Studies on a carbazole alkaloid from Murraya koenigii Spreng. and flavonoids from Pongamia glabra Vent.

l'hytnchcmkal invcsti~ations on the root hark of Murraya koenigii Spreng. (Fam. Rutuccae) afforded a new carbazole alk;lloid (I a), clumu~tcrized as ],6-climethyl-1-isopentenylcarlmzolc. on the lmsis of spccu·:•l dnta, and its. N-methyl derin1tin~ (I b) was :1lso syn thesized. In :1ddition, two known l'la\'onoids, pongmnol (2), and karanjin (]) were isolated from the seeds of l'ongamia glabrn Vent. (Fmn. Legmnino.~:lc) and these two t·ompnmuls were independently characterized by us with some new structun•l probability of pon~amol (2) on tht· hasis of spel'lral cvidt•m•t•s. All thc.~c compmmds were subjected to the compan1tivc larvicidal studies on the third instm· larvae of Culex quinqu~f"ascialus at IOU ppm concentration at an interval of 24 h; some at·ti\·ity was obscn-cd which w:1s related to their structun1l

M. koeni,~ii Spreng. is also a rich source of carbazole alkaloids. which are known to possess significant biological activities e.g. anti-biotic, anti-cancer, anti-viral, central nervous system. anti-inflammatory, pesticidal etc. 4 . Carbazole alkaloids e.g. mahanimbine, murrayanol, mahanine. ob-. tained from bioassay guided fractionation of the acetone extract of fresh leaves of M. koenigii Spreng. show mosquitocidal, anti-microbial activities and also exhibited topoisomci·ase I and ll inhibition activities 5 . Besides these, murrayanol shows anti-inflammatory act1 v1ty while mahanirnbine shows anti-oxidant property 5 . Murrayanine, girinimbine, rnahanimbine have anti-fungal property and in addition. mahanimbine also shows larvicidal activit/'. 9-Fonny 1-3-methylcarbazole, isolated from roots of M. koenigii Spreng .. displays weak cytotoxicity against both mouse melanoma B 16 anJ adrimycin resistant P388 mouse leukemia celllines 7 . This prompted us to further investigate the root bark of this plant for new bioactive compounds and our investigations furnished a new carbazole alkaloid, 3,6dimethyl-1-isopentenylcarbazole (la). Structure of this compound was elucidated on the basis of spectral data (UV, IR, 1 H and 13 C NMR and mass).
During previous investigations on various synthetic carbazole derivatives it was noticed that N-substituted derivatives are generally more toxic than the carbazoles, and it was particularly noticed that N-methyl carbazole is highly toxic on the larvae of Culex quinquefasciatus, while carbazole itself has little toxicitl. Besides that, it was observed that the antinemic activities of pure carbazole alkaloids e.g. koenimbine. koenidin, obtained from this plant increases due to N-methylation 9 . So, we converted the new carbazole alkaloid, 3,6-dimethyl-1-isopentenylcarbazole (Ia), to its N-methyl derivative (lb) by the al:tion of Jimethylsulphate and alkali in dry acetone medium with a view to make a comparative larvicidal study anJ its structure was confirmed on the basis of spectral data (UV, IR, 1 H and 13 C NMR and mass). Direct application of ethanol and sequential petroleum ether. chloroform, acetone and ethanol extract of seed of this plant revealed ant-intlammatory, analgesic and anti-ulcerogenic activities in rats 12 Antiviral properties of the seed e~tract was also investigated against herpes simplex virus in vitro studies in vero-cclls 13 . This plant is the reservoir ,lf large number of flavonoid type of compounds 10 and it is well known that flavonoids represent one of the most diverse and wide-spread groups of natural products. having . d t" b" I . I . . . 14 IS Th d a vane range o 10 og1ca act1V1t1es · ·. ese prompte us to investigate the seeds of Pongamia glabm Vent. for new bioactive compounds but we isolated two known flavonoids, pongamol (2) and karanjin (3), having similarity in their structures. Though these two compounds were knmvn 16 . in this communication, we have given a spe<.:ial method of isolation of th~se two compounds from the seeds of the plant and also independently chara<.:terized (2) and (3) on the basis of spectral data (UV. IR, 1 H and 1 'C NMR and mass). In addition to thut, we also want to explicate some special features of the structure of pongamol (2) in this communication.
Our previous investigations revealed that of the flavonoids type of <.:ompounds. like flavoncs and chalcones have larvicidal a<.:tivity, and their activity depends on the r.ature and position of the sub'itituent(s) present ir. these compounds. and it was also noticed that the chalcones were more active than tlavones 17 . So we included these two compounds in comparative larvicidal study to note whether in this case the chakone (2) is more active than the tlavone (3) or not.
The advantages of the allelochemics over the syntheti<.: pesticides are their low mammalian toxicity. easy biodegradability and minimal residual effect to the non-target animals. Moreover. the larvi<.:idal activity of many allelochemics has not been properly studied. Considering all these facts, we were interested to make a comparative larvicidal assessment of a new carbazole alkaloid. 3.6-dimethyl-1-isopentenylcarbazole (la) (isolated from the root bark of M. koenigii Spreng.)

