CN101402667B - Glycosylation modified nitric oxide donor type oleaolic acid compounds, preparation and uses thereof - Google Patents

Abstract

The invention relates to the field of medicaments, in particular to a group of glycosylated nitric oxide donating-oleanlic acid compounds (I), and also discloses preparation methods thereof, pharmaceutical compounds containing the compounds and pharmaceutically acceptable salt or ester, and application in preparing antineoplastic medicaments, wherein, the definition of R can be seen in the Instruction.

Description

Glycosylation modified nitric oxide donator type Oleanolic Acid compounds, Preparation Method And The Use

Technical field

The present invention relates to pharmaceutical field; Be specifically related to one type of glycosylation modified nitric oxide donator type Oleanolic Acid compounds (I); Also disclose they the preparation method, contain the medicinal compsns of these compounds or its pharmacy acceptable salt or ester, and the purposes in the preparation antitumor drug.

Background technology

Tumour is a kind of common major disease, the healthy and life security that serious threat is human.Though multiple antitumor drug is arranged clinically, owing to the complicacy of tumour pathological factor, the resistance of tumour and the reasons such as toxic side effect of antitumor drug, existing medicine still can not meet clinical needs.Therefore, the new type antineoplastic medicine that seek that curative effect is outstanding, persistent and toxic side effect is low is significant.

Nitrogen protoxide (being called for short NO, down together) as courier's material or effector molecule important in the body, is participated in multiple physiology and pathologic reaction.Experiment showed, that in a large number NO has dual regulation in tumour generation, tumor-blood-vessel growth, vascular function, tumor development and transfer process.This short knurl and anti-knurl dual function depend on expression, activity of nitricoxide synthase (be called for short NOS, down with) hypotype and distribute, and the concentration and the time length of NO release, cell is to the susceptibility of NO etc.It has been generally acknowledged that continue NO (no matter the being endogenic or ectogenic) apoptosis capable of inhibiting cell of lower concentration in the body, pair cell has the effect of protection and short its growth; The NO of high density can produce cytotoxicity in the body, and inducing apoptosis of tumour cell stops the diffusion and the transfer of tumour cell.The NO donor is one type and can be in vivo discharges the compound of a certain amount of NO through enzyme or non-enzyme effect.Bibliographical information, some NO donors can discharge the NO of high density specifically in tumour cell, kill to target property tumour cell (referring to Zhang Yihua; Tian Jide; Peng Sixun. the nitric oxide donors of targeting and related drugs thereof. Acta Pharmaceutica Sinica, 2006,41 (6): 481-486).

Oleanolic Acid (be called for short OA, down with) extensively is present in vegitabilia, and it has the liver provide protection to tetracol phenixin and other poisonous substance inductive rodent liver poisoning and chronic liver cirrhosis.Discover, mainly distribute and metabolism after the administration of OA whole body at liver.China clinically with OA as the Chinese medicine hepatoprotective, be mainly used in chronic hepatic diseases such as treatment hepatitis, liver cirrhosis.Recent study is found; OA can also suppress tumor neogenetic blood vessels and generate, and effects such as prevention invasion by tumor cells and transfer (referring to: Ovesna Z, Vachalkova A; Horvathova K; Et al.Pentacyclic triterpenoic acids:new chemo-protectivecompounds.Neoplasma, 2004,51 (5): 327-333).Yet there are shortcomings such as bioavailability is low, curative effect is undesirable in OA.

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen with following structural } Oleanolic Acid (CAS:875753-25-6 is called for short ZCVI4, down with),

And other nitric oxide donator type OA verivate mentions in the Chinese patent CN1594354A of bulletin on March 16th, 2005 that this patent is about using ZCVI4 and other a series of nitric oxide donator type OA derivatives for treatment hepatitis, liver cirrhosis and liver tumor.This patent will be drawn for referencial use at this comprehensively.

Summary of the invention

The present invention is lead compound with ZCVI4; Utilize 28 carboxyls of OA fragment and different glycosyl or substituted glycosyl couplings in the ZCVI-4 structure; The glycosylation nitric oxide donator type Oleanolic Acid compounds of design, synthetic a series of novel structures; Be used to treat particularly people's tumour of Mammals, comprise giving one or more compounds or its pharmacy acceptable salt or the ester of above-mentioned Mammals with antitumour activity dosage.But the invention also discloses a kind of preparation method of suitability for industrialized production of new type antineoplastic medicine.

The present invention relates to glycosylation modified nitric oxide donator type Oleanolic Acid compounds or its pharmacy acceptable salt or the ester shown in the following formula (I):

Wherein, R represents glycosyl or replaces glycosyl.

In the compound, described R represents glucone shown in the top formula (I), galactosyl, glucal acidic group, GS base, 2-deoxyglucose base; 2-deoxy-galactose base, xylosyl, Arabic glycosyl, fructosyl, rhamanopyranosyl; Ribosyl, mannose group, sorb glycosyl, sucrose base, malt-base; The cellobiose base, the lactose base, the cotton seed glycosyl, VISOSE three glycosyls, and as shown in the formula (II) or the represented group of formula (III):

The various glycosyls of wherein said R are that saccharide compound is taken off the group that any locational hydroxyl forms later on.

