Indian Journal o f Chemistry Vol. 40B, June 2001, pp. 490-494

Two new carbazole alkaloids from Murraya koenigii

B K Chowdhury*, S Jha. P Bhattacharyya & J Mukherjee+

*Department of Chemistry. School o f Tropical Medicine,Calcutta-700 073, India

+Department of Chemistry, University of Kalyani , Kalyani, West Bengl, India

Received 10 April 2000; accepted (revised) 9 August 2000

Two new alkaloids. 1-formyl-3-methoxy-6-methylcarbazole 1 and 6, 7 -dimethoxy-1-hydroxy-3-methylcarbazole 2, have been isolated from the leaves of Murraya koenigii. Both the compounds have been identified by spectral as well as chemical ev idences, and their structures confirmed by synthes is. Compound 2 is found to be active against Gram-positive and Gram-negative bacteria and fungi.

Murraya koenigii (L.) Spreng. (Rutaceae), reputed for its stimulant, stomachic and anti dysenteric activities 1,

is a fairly large genus of trees found in the Konkan region, Western Ghats, Tamilnadu and along the foot of the Himalayas from Kumaon to Sikkim. Thi s species has been proved to be a prolific source of carbazole alkaloids2

, and still continues to be the subject of chemical investigations in seaarch of new metabolites3

• In continuation of our quest for biologically active carbazole alkaloids from Murraya koenigiL-<, two new compounds 1 and 2 were obtained. The structural elucidation of the two compounds by chemical and spectral evidences as well as their sy nthesis are outlined herein. The results of screening the new metabolites for antimicrobial act ivities is also reported.

Compound 1, C 15H 13N02 ([Mt m/z 239) was found to be homogenous by TLC and mass spectrometry. Zinc-dust distillation of 1 led to the isolation of carbazole 3 and 3-methylcarbazole5

·6 4

indicating the presence of a 3-methylcarbazole residue in the molecule . Biogenetic considerations7

also suggested C-3 to be the most likely site for the methyl group. Compound 1 gave a positive 2,4-dinitrophenylhydrazine test and reduced ammoniacal Ag 0 3 indicating the presence of an aldehyde

function . Its UV spectrum A. ~;~11 227 (log E 4.78),

262 (4.1 ), 303 (4.4 1 ), 398 nm (3.87) was charac­teri st ic of l-formylcarbazole8 5. The 1R spectrum of 1 showed bands at 3405 ( H), 2760 (a ldehyde C-H), 1665 (C=O). 1605. 1580 (aromatic system) and 1208 cm·· 1 (aromatic ether). A signal at 8 I 0 . 15 (s) in the 1 H NMR and a signal at 8 191.6 (d) in the 13C NMR support the presence of a formyl group in 1.

R1 R2 RJ R-4

1 CHO OMe Me H

2 OH Me OMe OMe

3 H H H H

4 H Mo H H

5 CHO H H H

6 H OMe Me H

Moreover, the 1H NMR spectrum also showed signals at 9.61 (I H, br s, NH proton exchangeable with 0 20), 8.05 ( I H, d, 1=2.5 Hz, H-4) , 7.82 ( 1 H, d, 1=2.2 Hz, H-5), 7.45 (I H, d, 1=2.5 Hz, H-2), 7.35-7.28 (2H, m, H-7 and H-8), 4.03 (3H, s, ArOCH 3) and 2.32 (3H, s, ArCH 3 ).

Since H-4 and H-2 protons were not ortho-coupled, position 3 was substituted. The H-5 proton also was not ortho-coupled suggesting, thereby, a substitution at C-6. The formyl group could be placed at 1- or 8-position on the basis of UV data . From 1 H MR spectrum, the formyl and the methoxy g roups could be placed in the same ring. It is so because it was observed that due to the shielding effect of the adjacent methoxy group at C-3, the deshielding effect of the formyl group on H-2 and H-4 was less

CHOWDHURY et a/.: ALKALOIDS FROM MURRAY A KOENIG!! 491

pronounced. This justifies the placement of a methoxy group at C-3 and therefore the methyl group at C-6.

