Oxindole alkaloids from Neolaugeria resinosa

Pbytocbemistry, Vol. 32, No. 6, pp. 1587-1590,

1993

0031-9422/93S6.00+0.00 0 1993Pergamon Press Ltd

Printed in Great Britain.

OXINDOLE

ALKALOIDS

FROM NEOLAUGERIA

BERNARD WENIGER, YULIN JIANG, ROBERT ANTON, * JAUME BAsrIDA,t QUIRION$

RESINOSA

TERESA VAREA~ and JEAN-CHARLES

Lahoratoire de Pharruacognosie, Facultb de Pharruacie, Universitt Louis Pasteur Strashourg, B.P. 24,674Ol Illkirch cedex, France; TDepartament de Productes Naturals, Facultat de Farmacia, Universitat de Barcelona, 08028 Barcelona, Catalonia, Spain $Institut de Chimie des Substances Naturelles, 91190 Gif/Yvette, France (Received 21 August 1992)

Key Word Index-Neolaugeria resinosa; Rubiaceae; root bark; oxindole alkaloids; neolaugerine; isoneolaugerine; 15hydroxyisoneolaugerine.

Abstract-The new alkaloids neolaugerine, isoneolaugerine and 15hydroxyneolaugerine, possessing an original oxindole skeleton, have been isolated from the root bark of Neolaugeria resinosa. Their structures and stereochemistry were established by chemical and spectroscopic methods, including mass spectrometry, ‘H, i3C, 2D and NOE NMR experiments.

INTUODUCTION

Neolaugeria resinosa (Vahl) Nichols, formerly known as Laugeria resinosa Vahl, Laugeria densijloru (Griseb.) Hitchc., Antirhea resinosa (Vahl) Cook & Collins, Stenostomum densijlorum Griseb and Terebraria resinosa (Vahl)

Sprague, is a small- or medium-sized evergreen tree distributed throughout the Bahamas and West Indies from Cuba to Trinidad and Tobago Cl]. The genus is represented with three species in the Caribbean basin [2]. As part of our continuing interest in Caribbean plants in general and the chemotaxonomy of West Indian Rubiaceae in particular, we now report the isolation of new oxindole alkaloids from this species. RESULTS AND DISCUSSION

From the root bark, alkaloids were extracted by a routine work-up procedure (see Experimental) furnishing 3.1 g of crude material. Flash chromatography followed by TLC purification afforded three alkaloids as intense red-orange coloured compounds: neolaugerine (l), isoneolaugerine (2) and 15hydroxyisoneolaugerine (3). Compound 1 displayed a [M] ’ at m/z 312 (100%) which analysed for C,,H,,N20,. The UV spectrum indicated a highly conjugated system very similar to that of 2-indolinone [3]. The IR spectrum showed a broad NH band and a conjugated lactam carbonyl group at 1686 cm-‘. In the aliphatic part of the ‘H NMR spectrum, two singlets, corresponding to three hydrogens, at 63.89 and 2.27 indicated one methoxy and one N-methyl group. Moreover, five aromatic or olefinic protons were

*Author

to whom correspondence

should

be addressed.

observed; three belonging to an aromatic ring (67.29,6.99 and 6.81; J= 7.6 Hz), the other two being deshielded olefinic protons (67.62 and 6.82; J= 12.6 Hz). The spectrum also showed signals for one ethyl chain (60.90, Me, t, J=7.5 Hz and 61.78, m, CH,). The 13CNMR spectra exhibited a quaternary carbon signal (6 169.7), attributed to a conjugated lactam carbonyl function [4], five tertiary and five quaternary aromatic or olefinic carbons. Thus, it could be deduced that three of these quaternary carbons belong to the indolinone skeleton and the other two to the conjugated olefinic system. A ‘H COSY experiment showed the presence of a substituted N-methyl piperidine with a quaternary carbon and an ethyl chain at position 3. A iH-i3C COSY experiment allowed complete correlation between the two spectra and confirmed the presence of the substituted piperidine with the two methylene carbons tl to the nitrogen (657.6 and 60.4). From these data it appeared that neolaugerine was constituted of a conjugated indolinone system and a piperidine substituted ring. A small coupling (J = 1.8 Hz) observed between one of the olefinic protons (66.82) and an aliphatic proton (62.83) suggested that the piperidine ring could be linked to the aromatic ring through the ole6nic system as shown in structure A, which is in agreement with all the spectroscopic data. The remaining problem was to determine the stereochemistry of the double bonds and the position of the methoxy group on the aromatic ring. (E)- and (Z)-3-(3’methyl-2’-butenylidene)-2-indolinone have been previously isolated from Cimifuga dahurica [S]. Their structures were determined by X-ray analysis. The coupling constants for the two olehnic protons were different for the two isomers, J = 12 and 9 Hz for E and Z isomer, respectively. The value observed for neolaugerine