A kind of embodiment preferred of the present invention is in above-mentioned compound, and the various glycosyls of its R representative are that saccharide compound is taken off the group that the hydroxyl on 1 forms later on.

A kind of embodiment preferred of the present invention is in above-mentioned compound, and its R is a glucone, galactosyl, glucal acidic group, 2-deoxy-galactose base, 2-deoxyglucose base, lactose base, malt-base.

Further preferred version of the present invention is that its R is a galactosyl in the compound that such scheme uses.

Further preferred version of the present invention is that its R is that semi-lactosi is taken off the group that forms behind 1 hydroxyl in the compound that such scheme uses.

The another one aspect that the present invention relates to is glycosylation modified nitric oxide donator type Oleanolic Acid compounds and the pharmacy acceptable salt or the application of ester in the medicine of preparation prevention or treatment tumor disease of formula (I) expression.

Aforesaid tumor disease behaviour tumour.

Aforesaid glycosylation modified nitric oxide donator type Oleanolic Acid compounds and pharmacy acceptable salt or the application of ester in the medicine of preparation prevention or treatment tumor disease suc as formula (I) expression is characterized in that described tumor disease is liver tumor, liver cirrhosis, lung cancer, the esophageal carcinoma, cancer of the stomach, large bowel cancer, colorectal carcinoma, breast tumor, nasopharyngeal carcinoma, cervical cancer, lymphoma, tumor of prostate and white blood disease.

The invention still further relates to a kind of pharmaceutical composition of treating tumor disease, one or more that it is characterized in that comprising the glycosylation modified nitric oxide donator type Oleanolic Acid compounds shown in the formula (I) and pharmacy acceptable salt or ester are as effective constituent.Can also contain pharmaceutically acceptable carrier in addition, like Magnesium Stearate, sodium sulfite anhy 96, ten dimethyl cellulose sodium, micronization silica gel etc.

Among the present invention; Giving Mammals formula (I) compound and pharmacy acceptable salt or ester; And during the solvolyte of these compounds (being referred to as " medicine " here); Can use separately, perhaps preferably according to the pharmaceutical methods of standard with its be suitable for medicinal carrier or thinner and cooperate the back to use.Administering mode can be through various approach, comprise oral, parenteral administration or topical.Here the parenteral administration of indication includes, but are not limited to intravenous injection, intramuscular injection, abdominal injection, subcutaneous injection and transdermal administration.

Compound concrete structure and code name shown in the preferred formula (I) of the present invention's part is following:

The compound method of the compound shown in the formula of the present invention (I) is following: under the effect of pyridylacetic acid(HPAC) acid anhydride, the hydroxyl in the glycan molecule is carried out the acetylize protection; Then under red phosphorus/bromine water effect, carry out 1 bromo; With the ZCVI4 condensation, condensation product deacetylation in sodium methylate/ethanol/methylene system makes compound shown in the formula (I) then.

Concrete synthetic route:

Preparing method when the R of the compound shown in the general formula (I) is 2-deoxy-galactose base: semi-lactosi acetylize under the effect of pyridylacetic acid(HPAC) acid anhydride; Then under red phosphorus/bromine water effect, carry out 1 bromo; Under the effect of zinc powder, copper sulfate and sodium acetate, 1,2 of sugar is reduced to glycal again, the saturated benzole soln with HBr carries out addition reaction again, makes 3; 4; 5-O-ethanoyl a-D-2-deoxy-galactose bromo-derivative, again with the ZCVI4 condensation, deacetylation makes target compound (TM) in sodium methylate/ethanol/methylene system at last.

Concrete synthetic route:

Preparing method when the R of the compound shown in the general formula (I) is formula (II) or the described group of formula (III): under the effect at triethylamine; With 1; 3,4, amino a-D-glucose of 6-four-O-ethanoyl-2-and bromoacetyl bromide reaction; The midbody of gained again under the effect of salt of wormwood with the ZCVI4 condensation, last deacetylation makes target compound (TM).

Concrete synthetic route:

Description of drawings

Fig. 1 is that compound ZCVI4-2 is to human liver cancer cell SMMC7721 nude mice xenografts of human.

Embodiment

Below be the embodiment of The compounds of this invention, these embodiment also do not mean that limitation of the present invention.

Embodiment 1

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-β-D-glucopyranosyl-Oleanolic Acid (ZCVI4-1)

2,3,4,6-four-O-ethanoyl-a-D-Glucopyranose bromide reference literature [Yunnan Institute for nationalities's journal (natural science edition), 2004,13 (2): 83-85] preparation.

ZCVI4 reference literature (J.Med.Chem.2008,51,4834-4838) preparation.

ZCVI4852mg, 2,3,4,6-four-O-ethanoyl-a-D-Glucopyranose bromide 414mg, K ₂CO ₃200mg and cetyl trimethylammonium bromide 200mg are dissolved in 10mL water and 15mL CH ₂Cl ₂Mixing solutions in, vigorous stirring 48h tells organic layer under the room temperature, water layer CH ₂Cl ₂Extraction, the organic layer of merging concentrates; Rapid column chromatography gets pulpous state midbody 3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-28-O-[2; 3; 4,6-four-O-ethanoyl]-β-D-glucopyranosyl-Oleanolic Acid, productive rate 52%.

