Indian Journal of Chemistry Vol. 40B, June 200 1 , pp. 484-489

Structure and synthesis of glycoborine, a new carbazole alkaloid from the roots of Glycosmis arborea: A note on the structure of glycozolicine

Ajit Kumar Chakravarty *. a , Tapas Sarkar a , Kazuo Masudab , Tetsuya Takey b, Hirohisa Doib, Eiichi Kotani b & Kenji Shiojima b

a Indian Institute of Chemical Biology, Calcutta 700 032, India b Showa Pharmaceutical University, Machida, Tokyo 1 94-8543, Japan

Received 4 February 2000 ; accepted (revised) 12 June 2000

A new carbazole alkaloid, designated as glycoborine, has been isolated from the roots of Glycosmis arborea. Its structure has been elucidated unambiguously as 5-methoxy-3-methylcarbazole on the basis of 2D NMR spectral analyses and finally confirmed by its synthesis. The structure of glycozolicine, an alkaloid of Clycosmis pelltaphylia. reported earlier by another group as 5-methoxy-3-methylcarbazole is found to be erroneous. A comment has been put forward on the structure of glycozolicine.

Recently, we have reported the isolation of a number of carbazole and quinoline alkaloids from the roots of Glycosmis arboreal . Continued investigation on the roots of the plant resulted in the isolation of yet another carbazole alkaloid (0.000 1 %), designated as glycoborine 1. Detailed analyses of the 2 D NMR spectra of the compound unambiguously established i ts structure as 5-methoxy-3-methylcarbazole. Literature survey showed that a compound, designated as glycozolicine, possessing the same chemical structure was reported earl ier2. However, the I3C NMR data and melting point of the compound were totally different from those of 1 . We, therefore, tried to obtain an authentic sample of glycozolicine from the authors in order to confirm whether 1 is at all identical with the reported compound, and if not, to establish i ts structure by 2D NMR spectral analyses, but failed. Compound 1 was then synthesised to confirm its structure. A reassignment of the reported I3C chemical shifts of glycozolicine revealed that the compound might be 8-methoxy-3-methylcarbazole 2. Therefore, compound 2 was also synthesised and its mp and 13C chemical shifts were compared with those of glycozolicine. We report herein the structure elucidation of 1 , syntheses of both 1 and 2 , and a comment on the structure of glycozolicine.

Glycoborine 1, mp 1 55-56°C, was obtained as a minor compound from the petroleum ether extract of the roots of GlycoslIlis arborea. The compound showed in its high resolution mass spectrum, the molecular ion at II/lz 2 1 1 .0977 corresponding to the

1 Rl = OMe, R2 = R3 = R4 = H 2 R4 = OMe, Rl = R2 = R3 = H 7 R3 = OMe, Rl = R2 = R4 = H 8 R2 = OMe, Rl = R3 = R4 = H 9 Rl = R2 = R3 = R4 = H 1 0 R4 = OH, R l = R2 = R3 = H

molecular formula C I4H 1 3NO. Its I3C NMR spectrum

exhibited signals for 14 carbons, of which six are aromatic CH and six aromatic quaternary carbons, besides one each for CH3 and OCH 3 carbons (Table I). It was, therefore, presumed that the compound might belong to methylcarbazole skeleton. This contention was corroborated by i ts I H NMR spectrum (Table II ) which displayed, besides the singlets for an aromatic methyl (82.523) and an aromatic methoxyl (8 4.064) groups, the signals for six aromatic CH protons and an NH proton (87 .874, hr. s). The IH_ IH COSY spectrum of the compound revealed that (i) the one-proton broad singlet at 8 8 . 1 1 2 (H-4) was correlated to the CH) (8 2.523) and a CH (8 7 . 1 90, br. d, H-2) proton signals through longrange coupling, the latter in turn having vicinal

CHAKRA V ARTY et al. : STRUCTURE & SYNTHESIS OF GL YCOBORINE 485

Table 1- I 3C NMR s�ctra a (0, CDCI3, 1 25 MHz) of carbazole and tetrahydrocarbazole derivatives Carbon 1 2 Glycozolicineb 5 6

