- Email: [email protected]
A steroidal alkaloid from Buxus papillosa
Short Reports HCI and evapn the residue was taken in to boiling H,O and extracted with EtOAc. TLC (cellulose) of the EtOAc extract gave p-coumaric acid (co-chromatography with an authentic sample) and apigenin by prep. TLC (cellulose, CHCI,-HOAc-H,O, 10:9: 1, R, 0.70), by UV, ‘HNMR, EIMS. Acid hydrolysis. A small amount of 2 was refluxed with 3% and 10% HCI and the hydrolysis product was identified as described above.
Acknowledgements-We thank Dr E. Stefanou (Dept of Chemistry, University of Crete, Greece), Dr P. Christen (Dept of Pharmacy, University of Geneva, Switzerland) for providing necessary spectral facilities; and Dr S. Kokkini (Deptof Botany, University of Thessaloniki, Greece) for identification of the plant material. REFERENCES
M. J. (1986) Plant.
1. VillBr, A., Jimtnez,
M. J. and Alcaraz, Med. Phytothb-. 10, 31.
2. Diaz, R. M., Garcia-Granados,
A., Moreno, E., Parra, A., Quevedo-Sarmiento, J., Saenz de Buruaga, A. and Saenz de Buruaga, J. M. (1988) Planta Med. 54, 301.
683
3. Gonzalez, A., Fraga, B., Hernandez, M., Luis, J. and Larruga, F. (1979) Biochem. Syst. Ecol. 7, 115. 4. Kooiman, P. (1972) Acta Bot. NeerJ. 21. 417. Theodossiou, ‘P. (i962) Trav. Sot. Ph&m. Montpeflier 21, 221. K.okkalou, E. and Kapetanidis, I. (1988) Pharm. Acta HeJu. 63, 170. Karl, C., Miiller, G. and Pedersen, P. A. (1976) Phytochemistry 15, 1084. Ansari, F. R., Ansari, W. H., Rahman, W., Seligman, O., Chari, V. M., Wagner, H. and asterdahl, B. G. (1979) Planta Med. 36, 196. 9. Rao, L. J. M., Krishna Kumari, G. N. and Prakasa Rao, N. S. (1983) Phytochemistry 22, 1058. P. K. and Thakur, R. S. (1986) Phytochemistry 10. Chaudhuri, 25, 1770. 11. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970) in The Systematic ldentijication Of Flauonoids. Springer, Heidelberg. G., Markham, K. R., Ramachan12. Chopin, J., Dellamonica, dran Nair, A. G. and Gunasegaran, R. (I 984) Phytochemistry 23. 2106. 13. H&borne, J. B. (1983) in Phytochemical Methods. Chapman & Hall, London. 14. Barberan, F. A. T., Thomas, F. and Ferreres, F. (1985) J. Nat. Prod. 48, 28.
Phytuchemisrry,Vol. 29, No. 2, pp. 683 -685, 1990.
0
Printedin Great Britain.
A STEROIDAL ATTA-UR-RAHMAN,*
ALKALOID
ZAHIDA IQBAL, MUZAFFAR H.E.J. Research
Institute
FROM BUXUS
ALAM, M. IQBAL CHOUDHARY,
of Chemistry,
University
of Karachi,
0031.-9422/90 %3.00+0.00 19gOPergamonPressplc
PAPILLOSA HABIB NASIR
Karachi-75270,
and
TALAT FATIMA
Pakistan
(Receioed in revised form 26 May 1989)
Key Word Index-Buxus
Abstract-A its structure
new steroidal elucidated.
papillosa; Buxaceae;
steroidal
alkaloid, (-)-buxoxybenzamine,
INTRODUCTION
Buxus papillosa, C. K. Schneider (Buxaceae) is a commonly grown shrub of northern Pakistan. A number of steroidal alkaloids have been reported from this plant [l-5]. Continuing our investigations on the alkaloids of various Buxus species, we report here the isolation and structure of a new steroidal alkaloid (-)-buxoxybenzamine (1) from the leaves of B. papillosu.