Above-mentioned product is dissolved in 5mL CH ₂Cl ₂With 5mL CH ₃In the mixing solutions of OH, 0 ℃ of methanol solution that drips the sodium methylate of 1mol/L down, control pH is no more than 10; Be stirred to the raw material spot and disappear, the dichloromethane solution adjust pH that drips acetate is 7, concentrates; Rapid column chromatography gets white powder ZCVI4-1, productive rate 21%, m.p.101-103 ℃.ESI-MS1032[M+NH ₄] ⁺，1049[M+Cl] ^-； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.75(s，3H，CH ₃)，0.82(s，6H，2×CH ₃)，0.85(s，3H，CH ₃)，0.89(s，6H，2×CH ₃)，1.23(s，3H，CH ₃)，2.67(s，4H，2×COCH ₂)，2.77～2.88(m，1H，C ₁₈-H)，3.65(m，1H，H ₃)，3.78(m，1H，H ₅)，4.09(m，1H，H ₄)，4.18(brs，1H，3a-H)，4.21(t，2H，OCH ₂，J＝6Hz)，4.43(m，1H，H ₂)，4.76(d，1H，J＝4.5Hz，H ₆’)，4.83(d，1H，J＝6.0Hz，H ₆)，4.99～5.13(m，1H，OCH)，5.26(brs，1H，C ₁₂-H)，5.49(d，1H，J＝8Hz，H ₁)，7.61～7.64(m，2H，ArH)，7.73～7.76(m，1H，ArH)，8.06(d，2H，ArH，J＝8.2Hz)。

Embodiment 2

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-β-D-galactopyranose base-Oleanolic Acid (ZCVI4-2)

With reference to the preparation method of ZCVI4-1, with 2,3,4; 6-four-O-ethanoyl-a-D-galactopyranose bromide [Yunnan Institute for nationalities's journal (natural science edition), 2004,13 (2): 83-85] is a raw material; Make white powder compound ZCVI4-2, productive rate 28%, m.p.121-124 ℃.ESI-MS1032[M+NH ₄] ⁺，1049[M+Cl] ^-； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.67(s，3H，CH ₃)，0.74(s，6H，2×CH ₃)，0.83(s，3H，CH ₃)，0.87(s，6H，2×CH ₃)，1.1(s，3H，CH ₃)，2.53(s，4H，2×COCH ₂)，2.77～2.83(m，1H，C ₁₈-H)，3.51(m，1H，H ₃)，3.68(m，1H，H ₅)，4.28(m，1H，H ₄)，4.41(brs，1H，3a-H)，4.45(t，2H，OCH ₂，J＝6Hz)，4.51(m，1H，H ₂)，4.71(d，1H，J＝4.5Hz，H ₆’)，4.93(d，1H，J＝6.0Hz，H ₆)，4.99～5.03(m，1H，OCH)，5.16(brs，1H，C ₁₂-H)，5.21(d，1H，J＝8Hz，H ₁)，7.72～7.75(m，2H，ArH)，7.88～7.91(m，1H，ArH)，8.01(d，2H，ArH，J＝8Hz)。

Embodiment 3

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-β-D-glucal acidic group Oleanolic Acid (ZCVI4-3)

2,3,4-three-O-ethanoyl-a-D-glucuronic acid methyl ester bromide reference literature [organic chemistry, 1992,12,269-272] preparation.

2,3,4-three-O-ethanoyl-a-D-glucuronic acid methyl ester bromide 200mg, anhydrous K ₂CO ₃100mg and 414mgZCVI4 are dissolved in the 5mL dry DMF, N ₂Protection is stirring at room 48h down.Reaction solution is poured in the 50mL water into CH ₂Cl ₂Extraction (20mL * 3), organic layer are washed drying with the Hydrogen chloride of 1N again; Rapid column chromatography gets wax shape midbody 3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1; 2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-28-O-[2,3; 4-three-O-ethanoyl-β-D-] glucuronic acid methyl ester-Oleanolic Acid, productive rate 41%.This midbody is dissolved in the anhydrous THF of 4mL, adds LiOH20mg, stirred overnight at room temperature.Add anhydrous methanol 4mL again, 0 ℃ of methanol solution that drips the sodium methylate of 1mol/L down, control pH is no more than 10.After the TLC demonstration reacts completely, in reaction solution, drip the CH of acetate ₂Cl ₂The solution adjust pH be 7.Concentration of reaction solution, rapid column chromatography get wax shape ZCVI4-3, productive rate 15%.ESI-MS1027[M-H] ^-； ¹H?NMR(300MHz，DMSO)，d(ppm)：0.68(s，3H，CH ₃)，0.73(s，6H，2×CH ₃)，0.83(s，3H，CH ₃)，0.90(s，6H，2×CH ₃)，1.11(s，3H，CH ₃)，2.54(s，4H，2×COCH ₂)，2.67～2.93(m，1H，C ₁₈-H)，3.06-3.10(m，2H，H ₃，H ₄)，4.28(m，1H，3a-H)，4.39-4.43(m，2H，OCH ₂)，4.91-5.12(m，2H，OCH，H ₂)，5.12(d，2H，d＝5.7Hz)，5.16(brs，1H，C ₁₂-H)，5.22(d，1H，J＝8Hz，H ₁)，7.71～7.77(m，2H，ArH)，7.87～7.92(m，1H，ArH)，8.01(d，2H，ArH，J＝8Hz)。