<

.,. gel 9" .of 15 1 109.5 1 10.6 1 1 l . l ( 1 1 l . l ) 28.7 23.4 1 1 l . l 1 10.4 1 10.2 109.9 29.6 2 126.2 1 27. 1 1 25.5( 1 19.8) 36.8 32.3 1 26.3 1 27.2 1 27. 1 1 25.8 37.6 3 1 28.8 1 28.6 1 22.9( 1 22.9) 26.8 30.6 1 28.6 1 28.3 1 28.6 1 27 . 1 28.2 4 1 22.9 1 20.4 1 20. 1 ( 1 25.5) 45.8 30.3 1 19.9 1 20. 1 1 20.2 1 20.8 44.4 4a 1 22.8 123.8 1 23. 1 ( 1 23. 1 ) 1 04.2 1 09.5 1 24.4 1 23.6 1 23.4 1 22.7 92.8 4b 1 12.4 1 24.2 1 23. 1 ( 1 23. 1 ) 1 2 1 .8 123.2 1 17.6 1 23.5 123.1 1 23.9 142.0 5 1 56.2 1 12.8 1 15.7 ( 145.8) 1 54.9 1 18.4 1 2 1 .4 103.0 1 20.2 1 1 1 .2 1 15.9 6 100. 1 1 19.5 1 1 9.8( 105.9) 1 13.5 108.6 108.5 1 53.7 1 19. 1 1 19. 1 127.3 7 1 26.4 105.7 105.9 ( 1 20. 1 ) 1 30.7 1 56.6 1 59.9 1 14.9 1 25.6 1 10. 1 1 15.2 8 103.5 145.7 145.8( 1 15.7) 1 08.6 95.4 95.3 1 1 1 .3 1 10.5 143. 1 1 52.3 8a 14 1 .2 1 30. 1 1 34.9( 1 34.9) 1 38.5 1 38.2 142.6 1 34.7 1 39.7 1 29.4 143. 1 9a 1 36.8 1 37.4 1 40.7( 140.7) 1 38.5 1 33.4 1 39.2 1 38.5 1 37.6 1 36.6 1 82.8 3-Me 2 1 .4 2 1 .4 2 1 .5(21 .5) 20.9 22. 1 2 1 .4 2 1 .4 2 1 .4 2 1 .2 2 1 .0 OMe 55.4 55.4 55.4(55.4) 55.5 55.7 55.7 56.0 56.9

·Chemical shifts were assigned to specific carbons on the basis of analyses of 20 NMR, viz IH)H COSY, IH_l3C COSY, HSQC and HMBC spectra. bRevised assignments of the chemical shifts reported in ref.2. Values in the parentheses are the reported assignments based on 5-methoxy-3-methylcarbazole structure.

486 INDIAN J CHEM SEC B, JUNE 2001

Table III-One-bond ( 'H_ 13C COSY) and multiple-bond (HMBC) 'H- I 3C correlation data of 1 51 1 ppm One-bond Multiple-bond

correlation correlation 5e ppm 5ePpm

7.249 (H- I ) 109.54 (C- l ) 1 22.78 (C-4a) 7 . 190 (H-2) 126. 1 6 (C-2) 2 1 .43 (CH3-3) 8. 1 12 (H-4) 1 22.9 1 (C-4) 2 1 .43 (CH3-3) 6.646 (H-6) 100. 1 2(C-6) 103.50(C-8) 7.302 (H-7) 1 26.43 (C-7) 100. 1 2 (C-6) 6.98 1 (H-8) 1 03.50 (C-8) 100. 1 2 (C-6) 2.523 (CH3-3) 2 1 .43 (CH3-3) 122.9 1 (C-4) 4.064 (CH30-5) 55.37 (CH30-5) 1 56.20 (C-5)

