Actinomycetes 2215 ~ # Active Metabolite Separation And Purification Identification And Preparation

Chronic myelogenous leukemia (CML) is a clonal myeloproliferative disorder of the hematopoietic stem cell (HSC). CML is characterized by a chronic phase in which myeloid precursors and mature cells accumulate in the blood and extramedullary tissues. After 3-4 years, CML transform into a fatal blast crisis. The annual incidence of CML in our country is 0.36/100,000, which account for 20% of all leukemia. Glivec (or STI571), methanesulfonic acid;4-[(4-methylpiperazin-l-yl)methyl]-N-[4-methyl-3-[(4-pyridin-3-ylpyrimidin-2-yl)amino ]phenyl]benzamide, is a specific inhibitor of Bcr-Abl tyrosine kinase. It competitively inhibits the binding of adenosine triphosphate (ATP) to the kinase domain of Abl and induces apoptosis in a variety of Bcr-Abl-positive cell lines and Philadelphia-chromosome-positive (Ph+) cells. Glivec has become the front-line drug for CML since 2001, but resistance is increasingly encountered. So it is a need to develop some novel inhibitor for CML resistance.We established a set of methods for fermentation, extraction, and screening microbialmetabolite and screened 1000 species of soil microorganism, which collected from outlyingregions in our country. The metabolite of Streptomyces sp.2215 has been found to have a highinhibitory on K562 cells(IC₅₀=0.05μg/ml) and K562/G1c cells(resistant to Glivec, IC₅₀=0.036μg/ml), but weak inhibition on Vero cells (normal cell, IC₅₀=0.92 μg/ml).Streptomyces sp.2215 was fermented and the broth was isolated, guided by screening. A high active compound was obtained, it inhibited the growth of K562 cells (IC₅₀=0.43ng/ml) and K562/Glc cells (IC₅₀=0.39ng/ml), but inhibited that of normal Vero cells very weakly at the same dosage. Using 1D-NMR, 2D-NMR, FAB-MS, the active compound is identified to be Echinomycin,N-[3,11,13,16,24,26-hexamethyl-27-methylsulfany1-2,5,9,12,15,18,22,25-octaoxo-4,17-dipropan-2-y1-8-(quinoxaline-2-carbonylamino)-6,19-dioxa-28-thia-3,10,13,16, 23,26-hexazabicyclo[12.12.3]nonacos-21-yl]qumoxaline-2-carboxamide. A cyclic peptide antibiotic with strong inhibition on many tumor cells reported.In order to evaluate the perspective of application, a large-scale fermentation was...