Results and discussion
Structure elucidmions of carbazole alkaoid (la) and its N-methyl derivative (lb) : The petroleum ether extract of dried and finely pulver-ized root bark of M. koenigii Spreng. (Fam. Rutaceae) afforded compound (la). as a white crystalline solid, m.p. 168°C (yield 0.02%) (vide Experimental). Compound (la), C 19 H 21 N (number of protons and <.:arbon atoms were the same as obtained from its 1 H and 13 C NMR spectra), rvr+ 263 was found to be homogeneous by tic and mass spectrometry. TheIR spectrum (KBr) of compound (la) showed a sharp peak at 3317 (>N-H) along with the peaks at 1641 (CH=CH), 1600 (aromatic residue), 1459 (CH 1 ), 1403 (C-CH3) and 880 cm-1 (aromatic substitution) . . The olefinic protons were in the form of a doublet at 8 5.68 (H-1 0) and a triplet at 8 7.18 (H-11 ). The signals at 8 6.57 and 7.90 could be assigned to H-7 and H-8 respectively which are obtained as symmetrical doublet. The H-2 proton appeared at 8 7.22 and N-H proton at 8 7.81. The H-4 and H-5 protons appeared at 8 7.34 and 7.65 respe<.:tively.
Hence the 1 H NMR spectrum of this compound showed the presen<.:e of a N-H group. two aromatic methyl groups and one CH=CH-CH(CH 3 h group. The positions of these groups in compound (la) were further confirmed by its 13 C NMR spectrum (vide Experimental). All these data confirmed the structure of the unknown <.:arbazole alkaloid (la) as 3,6-dimethyl-1-isopentenylcarbazole.
The presence of >N-H group in this compound was further confirmed by the synthesis of its N-methyl derivative (lb) (m.p. 142°C, yield 50.0%) by shaking compound (la) with dimethylsulphate and concentrated caustic potash in acetone medium, followed by recrystallisation of the crude product from ethanol.
From analysis and mass spectral measurements, the molecular formula of N-methyl derivative of the compound (Ia) i.e. (lb) was obtained as C 20 H~3N; M+ 277 (numbers of protons and carbon atoms were the same as it was observed in its 1 Hand 13 C NMR). TheIR spectrum of this compound showed absence of >N-H peak and the presence of >N-C-H group present in the wmpound is indicated by the peak at 2917 cm-1 . Besides these, the peaks at 1635 (olefinic unsaturation, CH=CH), 1600 (aromatic residue) and 866 cm-1 (aromatic substitution) were obtained.
Comparison of the UV maxima with other carbazole compounds 1 x revealed the presence of a carbazole moiety in the compound. The 1 H NMR spectrum of compound From the comparison of the properties (m.p., analytical and spectral data) of compound (la) with various literature . c ata , 11 IS expected that compound (Ia) IS a new carbazole alkaloid, 3,6-dimethyl-1-isopentenylcarbazole, isolated from the root-bark of M. koenigii Spreng. (Fam. Rutaceae). Hence the probable structures of compound (Ia) and its N-methyl derivative (I b) are represented in the following figure as : 18 12 We have isolated two known tlavonoids, pongamol (2) and karanjin (3) from the seeds of the plant Pongcunia glabw Vent. (Fam. Leguminosae) by solvent extraction and chromatographic separation as given in the experimental part of this paper.
Structure c/ucidmions of" compound (2) and some Jnodi-.fications r1{ its structural features : The molecular formula of compound (2) was found to be e,sH 1 _.0 4 , from analysis and mass spectral measurements (numbers of protons and carbon atoms were the same as it was obtained in its 1 H and 13 e NMR). TheIR (KBr) spectrum of this compound shows a band at 1650-1640 cm-1 which is attributed to the a,p-unsaturated carbonyl group.  The comparative study of the spectral properties ( 1 H and 13 e NMR) and melting point with literature data 20 shows that the compound (2), which we have isolated from the seeds of Pongamia glabra Vent. was identical with a known compound, pongamol, obtained from the roots of Teplzrosia purpurea 20 and this compP• 10 was also obtained previously from different parts oft' ·same plant 16 . Though the enol form of pon :amol was previously reported as prominent structure:' our observations on the spectroscopic datn of pongam" . indicate that it is u tuutomer between keto and enol for1J• and the enol form becomes more prominent clue to th~ intra-molecular hydrogen bonded chalcone type structure.
Evidence for rhe enol form (hydrogen bonding in pongamol (2) (Fig. 2a) f : In pongamol. a band at 2700 em-' wtts obtained which is the characteristic of chelated hydroxyl group in enolic type of compounds 21 . So we may expect· following type of intra-molecular hydrogen bonding in pongamol.
Evidence for the kero form f keto-enol tautomerism in The presence of keto form of pongamol is supported by the following facts : (a) IR : P-Diketones 21 , which partly exist in the enol form, show carbonyl absorption in region 1640-1540 cm-1 . This compound also gave IR peaks in the region 1650-.1560 cm-1 • (b) 13 e NMR : ehalcan-P.P'-clione form can be readily differentiated from the enolic form due to the presence of an aliphatic methylene group (C-a) resonating at 55.3 22 . There was a prominent "e NMR signal at 0 54.3 for compound (2); which can not be explained on the basis of only enol ic structure of pongamol.
From these facts, it may be stated that pongamol is a keto-enol tau to mer (2b ), of which the enol form is stabilised by the intra-molecular hydrogen bonding. The molecular formula of compound (3) was found to be C 18 H 11 0~ from analysis unci mass spectral measurements (numbers of protons and carbon atoms were the same as it was obtained in its 1 H and "c NMR).   16H From the ANOVA-table, CD (critical difference) values were calculated for these compounds; CD at I% level was I 1.20 and at 5% level was 8. 13. From the CD vatues, the order of toxicity of these compounds can be determined. On the basis of CD. a suitable larvicide tmty be selected depending upon the availability and economy.
The carbazole alkaloid (la) and the flavone type compound (3) have moderate toxicity which is almost the same. But the N-rnethyl derivative of (la) i.e. compound (lb) is more toxic than the tlavonoids (2 and 3) and it is also more toxic than the carbazole alkaloid ( la). This supports our earlier findings that due toN-substitution by methyl group, the toxicity of carbazole compound enhances. The isolated chalcone, pongamol (2), has lowest toxicity in these cases.
Our previous study 17 showed that chalcones are generally more active than tlavones. But in the present study, it was noticed that the isolated falvone type compound, karanjin (3) has moderate toxicity and it was more toxic than the isolated chalcone type compound, pongamol (2). This may be due to the complexity of their structures. From previous study 17 , it was evident that activity oftlavone type compounds depends on the nature and positions of the substituents present in the compound and it was noticed that absence of substituents at 3, 7 and 8 positions and presence of substituent at 6 position enhanced the activity of the flavone type compounds. Karanjin (3) contains a furan ring fused at 7, 8 positions and it contains a methoxy group at 3 position. These may be the probable cause for its moderate activity. We also noticed earlier that, the toxicity of chalcone type compounds decreased due to the introduction of different gorups in the aromatic ring, adjacent to carbonyl group of chalcone 17 . Moreover, when the carbonyl group of chalcone type compounds was blocked by derivatisation [except in oxime derivative of benzalace-toneJ, the toxicity of the resultant compound also decreased (unpublished results). In the case of pongamol (2), a furan ring is attached at 3' and 4' positions and it has also substit-Note u.:nt at 2' position. Besides that, this compounJ has intramolecular hydrogen bonuing (Fig. 2a), which may Jem:tivnte the carbonyl group present in compound (2). This may be the probable cause of low activity of chalcone (2) than the tlavone (3).