was correlated through three bonds with the OMe proton signal (8 4.064). Moreover, H-7 signal also exhibited weak two- and four-bond correlations with C-6 and C-4b, respectively. Had the OMe group been attached to C-8 instead, the IH signal at 8 7 .302 should have shown three-bond correlation with the I3C signal at 8 1 1 2.42 (C-4b), and not with the quaternary carbon signal at 8 1 4 1 . 1 7 (C-8a). On the other hand, the aromatic methyl proton signal at 8 2.523 showed three-bond correlations with C-2 (8 126. 16) and C-4 (8 1 22.9 1 ), and H-2 proton signal at 8 7. 190 exhibited three - bond correlations with the quaternary carbon, C-9a (8 1 36.84), besides C-4 and CH3 carbons.

The assigned structure 1 for glycoborine was further supported by the NOE interactions observed in its NOESY spectrum as depicted in Figure 1. Based on the above observations, glycoborine was represented by 5-methoxy-3-methyIcarbazole structure 1.

The proposed structure of glycoborine 1 was finally confirmed by its synthesis (Scheme I). mMethoxyphenylhydrazine3 3 was condensed with 4-methyIcyclohexanone to give the product 4 which on treatment4 with glacial acetic acid at 95 °C yielded a mixture of 5-methoxy-3-methyl- l ,2,3,4-tetrahydrocarbazole 5 and 7-methoxy-3-methyl- l ,2,3,4-tetrahydrocarbazole 6. The compounds 5 and 6, after purification through column chromatography, on dehydrogenation5 in the presence of chloranil in xylene at 1 00°C furnished glycoborine 1 , identical (JR, IH NMR and I3C NMR) with the natural product, and 7-methoxy-3-methyIcarbazole 7, respectively in excellent yields.

A note on the structure of glycozolicine

Having unambiguously established the structure of glycoborine as 5-methoxy-3-methyIcarbazole 1, we

128.83 (C-3) 1 22.9 1 (C-4) 1 36.84 (C-9a)

1 12.42 (C-4b) 1 26. 1 6(C-2) 1 36.84 (C-9a) 1 12.42(C-4b) 1 22.78(C-4a) 1 56.20 (C-5) 1 12.42 (C-4b) 14 1 . 1 7 (C-8a) 156.20 (C-5) 1 12.42 (C-4b) 1 56.20(C-5) 1 26. 1 6 (C-2) 1 28.83 (C-3)

Figure 1 -NOE interactions observed in the NOESY of spectrum 1

concentrated our attention to the reported spectral data, particularly 13C NMR data, of glycozolicine which was claimed2 to have the same chemical structure as that of 1. It was evident from the reported I3C NMR data of glycozolicine (Table I) that the quaternary aromatic carbon attached to the OMe group resonated quite upfield at 8 145.8 (ef the chemical shifts of C-5 of 1 at 8 156.20, C-6 of 6-methoxy-3-methyIcarbazole 8 at 8 1 53.70 and C-7 of 7 at 8 1 59.94, Table I) . This up-field chemical shift of the oxygenated quaternary carbon demonstrated that the OMe group of glycozolicine may be located at C-8 as in 2, since in 3-methylcarbazole 9, C-I and C-8 resonated most up-field at - 8 1 10.06 . This contention was also supported by the shielding of the signal of the adjacent quaternary C-8a carbon (numbered as C- 1 3 in ref. 2) compared to the corresponding signal of 1, 7 or 8 (Table I). Similar upfield shift of C-8a signal was also reported for 8-hydroxy-3-methyIcarbazole7 10. We were, therefore, of the opinion that glycozolicine may possess a 8-methylcarbazole structure 2 instead of the claimed 5-methoxy-3-methylcarbazole structure 1, and accordingly compound 2 was synthesised from 0-methoxyphenylhydrazine 11 and 4-methyIcyclohexanone by following identical procedure as used in

CHAKRA V ARTY et ai. : STRUCTURE & SYNTHESIS OF GL YCOBORINE 487

�NHNH' R2 .

