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Use of compound scalarafuran

A compound, Ofuran technology, which is applied in the field of preparation of sesquiterpene compound Cetiofuran, can solve problems such as the mechanism is not clear, it is impossible to determine whether the compound has a direct effect, and IN inhibitors have no drugs on the market.

Inactive Publication Date: 2010-06-16
SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] Although there have been more than ten years of research on IN inhibitors, there are currently no drugs on the market for IN inhibitors, and only a few have entered the clinic
The reason is that there are two main limitations: one is the limitation of the activity assay method: the determination based on cell culture cannot determine whether the compound directly acts on IN, and there are too many false positives in molecular level screening; the other is the limitation of structural biology. Restrictions: the crystal structure of IN and its DNA-substrate complex have not been determined, and there are still many mechanisms in the integration of this complex biochemical process that have not been clarified
[0016] At present, the treatment of AIDS mainly adopts the combination drug strategy, that is, the combination of HIV reverse transcriptase and protease inhibitors, but the drug resistance caused by HIV virus mutation and the toxic and side effects of reverse transcriptase and protease inhibitors in the course of treatment serious

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Frozen fresh sponge (dry weight 138 g) was chopped, ultrasonically extracted with analytical grade acetone until colorless, the acetone extract was concentrated under reduced pressure (temperature below 30°C), the concentrate was suspended in 500 ml of distilled water, extracted with ether, The ether extract was concentrated under reduced pressure to obtain 5.4 grams of extract, which was subjected to silica gel (200-300 mesh) column chromatography [petroleum ether (60-90°C) / ethyl acetate gradient elution], and the eluents were combined Seven fractions were obtained, and the fifth fraction eluted by petroleum ether (60-90° C.) / ethyl acetate 78 / 12 was subjected to gel LH-20 (chloroform elution) column chromatography, and then passed through a silica gel column Chromatography, eluting with petroleum ether (60-90°C) / diethyl ether 75 / 25, isolated a pure compound as colorless crystals (8.2mg). 1 H-NMR and 13 The C-NMR data are as follows: 1 H NMR (CDCl 3 )δ: 7.53(s, 1H, H...

Embodiment 2

[0077] Construction of the fusion protein particle pGEX-4T-1-IN (F185K) of the HIV-1 integrase (IN, integrase) of embodiment 2GST mark:

[0078] (1) Experimental method:

[0079] 1) Obtain the DNA fragment of IN from the pUC18-IN plasmid by PCR technology (the gene sequence is based on AF040373 (GENEBANK)).

[0080] Primer: FW: 5′-ata tgg atc ctt ttta gat gga ata gat-3′

[0081] RV: 5′-ata tct cga gct aat cct cat cct g-3′

[0082] Denaturation at 94°C for 5min, then cycle: 94°C for 45s, 55°C for 45s, 72°C for 1min30s, repeat 29 cycles, then 72°C for 10min.

[0083] 1% agarose gel electrophoresis was used to identify PCR products and recover PCR fragments.

[0084] 2) Clone the IN PCR fragment into the vector pGEX-4T-1: the IN PCR product and pGEX-4T-1 were digested with BamHI and XhoI respectively; the digested product was recovered with a gel recovery kit; Ligation reaction; the ligation product was transformed into DH5α competent cells and spread on a plate contain...

Embodiment 3

[0093] Expression and purification of the HIV-1 integrase (GST-IN) of embodiment 3 GST markers:

[0094] (1) Experimental method

[0095] 1) Transfer the plasmid pGEX-4T-1-IN(F185K) into the expression strain BL21(DE3), select the correct clone and culture it in 10ml ampicillin LB medium (yeast extract 5g / L, tryptone 10g / L and NaCl10g / L, the concentration of ampicillin is 100mg / L), transferred to fresh ampicillin LB medium at a ratio of 1:100, and cultured with shaking until OD 600 When the value is between 0.6-0.8, add IPTG to a final concentration of 0.2mM and lower the culture temperature to 25°C to induce protein expression for 5-7 hours. Bacteria were collected by centrifugation at 5,000 rpm for 10 min, suspended and washed with PBS buffer, and then centrifuged again, and the collected bacteria were stored in a -70°C refrigerator until use.

[0096] 2) Suspend the bacteria to be used in 20ml PBS, sonicate the bacteria, centrifuge at 15,000rpm for 30min, take the superna...

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Abstract

The present invention discloses one kind of compound, citiofuran. The compound, citiofuran has very high combination activity, KD=16.4 microM, with HIV-1 integrase, and effective inhibition of the combination of the integrase to the substrate, IC50=120 microM, in substrate competition test. Therefore, the compound exhibits very high medicine preparing value.

Description

technical field [0001] The invention relates to the fields of medicinal chemistry and pharmacotherapeutics, in particular to a preparation method and application of a sesquiterpene compound scalarafuran. Background technique [0002] Since the first AIDS (acquired immunodeficiency syndrome) patient was discovered in the United States in 1981, AIDS has spread rapidly in more than 180 countries and regions around the world. At present, the total number of AIDS patients and virus carriers in the world has reached more than 42 million, of which nearly 1 million are in China, ranking second in Asia and 14th in the world. Moreover, in recent years, the number of AIDS cases in China has been increasing rapidly at an average annual rate of 30-40%, which has become an increasingly serious public security problem. [0003] As the pathogen of AIDS, HIV virus needs the participation of integrase (IN) to complete the replication process in the body after it infects the human body. IN c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K31/58A61P31/18
Inventor 沈旭郭跃伟蒋华良于志国杜丽
Owner SHANGHAI INST OF MATERIA MEDICA CHINESE ACAD OF SCI
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