Embodiment 4

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-β-D-2-acetylglucosamine base Oleanolic Acid (ZCVI4-4)

With reference to the preparation method of ZCVI4-1, with 3,4,6-three-O-ethanoyl-2-N-ethanoyl-a-D-GS bromide [J.Org.Chem.2006,71,3619-3622] is a raw material, makes white powder ZCVI4-4, productive rate 36%, m.p.142-146 ℃.ESI-MS1078[M+Na] ⁺； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.73(s，3H，CH ₃)，0.77(s，6H，2×CH ₃)，0.89(s，3H，CH ₃)，0.91(s，6H，2×CH ₃)，1.21(s，3H，CH ₃)，2.59(s，4H，2×COCH ₂)，2.73～2.99(m，1H，C ₁₈-H)，3.17(m，3H，COCH ₃)，3.40～3.51(brs，1H，NH)，3.64～3.67(m，2H，H ₃，H ₅)，4.32～4.36(brs，1H，3a-H)，4.40～4.47(m，4H，OCH ₂，H ₂，H ₄)，4.99(d，1H，J＝5.5Hz，H ₆)，5.03-5.05(m，2H，OCH，H _6’)，5.20(brs，1H，C ₁₂-H)，5.26(d，1H，J＝8.9Hz，H ₁)，7.75～7.78(m，2H，ArH)，7.91～7.94(m，1H，ArH)，8.02～8.04(m，2H，ArH)。

Embodiment 5

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-β-D-2-deoxyglucose base Oleanolic Acid (ZCVI4-5)

With reference to the preparation method of ZCVI4-1, with 3,4,6-three-O-ethanoyl-a-D-2-deoxyglucose bromide [CarbohydrateResearch, 2005,340:2670-2674] is a raw material, makes pulpous state compound ZCVI4-5, productive rate 27%.ESI-MS997[M-H] ^-； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.57(s，3H，CH ₃)，0.69(s，6H，2×CH ₃)，0.77(s，3H，CH ₃)，0.81(s，6H，2×CH ₃)，0.98(s，3H，CH ₃)，2.57(s，4H，2×COCH ₂)，2.87～2.93(m，1H，C ₁₈-H)，4.43～3.51(m，1H，H ₄)，3.62-3.79(3m，3H，H ₅，H ₆，H _6’)，3.86～4.01(m，1H，H ₃)，4.42(brs，1H，3a-H)，4.43(t，2H，OCH ₂，J＝6Hz)，4.68～4.98(m，1H，OCH)，5.18(brs，1H，C ₁₂-H)，5.24(dd，1H，H ₁)，7.73～7.74(m，2H，ArH)，7.88～7.95(m，1H，ArH)，7.99～8.07(m，2H，ArH)。

Embodiment 6

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-β-D-2-deoxy-galactose base Oleanolic Acid (ZCVI4-6)

With reference to the preparation method of ZCVI4-1, with 3,4,6-three-O-ethanoyl-a-D-2-deoxy-galactose bromide [Joumal ofcarbohydrate chemistry, 1996,15 (8): 955-964] is a raw material, makes pulpous state compound ZCVI4-6, productive rate 21%.ESI-MS1016[M+NH ₄] ⁺； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.61(s，3H，CH ₃)，0.71(s，6H，2×CH ₃)，0.82(s，3H，CH ₃)，0.85(s，6H，2×CH ₃)，1.2(s，3H，CH ₃)，2.58(s，4H，2×COCH ₂)，2.67～2.81(m，1H，C ₁₈-H)，4.53～3.61(m，1H，H ₄)，3.65～3.82(m，3H，H ₅，H ₆，H _6’)，3.90～4.0(m，1H，H ₃)，4.41(brs，1H，3a-H)，4.45(t，2H，OCH ₂，J＝6Hz)，4.78～5.00(m，1H，OCH)，5.14(brs，1H，C ₁₂-H)，5.21(dd，1H，H ₁)，7.72～7.74(m，2H，ArH)，7.88～7.91(m，1H，ArH)，7.99～8.01(m，2H，ArH)。

Embodiment 7

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-β-D-xylosyl-Oleanolic Acid (ZCVI4-7)

With reference to the preparation method of ZCVI4-1, with 2,3,5-three-O-ethanoyl-a-D-wood sugar bromide [Carbohydrate Research, 2005,340:2670-2674] is a raw material, makes white powder compound ZCVI4-7, productive rate 20%, m.p.103-106 ℃.ESI-MS1007[M+Na] ⁺，1019[M+Cl] ^-； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.65(s，3H，CH ₃)，0.74(s，6H，2×CH ₃)，0.85(s，3H，CH ₃)，0.91(s，6H，2×CH ₃)，1.18(s，3H，CH ₃)，2.54(s，4H，2×COCH ₂)，2.62～2.73(m，1H，C ₁₈-H)，3.68～3.78(m，2H，H ₃，H ₄)，4.28～4.34(m，1H，3a-H)，4.48～4.55(m，2H，OCH ₂)，4.67～4.81(m，2H，H ₅，H _5’)，4.91～5.00(m，1H，OCH)，5.12～5.22(m，1H，H ₂)，5.28(brs，1H，C ₁₂-H)，5.32(d，1H，J＝5Hz，H ₁)，7.71～7.76(m，2H，ArH)，7.89～7.90(m，1H，ArH)，7.99～8.02(m，2H，ArH)。

