CN1067052C - Isocedrela acid methyl ester and method for preparation - Google Patents

Abstract

The present invention belongs to isocedrela acid methyl ester obtained by extraction and separation from a sponge, and chemical reaction, and a preparation method thereof. A plurality of organic compounds with unique structures, such as terpene, sterol, glycoside, alkaloid, polypeptide, polyether, etc., are discovered in sponge biology, wherein a plurality of organic compounds have the physiological activity of tumor resistance, virus resistance, cardiovascular disease resistance, etc. The isocedrela acid methyl ester of the present invention is called the methylester of rhabdastrellicacid, and the compound is obtained by the separation and the extraction from the sponge, and the chemical reaction and has a new chemical structure, obvious antineoplastic activity, significant theoretical value and application prospects.

Description

Methyl isoailanthic acid and preparation method thereof

The present invention belongs to methyl isoailanthic acid obtained by extraction, separation and chemical reaction from sponge.

Sponges are the most primitive group of multicellular animals, and many structurally distinct organic compounds have been discovered from sponge organisms: terpenes, sterols, glycosides, alkaloids, polypeptides, polyethers, macrolides and the like, wherein many of the terpenes, sterols, glycosides, alkaloids, polypeptides, polyethers, macrolides have physiological activities such as anti-tumor, anti-cardiovascular disease, antibacterial and antiviral activities, and many of the compounds have potential clinical application values.

Chinese patent (application No. 95116029. X, publication No. CNl130619A) discloses an isoailanthic acid triterpene compound, wherein the structure of the isoailanthic acid methyl ester provided by the invention is added with a CH₃The chemical structural formula is as follows:

the compound name is: (13E,15E,17E,22E,24E) -3, 12-dioxo-13, 15,17,22, 24-pentaene-26-isoailanthic acid methyl ester: the chemical name of English is: methyl (13E,15E,17E,22E,24E) -3, 12-dioxosomalaberica-13, 15,17,22,24-pentaen-26-oate English trivial name: methyl-rhabdate, i.e., methyl rhodanate. The physical constants of the compounds are as follows:

yellow disk crystal, m.p. 102-104 ℃ [ α]]²⁰ _D(C=0.29,CH₂Cl₂):-17.2℃:λ_max(C=0.29,CH₂Cl₂)nm:296(logε=3.69),381(logε=4.07),395(logε=4.13),418(logε=4.08)；v^KBr _max(cm^-1):2950,1702,1614,1577,1538,1434,1382,1270,1230,1168,1101,968；δ¹H(CDCl₃):7.29(1H,d,J=11.09Hz),7.05(1H,dd,J=11.44,14.91Hz),6.99(1H,dd,J=11.44Hz,15.25Hz),6.73(d,J=14.90Hz),6.64(1H,d,J=14.91Hz),6.39(1H,d,J=11.44Hz),3.79(3H,s),2.72(1H,m),2.40(1H,m),2.38(1H,m),2.35(3H,s),2.23(2H,d,J=10.05Hz),2.17(1H,m),2.16(2H,m),2.04(3H,s),2.02(3H,s),1.86(1H,t,J=10.4Hz),1.62(1H,m),1.51(2H,m),1.44(3H,s),1.13(3H,s),1.06(3H,s),0.86(3H,s)；δ¹³C(CDCl₃):31.33(C₁),33.44(C₂),219.21(C₃),46.84(C₄),45.47(C₅),19.72(C₆),38.45(C₇),44.98(C₈),47.86(C₉),34.76(C₁₀),36.64(C₁₁),207.11(C₁₂),146.50(C₁₃),143.28(C₁₁),134.24(C₁₅),131.94(C₁₆),134.87(C₁₇),14.50(C₁₈),23.42(C₁₉),138.54(C₂₀),12.89(C₂₁),143.76(C₂₂),124.84(C₂₃),138.75(C₂₄),128.28(C₂₅),168.88(C₂₆),12.98(C₂₇),29.17(C₂₈),19.34(C₂₉),25.88(C₃₀),50.71(OMe)；m/z:478(M⁺,6.5),463(4.8),431(6.2),365(100),313(18.6),241(19.9),159(11.0),91(11.3),55(11.0),43(13.3).

The method for extracting, separating and chemically reacting the compound comprises the following steps: sun-drying chopped sponge of south China sea (Rhabdstralla SD.) and extracting with ethanol, concentrating the obtained extract to obtain a slurry, dispersing the slurry in a proper amount of water, extracting with ethyl acetate to obtain an ethyl acetate soluble substance, performing silica gel column chromatography, performing gradient leaching with petroleum ether-ethyl acetate to obtain a semi-solid with medium polarity, performing repeated column chromatography with silica gel column and sephadex column, performing leaching with petroleum ether-ethyl acetate and chloroform-methanol as eluent to obtain a golden yellow solid, and recrystallizing in petroleum ether-acetone system to obtain golden yellow round plate-shaped crystal. The crystal is subjected to diazotization reaction to obtain yellow solid, namely the compound related to the invention.

The compound has a novel chemical structure and has obvious antitumor activity. If the compound is observed to have obvious inhibition effect on ehrlich ascites carcinoma solid tumors by oral administration of the compound to mice, the inhibition rates of 50,10 and 5ug/kg of the compound to the ehrlich ascites carcinoma solid tumors of the mice are respectively 64.7 percent, 58.8 percent and 45.1 percent, pharmacological tests show that three doses of methyl rotecate have obvious inhibition effect on the ehrlich ascites carcinoma solid tumors, and the inhibition rate is up to 65 percent, and experiments show that the compound is an organic compound with stronger anti-tumor effect and has good application prospect.