RESULTS AND DISCUSSJON
The ethanolic
extract of air-dried
leaves of B. papillosa
alkaloids;
buxoxybenzamine;
ZD-NMR
has been isolated form the leaves of Buxus papillosa and
was evaporated and partial separation of the alkaloids was carried out by extraction into chloroform at different pH values. The fraction obtained at pH 8.5 was loaded on a silica gel column. Elution was carried out with chloroform-methanol. Further purification of the fraction eluted in 96% chloroform-4% methanol by prep. TLC resulted in the isolation of 1. (-)-Buxoxybenzamine (l), C,,H,,N*O,, partially soluble in chloroform, showed UV absorption at 224 nm, characteristic of a secondary benzamidic chromophore [6]. The IR spectrum displayed absorptions at 3400 (OH and NH), 1730 (ester carbonyl), 1640 (amide carbonyl) and 1540 (C=C) cm-’ [3].
Short Reports
684
1
The ‘HNMR spectrum of 1 (CDCI,+few drops of CD,OD, 400 MHz) showed four tertiary methyl singlets at 60.78, 0.81, 0.83 and 1.01. A doublet at 60.82 (J,,,,O = 8.1 Hz) was assigned to the 21secondary methyl group. while the vicinally coupled C-20 methine proton appeared at 62.44 and was further coupled to the C-17~ proton which appeared at b 1.78. A three-proton singlet at 6 1.98 was due to the acetate methyl protons, while a 6H singlet at 62.13 was assigned to the N(Me), protons. A multiplet centred at 64.70 was assigned to the C-6 methine proton geminal to the r-acetoxy group [6], while the neighbouring C-5x proton appeared as a doublet of doublet at 62.50 (J,,, = 11.0 Hz, J,.,,=9.5 Hz). The characteristic [runs-diaxial coupling constant of 11.0 Hz indicated the r orientation of the C-6 acetoxy group. A broad doublet resonating at 63.93 (J28,3Z= 8.7 Hz) was assigned to the C-2fl proton geminal to the hydroxy group. A multiplet centred at 63.82 was assigned to the C-3 proton r to the amide nitrogen, while the NH proton resonated at 66.84 (J NH.3z= 9.5 Hz) as a broadened doublet. This doublet sharpened when the spectrum was run in pure CDCl,. An interesting doublet resonating at 62.96 (J 11,I2p= 2.0 Hz) was assigned to the C-12 proton geminal to the C-9,1 I-epoxide. The J value indicated that the epoxide has p-orientation, because in an r-orientation the C-11/I proton would have afforded a doublet with -3 5 Hz [7]. A doublet of doublet at (r 1.99 :;,r;;.‘;$= i3.8 Hz, J 12z., la = 2.0 Hz) was assigned to C12~ proton while the geminal C-12/I proton appeared at 6 1.75. A broad singlet at 65.56 was due to the C-l olefinic proton while multiplets centred at 67.34 (2H), 7.42 (1H) and 7.71 (2H) were assigned to the five aromatic protons at C-2’, C-6’, C-4’ and C-3’,C-5’ respectively. Two clean AB doublets at (i2.70 and 1.65 (J,9a,,98= 13.5 Hz) were due to the 19a and 19,%methylene protons. The assignments of the resonances to various protons were facilitated by homonuclear 2D J-resolved, COSY-45” and NOESY experiments [S, 93. In the COSY-45” spectrum coupling of C-l 1 H (62.96) was observed with C-19 rH (62.70), C-19 /IH (61.65) C-12aH (61.99) and C-l2/IH (61.75). Similarly C-3aH (63.82) was coupled with C-2BH (b4.00) as well as with the amide NH proton at 66.28. Its three-bond coupling with C-5srH was also observed. The assignment to C6 /3H at 64.70 was confirmed by its COSY-45’ interaction with C-5aH (fi2.50), the latter showing allylic coupling with the C-l vinylic proton at 65.56 and homoallylic
