Novel Tandem Biotransformation Process for the Biosynthesis of a Novel Compound, 4-(2,3,5,6-Tetramethylpyrazine-1)-4′-Demethylepipodophyllotoxin

Abstract

According to the structure of podophyllotoxin and its structure-function relationship, a novel tandem biotransformation process was developed for the directional modification of the podophyllotoxin structure to directionally synthesize a novel compound, 4-(2,3,5,6-tetramethylpyrazine-1)-4-demethylepipodophyllotoxin (4-TMP-DMEP). In this novel tandem biotransformation process, the starting substrate of podophyllotoxin was biotransformed into 4-demethylepipodophyllotoxin (product 1) with the demethylation of the methoxyl group at the 4 position by Gibberella fujikuroi SH-f13, which was screened out from Shennongjia prime forest humus soil (Hubei, China). 4-Demethylepipodophyllotoxin (product 1) was then biotransformed into 4-demethylpodophyllotoxone (product 2) with the oxidation of the hydroxyl group at the 4 position by Alternaria alternata S-f6, which was screened out from the gathered Dysosma versipellis plants in the Wuhan Botanical Garden, Chinese Academy of Sciences. Finally, 4-demethylpodophyllotoxone (product 2) and ligustrazine were linked with a transamination reaction to synthesize the target product 4-TMP-DMEP (product 3) by Alternaria alternata S-f6. Compared with podophyllotoxin (i.e., a 50% effective concentration [EC 50 ] of 529 M), the EC 50 of 4-TMP-DMEP against the tumor cell line BGC-823 (i.e., 0.11 M) was significantly reduced by 5,199 times. Simultaneously, the EC 50 of 4-TMP-DMEP against the normal human proximal tubular epithelial cell line HK-2 (i.e., 0.40 M) was 66 times higher than that of podophyllotoxin (i.e., 0.006 M). Furthermore, compared with podophyllotoxin (i.e., log P ‫؍‬ 0.34), the water solubility of 4-TMP-DMEP (i.e., log P ‫؍‬ 0.66) was significantly enhanced by 94%. For the first time, the novel compound 4-TMP-DMEP with superior antitumor activity was directionally synthesized from podophyllotoxin by the novel tandem biotransformation process developed in this work.Podophyllotoxin (PTOX) is a naturally occurring aryltetralin lignan, and interest in it has been heightened by its potential antitumor activity (3, 16) but has failed to be used in human clinical trials because of unacceptable gastrointestinal toxic side effects (3). In order to overcome these limitations, numerous studies of Podophyllum lignans currently focus on the structural modification of the cycloparaffin (C ring) in the tetranap skeleton and the benzene with three methoxyl groups (E ring) to generate derivatives with superior pharmacological profiles (25). Aromatic heterocyclic compounds substituted with a carbon-nitrogen (CON) bond at the 4 position (14) may be a very effective pathway for improving the antitumor activity of PTOX, and most of these derivatives were more active than etoposide (1,5,7,8,15,20), which is a clinically important anticancer drug. Furthermore, previous investigations indicated that a PTOX derivative in which the para-position methoxyl group (4Ј-MeO) of PTOX was demethylated shows stronger anticancer activity and solubility than PTOX (29, 39). All of these analyses suggested that...

Accelerated green process of tetramethylpyrazine production from glucose and diammonium phosphate

“…This alkaloid has been widely used in China to treat several diseases [8,9]. TMP can also serve as the precursor in the synthesis of several other biological materials of value [10][11][12].…”

Novel Biotransformation Process of Podophyllotoxin to 4 -Sulfur-Substituted Podophyllum Derivates with Anti-Tumor Activity by Penicillium purpurogenum Y.J. Tang

“…In our previous work, we biotransformed PTOX to 4 -demethylpodophyllotoxin (DMEP) using Gibberella fujikuroi SHf13, wherein the benzyloxy at the 4 position of PTOX was demethylated and epimerized at the 4 position while maintaining chirality at all other stereocenters [26]. The anti-tumor mechanism of DMEP is different from that of PTOX, in that DMEP induces the breakage of single-and double-stranded DNA as the initial step in a series of biochemical transformations that eventually lead to cell death.…”

“…A total of 21 endophytes were isolated from Diphylleia sinensis Li, which is rich in podophyllum compounds. Finally, the strain of DSL-f3 with PTOX biotransformation ability was obtained using the screening strategy previously described by Tang et al, [26] and was then selected for the following 4 -sulfur-substitution.…”

“…4 -demethylepipodophyllotoxin (DMEP) was biotransformed into 4 -demethylpodophyllic acid with a hydrolysis reaction by Bacillus fusiformis CICC20463 [24,25]. Furthermore, a novel tandem biotransformation process was developed for the directional modification of PTOX structure to biosynthesize a novel compound, 4-(2,3,5,6-tetramethylpyrazine-1)-4 -demethylepipodophyllotoxin with superior activity against the tumor cell line BGC-823 [26]. This work demonstrated that PTOX and its analogue DMEP could be transformed into four novel sulfur substituted derivatives, and then the derivatives were evaluated for the anti-tumor activity (i.e., BGC823, A549 and HepG2) and the toxic side-effect on HK-2 cell line.…”

Recent Advances in the Chemistry and Biology of Podophyllotoxins

“…Finally, tetramethylpyrazine was coupled with 8 to synthesize 429 , which showed nearly 4815 times more potent cytotoxicity than 1 against BGC‐823 cells (EC 50 : 0.11 μ m ( 429 ) vs. 529.66 μ m ( 1 )). Meanwhile, the toxic effect of 429 (EC 50 : 0.4 μ m ) on the normal human cell line HK‐2 was sharply reduced compared with that of 1 (EC 50 : 0.006 μ m ) . The reaction conditions for the biotransformation of 23′ into 429 were systematically investigated, and the optimal conditions were determined.…”