Compound 1, on decarbonylation with Pd-C, gave a compound 6 which was identified as g lycozoline9 by direct comparison with an authentic sample. Since the structure of g lycozoline has already been established as 3-methoxy-6-methylcarbazole, 1 must have a methoxy group at C-6 and a methyl group at C-3. From all these evidences the structure of 1 was established at l-formyl-3-methoxy-6-methylcarbazole which was further supported by its 13C NMR data (see Experimental). Finally, the structure of 1 was confirmed by its synthesis (Scheme 1). 2-Amino-5-methoxybenzaldehyde 10 7 was converted to 9 via the N-acetyl derivative 8. Compound 9 on condensation with 1-bromo-4-methylbenzene 10 in nitrobenzene in the presence of Cu and K2C03 furnished the diphenylamine derivative 11 11. Hydro lys is of 11 followed by cycl isati on with palladium acetate 12

furni shed 1. The synthetic 1 was found to be identical with natural! in all respects (mp, mmp, UV, IR) .

Compound 2, C1 5H1 5N03 ([Mt m/z 257), was readily solubl e in alkali and showed a violet colour with ethanolic FeCI3, suggesting the presence of a phenolic hydroxy l group. The presence of a hydroxyl group was further confirmed by a strong absorption at

3305 cm-1 in its IR spectrum. Its UV spectrum A. ~;~11

235 (log£ 4.65), 285(4.18), 305(4.32), 330 nm (3.50) was characteristic of a carbazole nucleus. On zinc­dust di stillation 2 furnished carbazole and 3-methylcarbazol e5·6 indicating the presence of a 3-methylcarbazol e residue in the molecule. The 1H NMR spectrum showed signals at 8 11.02 (I H, s, Ar­OH, exchangeable with 0 20) , 9.6 (I H, s, NH,

Jlo~ Jl~ '-'::: .. .. (ii) AcHN

H2 (i) AcH

0 0

7 8

exchangeable with 0 20), 7.64 (I H, d, 1=2 Hz, H-4), 7.45 (I H, s, H-5), 6 .85 (I H, s, H-8), 6.80 (1 H, d, 1=2 Hz, H-2), 3.80 (3H, s, Ar-OCH3), 3.77 (3H, s, Ar­OCH3) and 2.34 (3H, s, Ar-CH3). The 1H NMR signal for the hydroxyl proton of 2 was similar to that of l­hydroxycarbazole1 3. Isolation of a 3-methylcarbazole suggests the presence of a methyl group at C-3 or C-6. In carbazole alkaloids H-4 and H-5 protons appear at

higher fields . The protons at 8 7 .64 and 7.45 were therefore attributed to H-4 and H-5. However, the

proton at 8 7.45 is comparatively more shielded. This suggests the placement of a methoxy group ortho to it. The singlet nature of this proton also suggests the presence of the two methoxy groups at adjacent positions. The proton at 8 7 .45 is therefore attribu ted to H-5 and the meta-coupled doublet at 8 7.64 is attributed to H-4. Consequently, the methyl group could be placed at C-3. The singlet at 8 6.85 ass igned to H-8 and a //l eta-coupled doublet at 8 6.80 ass igned to H-2 place the hydroxyl group at 1-position. From these evidences the structure of 2 was established as 6,7-dimethoxy-1-hydroxy-3-methylcarbazole which was further supported by its 13C NMR data. The sy nthesis of 2 has been reported4 as an intermediate in the synthesis of Koeni gine-quinone B. Compound 2 was sy nthesised by the method reported earl ier and sy nthetic 2 was found to be identical with natural 2 in all respects (mp, mmp, UV, IR).

Antimicrobial activity Prompted by the reported 14 antibacterial properties

of the benzo[b ]carvazoles kinamycins A-D, both 1 and 2 were screened for antimicrobial activity. Compound 1 showed low activity against Gram-

~ (iii) CH(0Me)2

9

Me~OMe

~~ ...... ,___

11

1 (iv)

MeV'i1HOJI/e ~ 1.&

H CHO CHO {V)

1 12

Scheme 1- Reagents and Conditions- (I) Ac20/Ac0H, retluxed for 30 min ; (ii ) anhyd MeOH/conc HCI , room temp, 120 hr; (iii ) p­bromoroluene/K2COiCu powder. retluxed in nitrobenzene for 16 hr; (iv) H20/conc HCI , healed for 30 min ; (v) Pd(Oac)z/AcOH, retluxed for I hr.

492 INDIAN J. CHEM., SEC. 8, JUNE 2001

negative bacteria and fungi, while compound 2 was found to be highly active against both Gram-positive and Gram-negative bacteria and fungi. The minimum inhibitory concentrations (MIC) of 1 and 2 were determined by the agar dilution method. The MIC were studied up to I 00 Jlg mL-1 and incubations were done at 37°C. The MIC values of standard compounds streptomycin (100 J..tg mL-1

), peni cillin (2 J..tg mL-1 ),

nystatin and griseofulvin (I J..tg mL-1) were also

studied (cf. Table 1).