1587

B. WENIGER et al.

1.588

Et

MC0

1

2

K=H

3

R=OH

W /

CH, /

/”

I

2

3

Fig. 1. NOES observed for neolaugerine (l), isoneolaugerine (2) and 15-hydroxyisoneolaugerine (3).

(J = 12.6 Hz) was in favour of an E stereochemistry for the C-3/C-8 double bond. This hypothesis was confirmed by

a NOESY experiment which established the Z stereochemistry of the C-9/C-10 bond (Fig. 1). Moreover, the correlation between H-6 and the protons of the methoxy group indicated that the methoxy group was located at position 7 of the indolinone group. The second alkaloid isoneolaugerine (2) was isomeric with neolaugerine. Their mass spectra displayed [M] + at m/z 312 and common fragments at m/z 283,269,254,240

and 98. The UV and IR spectra of 2 were very similar to those of 1. The aromatic part of the ‘H NMR spectrum showed the same signals for 1 and 2 with the exception of H-9 which appeared as a doublet (J = 12.5 Hz) suggesting differences in the stereochemistry of the C-9/C-10 double bond. The aliphatic part was more difficult to interprete due to overlapping of protons (2 x H-l 1, H-12, H-14 and H-15). In the 13CNMR spectrum, the only significant differences were observed for C- 15 and C- 16. From these data, it was concluded that 2 is the (E) 9-10 isomer of

Oxindole alkaloids from Neohgeria resinosa

1589

N

/

..I,,, /

I

-\

0 HQ

f: CHO

OMe

OMc 6

OMe

OMe 8

Fig. 2. Possible biosynthesis of neolaugerine-type alkaloids.

neolaugerine. This hypothesis was confirmed by a NOESY experiment which showed NOE between H-8 and H-11 and between H-9, H-4 and H-15 (Fig. 1). Alkaloid 3 exhibited a [M]’ at m/z 328 suggesting the presence of an hydroxyl group. This was confirmed by an IR spectrum which showed a broad hydroxyl band at 3450 cm- ‘. The UV spectrum showed the same maxima as those observed for 1 and 2. The 13CNMR spectrum was particularly instructive. No differences were observed in the low field part of the spectrum. The aliphatic part showed a new quaternary carbon (674.5) and the disappearance of the C-15 of neolaugerine and isoneolaugerine. The presence of the hydroxyl group on C-15 was confirmed by ‘HNMR. All the signals were assigned through homo- and heteronuclear COSY spectra. The two H-14 appeared as two doublets 62.68 and 2.15 (J = 10.8 Hz), indicating the presence of two substituents on C-15. A NOESY experiment established the stereochemistry of the two double bonds (Fig. 1). The same correlations as those observed for 2 could be observed, indicating that alkaloid 3 is 15-hydroxyisoneolaugerine.

The formation of the neolaugerine skeleton could be explained by a biogenesis similar to those of Cinchona alkaloids (Fig. 2) [6]. Methoxydihydrocorynantheol (4) could be oxidized to oxindole (5) which could be cyclized to quinuclidine (6) in a similar way to that proposed for Cinchona alkaloids. Then a fragmentation could occur leading to intermediate 7. After methylation compound 8 could be fragmented with loss of the C-5/C-6 chain to furnish the neolaugerine skeleton. A similar mechanism, with the exception of the oxindole rearrangement, seems to be involved in the formation of cinchonaminone, an alkaloid isolated from C. succirubra [7].