coupling with C-2 /IH at 6 3.98. The C-20 methine proton (62.44) showed a cross-peak with C-17 rH at 61.72. The upfield value of C-17xH suggested the lack of any electron withdrawing substituent at C-16. On irradiation at 63.98 (H-2/?). NOE was observed at H-32 (63.82) and at the C-l olefinic proton at S5.56. On the other hand when irradiation was carried out at 65.56, NOES were seen at 63.98 (H-2/$, 3.82 (H-3x) and 62.70 (H-19x). These results unambiguously indicate that the H-28 and H-3x are close in space. while the H-l olehnic proton is also in close proximity. The a-stereochemistry of the acetate group at C-6 can be deduced from the NOE results, as follows: on irradiation of the C-68 proton (fi4.70), NOES were seen at H-19/1 proton (62.70) and at the H-7% and /I protons (6 1.95 and 1.54). Moreover, when H-5% (62.50) is saturated no NOE was observed at 64.70 (H-6/I). This finding is consistent with the r-stereochemistry of the C-6 acetate group. The observed NOES are shown in Table 1. DEPT experiments established the multiplicity of the 13C signals (Table 2). Further insight into the structure of 1 was provided by 2D one-bond ‘H-“C correlation (HETCOR) experiment (Table 2) which permitted the assignment ofall the protonated carbons in the 13C NMR spectrum of 1. The presence of an epoxide function could be deduced from the signal at 362.48 which is consistent with a quaternary carbon of an oxirane system and assignable to C-9; the other carbon of the oxirane system resonated at 663.36 and was assigned to C-l 1. The high resolution mass spectrum of 1 showed the molecular ion at ml: 578.3721 corresponding to the molecular formula C35H50N205. indicating the presence of 12 double bond equivalents in the molecule. The [M] + was further confirmed by FAB and FD techniques. A peak at m/z 563.3480 was due to the loss of a methyl group from the molecular ion while a large peak at m/z 105.0331 corresponded to the benzoyl cation. Compound 1 showed a base peak at m,/z 72.0813 which arose by cleavage of the nitrogen-containing ring D side chain [lo]. A peak at m/z 175.1 167 was the result of a retroDiels-Alder cleavage of ring A. On the basis of the above spectroscopic studies. structure 1 was assigned to ( -)buxoxybenzamine.
Table 1. Connectivities established by NOE difference spectra ___--___ Proton irradiated
Chemical shift (d)
NOE connectivity (s)
“4, NOE
H-l
5.56
H-2/1
3.98
H-51 H-60
2.50 4.70
H-20 H-8/I
2.36 2.00
H-2p (3.98) H-3x (3.82) H-19/I(1.65) H-3x (3.82) H-l (5.56) H-31 (3.82) H-198(2.70) H-7/J(l.9.5) H-16/$(1.35) H-19/j (1.65) H-68 (4.70) 30-CH, (1.01) H-7/?(1.95)
10.0 6.0 7.5 7.0 5.0 5.0 8.0 5.0 1.8 3.0 5.0 6.0 3.0
Short Reports
Table
2. 13C NMR,
multiplicities (DEPT) nectivities (HETCOR)
C
(6°C)
DEPT
1 2 3 4 5 6 7 8 9 10 11 12
134.09 67.48 61.13 38.52 53.35 77.89 35.54 41.07 62.48 131.78 63.36 35.11
CH CH CH C CH CH CH, CH C c CH CH,
13 14 15
42.94 38.65 26.8
C C CH,
16 17 18 19