Experimental Section General. Melting points are uncorrected. UV and

IR spectra (in KBr discs) were recorded on a Shimadzu 160A and NICOLET 205 Fr-IR instruments, respectively; 1H NMR (100 MHz) and 13C NMR (25 MHz) spectra on a JEOL Ff 100 spectrometer; and mass spectra on a JEOL JMS-D 300 instruments. Elemental analyses were done on a Carlo-Earba EA 1108 analyser. HPLC experiments were conducted by using two Waters Assoc. Model 510 pumps for solvent delivery controlled by a Waters Assoc. Model 680 solvent programming unit. Waters Assoc. Model 484 absorbance detector and Waters Assoc. Model 746 data module integrator were also used . GC separations were achieved with a Hewlett-Packard 5880A instrument. In all the cases, silica gel (60-120 mesh, Qualigens, India) were used for CC and silica gel G (E. Merck, India) for TLC.

Plant material. Powdered leaves of M. koenigii (Linn.) Spreng. were purchased from a local supplier in January , 1998 and identified by Dr A.K. Das of the Department of Botany, Presidency College, Calcutta.

Isolation of compounds. Air-dried, finely powdered leaves of Murraya koenigii (I kg) were extracted with pet.ether (60-80°C) in a Soxhlet for 48 hr, and the solvent was distilled off. The residue was taken up in Et20 and fractionated into neutral, acidic and basic fractions in the usual way. The neutral fraction was chromatographed over silica gel (300 g) eluting with pet.ether (60-80°C) and pet.ether -CH2Cl2 in the proportions 5:1, 3:1, 2:1 and 1: 1, respectively. The pet.ether -CH2CI2 (3: I) fraction furnished a light yellow oil (170 mg) which was rechromatographed over silica gel ( 15 g). The pet.ether -CH2Ch (2: I) eluates gave on removal of the solvent, a light yellow solid which was further purified by preparative TLC (silica gel G, 1 mm, hexane-benzene-ethyl acetate 3:2: I). The band of R 0.55 was separated and extracted with 3% MeOH in CH2Ch . The residue on removal of the solvent gave a solid (II 0 mg) which on crystallisation from benzene - pet.ether furnished 1 as light yellow crystals. The acidic fraction was concentrated and chromatographed over silica gel (400 g). The column was eluted with same solvents as described above. From the pet. ether -CH2Ch (l: I) eluate compound 2 (78 mg) was obtained which was crystallised from benzene.

1-Formyl-3-methoxy-6-methylcarbazole 1. Light yellow crystals; mp 181-82°C (Found : C, 75.48; H, 5.49; N, 5.94. Calcd. for C 15 H13N02 : C, 75.31; H, 5.43; N, 5.85%). UV (nm) (log E): 227 (4.78), 262 (4.1), 303 (4.41) 398 (3.87); IR: 3405, 2760, 1665, 1605, 1580, 1208 cm·1

• 1H NMR (100 MHz, CDCI3 ):

o 10.15 (IH, s), 9.61 (IH, br s), 8.05 (IH, d, 1=2.5

Table ! - Minimum inhibitory concentrations of compounds

SINo Name of the Organi sm Compd 1 Compd 2 p s G N

Escherichia coli ST 203 40 8 5

2 Becillus subtilis ST 204 > 100 14 0.01

3 Salmonella typhi ST 288 > 100 12 10

4 Pseudomonas aemginosa ST 243 16 14 6

5 Staphylococcus au reus >100 1.3 0.03 MC 27927

6 Candida a/hicans ST 388 16 5 0.05

7 Tndwphy10n mbrum ST 389 5 2 0.02

-=Not tested: P =Penicillin: S =Streptomycin: G =Griseo fulvin: N =Nystatin.

CHOWDHURY eta/.: ALKALOIDS FROM MURRAY A KOENIG/I 493

Hz), 7.82 (I H. d, 1=2.2 Hz), 7.45 (IH, d, 1=2.5 Hz), 7.35-7 .28 (2H, m), 4.03 (3H, s), 2.32 (3H, s); 13C NMR (25 MHz,CDCb): 8 112.1 (s, C-1), 106.1 (d, C-2), 148.3 (s, C-3), 115.1 (d, C-4), 118.2 (s, C-4a), 120.3 (s, C-4b), 126.4 (d, C-5), 124.1 (s, C-6), 119.8 (d, C-7), 111.5 (d, C-8), 139.5 (s, C-8a), 140.7 (s, C-9a), 191.6 (d, CHO), 55.1 (q, 3-0Me), 21.1 (q, 6-Me). MS: m/z 239 (M+).