Experimental
The samples were dried over P 2 0~;1KOH at 80"C under  /so/arion of pongamof (2) and karanjin (3) kg of dried and crushed seeds of Pongamia glabra Vent. (Fam. Leguminosae) were extracted in a soxhlet with alcohol for two '' ..:'c:ks at room temperature. The solvent was removed from the extract by cautious distillation under reduced pressure. whereby a gummy mass was obtained. This was dissolved in 40% alcohol and fractionated by three solvents, viz. n-hexane. methylenechloride. and ethyl acetate, one after another. The methylenechloride fraction was made free from the solvent by evaporation under reduced pressure, whereby a gummy mass was obtained. This gummy mass was dissolved in methanol and partitioned between petroleum ether ( 40-60"C) and methanol (90% ).
The epi-phase i.e. the petroleum ether (40-60"C) layer furnished no appreciable result after complete removal of the solvent and repeated crystallizations in different solvent mediums. The hypo-phase i.e. the methanol layer. after complete removal of the solvent under reduced pressure, gave a gummy mass which was subjected to column chromatography over silica gel. 60-120 mesh, and the column was eluted by different solvents and their mixtures in different proportions. viz. petroleum ether, petroleum ether and benzene mixture. benzene, benzene. I 00 ml of eluent was collected in each fraction.

Method of bio-assay :
Toxicity tests of the pure compounds (la-b, 2 and 3) were performed on the 3rd instar larvae of Culex quinquefascimus in water medium. 0.5% of the alcoholic solution of each compound was added in thin stream with gentle stirring into the beaker containing 25 third instar mosquito larvae in water, so that the final concentration of the compound became I 00 ppm at 30 ± 2°C. In the similar fashion, same amount of alcohol was added to the control. Few drops of emulsifier were also added to maintain the uniformity of the concentration of the compound in the solution and similar addition was also done in control. Five replications were performed for each set. Percent mortality was calculated after 24 h. No mortality was observed in control. Analysis of Variance (ANOV A) calculations were performed for those compounds, which were apparently found not to be equivalent. The order of toxicity was determined by Critical Difference (CD) values.