EXPFBUMENTAL

NMR ‘H (300 MHz) and 13C (75 MHz), chemical shifts given in ppm relative to TMS (6 =O), coupling constants (J) are given in Hz. COSY spectra and ‘H-13C correlations were performed using the Bruker library of microprograms.

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B. WENIGER et al,

Plant material. Root material of N. resinosa was collected in Maricao, 18 miles south east of Mayagiiez, Puerto Rico, in December 1990. A voucher specimen (Caminero & Garcia N”391) is deposited at the Herbarium of the University of Puerto Rico in Mayagiiez. Extraction and isolation. Dried and ground root bark (250 g) was moistened with 50% NH,OH and extracted exhaustively with CHQ, using a Soxhlet apparatus. The organic soln was extracted with 10% HCI until a Valser Mayer’s test was negative. The acid layer was sepd, made alkaline with 50% NH,OH and extracted with CH,Cl,. The CH,Cl, layers were washed with H,O, dried (Na,SO,) and evapd in uacuo to give 3.1 g of crude alkaloid mixt. (AM), yield 1.54%. Crude AM was fractionated by flash CC on a silica gel column (63-200 pm) using CH,Cl,-MeOH (4:l) to afford 8 frs (I-VIII). The less polar fr., fr.1, and frs IV and V were further purified by TLC (silica gel; EtOAc-MeOH, 9: 1) to afford neolaugerine (750 mg), isoneolaugerine (54 mg) and 15-hydroxyisoneolaugerine (75 mg), respectively. Neolaugerine (1). Amorphous orange-red powder. [a]:: +89” (MeOH; c 0.60). IR v$!:‘~ cm-‘: 3500, 1686, 1609, 1460, 1280. UV e:” nm (log 6): 256 (3.90), 264 (3.97), 286 (3.92), 296 (3.87), 344 (4.13). MS: m/z (rel. int.): 312 (lOO), 283 (17), 269 (48), 254 (22), 240 (36), 226 (15), 124 (24), 98 (33), 70 (40), 57 (33); HMRS: [M]’ (found: 312.1838; C19H,,N,02 requires: 312.1838). ‘H NMR (300 MHz, CDCl,) 6: 7.62 (d, J = 12.6 Hz, H-8X 7.59 (br s, NH), 7.29 (d, J = 7.6 Hz, H-4), 6.99(t,J=7.6Hz, H-5), 6.82(&,5=12.6, 1.8Hz, H-9), 6.81 (d,J=7.6Hz, H-6), 3.89(s,OMe), 3.10-3.0(m,H-15 and H12eq), 2.94 (dt, J= 11.3, 1.9 Hz, H-14eq), 2.83 (tdd, J= 12.6, 5.7, 1.8 Hz, H-llax), 2.27 (s, NMe), 2.26 (ddd, J= 12.6, 3.0, 1.5H~H-lleq),2.07(dd,J=11.3,3.6Hz,H-14ax),2.OO(ddd, J= 12.6, 10.6, 3.0Hz, H-12ax), 1.78 (quint, J=7.5 Hz, 2xH16), 0.90 (t, J= 7.5 Hz, Me-17). 13CNMR (see Table 1). Isoneolaugerine (2). Amorphous orange-red powder. [a]gs+34o (MeOH; c 0.80). IR v%:‘~ cm-‘: 1717, 1616, 1215, UV E.z$‘” nm (log E):256 (3.83), 264 (3.89), 285 (3.86), 296 (3.81), 341 (4.00). MS: m/z (rel. int.): 312 (lOO), 283 (14), 269 (46), 254 (19), 240 (31), 226 (13), 124 (35). 98 (46), 70 (50), 57 (50); HMRS: [M]’ (found: 312.1841; C,9H,,N,0, requires: 312.1838). ‘H NMR (300 MHz, CDCl,) 6:7.80 (br s, NH), 7.65 (d, J=12.5Hz, H-8), 7.23 (d, J=7.7 Hz, H-4), 6.96 (t, J=7.7 Hz, H-5), 6.80 (d, J=7.7Hz, H-61, 6.75 (d, J = 12.5 Hz, H-9), 3.89 (s, OMe), 2.80-2.65 (in, 2xH-1 l), 2.60 (m, H-14eq and H-12eq), 2.5c2.35 (q H-14ax and H-12ax), 230 (s, NMe), 2.30 (171,H-15), 1.85 (m, H-16) 1.60 (m, H-16), 0.95 (t, J= 7.0 Hz, Me-17). 13CNMR (see Table 1). 15-Hydroxyisoneolaugerine (3). Amorphous orangered powder. [a]k5 +3” (MeOH; c 0.20). IR v$!:‘~ cm-‘: 3450, 1682, 1608, 1217. UV n=” nm (log E): 256 (3.86), 264 (3.94), 286 (3.91), 297 (3.86), 343 (4.11). MS: m/z (rel. int.): 328 (68), 297 (6), 271 (lOO), 228 (34), 140 (30), 96 (30), 83 (28), 58 (75); HMRS: CM] + (found: 328.1792; C,9H,,N,03 requires: 328.1787). ‘HNMR (300 MHz, CDCI,) 6: 8.40 (br s, NH), 7.62 (d, J= 12.6 Hz, H-8), 7.38