Embodiment 8

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of the Arabic glycosyl Oleanolic Acid of 28-O-β-D-(ZCVI4-8)

With reference to the preparation method of ZCVI4-1, with 2,3,5-three-O-ethanoyl-a-D-pectinose bromide [CarbohydrateResearch, 2005,340:2670-2674] is a raw material, makes white powder compound ZCVI4-8, productive rate 25%, m.p.117-120 ℃.ESI-MS983[M-H] ^-，1019[M+Cl] ^-； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.68(s，3H，CH ₃)，0.79(s，6H，2×CH ₃)，0.81(s，3H，CH ₃)，0.89(s，6H，2×CH ₃)，1.08(s，3H，CH ₃)，2.52(s，4H，2×COCH ₂)，2.67～2.73(m，1H，C ₁₈-H)，3.68～3.70(m，2H，H ₃，H ₄)，4.29～4.30(m，1H，3a-H)，4.41～4.44(m，2H，OCH ₂)，4.57～4.71(m，2H，H ₅，H _5’)，4.98～5.02(m，1H，H ₂)，4.99～5.08(m，1H，OCH)，5.21(brs，1H，C ₁₂-H)，5.33(d，1H，J＝5Hz，H ₁)，7.72～7.76(m，2H，ArH)，7.88～7.89(m，1H，ArH)，7.99～8.01(m，2H，ArH)。

Embodiment 9

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-β-D-malt-base Oleanolic Acid (ZCVI4-9)

With reference to the preparation method of ZCVI4-1, with 2,3,6,2 ', 3 ', 4 ', 6 '-seven-O-ethanoyl-a-D-SANMALT-S bromide [Carbohydrate Research, 2005,340:2670-2674] is a raw material, makes waxy compound ZCVI4-9, productive rate 29%.ESI-MS1194[M+NH ₄ ⁺] ⁺，1199[M+Na] ⁺； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.69(s，3H，CH ₃)，0.71(s，6H，2×CH ₃)，0.81(s，3H，CH ₃)，0.85(s，6H，2×CH ₃)，1.1(s，3H，CH ₃)，2.53(s，4H，2×COCH ₂)，2.71～2.83(m，1H，C ₁₈-H)，3.41～3.43(m，1H，H ₄)，3.61～3.67(m，2H，H ₅’，H _2’)，3.71～3.78(m，1H，H ₂)，4.13～4.32(m，2H，H ₅，H _4’)，4.38～4.46(m，2H，H ₃，H _3’)，4.54～4.61(m，1H，H _6b’)，4.67～4.83(m，1H，H _6b)，5.00～5.03(m，2H，H _6a，H _6a’)，5.09～5.21(m，4H，OCH，3a-H，OCH ₂)，5.23～5.31(m，2H，C ₁₂-H，H _1’)，5.62(d，1H，J＝8Hz，H ₁)，7.72～7.75(m，2H，ArH)，7.88～7.91(m，1H，ArH)，7.98～8.00(m，2H，ArH)。

Embodiment 10

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-β-D-cellobiose base Oleanolic Acid (ZCVI4-10)

With reference to the preparation method of ZCVI4-1, with 2,3,6,2 '; 3 ', 4 ', 6 '-seven-O-ethanoyl-a-D-cellobiose bromide [Carbohydrate Research, 2005; 340:2670-2674] be raw material, make white powder compound ZCVI4-10, productive rate 23%, m.p.126-129 ℃.ESI-MS1175[M-H] ^-； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.65(s，3H，CH ₃)，0.78(s，6H，2×CH ₃)，0.89(s，3H，CH ₃)，0.95(s，6H，2×CH ₃)，1.13(s，3H，CH ₃)，2.53(s，4H，2×COCH ₂)，2.76～2.83(m，1H，C ₁₈-H)，3.42～3.43(m，1H，H ₄)，3.62～3.67(m，2H，H _5’，H _2’)，3.74～3.78(m，1H，H ₂)，4.23～4.33(m，2H，H ₅，H _4’)，4.38～4.46(m，2H，H ₃，H _3’)，4.50～4.61(m，1H，H _6b’)，4.67～4.77(m，1H，H6b)，5.00～5.03(m，2H，H _6a，H _6a’)，5.09～5.18(m，4H，OCH，3a-H，OCH ₂)，5.27～5.31(m，2H，C ₁₂-H，H _1’)，5.61(d，1H，J＝8Hz，H ₁)，7.72～7.75(m，2H，ArH)，7.88～7.91(m，1H，ArH)，7.98～8.00(m，2H，ArH)。

Embodiment 11

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-β-D-lactose base Oleanolic Acid (ZCVI4-11)