32.41 49.91 16.63 44.41
CH, CH Me CH,
61.51 9.70 17.29 27.22 17.01 134.41 128.33 126.95 131.42 126.95 128.33 39.56 21.61 169.98 169.76
CH Me Me Me Me c CH CH CH CH CH Me Me C C
20 21 30 31 32 1’ 2 3’ 4 5’ 6 -N (Me), O-XX& &Me<0 3-HN-CO-Ar
‘H-i3C (6iH)
and
iH-13C
con-
connectivities
5.56 3.98 3.82 2.50 4.70 (61) 1.54 (H-7a), 2.00
1.95 (H-7B)
2.96 1.98 (H-12a) 1.75 (H-12b)
1.80 (H-15a) 1.44 (H-15/1) 1.38(H-16a),(l.35)(H-16p) 1.72 0.78 2.70 (H-19a) 1.65 (H-198) 2.44 0.82 0.83 1.01 0.81 7.34 7.71 1.42 7.71 7.34 2.13 -
EXPERIMENTAL
The leaves of B. papillosa (dry wt 50 kg) were collected from the northern regions of Pakistan. The EtOH extracts of the airdried leaves was evapd to a gum. The total alkaloids were
685
obtained by extraction into 10% HOAc. Partial separation of the alkaloids was achieved by extraction with CHCI, at different pH values. The fraction obtained at pH 8.5 (20 g) was loaded on a silica gel column (1.0 kg). Elution with 96% CHCI,4% MeOH afforded fraction A (1.0 g). Fraction A was further purified by prep. TLC (silica gel) in C,H,-CHCI,-MeOH (20:20:3) to afford (-)-buxoxybenzamine as an amorphous solid (15 mg), [a];‘= -22” (MeOH); UV 1”,$” nm: 224; IR v!,!,f; cm-‘, 3400 (NH and OH); 1730 (ester carbonyl), 1640 (amide carbonyl) and 1540 (C=C); ‘H NMR (CDCI,+few drops of CD,OD, 400 MHz) 6: 0.78(38, s, Me), 0.81 (3H, s, Me), 0.82 (3H, d, J,,,,, =8.1 Hz, 21-Me), 0.83 (3H, s, Me), 1.01 (3H, s, Me), 1.98 (3H, s, OAc Me), 1.65 (lH, d, 5198,,9.=13.88 Hz, H-19&, 1.75 (lH, m, H-12a), 1.72 (lH, m, H-17), 1.99 (lH, m, H-12/3), 2.13 (6H, s, N(CH,),),2.44(1H,dd,J,,,,,=8.1 Hz,J,,,,,= 10.13 Hz,H-20), 2.50 (lH, d, J,,,,B=ll.O Hz, .I,,.,,=95 Hz, H-5), 2.70 (lH, d, J 16n.198=13.85 Hz H-19a), 2.96 (lH, d, J,,,~,z8=2.0 Hz, C-11) 3.82 (lH, br d, J 3=,aa=8.7 Hz,C-3a), 3.98(1H,d,J,,*,,=8.7 Hz, C-2~),4.70(1H,m, H-6/3), 5.56,(1H, br s, H-l), 6.28(1H,d,.lNH.a. =9.5 Hz, NH), 7.34-7.71 (5H, m, Ar-H); MS m/z (formula rel. int.,): 578.3721 (C,,H,,N,O,, calcd. 578.3729, (2)) 563.3480 (C 34H 47N 2O,,calcd, 563.3475,(4)), 175.1167(C,,H,,NO,calcd. 175.1259, (6)), 119.1013 (C,H,NO, calcd. 119.1102, (5)), 105.0331 (C,H,O, calcd. 105.0340, (lo)), 72.0813 (C,H,ON, calcd. 72.0810, (loo)). REFERENCES
1. A.-ur-Rahman, Alam, M., Ahmad, G. and Choudhary, M. I. (1988) Heterocycles 27, 89. 2. A.-ur-Rahman, Choudhary, M. I. and Nisa, M. (1985) Hetero&es, 23, 1951. M. I., A.-ur-Rahman, Freyer, A. J. and Shamma, 3. Choudhary, M. (1987) J. Nat. Prod. 50, 84. Alam, M. and Choudhary, M. I. (1988) 4. A.-ur-Rahman, Phytochemistry 27, 3342. Alam, M. and Choudhary, M. I. (1988) J. 5. A.-ur-Rahman, Nat. Prod. 51, 309. 6. Kupchan, S. M., Kennedy, R. M., Schleigh, W. R. and Ohta, G. (1967) Tetrahedron 23,4563. 7. Bhacca, N. S. and Williams, D. H. (1966) Application of NMR Spectroscopy in Organic Chemistry, p. 101. HoldenDay, San Francisco. (1986) Nuclear Magnetic Resonance Spectro8. A.-ur-Rahman scopy, p. 202. Springer, New York. (1989) One and Two-Dimensional NMR Spec9. A.-ur-Rahman troscopy, p. 359. Elsevier, Amsterdam. 10. Wailer, G. R. and Dermer, 0. C. (1980) Biochemical Applications of Mass Spectrometry, p. 783. Wiley Interscience, New York.
