0.26±0.14 |
| 7 | 2.90±0.28 | 0.95±0.12 | 3.33±0.82 |
| 8 | >200 | >200 | 39.23±5.02 |
| MA (maslinic acid) | 48.70±9.93 | 9.75±0.79 | 10.85±0.89 |
Note: the experimental data are the average of 3 experiments. IC (integrated circuit)50Lower values indicate greater activity of the compound in inhibiting tumor cell growth.
Claims (10)
1. L-phenylalanine modified maslinic acid represented by the following formulas 1 to 7:
2. the method of synthesizing L-phenylalanine modified maslinic acid as claimed in claim 1, comprising:
a method of synthesizing compound 1 and compound 2, comprising the steps of: dissolving benzyl maslinic acid in a first organic solvent, adding Boc-L-phenylalanine, dicyclohexylcarbodiimide and 4-dimethylaminopyridine, reacting under heating or non-heating conditions, filtering the obtained material after the reaction is finished, collecting filtrate, recovering the solvent, performing silica gel column chromatography on the obtained residue, eluting with an eluent, and detecting combined fractions by using thin layer chromatography to obtain a compound 1 and a compound 2 respectively;
a method of synthesizing compound 3, comprising the steps of: dissolving the compound 1 in a second organic solvent, adding ammonium formate and a palladium-carbon catalyst, reacting under a heating condition, cooling to normal temperature after the reaction is finished, filtering, collecting filtrate, recovering the solvent, separating the residue with a third organic solvent, collecting a water phase, extracting with an extracting agent, collecting an organic phase, washing, drying and spin-drying to obtain a crude compound 3;
a method of synthesizing compound 4, comprising the steps of: dissolving a compound 4 in a first organic solvent, adding a saturated hydrogen chloride ethyl acetate solution, reacting under heating or non-heating conditions, recovering the solvent after the reaction is finished, dispersing obtained residues in a composition of ethyl acetate and water, adjusting the pH value of the system to be alkaline, separating liquid, collecting an organic phase, washing, drying and spin-drying to obtain a crude compound 4;
a method of synthesizing compound 5, comprising the steps of: dissolving the compound 1 in a first organic solvent, adding a saturated hydrogen chloride ethyl acetate solution, reacting under a heating condition or a non-heating condition, and recovering the solvent after the reaction is finished to obtain a crude compound 5;
a method of synthesizing compound 6, comprising the steps of: taking the compound 2 as an initial raw material, and synthesizing according to the synthesis method of the compound 3 to obtain a crude product of the compound 6;
a method of synthesizing compound 7, comprising the steps of: taking the compound 6 as an initial raw material, and synthesizing according to the synthesis method of the compound 5 to obtain a crude product of a compound 7;
wherein the content of the first and second substances,
the first organic solvent is one or the combination of more than two of dichloromethane, chloroform, methanol, tetrahydrofuran and ethyl acetate;
the eluent is a mixed solvent consisting of petroleum ether and ethyl acetate;
the second organic solvent is one or the combination of more than two of methanol, ethanol and tetrahydrofuran;
the third organic solvent is a combination of water and one selected from dichloromethane, chloroform and ethyl acetate;
the extractant is dichloromethane, chloroform or ethyl acetate.
3. The method of synthesis according to claim 2, characterized in that: in the synthesis method of the compound 1 and the compound 2, the reaction is carried out at 50 ℃ or lower.
4. The method of synthesis according to claim 2, characterized in that: in the synthesis method of the compound 1 and the compound 2, the volume ratio of petroleum ether to ethyl acetate in the composition of the eluent is 20-5: 1.
5. the method of synthesis according to claim 2, characterized in that: in the synthesis method of the compound 3 and the compound 6, the reaction is carried out at the temperature of 40-80 ℃.
6. The method of synthesis according to claim 2, characterized in that: in the synthesis methods of compound 3 and compound 6, the third organic solvent has a composition in which the volume ratio of water to dichloromethane, chloroform, or ethyl acetate is 1: 1-3.
7. The method of synthesis according to claim 2, characterized in that: in the synthesis of compound 5 and compound 7, the reaction is carried out at 60 ℃ or lower.
8. The method of synthesis according to any one of claims 2-7, characterized in that: in the synthesis method of the compounds 3-8, a purification step of purifying crude compounds is also included.
9. Use of a compound according to claim 1 for the preparation of an antineoplastic medicament.
10. A pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claim 1.
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| Ren＇e Csuk等.Synthesis and Cytotoxic Activity of Methyl Glycyrrhetinate Esterified with Amino Acids.《Z. Naturforsch》.2012,第67b卷第732、734页. * |
| 氨基酸类衍生物抗肿瘤作用的研究进展;叶因涛等;《会议论文》;20111104;全文 * |
| 齐墩果酸的结构修饰与生物活性研究进展;唐初等;《Chin.J.Org.Chem.》;20131231;第33卷;第46-65页 * |
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