(i) 3 Rt :s OMe, R2 = H

11 Rt " H, R2 - OMe 4 Rt = OMe, R2 = H 12 RJ = H, R2 = OMe RJ �CH3 ��M·{iii)

RJ From 4 & 12 From 4 #YCH3 CH3 �CH3

o � (iii)� 0 0 N M� N H M H H R2

1 RJ = OMe, R2 = H 1 Rt " H, R2 - OMe R2 5 Rt = OMe, R2 = H 13 Rt = H, R2 = OMe 6 7

(i) C6H6, (ii) AcOH, (iii) Chloranil � xylene, lO()O C

Scheme I - Synthesis of 1, 2 and 7 the synthesis of 1 and 7 (Scheme I). The I3C and J H NMR chemical shifts of synthetic 2 were unambiguously assigned by 20 NMR spectral analyses and are summarised in Tables I and II, respectively. It can be seen from Table I that the oxygenated quaternary carbon of 2 indeed resonated at the same frequency as that reported2 for glycozolicine (8 145.8). However, sOMe deviations from the reported values were observed in the chemical shifts of C-3,C-5, C-8a and C-9a by 3-5 ppm (Table I). On the other hand, the mp ( 137-38 0c) of the synthetic 2 was very close to that reported ( 135 °C) for glycozolicine. The structure of glycozolicine as 2, therefore, could neither be confirmed nor be rejected. Direct comparison of the synthetic 2 with glycozolicine could also not be done due (0 the nonavailability of the authentic specimen.

It is noteworthy to mention that while elucidating the structure of glycozolicine, Jash et al. 2 reportedly synthesised 5-methoxy-3-methylcarbazole 1 which was claimed to be identical with natural glycozolicine. However, we have proved beyond doubt that glycozolicine cannot be represented by 5-methoxy-3-methylcarbazole structure. Its reported mp and I3C NMR data have been found by us to be closer to those of 8-methoxy-3-methylcarbazole 2 rather than those of

1. It can, therefore, be concluded that the reported structure of glycozolicine as 1 was not only erroneous but also confusing.

Oxidative rearrangement of tetrahydrocarbazoles. During the syntheses of 1 and 2, it was observed that the respective intermediate tetrahydrocarbazoles 5 and 1 3 were very unstable and new products were formed either in presence of CHCh or silica gel. A solution of 5 in CHCh when stirred for 3 days at room temperature yielded spiro[4'-methoxy-3-methylcyclopentane- l ,2'-indolin-3'-one] 14. On the other hand, when 8-methoxy-3-methyl- l ,2,3,4-tetrahydrocarbazole 1 3 was subjected to silica gel chromatography and eluted with CHCI 3 -hexane ( 1 :5), 8-methoxy-3-methyl -4a-hydroperoxy- l ,2,3,4-tetrahydro-4aH-carbazole 1 5 was obtained in 35% yield (Scheme II). Recently, photooxidation of tetrahydrocarbazoles to hydroperoxy derivatives followed by rearrangement of the latter to spiroindolinones were reported 8.9.

Experimental Section General. Melting points are uncorrected. Low

resolution ElMS (JEOL JMS HX- l l O) and high resolution ElMS (JEOL JMS 0-300) were obtained

488 INDIAN J CHEM SEC B, JUNE 2001

OMc OMo ©o:YCH3 CH) H 5 14

ooH , CH3 �CH3 � Silica gel a 0 . JCe�- � 0 c

13 ..

Scheme II

by a direct inlet system at 30 eV. IH, J 3C and 20 NMR spectra were taken on 300 MHz (JEOL LAMBDA 300) and 500 MHz (JEOL ALPHA 500) instruments.

Plant material. The roots of Glycosmis arborea were collected from MIs United Chemical and Allied Products, 1 0, Clive Row, Calcutta 700 001 and a voucher specimen is available in the herbarium of the company.

