New bisbenzylisoquinoline alkaloid, isolation and structure elucidation from Cocculus pendulus

Abstract This paper describes phytochemical studies on aerial part of the Cocculus pendulus, which led to the identification of a new ingredient namely, 2 N-methylkohatine (1) along with four reported compounds Kohatine (2), 1,2-Dehydrokohatine (3) and 5′Hhydroxyapateline (4). Structures of the all Compounds were elucidated by using 1 D and 2 D NMR and mass spectroscopy furthermore the structure of new Compound 1 was also proved by synthesize from compound 2 (Kohatine). Graphical Abstract


Introduction
Cocculus pendulus (Forst.& Diels), is a climbing shrub growing in both northern and southern parts of Pakistan.The roots of the plant are commonly used in Pakistan, for the treatment of intermittent fever, and as a tonic (Hussain et al. 1984).Number of bisbenzylisoquinoline alkaloids being reported (Hussain et al. 1984) in recent years that discussed there antitumor, anti-inflammatory, antitubular activities (Barbosa-Filho et al. 2000).The first comprehensive review on bisbenzylisoquinoline alkaloids, published by Guha et al. in 1979(Guha et al. 1979), which reported 186 alkaloids.Since then a number of reviews have been published (Schiff 1983;Schiff 1987;Schiff 1991;Schiff 1997).Extracts of the Cocculus pendulus have also been investigated for hypotensive and anticancer activities (Bhakuni et al. 1969), As it is a rich source of bisbenzylisoquinoline alkaloids that may be the main cause of its biological activity for-instance Tetrandrine obtained from cocculus sarmentosus, is known to possess antitumor, anti-inflammatory, antitubular activities and protects silicoses, histamine induced effects, as well as causes lowering of blood pressure (Barbosa-Filho et al. 2000).Oxycanthine and cepharanthine have been found highly effective against the human tuberculosis and leprosy (Burger 1970).
Tabular listing of bisbenzylisoquinoline alkaloids in alphabetical order has been given by Buck in his review in the series of the 'Alkaloids' by Manske (Buck 1987).These tables include a comprehensive survey of the pharmacological activity shown by the bisbenzylisoquinoline alkaloids.
The discovery of new Bisbenzylisoquinoline alkaloids continues linked to investigation of the metabolic pathways that control their biosynthesis, coupled with the future isolation of the enzymes/genes that control specific steps.This will provide new pharmacological tools, as well as medicinal and agricultural products.

Results and discussion
Compound 1 was purified from the fraction F-4 of pH-7 extract.Its UV spectrum exhibited absorption characteristics of a bisbenzylisoquinoline system, with absorption at k max 205 (log e 3.60), 236 (log e 3.40) and 292 nm (log e 2.62).The IR spectrum of the substance showed bands at 1593 and 1505 cm -1 (Aromatic C ¼ C) and 3574 cm -1 (OH).High Resolution Mass Spectroscopy (HRMS) exhibited the exact mass to be 578.2624,which corresponded closely with the mass calculated (578.2417) for the formula C 35 H 34 N 2 O 6 .The mass spectrum exhibited a strong molecular ion peak at m/z 578.Another prominent peak in the spectrum arose at m/z 366, due to double benzylic cleavage.Loss of a proton from the later gave rise to the base peak at m/z 365 (exact 365.1469, calculated 365.1501), corresponding to the formula C 21 H 21 N 2 O 4 .Another characteristic peak, at m/z 351, appeared due to the initial loss of a CH3 group from 6 0 -OCH3 followed by a double benzylic cleavage, corresponding to the formula C 20 H 19 N 2 O 4 .The fragment ion at m/z 183 (183.0796,calculated 183.0789) and at m/z 175 (175.0511,calculated 175.0633) 66 were due to the doubly charged bisisoquinoline unit (Figure 1).
The other two aromatic singlets in the 1 H-NMR spectrum at d 6.10 and d 6.52 could only be assigned to H-8 and H-5, respectively (Guinaudeau et al. 1987).
Complete assignment of all protons and carbons was possible in 1 with the help of 1 D and 2 D-NMR techniques (Table 1).The 13 C-NMR spectra (broad band decoupled, DEPT, CDCl 3 þ CD 3 OD).The broad-band spectrum showed 35 carbon signals.The DEPT spectrum showed the presence of three methyl signals (two upfield signals at d 40.08 and d 40.87 were assigned to 2 N-CH 3 and 2 0 N-CH 3 , respectively and the downfield signal at d 61.17 was due to 6 0 O-CH 3 group).6 Methylene and 11 methine carbon signals were also visible.Remaining 15 carbon signals were assigned for quaternary carbons.COSY-45 spectrum helped establish the assignments for C-a and C-a 0 protons, where vicinal coupling was clearly seen between H-1 0 (d 3.98) and the signal at d 2.53 and 3.25 assigned to H-a 0 .This left C-a (d 50.78) protons at d 2.78 (H-a a ) and d 2.87 (H-a b ).These assignments were further confirmed by HMBC spectrum as it exhibited cross-connectivities between H-10 0 (d 7.04) and C-a 0 (d 41.11) and H-14 (d 6.73) and C-a (d 50.78).2 N 0 -CH 3 proton (d 2.48) showed strong cross-peaks with C-1 0 (d 60.07) and C-3 0 (d 43.63).C-3, C-4 and C-4 0 were established on comparison with reported values.NOEDS experiments were carried out to ensure these assignments.Irradiation of d 6.10 signal (H-8) caused a 28% enhancement in the meta-coupled doublet at d 6.45 (H-10).Reverse was the case when H-10 was irradiated.The other aromatic singlet in the 1 H-NMR at d 6.52 could only be placed at C-5 on comparison with literature (Madyastha 1994).
The stereochemistry of the compound is 1S-1 0 S.This was deduced by a positive optical rotation of the compound (Cassels 1980) and the presence of H-1 and H-1 0 at d 3.22 and d 3.98, respectively (Madyastha 1994).Moreover the chemical shift differences between 2 N-CH 3 and 2 0 N-CH 3 were also helpful.The final proof for the structure 1 was obtained by treating Kohatine (2) with formaldehyde in the presence of sodium cyanoborohydride, which afforded 2 N-methylkohatine (1) as the only product.The spectral characteristics of the product were found identical with 2 N-methylkohatine 1.