With reference to the preparation method of ZCVI4-1, with 2,3,6,2 '; 3 ', 4 ', 6 '-7-O-ethanoyl-a-D-lactose bromide [CarbohydrateResearch, 2005; 340:2670-2674] be raw material, make white powder compound ZCVI4-11, productive rate 23%, m.p.150-153 ℃.ESI-MS1175[M-H] ^-； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.65(s，3H，CH ₃)，0.78(s，6H，2×CH ₃)，0.89(s，3H，CH ₃)，0.95s，6H，2×CH ₃)，1.13(s，3H，CH ₃)，2.53(s，4H，2×COCH ₂)，2.76～2.83(m，1H，C ₁₈-H)，3.39～3.43(m，1H，H ₄)，3.59～3.67(m，2H，H _5’，H _2’)，3.74～3.80(m，1H，H ₂)，4.13～4.33(m，2H，H ₅，H _4’)，4.36～4.46(m，2H，H ₃，H _3’)，4.50～4.67(m，1H，H _6b’)，4.67～4.77(m，1H，H6b)，4.89～5.02(m，2H，H _6a，H _6a’)，5.09～5.16(m，4H，OCH，3a-H，OCH ₂)，5.27～5.32(m，2H，C ₁₂-H，H _1’)，5.54(d，1H，J＝8Hz，H ₁)，7.72～7.76(m，2H，ArH)，7.88～7.91(m，1H，ArH)，7.98～8.01(m，2H，ArH)。

Embodiment 12

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-acetyl-(2-deoxyglucose) amino Oleanolic Acid (ZCVI4-12)

With reference to the preparation method of ZCVI4-3, with N-acetyl bromide-1,3,4,6-four-O-acetylamino glucose [J.Med.Chem.1978,21 (12): 1222-1225] is raw material, makes waxy compound ZCVI4-12, productive rate 19%.ESI-MS1071[M-H] ^-； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.67(s，3H，CH ₃)，0.77(s，6H，2×CH ₃)，0.83(s，3H，CH ₃)，0.89(s，6H，2×CH ₃)，1.2(s，3H，CH ₃)，2.58(s，4H，2×COCH ₂)，2.79～2.83(m，1H，C ₁₈-H)，3.52～3.59(m，2H，H ₅，H ₃)，3.72～3.74(m，1H，H ₄)，3.81(m，1H，H ₂)，4.28(m，1H，H ₄)，4.44～4.45(m，4H，3a-H，OCH ₂，H ₆，H _6’)，4.93～4.94(m，1H，OCH)，5.00(brs，1H，C ₁₂-H)，5.21(m，1H，H ₁)，7.72～7.76(m，2H，ArH)，7.88～7.91(m，1H，ArH)，8.00(d，2H，ArH，J＝7.8Hz)。

Embodiment 13

3-{ [4-(1-methyl-3-{ [5-oxygen-4-(benzenesulfonyl)-1,2,5-oxazole-3-] oxygen } propoxy-)-4-oxygen succinyl] oxygen }-preparation of 28-O-acetyl-(2-deoxy-galactose) amino Oleanolic Acid (ZCVI4-13)

With reference to the preparation method of ZCVI4-3, with N-acetyl bromide-1,3,4,6-four-O-ethanoyl-galactosamine [J.Med.Chem.1978,21 (12): 1222-1225] is a raw material, makes waxy compound ZCVI4-13, productive rate 30%.ESI-MS1071[M-H] ^-； ¹H?NMR(500MHz，DMSO)，d(ppm)：0.68(s，3H，CH ₃)，0.78(s，6H，2×CH ₃)，0.84(s，3H，CH ₃)，0.89(s，6H，2×CH ₃)，1.1(s，3H，CH ₃)，2.58(s，4H，2×COCH ₂)，2.57～2.83(m，1H，C ₁₈-H)，3.53～3.59(m，2H，H ₅，H ₃)，3.72～3.74(m，1H，H ₄)，3.92(m，1H，H ₂)，4.34(m，1H，H ₄)，4.34～4.45(m，4H，3a-H，OCH ₂，H ₆，H _6’)，4.80～4.94(m，1H，OCH)，5.10(brs，1H，C ₁₂-H)，5.21(d，1H，H ₁，J＝1.5Hz)，7.72～7.76(m，2H，ArH)，7.88～7.91(m，1H，ArH)，8.00(d，2H，ArH，J＝7.8Hz)。

The external pharmacodynamic study of embodiment 14ZCVI4-2

Purpose: select 4 kinds of tumor cell of liver strains that ZCVI4-2 is carried out the extracorporeal anti-tumor function screening.

Sample and reagent: prepare during the test of ZCVI4-2 injection liquid temporarily; Hydroxycamptothecin injection liquid (Hubei Hua Yuan century medicine company); Cell culture medium RPMI-1640, F12, MEM, DMEM (high sugar) are available from GIBCO company; Foetal calf serum (clear ltd of the Hangzhou four seasons); Tetramethyl-azo azoles blue (MTT) and DMSO are all available from Sigma company; Other common agents is commercially available analytical pure.

Cell strain: human hepatoma cell strain SMMC-7721, human hepatoma cell strain HepG2, human hepatoma cell strain IM3, human hepatoma cell strain BEL-7402 are all available from Chinese Academy of Medical Sciences's cell bank and Shanghai cell institute of the Chinese Academy of Sciences.

