New furocarbazole alkaloids from Lonicera quinquelocularis

Two new furocarbazole alkaloids, 3-formyl-6,7-dimethoxy-furo[1,2]carbazole (1) and methyl-6,7-dimethoxy-furo[1,2]carbazole-3-carboxylate (2), along with two known carbazole alkaloids, 3-formyl-2-hydroxy-7-methoxycarbazole (3) and methyl 2,7-dimethoxycarbazole-3-carboxylate (4) were isolated from the ethyl acetate soluble fraction of Lonicera quinquelocularis. Their structures were established on the basis of spectroscopic analysis.


Introduction
The genus Lonicera belongs to the family Caprifoliaceae comprises of 12 genera and 450 species, found in temperate region of Northern Hemisphere. Plants of this genus are used for the treatment of acute fever, headache, respiratory infections (Houghton et al. 1993), antibacterial, antioxidant, cytoprotective, hepatoprotective, antiviral, anti-tumour and anti-inflammatory activities (Shi et al. 1999;Kähkönen et al. 2001;Puupponen-Pimia et al. 2001;Yoo et al. 2008;Wang et al. 2009). Previous literature of this plant showed the isolation of various phytoconstituents such as iridoids, bisiridoids, sulphur-containing monoterpenoids, alkaloidal glycosides, triterpenoids, saponins, coumarin glycosides and flavone glycosides (Souzu & Mitsuhashi 1969, 1970Machida et al. 1995). Lonicera quinquelocularis belongs to this genus mostly found in dry sunny places between 750 and 3000 m in many countries of Asia. Previous phytochemical study on this plant has found the isolation of triterpenoids, lonicerin, loganins, coumarins, iridoide glycosides, phthalates and benzoates (Kumar et al. 2000;Ali et al. 2013;Khan, Afzal, et al. 2014;) from this plant. The diverse medicinal importance of genus Lonicera has prompted us to investigate the constituents of L. quinquelocularis. Herein, we reported the isolation and identification of two new furocarbazole alkaloids namely, 1 and 2 along with two known alkaloids 3 and 4 ( Figure 1).
Compound 1 was isolated as yellow crystals. The UV spectrum [l max 204 (4.10), 234 (4.37), 245 (4.22), 278 (4.34), 285 (4.21), 332 (3.96) and 361 (3.90) nm] and the IR spectrum (y max at 965,1190,1510,1578,1631,1658,3263 and 3400 cm 21 ) indicated a 3-formyl furocarbazole framework. 1 H NMR spectrum ( Figure S1) displayed signals at d 8.54 (1H), 7.59 (1H) and 7.02 (1H) all singlet were characteristics of substituted aromatic A and C ring of the carbazole moiety. The peaks resonated at 7.83 (1H, d, J ¼ 7.3 Hz) and 7.36 (1H, d, J ¼ 7.3 Hz) each doublet (J ¼ 7.4 Hz) were characteristics of furan ring fused to carbazole nucleus. The presence of 1 H NMR signal at 10.09 along with the 13 C NMR peak ( Figure S2) at 187.56 gave assignment about the presence of aldehyde group which was further confirmed by the most deshielded signal at d 8.54 assigned to H-4 due to the ortho-formyl group. Two singlets each for 3H found at d 3.82 and 3.78 were assigned to two methoxy groups attached to the aromatic rings. The presence of a formyl group was further confirmed by the observation of a mass fragmentation ion at m/z 267 (M-CO). In addition, a mass spectral fragment at m/z 265 (M-OCH 3 ) and 235 (M-2OCH 3 ) suggested the presence of two aromatic methoxy groups. The HMBC correlations ( Figure S5) were in conformity with the assigned structure of compound 1. The data were in close agreement with the available literature (Ito & Furukawa 1990).
The IR spectrum of compound 2 showed bands for NH (3440 cm 21 ) and COOMe groups (1705 cm 21 ), and for an aromatic system (1615, 1580, 1227, 1195, 1040, 815 and 728 cm 21 ) indicating the carbazole framework. The methyl ester was indicated by a 1 H NMR ( Figure S3) displayed singlet at d 3.92 for the methoxy group and in the 13 C NMR spectrum (Figure S4 signals at d 7.85 and 7.29 each one proton doublet (J ¼ 7.6 Hz). The data coincide with the literature (Wu et al. 1997).

Experimental 3.1. General experimental procedures
Melting point was determined by using the Kofler hot-stage apparatus (Reichert, Vienna, Austria). Aluminium TLC plates (20 £ 20, 0.5 mm thick) pre-coated with silica gel 60 F 254 (0.2 mm layer thickness; E. Merck, Darmstadt, Germany) were used for TLC to check the purity of the compounds. Column chromatography (CC) was carried out using silica gel of 230-400 mesh (E. Merck). Preparative TLC glass plates (20 £ 20, 2 mm thick) pre-coated with silica gel 60 F 254 (0.5 mm layer thickness; E. Merck) were used for the purification of semi-pure compounds. Ceric sulphate and potassium permanganate solutions were used as visualisation reagents. The UV spectra (l max nm) were recorded on Shimadzu UV-2700 spectrophotometer (Shimadzu, Kyoto, Japan) in EtOH. Mass spectra were recorded on Bruker TOF Mass spectrometers (Billerica, MA, USA) using electrospray ionisation (ESI). The 1 H NMR and 13 C NMR spectra were recorded on a Bruker DPX-400 NMR spectrometer (400 MHz for 1 H and 100 MHz for 13 C NMR), using CDCl 3 as solvents. Further assignments were made by DEPT, COSY, HMQC and HMBC experiments.

Plant material
The whole plant of L. quinquelocularis was collected from Bara Galli, Hazara division, District Mansehra. It was identified by Professor Dr Manzoor Ahmad, Plant Taxonomist, Department of Botany, Government Degree College Abbotabad where a voucher specimen has been deposited in herbarium (Accession No. C-0013).
The subfraction E gave two spots on TLC with good R f value and hence were separated by preparative TLC eluted with n-hexane: CH 2 Cl 2 (9:1 -2:8) to get the two spots separately which were placed in fume hood to get fine crystals of compounds 1 and 2.
The subfraction F on placing in fume hood spontaneously converted into fine crystals with traces of impurities which was washed with n-hexane: EtOAc (20:1) till a single spot was obtained on TLC. This was again recrystallised using double distilled hexane to get fine and pure crystals which was confirmed as compound 3 by spectroscopic analysis.