General experimental details
The melting points were determined on Buchi 510 apparatus.The optical rotations were measured on JASCO 360 digital polarimeter.Some of the optical rotations were also recorded on polatronic D instrument.The pH values were measured on model 25 pH meter (Shanghai Kanchon, Peoples Republic of China).The infrared spectra were recorded on a Shimadzu IR-460 FT Spectrophotometer or on a JASCO IRA-1 IR Spectrophotometer using CHCl 3 as solvent.Some samples were analyzed through Chromatograms were observed with ultraviolet light at 254 and 366 nm.Dragendorff's reagent was used for the detection of alkaloids.

Plant material
The present study was carried out on 40 kg of the aerial parts and leaves of the plant, collected from the Malir District in the suburban areas of Karachi.A herbarium specimen (Voucher # 12760) was also deposited in the Department of Botany, University of Karachi.

Fractionation
40 kg of the aerial parts and leaves of the plant were air dried for 15 days, leaving 15 kg material.It was chopped and finally crushed with an ultra-turex and then soaked in 60 liters of methanol.The combined filtrates were evaporated under vacuum to afford a crude gum (1.5 kg).The resulting methanolic extract was dissolved in 10% aqueous solution of HCL (10 lit) and defatted with n-Hexane (15 L overall).The acidified aqueous layer so obtained, containing chloride salts of the bases, was basified with the help of NH 4 OH upto pH-11.Free alkaloids were then extracted with CHCl 3 (20 L overall) and ethyl acetate (10 L), the resulting organic extracts (from CHCl 3 and EtOAc) were combined and solvents evaporated to yield the crude alkaloids (70 g).The alkaloids were re-dissolved in a 5% acetic acid solution (3 L).The pH adjusted with ammonium hydroxide, and the alkaloids were extracted with CHCl 3 , at pH-3 (12.29), pH-5 and pH-7 (27 g).The various chloroform layers were separated from the aqueous layer and dried over anhydrous sodium sulfate.The chloroform was evaporated at reduced pressure and pH 7 fraction was subjected to repeated column chromatography.
F-2 was again adsorbed on flash silica (230-400 mesh 0.85 g) and chromatographed over a column packed with flash silica (230-400 mesh, 13.5 g).The column was run in 9.8: 0.2 (chloroform: methanol) along with traces of aqueous ammonia. 1, 2 and 3 were the less polar compounds separated from 4 (13 mg).The slow moving, highly polar alkaloids 1, 2 and 3 from F-2 were combined in a fraction F-4 (150 mg).The later was again absorbed and loaded on silica gel (230 À 400 mesh, 450 mg and 7.25 g).The column was run in 9.85:1.5,chloroform: methanol along with traces of NH 4 OH.The fast moving component 1 (20 mg) was first eluted, and then on increasing the polarity upto 9.8: 0.2 and running the column very slowly along with traces of NH 4 OH, 2 and 3 got departed.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.
, orthocoupled with H-14) were assigned to H-10 and H-13, respectively.This leaves only with one possible position in ring C for substitution of OH group.Four double doublets appearing at d 6.83 (

Table 1 .
1 H-NMR (400 MHz) and 13 C-NMR (125 MHz) Data for Compound 1 (J ¼Hz)KBr disc as well.UV experiments were carried out on Hitachi U-3200 spectrophotometer or on a Shimadzu UV 240 instrument using analytical grade methanol (Merck).Electron Impact (EI), peak matching, Field Desorption (FD) and Fast Atom Bombardment (FAB) experiments were performed on a Varian MAT 312 double focusing mass spectrometer.The instrument was connected to a MAT 188 data system with PDP 11/34 DEC computer system.High Resolution Electron Impact Mass Spectra (HREIMS) were recorded on Jeol-JMX HX-110 mass spectrometer.The 1 H-NMR were recorded on a Bruker AMX-300, AM-400 and AMX-500 Nuclear Magnetic Resonance Spectrometer in CDCl 3 , CD 3 OD or a mixture of both, while C 5 D 5 N in cases of 4. 13 C-NMR spectras were recorded on Burker AMX-500 or on a Bruker AM 400 Spectrometer at 125 MHz or 100 MHz in CDCl 3 or CD 3 OD.The reagents and solvents used in various experiments were purchased from E. Merck and Fluka for Thin Layer Chromatography (TLC) and was carried out using precoated silica gel 60-F 254 preparative plates (20-x 20 cm) of E. Merck (Art 5715) DC.Karten Si F 254 (20 Â 20 cm).RP-DC Fertig platter RP-18 F 254 S (5 Â 10 cm).Silica gel type-60 of E-Merck (Art.7734, 70-230 mesh) was used for the column chromatography.Flash chromatography was done with silica gel (type-60, Art.9385, 230-400 mesh).