Rustmicin, a new macrolide antibiotic active against wheat stem rust fungus.

Sir: Stem rust (Puccinia graminis f. sp. tritici) is a serious wheat disease causing decrease of wheat production in many areas of the world1) and has been partially controlled so far by the use of resistant races2), eradication of the alternate host, i.e. barberry3), or by synthetic fungicides4~7) We undertook the screening of new antibiotics active against wheat stem rust fungus using an inhibition assay of the germination of uredospores in vitro and pot test in green house. During the course of this screening, rustmicin (I) (formerly called P-59B18)) was obtained from the cultured broth of Micromonospora narashinoensis 980-MC,. The present communication describes the isolation and structural elucidation of I. Fermentation was carried out at 27°C with agitation of 400 rpm and aeration of 25 liters/ minute for 4 days in two 50-liter jar fermentors each containing 25 liters of a medium consisting of soluble starch 2.5 %, soybean meal 1.5 %, dry yeast 0.2% and CaCO3 0.4% (pH 7.4). The activity against wheat stem rust fungus was monitored by an in vitro method, which assayed the germination of uredospores on the agar containing a sample 2 hours after inoculation at 20°C, throughout the purification. The active substance was isolated from the filtrate as described below. The cultured broth (ca. 47 liters) was harvested, adjusted to pH 7.0 with 2 N HCI and filtered. The filtrate was adsorbed on a Diaion HP20 column (5 liters) which was washed successively with H2O and 50% aq MeOH (10 liters each) and finally the active metabolite was eluted with MeOH (10 liters). The MeOH concentrate (0.5 liter) was partitioned between H2O and EtOAc three times. The combined organic layer (1 liter) was washed successively with 5 % NaHCO3, 0.01 N HCl and H2O, dried over anhydrous Na2SO4 and concentrated in vacuo to afford a brownish oil (350 mg). This crude material was chromatographed over a Sephadex LH-20 column with MeOH and the active fractions were combined and evaporated in vacuo to dryness. The final purification of the antibiotic was achieved by reversed phase HPLC (Waters Radial PAK 8C18) eluting with 70% aq MeOH to yield 7.5 mg of I in a pure form. I is a neutral colorless oily material possessing the following physico-chemical properties: UV AMeOHmax nm (s) 215 (9,500) and 240 (8,000); IR v CHCl3max3600 (OH), 1730 and 1710 cm-1 (COO and C=O); EI-MS m/z 380 (M+). Its molecular formula was established to be C21H32O6 by high resolution mass spectral data (found 380.2196; calcd 380.2200). The signals in the 13C NMR spectrum of I taken in CDCl 3 accounted for 21 carbons and 30 nonexchangeable protons (see Table 1). Two protons at 5H 2.31 (1H, dd, J=11.0 and 2.0 Hz) and 3.46 (1H, br s) in the 1H NMR spectrum of I revealed the presence of a primary and a tertiary alcohol, respectively. In the 13C NMR spectrum, the resonance at o, 169.2 was assigned to an ester carbonyl due to an IR absorption band at 1730 cm-1. The presence of a saturated ketone at o, 209.2 indicated that the UV absorption max 240 nm) is due to a conjugated diene system. Thus, the functional groups in I are summarized as follows: CH3 x 4, CH2 x 3, CH x 2, CH3O x 1, CH2OH x 1, CH-O x 1, COH x 1, CH2 x 1, =CHx2, =Cx3, COO-XI and C=Oxl. These data suggested the presence of one ring structure in I. The structure of I was determined mainly based on spin decoupling and NOE experiments. The olefinic proton resonating at off 5.77 (H-11; 1H, br s) showed long range couplings with a methyl at off 1.81 and two protons at off 4.91 and 4.97; irradiation of H-11 sharpened these three signals. The proton at off 4.91 was coupled to the signal at off 5.08 by a small coupling constant (J=2.2 Hz). These two resonances were ascribed to an exomethylene whose 13C resonance was observed at o, 116.9. The conjugated diene system in I proved to be located Fig. 1. Structure of rustmicin (I).