6, 7 -Dimethoxy-1-hydroxy-3-methylcarbazole 2. Colourless needles, mp 2!6°C (Found: C, 70.1 0; H, 5.90; N, 5.48 .Calcd for C1 5H1 5N03 : C, 70.02; H, 5.88; N, 5.44%); UV (nm) (log c:) : 235 (4.65), 285 (4.18), 305 (4.32), 330 (3.50). IR : 3390, 3305, 1600, 1500, 1390, 12 10,985, 850 cm-1; 1H NMR (100 MHz, CDC13 ) : 8 11.02 (1 H, s), 9.6 ( I H, s), 7.64 (I H, d, 1=2 Hz), 7.45 (I H, s), 6.85 (I H, s), 6.80 (I H, d, 1=2 Hz), 3.80 (3H, s), 3.77 (3H, s), 2.34 (3H, s) . 13C NMR (25 MHz, CDC13 ) : 8 145.1 (s, C-1 ), 106.8 (d, C-2), 124.5 (s , C-3), 121.1 (d, C-4), 122.9 (s, C-4a), 122.6 (s, C-4b), 110.5 (d, C-5) , 154.0 (s, C-6), 154.2 (s, C-7), 102.8 (d, C-8), 135 .5 (s, C-8a), 140.4 (s, C-9a), 56.3 (q, 6-0Me), 57. 1 (q, 7-0Me), 21.3 (q, 3-Me). MS : mlz 257 (M+) .

Zinc-dust distillation of 1. Compound 1 (50 mg) was thoroughly mixed with Zn-dust (5 g) previously dried at 250°C and heated in a sealed tube for 2 hr. The ether soluble portion of the reaction product was dissolved in benzene and chromatographed over alumina (5 g). Elution with benzene-petrol furnished solid (3 mg) which showed the presence of two compounds by TLC on silica gel (petroi-HOAc, 10: I, Rr 0.55 and 0.65, respectively). The two compounds were identifi ed as carbazole (mp 242°C) and 3-methylcarbazole (mp 207°C), respectively by GC and HPLC5

·6

.

Decarbonylation of 1. Compound 1 (25 mg) was mixed with Pd-C (1 0 mg) and heated in a sealed tube with 2 mL of dry EtOH for 20 min at 270°C under vacuum. The residue obtained after removal of the solvent from the EtOH extract of the reaction product on crystallisation from benzene-pet. ether furnished crystals of 6 ( I 0 mg), mp 180°C. The compound was fo und to be identical with glycozoline in all respects (mp, mmp, UV, IR).

Acetylation of 7. To compound 7 (16.5 g) were added acetic anhydride (I 0 mL) and glacial aceti c ac id (I 0 mL) and the mixture was refluxed on a water-bath for 30 min. After the reaction was complete the mixture was poured into ice-H20 (500 g) when a solid was obtained. The solid was filtered and

washed with cold water. It was then dried and recrystalli sed from benzene-pet.ether to give needles of 8 (14.64 g, 69.4%), mp 139°C (Found : C, 62 .55 ; H, 5.76; N, 7.39. Calcd for C 10H 11 N03 : C, 62 .17; H, 5.69; N, 7.25%); IR : 3320 (NH), 1670 (C=O), 1652 (CO), 1595, 1510 cm·1 (aromatic system).

Dimethylacetal derivative of 2-(N-acetyl)amino-5-methoxybenzaldehyde 9. Compound 8 ( 13 .5 g) in anhydrous MeOH (60 mL) and cone. HCI (0.5 mL) was allowed to stand at room temperature for 5 days . A solution of MeONa in MeOH was added until the solution was just alkaline to moistened litmus paper. The MeOH was removed by distillation on a steam­bath, the residue was cooled to room temperature and treated with cool H20 to remove inorganic substances. The aqueous solution was separated and extracted with two 30 mL portions of ether and the extracts were added to the crude acetal. The solution was dried over anhydrous MgS04 . After ether had been removed by distillation on a steam-bath, the residue was distilled under reduced pressure at 127-29°C/l mm of Hg to give 9 (12.08 g, 72.3%). (Found: C, 60.72; H, 7.33 ; N, 5.70. Calcd for C 1 2H 1 1NO~: C, 60.25; H, 7.11 ; N, 5.89%); IR: 3315 (NH), 1660 (CO) and 1600, 1512 cm·1 (aromatic system).