Isolation of glycoborine 1. Air-dired and milled roots (5.5 kg) of G. arborea were extracted with petroleum ether (60-80°C). The extract was concentrated and the concentrate on repeated column chromatography over neutral alumina furnished 1 (6 mg, 0.0001 %), besides other compounds already reported by us. It was recrystalIised from pet. etherchloroform in fine needles, mp 1 55-56 °C ; UV: 244, 287, 324, 337 nm; EI-MS: mlz 2 1 1 .0977 (M+, 1 00%), 196 (2 1 ), 1 68 (63), 1 67 ( 1 6) ; J 3C NMR : see Table I; IH NMR: see Table II.

5-Methoxy-3-methyl-1,2,3,4 tetrahydrocarbazole 5 and 7-methoxy-3-methyl-1,2,3,4-tetrahydrocarbazole 6. To a stirred mixture of 3N NaOH ( 10 mL) and ether (50 mL) cooled to O°C, was added mmethoxyphenylhydrazine hydrochloride3 ( 1 .75 g, 10 mmoles). The aqueous layer was saturated with NaCI, separated from the ether layer and extracted twice with ether. The combined ether extract was washed once with saturated NaCI solution, dried over anhyd Na2S04 and evaporated to dryness under reduced pressure at room temp. to get m-methoxyphenylhydrazine as light yellow oil. It was dissolved in benzene (25 mL) and 4-methYlcyclohexanone (2.24 g, 20 mmoles) was added to it, and the reaction mixture was kept as such for 15 min. After removal of the solvent, glacial AcOH (2.mL) was added to the residue and the

mixture was heated on a steam-bath until vigorous reaction started. When the reaction subsided, the reaction mixture was refluxed for 1 0 min and AcOH was removed. Ice-water was added to the residue and the mixture was extracted with CHCh. The extract was washed with water, dried over anhyd Na2S04 and evaporated. The residue was chromatographed over silica gel to afford the tetrahydrocarbazoles 5 ( 1 29 mg, 6%) and 6 ( 1 . 1 6 g, 54%).

5-Methoxy-3-methyl-1,2,3,4-tetrahydrocarbazole 5. Recrystallised from ether-hexane, mp 141 -42 °c , IR (KBr): 3349 (NH), 1 625, 1 589 cmol ; 13C NMR: see Table I; IH NMR: see Table II. HR-MS: M+ 2 15 . 1 325; Calcd for CI4H17NO: 2 1 5. 1 3 10. Anal. Calcd for C I4H17NO : C, 78. 1 0; H, 7.96; N, 6.5 1 . Found: C, 78.05; H, 7.94; N, 6.55%.

7 -Methoxy-3-methyl-1,2,3,4-tetrahydrocarbazole 6. Recrystallised from MeOH as colourless needles, mp 138-39 DC; IR (KBr): 3408 (NH), 1629, 1590 cmol ; I3C NMR: see Table I; IH NMR : see Table II. HR-MS: M+ 2 1 5 . 1 333; Calcd for C I4H17NO: 2 15. 1 3 10. Anal Calcd for CI4H17NO: C, 78. 1 0; H, 7 .96; N, 6.5 1 . Found: C, 78. 1 5 ; H, 7.99; N, 6.56%.

Glycoborine 1 from 5. To a solution of the tetrahydrocarbazole 5 ( l 08 mg, 0.5 mmole in xylene (5 mL), chloranil (246 mg) was added and the mixture was stirred at 1 00° C for 2hr. The reaction mixture was then cooled, diluted with ether (20 mL), washed first with 0. 1 M NaOH solution and then with water, dried over anhyd Na2S04, and evaporated. The residue on chromatography over silica gel yielded glycoborine 1 (90 mg, 85%), recrystallised from ether-hexane as fine needles, mp 1 38-39°C, identical in all respect (mmp, IH and J3C NMR) with the natural product.

7-Methoxy-3-methylcarbazole 7 from 6. Compound 7 was prepared from 6 by the identical procedure as described above in 90% yield. It was recrystallised from benzene in fine needles, mp 241 -42°C; IR (KBr): 3398 (NH), 1 6 14, 1 573 cmo l ; J 3C NMR: see Table I; IH NMR: see Table II. HR-MS: M+ 2 1 1 . 1 034; Calcd for C I4H J3NO : 2 1 1 .0996. Anal. Calcd for CI4H J 3NO: C, 79.59; H, 6.20; N, 6.63. Found: C, 79.64; H, 6.26; N, 6.65%.