The cultivation of cell strain: each experimental cell strain (Shanghai Inst. of Cytobiology, Chinese Academy of Sciences provides) is gone down to posterity frozen by this laboratory.Cell is at 37 ℃, 5%CO ₂The conventional cultivation in the incubator of saturated humidity.Nutrient solution is for containing 10% heat-inactivated fetal bovine serum, the RPMI1640 cell culture medium of penicillium mould 100u/ml and Streptomycin sulphate 100u/ml.48h changes nutrient solution, when cell converges, goes down to posterity with 0.25% tryptic digestion.Experiment all is in logarithmic phase with cell, and the trypan blue exclusion method shows cell viability>95%.Experimental technique: get and be in one bottle in logarithmic phase cell in good condition, add Digestive system (0.125% trypsinase+0.01%EDTA) digestion, counting 2～4 * 10 ⁴Individual/ml, process cell suspension inoculation on 96 orifice plates, 180 μ L/ holes are put in the constant temperature CO2 incubator and were cultivated 24 hours.Change liquid, adding receives the reagent thing, and cultivated 72 hours in 20 μ L/ holes.MTT is added in 96 orifice plates, and hatched in the incubator 4 hours in 20 μ L/ holes.Supernatant is removed in suction, adds DMSO, 150 μ L/ holes, and jolting is 10 minutes on the dull and stereotyped shaking table.Tried thing and investigated 3 concentration (0.1,1.0,10 μ M), use the enzyme linked immunological monitor, calculated the cell inhibitory rate under each concentration respectively in the absorbancy of wavelength as the every hole of mensuration, 570nm place.

The inhibiting rate method of calculation:

The absolute OD value of the relative OD value=negative control hole of the negative control hole-absolute OD value in blank hole

The relative OD value in the susceptibility hole=absolute OD value in susceptibility hole-absolute OD value in blank hole

Experimental result

Can know that from table 1 ZCVI4-2 has the anti-liver tumor effect of remarkable vitro, obviously is better than positive control drug Hydroxycamptothecin (HCPT).

Table 1.ZCVI4-2 is to 4 kinds of human liver tumor cell's restraining effect results

Pharmacodynamic study in the body of embodiment 15ZCVI4-2

1, the ZCVI4-2 intraperitoneal administration is to the experimental therapy effect of mouse H22 liver cancer

Purpose: test ZH-2 is to the growth-inhibiting effect of mouse H22 liver cancer.

Animal: the ICR mouse, 18-22g is provided by animal housing of China Medicine University, conformity certification number: kinoplaszm SCX (Soviet Union) 2002--0011 of Soviet Union.

The knurl kind: mouse H22 knurl kind is provided by institute of materia medica, Chinese Academy of Sciences Shanghai.

Tester: the ZCVI4-2 injection liquid is provided by medicament teaching and research room of China Medicine University Zhang Jianjun.

The medication and the course of treatment:

Select for use 18-22 to restrain the H22 knurl kind of female ICR mouse and well-grown 7-11 days, it is subcutaneous to be inoculated in the right side of mice armpit, about 4.5-5 * 10 ⁶Cell/only, inoculate and divide cage at random, intraperitoneal injection after 24 hours.Pressed the various dose successive administration 7 days.Put to death animal on the 10th day, weigh, knurl is heavy, calculate that respectively to organize average knurl heavy, press formula and obtain tumor control rate and carry out the T check.

Result and conclusion

As shown in table 2, ZCVI4-2 has certain growth-inhibiting effect to mouse H22 liver cancer.

Table 2.ZCVI4-2 is to the growth-inhibiting effect of mouse H22 liver cancer

2, the ZCVI4-2 intravenously administrable is to the experimental therapy effect of mouse S180 sarcoma

Experimental technique

Select for use 18-22 to restrain the S180 knurl kind of female ICR mouse and well-grown 7-11 days, it is subcutaneous to be inoculated in the right side of mice armpit, about 4.5-5 * 10 ⁶Cell/only, inoculate and divide cage at random after 24 hours, the tail intravenously administrable.Pressed same dosage successive administration 7 days.Put to death animal on the 8th day, weigh, knurl is heavy, calculate that respectively to organize average knurl heavy, press formula and obtain tumor control rate and carry out the T check.

Experimental result

Like table 3 data presentation, compare with blank control group and control group, ZCVI4-2 is inhibited to the growth of mouse S180 sarcoma.

Table 3.ZCVI4-2 is to the growth-inhibiting effect of mouse S180 sarcoma

Annotate: D1, D8: inoculation back the 1st, 8 day

3, the ZCVI4-2 intravenously administrable is to the experimental therapy effect of people's liver cancer SMMC-7721 Nude Mice

Because animal knurl and human tumor are not quite identical, animal knurl test-results exist and clinical effectiveness between difference.For this reason, we have selected people's liver cancer SMMC7721 Nude Mice to carry out ZCVI4-2 anti-tumor in vivo effect research on the basis of above-mentioned two kinds of animal knurls test.