Example (b): 1. extraction and separation of rothiolate

Sun-dried south sea sponge Rhabdastrella sp. (3.0kg), minced, extracted three times with 95% industrial ethanol (5kg) at room temperature, the extracts were combined and concentrated under reduced pressure to a slurry, the slurry was uniformly dispersed in 500ml of distilled water, extracted three times with ethyl acetate (500ml), the extracts were combined and concentrated under reduced pressure to give 71g of brown slurry.

The above slurry was subjected to silica gel (200g, Qingdao Seawa chemical plant, silica gel H, 10-40. mu.) column chromatography (φ = 3.6X 48cm), gradient elution was carried out with 500ml of ethyl acetate-petroleum ether (10%, 15%, 20%, 30%) and 500ml of 30% acetone-petroleum ether at different ratios to obtain five eluates of different polarities, wherein 10.0g of a solid was obtained by subjecting the fraction of 30% ethyl acetate-petroleum ether to pressure reduction, and crude fraction A (3.7g) was obtained by subjecting the solid to silica gel (100g, Qingdao Seawa chemical plant, 300-400 mesh) column chromatography (φ = 2.4X 48cm, eluent: 15% acetone-petroleum ether). Subjecting fraction A to Sephadex LH-20 column chromatography with 30% chloroform-methanol eluent to obtain 3.2g of yellow-green solid, subjecting the solid to silica gel (100g, Qingdao Kaiyaku, silica gel H, 10-40 μ) column chromatography (phi =2.4 × 48cm, eluent: 20% acetone-petroleum ether) to obtain 2.5g of yellow solid, dissolving the solid in petroleum ether-acetone system, and standing at low temperature (-5-10 deg.C) to obtain 200mg of golden yellow round sheet crystal, i.e. rogli acid. 2. Preparation of diazomethane:

the reaction formula is as follows: a.

b．

c．

d． ① A500 ml three-necked flask with a weighed excess was charged with 100g (0.75mol) of methylamine in water and the solution was chargedConcentrated hydrochloric acid was added dropwise to make the methyl red (about 75ml of concentrated hydrochloric acid was required).

② Water was added to make the total weight 250g, then 150g (2.5mol) urea was added and allowed to reflux and simmer for 2 hours 45 minutes, and finally vigorously refluxed for 15 minutes.

③ mixing the obtained solutionThe methylurea solution was cooled to room temperature and 55g (0.75mol) of 95% NaNO was dissolved₂Cooling to 0 deg.C with ice salt bath, adding 300g of ice and 50g (0.5mol) of concentrated sulfuric acid into a 1.5L beaker, coating the beaker with ice salt bath, and dropping the 0 deg.C methylurea-sodium nitrite mixed solution into the beaker under mechanical stirring at a speed of not more than 0 deg.C. About one hour, the solution addition was complete. Nitrosomethylurea is precipitated in a crystalline foam, floats on the liquid surface, is immediately filtered by a Buchner funnel, is compacted by the back of a glass plug, is washed for 2 to 3 times by 50ml of cold water, is weighed after being drained, and 58g of the product is obtained.

④ preparing 30ml of 50% KOH aqueous solution, pouring into a 250ml round bottom flask, adding 100ml of diethyl ether, cooling to 5 ℃, adding 10g N-nitroso-N-methylurea while shaking, installing a condenser on the flask for distillation, the lower end of the condenser being connected to a receiver, which is immersed into a 150ml conical flask containing 30ml of diethyl ether through a two-hole rubber stopper, distilling the diethyl ether in a water bath at 50 ℃, the distillate becoming colorless after typically two thirds of the diethyl ether are distilled off, in any case not all the diethyl ether can be distilled off, about 80ml of distillate, about 35mg/ml in concentration, yellow, storing in a refrigerator, 3 preparing methyl rothiolate:

the reaction formula is as follows:

the method comprises the following steps: in a 25ml egg-shaped flask were added crystal of rothiric acid 40mg, 2ml of diethyl ether and 1ml of chloroform. 2ml of diazomethane in ether solution was added to the egg-shaped flask, and the reaction was magnetically stirred for 2 to 3 hours (judged complete by TLC) under a room-temperature sealed system. The solvent was removed under reduced pressure, and the product was purified by column chromatography on silica gel (silica gel 300-400 mesh, eluent: 20% acetone-petroleum ether), concentrated and dried to give 34.7mg of a yellow solid of methyl rothite in 83.0% yield and 0.0055% total yield calculated as rothite. The chemical structure is determined by methods such as infrared spectrum, mass spectrum, nuclear magnetic resonance spectrum and the like as follows:

Claims (3)

1、(13E,15E,17E,22E24E) -3, 12-dioxo-13, 15,17,22, 24-pentaene-26-methyl isoailanthic acid, and the chemical structural formula is as follows.

2. A method for preparing the compound of claim 1, characterized in that the ethanol extract concentrate of sponge is dispersed in water, extracted with ethyl acetate, the extract concentrate is treated with repeated silica gel column chromatography and gel column chromatography, petroleum ether-ethyl acetate, petroleum ether-acetone and chloroform-methanol are respectively used as eluent, the obtained solid is placed in a petroleum ether-acetone system at a low temperature of-5 to-10 ℃ to obtain golden yellow flaky crystals, and the crystals are subjected to diazotization reaction to obtain yellow solid.

3. The method of claim 2, wherein the sponge is a south sea sponge.