Investigation Of Antitumor Constituents From Peganum Harmala L. Seeds

Extensive structural and biophysical evidence confirmed that certain G-rich DNA(and RNA)sequences can fold into four stranded DNA secondary structures,called G-quadruplexes.G-quadruplex structures arised from the self-stacking of two or more guanine quartets connected by Hoogsteen hydrogen bonds and stabilized by monovalent cations.The high thermodynamic stability of G-quadruplexes under near-physiological conditions suggests that these structures may form in genomic DNA in vivo.In fact,these structures have been very rcecently visualized in both the DNA and RNA of human cells,and even been marked in human regulatory chromatin.G-quadruplex structures have been linked to transcription,replication,and genome instability,and are implicated in cancer and other diseases.G-quadruplex structures are enriched in the telomerir DNA,promoters and 5’ UTRs of highly transcribed genes,particularly in genes related to cancer,which have emerged as attractive targets for anticancer drugs with a novel mechanism of action.The molecules,which can stabilize or regulate the conformation of G-quadruplex or induce the formation of G-quadruplex from single-stranded DNA,have the potential for preventing the growth of cancer cells through the disruption of telomere maintenance or the alteration of oncogene expression levels,even as the most attractive frontiers in the development of novel anticancer therapeutics.In our preliminary screening of G-quadruplex ligands from 17 medicinal plants by 1H NMR,the crude extract of the seeds of L.showed most potential binding activity to d(TTGGGTT)4 G4DNA and specific cytotoxic effect against PC-3 cell lines with an IC50 value of 13.65 μg/mL.The studies on chemical constituents of Peganum harmala L.and their antitumor activities were carried out to look for the natural products which may have G-quadruplex binding activities.Peganum harmala L.,a plant belonging to the family of Zygophllaceae,is mainly distributed in arid and semi-arid region of northwest China,including Xinjiang,Qinghai,Gansu,the northern edge of the Ningxia and most parts of Inner Mongolia.Its main active ingredients are alkaloids,which showed significant pharmacological effects on the central nervous system,cardiovascular system,respiratory system,antitumor,anti-aging and so on.In clinical trials,the powder of the P.harmala L.had been used for the treatment of stomach cancer patients and had achieved good results.The chemical constitutents of P.harmala L.seeds were investigated with various column chromatography on silica gel,Sephadex LH-20,ODS,and HPLC.Their structures,including stereochemistry,were elucidated through spectroscopic analyses,quantum chemistry calculations,and single-crystal X-ray diffraction.Seventy-one alkaloids including 43 new compounds were isolated.Importanly,twelve new compounds bearing new skeleton were first reported.Peganumine A(1)represent a pair of partially racemized dimeric β-carboline alkaloid characterized by a unique 3,9-diazatetracyclo-[6.5.2.01,9.03,8]pentadec-2-one scaffold.Pegaharmines A-D(3-7,13)are four pairs of partially racemized β-carboline alkaloids characterized by the rare carbon skeleton of a tetrahydro-β-carboline fused with five-membered amide ring.Pegaharmaline A(11)and(±)-peharmaline A(9)represent novelβ-carboline-vasicinone hybrid alkaloids with unique hybrid dimeric systems.Pegaharmaline B(12)is a perfect polymerized β-carboline alkaloid featuring a unique and highly conjugated C24 skeleton.(±)-Peganines A-B(15-17)are two pairs of unique racemic pyrroloindole alkaloids.Peganumals A-B(19-20)are two rare thiazole derivatives.Pegaharmines F(21)is a new β-carboline alkaloids bearing a unique C-1 α,β-unsaturated ester motif.Pegaharmines G-K(22-26)are new β-carboline alkaloids.Peganumaline A-G(39-48,50,52)and Pegaharmine E(49)are new indole alkaloids.Peganumines B-E(53-56)are two pairs of new quinazoline alkaloid glycoside epimers.These structurally diverse alkaloids greatly enriched the diversity of the P.harmala L.alkaloids and some of which characterized by partially racemized alkaloids were first reported in the genus Peganum.These novel molecules provide insights into the biosynthetic pathway of β-carboline alkaloids,which implies that biogenetic mechanisms for creating distinct enantiomers may be widely expressed in this plant,and contribute important reference data for potential anticancer lead compound.Their antiproliferative effects against three cancer cell lines and G-quadruplex binding activities were evaluated using the trypan blue method and the MTT method,and 33β-carboline alkaloids showed significant cytotoxic activities which means that the β-carboline alkaloids are the main antitumor constituents from P.harmala L..The data indicated the dimerization and extended electron-deficient π-aromatic surface are important for the antitumor activity and the molecule which have good fused plane rings have better antitumor activity.We then investigated the binding activities of these alkaloids with the G-quadruplexes.The β-carboline alkaloids exhibited good binding activities and the compounds 3,27,29,31,and 32 showed the strongedt binding activity.Intriguingly,the G-quadruplex interactions appeared to be positively correlated with antiproliferative activities in cancer cell lines,suggesting that these β-carboline alkaloids may exert their anticancer effects through G-quadruplex interactions.For example,the most cytotoxic pegaharmine D(3)appeared to be the most potent G-quadruplex interactive compound.Moreover,pegaharmine D(3)could selectively bind to the biologically relevant intramolecular parallel G-quadruplexes formed in the promoter regions of c-MYC,BCL-2,and VEGF genes,but not to the hybrid-type(3+1)human telomeric G-quadruplexes,or double-and single-stranded DNA.Collectively,this work contributed a practical strategy for the discovery of novel G-quadruplex ligands from natural products and provided potential insights for usingβ-carboline alkaloids as anticancer lead compounds specifically targeting G-quadruplexes.