Dimethylacetal derivative of (2-formyl-4-methoxyphenyl)( 4'-methylphenyl) amine 11. A mixture of compound 9 (II g), p-bromotoluene (7 .8 g) , K2C03 (6.35 g), nitrobenzene (70 mL) and copper powder (2 g) was stilTed with magnetic stirrer and boiled for 16 hr. The reaction mixture was cooled and nitrobenzene was removed by steam di still at ion . The mixture, after removal of nitrobenzene, was extracted with CH2Cb (3x50 mL). The CH2Cb extract was washed with water (2x50 mL) and dri ed over anhydrous Na2S04. On removal of the solvent a yellow liquid was obtained. The liquid was distilled under reduced pressure at I 12-l7°C/5 mm of Hg to yield 11 (8.4 g, 63.6% ). (Found : C, 71.16; H, 7 .40; N, 4.96. Calcd for C 17H21N03 : C, 71.08; H, 7.31 ; N, 4.87%); IR: 3380 (NH) and 1602, 151 4 cm·1

(aromatic system). (2-Formyl-4-methoxyphenyl) ( 4-methylphenyl)­

amine 12. Compound 11 (7 .5 g) was added to water (70 mL) containing Cone HCl (5 mL) and the mixture was heated on a steambath with occasional shaking for 30 min. The solution was cooled, neutrali sed by dropwise addition of NH40H, and then extrac ted with CH2Cl2 (3x30 mL). The CH2Cl2 ex tract was washed with water (2x30 mL), dried over anhydrous Na2SO~. Removal of the solvent yi elded compou nd 12 (4.5 g,

494 INDIAN J. CHEM .. SEC. B, JUNE 200 1

71.5 %), mp 79°C. (Found: C, 74.31; H, 6.13; N, 5.71. Calcd for C15H15N0 2: C, 74.68; H, 6.22; N, 5.80%); IR 3370 (NH), 1670 (C=O), 1600, 1510 em·' (aromatic system).

1-Formyl-3-methoxy-6-methylcarbazole 1. Compound 12 (4.3 g) was cyclised to compound 1 by retluxing the res idue with palladium acetate (4 g) in acetic ac id (25 mL) for I hr. The reaction was monitored by TLC. The solvent was dist illed off, the residue di ssolved in CH2Cb and chromatographed over silica gel (20 g). The column was eluted with pet.ether, pet. ether -CHzCh ( 4: I, 2: I) and CH2Ciz. From pet. ether-CH2CI2 (2: I) eluate a solid was obtained whi ch was recrystallised from benzene­pet.ether to yield light ye llow needles (2.9 g), mp I78°C. This was fi nally purified by sublimation under reduced pressure to furnish compound 1 (2 .23 g, 52.3% ), mp 18 1 °C.

Zinc-dust distillation of 2. Compound 2 (50 mg) was subjected to zinc-dust distillation by the same method and under the same condi tions as described above in case of compound 1. Two compounds were obtained which were identified as carbazole and 3-methy lcarbazole by GC and HPLC.

MIC test for bacteria. Suspensions of the cultures of bacteria were prepared on solid nutri ent agar medium by adding the bacteria to sterile distilled water until it appeared faintly turbid. Different concentrations ( I 00, 80, 60, 40, 20, I 0 and 5 )lg/mL) of the test compounds were incorporated in pl ates of nutrient agar. Inocul ation of the bacterial suspensions were made by the streak method on the surface of each plate. The plates were incubated for 48 hr at 37°C. The lowest concentration of the compound that entirely prevented the growth of the bacteria on the plates was the minimum inhibitory concentration of each of the compound for the particul ar bacteria.

MIC test for fungi. M IC of the test compounds were ascertained by plate method. A series of plates

of Sabouraud 's dextrose agar medium were prepared. Each plate was inoculated from the 10-fold diluted culture of young slant of each fungus. The plate containing 0.1 mL diluted culture was then allowed to dry and four holes were cut on the surface of the solid medium with a sterile cork borer. Different concentrations of the test compounds were incorporated into the cup and the plates were incubated for 72 hr at 28°C. Similar experiments were done using the different concentrations of the standard drug. The diameters of the zones of inhibition were measured. The lowest concentration of the compounds that entirely prevented the growth of the fungi on the plates was the minimum inhibitory concentrati on of the compound for that particular fungus.

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