TWO new chromone derivatives, 2,2-dimethyl-5-amino-6-(3'-N-formyl-4'-O-hydroxylbucyryl)-4-chromone [3] and 2,2-dimethyl-5-amino-6-(3'-N-acetyl-4'-O-acetylbutyryl)-4-chromone [4], were isolated from the rice culture of Fusarium equiseti. Their structures were deduced from chemical and spectral data. The chromone derivative, fusarochromanone [1], has been found in cultures of Fusarium equiseti (Corda) Sacc. (previously identified as Fusarium roseum gramineurum) (1-4) and chicken feed samples (1). This mycotoxin causes tibia dyschondroplasia, a common bone disease in chicks (2,5) and reduced hatchability of fertile chicken eggs (2) under experimental conditions. In a previous report (3), we described the isolation and identification of a monoacetate 2 of fusarochromanone from the rice culture of F. equiseti. Further fractionation of the crude extract allowed us to isolate a minor metabolite 3 as a yellow oil from polar fractions and another metabolite 4 as needle-shaped colorless crystals from nonpolar fractions. Both compounds exhibit blue fluorescence under uv. Eims of 3 yielded an [M]+ at m/z 320 and [M - H2O]+ at m/z 302 indicating the presence of an -OH group. Acetylation of 3 yielded a monoacetate 5 with an [M]+ at m/z 362 in eims, which further supported the presence of an -OH group. Fabms of 3 yielded an [M + H]+ at m/z 321 and [M + Na]+ at m/z 343; negative cims of 3 yielded [M]- at m/z 320, further supporting the mol wt as 320 amu. Hreims of 3 yielded an [M]+ at m/z 320.1355 (calcd 320.1372 for a molecular formula of C16H20N2O5, which was compatible with 8 double bond equivalents). Eims of 3 was very similar to those of 1 and 2 (3), indicating closely related structures. Mass measurement of fragments at m/z 275.1167 (calcd 275.1158 for C13H15NO4) and m/z 257.1034 (calcd 257.1051 for C13H13NO3) suggested structures 6 and 7. A signal at δ 8.14 (1H, d, J=1.6) in the 1H-nmr spectrum of 3 indicated the presence of an -NHCHO group. Hydrolysis of 3 in HCl yielded 1, which was confirmed by tlc and eims. Compound 3 was obtained by reacting 1 with HCO2H and the structure was confirmed by eims, fabms, 1H-nmr, and tlc. Therefore, the structure of 3 was established as 2,2-dimethyl-5-amino-6-(3'-N-formyl-4'-O-hydroxylbutyryl)-4-chromone or 3'-N-formyl fusarochromanone. The eims of 4 yielded an [M]+ at m/z 376. Fabms yielded an [M + H]+ at m/z 377 and [M + Na]+ at m/z 399, and negative cims yielded [M]- at m/z 376. The hreims indicated a molecular ion at m/z 376.1661 (calcd 376.1634 for C19H24N2O6). Signals at δ 4.14 (1H, dd, J=5.3, 11) and 4.32 (1H, dd, J=6.1, 11) in the 1H-nmr spectrum of 4 indicated a downfield shift, compared with 3.70 (2H, m) in the previously reported spectrum of 2 (3). This suggested acetylation of the -OH at C-4'. An absorption band at 1734 cm-1 in the ir spectrum also supported the presence of acetyl groups. Hydrolysis of 4 in HCl yielded 1 and 2, confirmed by tlc and eims. Acetylation of 2 with N-acetylimidazole yielded 4, which was confirmed by tlc and eims. Therefore, the structure of 4 was determined as 2,2-dimethyl-5-amino-6-(3'-N-acetyl-4'-O-acetylbutyryl)-4-chromone or 3'-N-acetyl-4'-O-acetyl fusarochromanone.