Table 1. 13C NMR spectral data of compounds 1-3 (75 MHz, CDCI,) C 2 3 3a 4 5 6 7 7a 8 9 10 11 12 14 15 16 17 N-Me O-Me

1

2

3

169.7 125.0 123.9 116.6 122.4 111.2 144.0 130.0 131.7 120.3 158.0 25.7 57.6 60.4 39.9 34.5 12.2 46.3 55.9

169.9 125.4 123.6 116.4 122.3 111.2 144.0 130.2 131.7 118.3 157.3 27.5 56.8 61.5 47.1 24.2 12.0 45.9 55.8

169.9 126.4 123.6 116.8 122.5 111.3 143.9 130.0 131.4 117.1 157.2 26.7 56.2 67.3 74.5 30.4 7.3 45.6 55.8

(d, J=7.6 Hz, H-4), 7.13 (d, J=12.6 Hz, H-9), 6.93 (t, J =7.6 Hz, H-5), 6.78 (d, J=7.6 Hz, H-6), 3.89 (s, OMe), 2.98 (dt, J= 14.5, 4.0 Hz, H-lleq), 2.73 (dtd, J=10.8, 5.4, 1.0 Hz, H-12eq), 2.68 (dd, J= 10.8, 1.0 Hz, H-14eq), 2.45 (ddd, J=14.5, 10.8, 4.8 Hz, H-llax), 2.30 (s, N-Me), 2.18 (td, J= 10.8, 4.0 Hz, H-12ax), 2.15 (d, J=10.8 Hz, H14ax), 2.03 (dq, J= 15.0, 7.5 Hz, H-16), 1.75 (dq, J= 15.0, 7.5 Hz, H-16), 0.90 (t, J=7.5 Hz, Me-17), 13CNMR (see Table 1). Acknowledgements-The authors wish to thank G. Caminero and R. Garcia (University of Puerto Rico, Mayagiiez) for collection of plant material. REFERENCES

1. Little, Jr., E. L. and Wadsworth, F. H. (1964) Common Trees of Puerto Rico and the Virgin Islatuis, p. 524. U.S. Department of Agriculture, Washington DC. 2. Nicolson, D. H. (1979) Brittonia 31, 119. 3. Hinman, R. L. and Bauman, C. P. (1964) J. Org. Chem. 29, 2431 4. Pretsch, E., Clerc, T, Seibl, J. and Simon, W. (1983) Tables of Spectral Data for Structure Determination of Organic Compounds. Springer, Berlin. 5. Baba, K., Kozawa, M., Hata, K., Ishida, T. and Inoue, M. (1981) Ckm. Pharm. Bull. 29, 2182 6. Verpoorte, R, Schripsema, J. and van der Leer, T. (1988) 7’he Alkaloids Vol. 34 (Brossi, A., ed.), p. 331. Academic Press, New York. 7. Mitsui, N., Nero, T., Kuroyanagi, M., Miyase, T., Umehara, K. and Ueno, A. (1989) CXem. Pharm. Bull. 37, 363.