Synthesis of 8-methoxy-3-methylcarbazole 2. The reaction between o-methoxyphenylhydrazine 11 and 4-methylcyc1ohexanone was carried out as described above to obtain crude 8-methoxy-3-methyl-l ,2,3,4-tetrahydrocarbazole 13 which was immediately treated with chloranil in benzene under nitrogen atmosphere

CHAKRA Y ARTY et al. : STRUCTURE & SYNTHESIS OF GL YCOBORINE 489

and the reaction mixture was refluxed for 6 hr in dark. Usual work-up and purification of the product through column chromatography yielded 2 in 15% yield. It was recrystallised from hexane as fine needles, mp 1 37-38° C; IR (KBr): 341 2 (NH), 1 6 1 2, 1578 cm· l ; l 3C NMR: see Table I; IH NMR : see Table II. HR-MS: M+ 2 1 1 . 1004; Calcd for CI4HI3NO: 2 1 1 .0934. Anal.Calcd for CI4H l3NO: C, 79.59; H, 6.20; N, 6.63. Found: C, 79.60; H, 6.23 ; N, 6.66%.

8-Methoxy-3-methyl-4a-hydroperoxy-l,2,3,4 tetrahydro-4aH-carbazole 15 from 13. The crude 13 prepared above was subjected to column chromatography over silica gel. Elution with CHCI3-hexane ( 1 : 15 , v/v) furnished 15 (yield 35%), recrystallised from acetone as fine needles, mp 128-29° C (dec.); IR (KBr): 3056, 2992, 2746, 16 15, 1 589, 1487 cm·l ; l3C NMR: see Table I; IH NMR: see Table II. HR-MS: M+ 247. 1236; Cal cd for CI4H1703N: 247 . 12 16 . Anal. Calcd for CI4H 1703N: C , 67.99; H , 6.93; N , 5 .66. Found: C, 67.83; H, 6.83; N, 5 .76%.

Spiro[ 4' -methoxy-3-methylcyclopentane-l,2' -indolin-3' -one] 14 from 5. A solution of 5 ( 108 mg) in CHCh ( 10 mL) was stirred at room temperature for 3 days. After removal of the solvent, the residue was subjected to flash chromatography over silica gel using EtOAc-hexane ( 1 :5 , v/v) as the mobile phase to obtain 14 as an oil in 12% yield. IR (KBr): 3320, 1674, 1 6 14, 1 5 86, 1 50 1 , 1 46 1 , 746 cm - I ; IH NMR

(CDCh, 300 MHz): 0 1 .08 (3H,d, 1 = 6.6 Hz, CH3-3), 1 .25- 1 .60 (3H, m aliphatic-H), 2.06-2 . 1 8 (2H, m aliphatic-H), 2.3 1 -2.42 (2H, m, aliphatic-H), 3.9 1 (3H, s, CH30-4 '), 4.75 ( 1H, br. s, NH), 6. 19 ( 1H, d, 1= 8 . 1 Hz, H-5' ) , 6.36 ( 1H, d, 1= 8 . 1 Hz, H-7'), 7 .34 ( 1 H, t, 1 = 8 . 1 Hz, H-6' ) ; 13C NMR (CDCh, 75 MHz): 0 74.5 (C- l ), 45 .8 (C-2), 34. 1 (C-3), 35.0 (C-4), 29.7 (C-5), 205 .7 (C-3'), 1 09.5 (C-3'a), 1 58.5 (C-4' ), 99.7 (C-5' ), 1 38.5 (C-6'), 1 04.2 (C-7' ), 1 62.5 (C-7' a), 20.9 (CH3-3), 55.7 (CH30-4'); ElMS: m/z 23 1 (M+).

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