Cytotoxic and antibacterial polyketide-indole hybrids synthesized from indole-3-carbinol by Daldinia eschscholzii.

Acta Pharmaceutica Sinica B 2019;9(2):369–380 Chinese Pharmaceutical Association Institute of Materia Medica, Chinese Academy of Medical Sciences Acta Pharmaceutica Sinica B www.elsevier.com/locate/apsb www.sciencedirect.com ORIGINAL ARTICLE Cytotoxic and antibacterial polyketide-indole hybrids synthesized from indole-3-carbinol by Daldinia eschscholzii Liping Lina,b, Nan Jiangc, Huimin Wub, Yaning Meid, Jie Yange, Renxiang Tana,b,n a State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210046, China State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023 China c School of Pharmacy, Nanjing Medical University, Nanjing 210029, China d Department of Clinical Laboratory, the First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China e Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing 210028, China b Received 10 July 2018; received in revised form 25 August 2018; accepted 7 September 2018 KEY WORDS Polyketide-indole hybrids; Indole-3-carbinol; Daldinia eschscholzii; 8-Amino-7-oxononanoate synthase; Decarboxylative Claisen condensation; Antibacterial; Anticancer Abstract Two skeletally undescribed polyketide-indole hybrids (PIHs), named indolchromins A and B, were generated from indole-3-carbinol (I3C) in the fungal culture (Daldinia eschscholzii). The indolchromin structures were elucidated mainly by their 1D and 2D NMR spectra with the former confirmed by the single-crystal X-ray crystallographic analysis. Each indolchromin alkaloid was chirally separated into four isomers, whose absolute configurations were assigned by comparing the recorded circular dichroism (CD) spectra with the electronic CD (ECD) curves computed for all optional stereoisomers. Furthermore, the indolchromin construction pathways in fungal culture were clarified through enzyme inhibition, precursor feeding experiment, and energy calculation. The cascade reactions, including decarboxylative Claisen condensation catalyzed by 8-amino-7-oxononanoate synthase (AONS), C(sp3)-H activation, double bond migration, and Michael addition, all undergone compatibly during the fungal cultivation. In an MIC range of 1.3–8.6 μmol/L, (2S,4R)- and (2R,4S)-indolchromin A and (2R,4S)indolchromin B are inhibitory against Clostridium perfringens, Clostridium difficile, Veillonella sp., Bacteroides fragilis, and Streptococcus pyogenes. (2R,4S)-Indolchromin A and (2S,4S)-indolchromin B were cytotoxic against the human breast cancer cell line MDA-MB-231 with IC50 values of 27.9 and 131.2 nmol/L, respectively, with the former additionally active against another human breast cancer cell line MCF-7 (IC50 94.4 nmol/L). n Corresponding author at: State Key Laboratory Cultivation Base for TCM Quality and Efficacy, Nanjing University of Chinese Medicine, Nanjing 210023, China. E-mail address: (Renxiang Tan). Peer review under responsibility of Institute of Materia Medica, Chinese Academy of Medical Sciences and Chinese Pharmaceutical Association. https://doi.org/10.1016/j.apsb.2018.09.011 2211-3835 & 2019 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 370 Liping Lin et al. & 2019 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction Indole alkaloids are an important class of organic molecules that contain one or more indole or indoline motifs. They widely distribute as secondary metabolites in bacteria, fungi, plants and animals1–3. Some indole alkaloids have been demonstrated to be cytotoxic (e.g., vintafolide and lestaurtinib4), analgesic (e.g., physostigmine5), antibacterial (e.g., dalesindole6), neuritogenic (e.g., manzamines7), immunosuppressive (e.g., sotrastaurin and tivantinib4), stem cell differentiation regulatory (e.g., kenpaullone and stauprimide8), and others. Therefore, the indole motif is present in diverse pharmacophores and many research groups have been motivated to discover more bioactive new alkaloids branded with such motifs9–11. Polyketides are among the most diverse natural product categories12, but few polyketide-indole hybrids (PIHs) have been synthesized chemically or characterized from nature except for the tryptophan-derived cytochalasans3. A presumable underlying reason for the PIH rarity arises from the fact that the PIH synthesis faces a suite of challenges, such as the complicated reaction steps (including iterative/inevitable procedures for protection and deprotection) and/or the utilization of toxic/expensive reagents13,14. Strategically, the generation of new PIHs usually includes cascade reactions such as C–C(N/O) bond formation15 and selective functionalization of unactivated aliphatic C(sp3)–H bonds16. However, these reactions largely proceed in harsh reaction conditions17. Thus, the greener approach is highly desired for the access to undescribed PIH molecules, and preferably, the PIH-constructing reactions could be completed in one pot in an eco-friendly manner. Indole-3-carbinol (I3C, 1) is a cancer-preventive agent18 released via the degradation of indole glucosinolate in cruciferous vegetables19,20. After its oral administration to mice, 1 is metabolized into indole alkaloids including indole-3-carbaldehyde (I3A), indole-3carboxylic acid (I3CA, 2), 1-(3-hydroxymethyl)-indolyl-3- Scheme 1 Indolchromins A (4) and B (5) resulting from the coupling of fungal polyketide with I3C (1) catabolites. (A) Proposed polyketideindole hybridization leading to 4 and 5 in the I3C exposed fungal culture. (B) Reaction energy profile for the generation of 4 and 5 computed by DFT method at B3LYP/6-31þG(d,p) level in polarizable continuum model (PCM, dielectric constant ε ¼ 78.39 for H2O). The relative energies in kcal/mol are calculated from the sums of the respective total energies (Esol) of all species (Supporting Information Table S2). AONS, 8-amino-7oxononanoate synthase; I3CA (2), indole-3-carboxylic acid. Polyketide-indole hybrids synthesized from indole-3-carbinol by Daldinia eschscholzii indolylmethane (HI-IM), 3,3'-diindolylmethane (DIM), indolo[3,2b] carbazole (ICZ), and 2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr1)21. The tumor therapeutic effect of DIM as a drug candidate under the phase III clinical trial (http://www.clinicaltrials.gov) could be alternatively formed by the decarboxylative Claisen condensation between I3A and I3CA (2), which was catalysed by 8-amino-7oxononanoate synthase (AONS) in Daldinia eschscholzii6. Encouraged by the observation, we wondered whether 1 could be further metabolized and hybridized with polyke (...truncated)