Tried thing: the ZCVI4-2 injection liquid provides lot number by preparation teaching and research room of China Medicine University Zhang Jianjun: 070327.The blank solvent contrast provides lot number by preparation teaching and research room of China Medicine University Zhang Jianjun: 070327-0.Positive control drug is 5 FU 5 fluorouracil (5-Fu), and Tianjin gold credit amino acid ltd produces lot number: 0505241, be diluted to administration concentration with saline water before using.Transplanted tumor: select human liver cell property liver cancer SMMC-7721 Nude Mice for use, it is subcutaneous and set up to be inoculated in nude mouse by human liver cell property liver cancer SMMC-7721 cell strain.The cell inoculation amount is 2 * 10 ⁶, inoculation is used after forming and in the nude mouse body, passing for 3 generations again behind the transplanted tumor.

Animal: female BALB/cA nude mouse, age in days 35-40 days, body weight 18-22g was provided by Chinese Academy of Sciences's Shanghai Experimental Animal Center.The conformity certification numbering: Shanghai is moving closes the card word No. 122.12 of the negative control groups of every treated animal number, 6 of administration groups.

Experimental technique

The tumor tissue of getting the growth animated period cuts into 1.5mm ³About, under aseptic condition, it is subcutaneous to be inoculated in nude mouse right side armpit.Nude Mice is treated tumor growth to 100～300mm with vernier caliper measurement transplanted tumor diameter ³After with the animal random packet.Use the method for measuring the knurl footpath, dynamic observe by the antineoplastic effect of examination thing.The measurement number of times of diameter of tumor is that each the measurement need claim also that simultaneously mouse is heavy 3 times weekly.The administration group is intravenously administrable 3 times weekly, and administration volume 0.4ml/ only.Positive controls is intravenously administrable 3 times weekly, and negative control group is given equivalent saline water simultaneously.

Detect index and method of calculation

(1) (tumor volume, TV), calculation formula is gross tumor volume: TV=1/2 * a * b ²

Wherein a, b represent length and width respectively.

(2) relative tumour volume (relative tumor volume, RTV), calculation formula is:

RTV＝TV _t/TV ₀。

TV wherein ₀(d during for minute cage administration ₀) gross tumor volume, TV _tGross tumor volume when measuring each time.

(3) relative tumor proliferation rate T/C (%), calculation formula is:

$T/C=\frac{T_{\mathrm{RTV}}}{C_{\mathrm{RTV}}}\×100\%$

T _RTV: treatment group RTV; C _RTV: negative control group RTV.

Test-results is with the evaluation index of relative tumor proliferation rate T/C (%) as anti-tumor activity.

Experimental result:

With shown in Figure 1, the ZCVI4-2 injection liquid has certain growth-inhibiting effect to human liver cell property liver cancer SMMC-7721 Nude Mice like table 4.ZCVI4-2 injection liquid 25mg/kg and 12.5mg/kg dose groups are respectively 44.1% and 50.0% to the T/C of SMMC-7721.

Table 4.ZCVI4-2 injection liquid is to the experimental therapy effect of people's liver cancer SMMC-7721 Nude Mice

D0: divide the cage administration time

Claims (6)

1. glycosylation modified nitric oxide donator type Oleanolic Acid compounds and the pharmacy acceptable salt thereof shown in following general formula I:

Among its Chinese style I, R represents the group of glucone, galactosyl, glucal acidic group, GS base, 2-deoxyglucose base, 2-deoxy-galactose base, xylosyl, Arabic glycosyl, malt-base, cellobiose base, lactose base or following formula II and formula III:

Described glucone, galactosyl, glucal acidic group, GS base, 2-deoxyglucose base, 2-deoxy-galactose base, xylosyl, Arabic glycosyl, malt-base, cellobiose base, lactose base are that corresponding saccharide compound is taken off the group that any locational hydroxyl forms later on.

2. glycosylation modified nitric oxide donator type Oleanolic Acid compounds and the pharmacy acceptable salt thereof represented of general formula I according to claim 1 is characterized in that described R is glucone, galactosyl, glucal acidic group, 2-deoxy-galactose base, 2-deoxyglucose base, lactose base, malt-base.

3. glycosylation modified nitric oxide donator type Oleanolic Acid compounds and the pharmacy acceptable salt thereof represented like the said general formula I of claim 2 is characterized in that described R is a galactosyl.

4. glycosylation modified nitric oxide donator type Oleanolic Acid compounds and the pharmacy acceptable salt thereof represented of general formula I according to claim 1 is characterized in that described glucone, galactosyl, glucal acidic group, GS base, 2-deoxyglucose base, 2-deoxy-galactose base, xylosyl, Arabic glycosyl, malt-base, cellobiose base, lactose base are that corresponding saccharide compound is taken off the group that 1 hydroxyl forms later on.

5. glycosylation modified nitric oxide donator type Oleanolic Acid compounds and the pharmacy acceptable salt thereof represented like the said general formula I of claim 3 is characterized in that described galactosyl is that semi-lactosi is taken off the group that forms behind 1 hydroxyl.

6. the glycosylation modified nitric oxide donator type Oleanolic Acid compounds represented of general formula I and pharmacy acceptable salt thereof the application in the medicine of preparation prevention or treatment tumor disease according to claim 1, wherein said tumor disease is a liver tumor.

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