The remaining methoxyl groups position at C-5 position was established by its cyclization and alkaline degradation, when 8-methoxy-2,2-dimethyl-chroman-6-carboxylic acid, m.p. 179–180°, molecular formula C13H16O4 and M+ 236 (CIMS) was obtained. The appearance of chemical shift at δ 1.35 (6H, br, s), 3.66 (1H, m, –C1, –H) 3.70 (1H, m, –C1, H) and 5.11 (1H, br t, J = 7 Hz, C2, –H) in the 1H NMR spectrum of RS-2 were characteristic OSI-744 cell line of the presence of prenyl unit in the aglycone as portrayed in Graph 3. The position and nature of the prenyl unit was confirmed by the further analysis of RS-2(A). The chemical shift

at δ 6.84 (1H, s) in the (1H, s) in the 1H NMR spectrum of the aglycone indicated the presence of hydrogen at C-8. 10 Because of the presence of hydroxyl groups at C-5 and C-7 positions and a methoxyl group at C-6 which has already been proved and therefore it was concluded that the prenyl unit was not attached with ring C. Also the presence of methoxyl group at C-3, ruled out

the possibility of presence of prenyl group in ring A of RS-2(A). Thus based on the above fact it is clearly inferred that there was the only option of presence of prenyl unit in ring B. Based on the above deliberations, the C-4 position has been proved to be occupied by the –OH group, whereas, the presence of –OCH3 group at C-5 in RS-2(A) was confirmed by the cyclization followed by oxidation of the cyclized product. As such the only position left for the presence of prenyl unit were

C-2, C-6 Roxadustat chemical structure or C-3. Out of the above three possibilities, on critical crotamiton examination the position C-2 and C-6 were excluded on the ground that signals in 1H NMR spectrum of the aglycone at δ 7.76 (1H, d, J = 2.6 Hz) and δ 7.38 (1H, d, J = 2.6 Hz) indicated the presence of hydrogen atoms at C-2 and C-6 respectively thereby ruling out, the possibility of the presence of prenyl unit at C-2 and C-6. Therefore the only position left for the presence of prenyl unit was C-3 in the ring B. The position C-3 for the prenyl unit was also confirmed on the basis of the fact on cyclization followed by oxidation in the presence of formic acid, RS-2(A) yielded 8-methoxy-2,2-dimethyl-chroman-6-carboxylic acid. The chemical ionization mass spectrum study of RS-2(A) produced fragment ion peaks at m/z 373 and 372 by the loss of M-55 and M-56 suggesting the prenylation adjacent to –OH group and thus further established the presence of prenyl unit at C-3 in RS-2(A). The above deliberation clearly established the nature of substitution pattern in the ring B as; 4-hydroxy, 5-methoxy, 3-(3-methyl-but-2-enyl) finally. Keeping all the facts together the structure to the prenylated aglycone RS-2(A) was established, as; 5,7,4-trihydroxy 3-(3-methyl-but-2-enyl) 3,5,6-trimethoxy-flavone as depicted in Fig. 4.