Acknowledgements-We thank Dr E. Stefanou (Dept of Chemistry, University of Crete, Greece), Dr P. Christen (Dept of Pharmacy, University of Geneva, Switzerland) for providing necessary spectral facilities; and Dr S. Kokkini (Deptof Botany, University of Thessaloniki, Greece) for identification of the plant material. REFERENCES
M. J. (1986) Plant.
1. VillBr, A., Jimtnez,
M. J. and Alcaraz, Med. Phytothb-. 10, 31.
2. Diaz, R. M., Garcia-Granados,
A., Moreno, E., Parra, A., Quevedo-Sarmiento, J., Saenz de Buruaga, A. and Saenz de Buruaga, J. M. (1988) Planta Med. 54, 301.
683
3. Gonzalez, A., Fraga, B., Hernandez, M., Luis, J. and Larruga, F. (1979) Biochem. Syst. Ecol. 7, 115. 4. Kooiman, P. (1972) Acta Bot. NeerJ. 21. 417. Theodossiou, ‘P. (i962) Trav. Sot. Ph&m. Montpeflier 21, 221. K.okkalou, E. and Kapetanidis, I. (1988) Pharm. Acta HeJu. 63, 170. Karl, C., Miiller, G. and Pedersen, P. A. (1976) Phytochemistry 15, 1084. Ansari, F. R., Ansari, W. H., Rahman, W., Seligman, O., Chari, V. M., Wagner, H. and asterdahl, B. G. (1979) Planta Med. 36, 196. 9. Rao, L. J. M., Krishna Kumari, G. N. and Prakasa Rao, N. S. (1983) Phytochemistry 22, 1058. P. K. and Thakur, R. S. (1986) Phytochemistry 10. Chaudhuri, 25, 1770. 11. Mabry, T. J., Markham, K. R. and Thomas, M. B. (1970) in The Systematic ldentijication Of Flauonoids. Springer, Heidelberg. G., Markham, K. R., Ramachan12. Chopin, J., Dellamonica, dran Nair, A. G. and Gunasegaran, R. (I 984) Phytochemistry 23. 2106. 13. H&borne, J. B. (1983) in Phytochemical Methods. Chapman & Hall, London. 14. Barberan, F. A. T., Thomas, F. and Ferreres, F. (1985) J. Nat. Prod. 48, 28.
Phytuchemisrry,Vol. 29, No. 2, pp. 683 -685, 1990.
0
Printedin Great Britain.
A STEROIDAL ATTA-UR-RAHMAN,*
ALKALOID
ZAHIDA IQBAL, MUZAFFAR H.E.J. Research
Institute
FROM BUXUS
ALAM, M. IQBAL CHOUDHARY,
of Chemistry,
University
of Karachi,
0031.-9422/90 %3.00+0.00 19gOPergamonPressplc
PAPILLOSA HABIB NASIR
Karachi-75270,
and
TALAT FATIMA
Pakistan
(Receioed in revised form 26 May 1989)
Key Word Index-Buxus
Abstract-A its structure
new steroidal elucidated.
papillosa; Buxaceae;
steroidal
alkaloid, (-)-buxoxybenzamine,
INTRODUCTION
Buxus papillosa, C. K. Schneider (Buxaceae) is a commonly grown shrub of northern Pakistan. A number of steroidal alkaloids have been reported from this plant [l-5]. Continuing our investigations on the alkaloids of various Buxus species, we report here the isolation and structure of a new steroidal alkaloid (-)-buxoxybenzamine (1) from the leaves of B. papillosu.
RESULTS AND DISCUSSJON
The ethanolic
extract of air-dried
leaves of B. papillosa
alkaloids;
buxoxybenzamine;
ZD-NMR
has been isolated form the leaves of Buxus papillosa and
was evaporated and partial separation of the alkaloids was carried out by extraction into chloroform at different pH values. The fraction obtained at pH 8.5 was loaded on a silica gel column. Elution was carried out with chloroform-methanol. Further purification of the fraction eluted in 96% chloroform-4% methanol by prep. TLC resulted in the isolation of 1. (-)-Buxoxybenzamine (l), C,,H,,N*O,, partially soluble in chloroform, showed UV absorption at 224 nm, characteristic of a secondary benzamidic chromophore [6]. The IR spectrum displayed absorptions at 3400 (OH and NH), 1730 (ester carbonyl), 1640 (amide carbonyl) and 1540 (C=C) cm-’ [3].
Short Reports
684
1
The ‘HNMR spectrum of 1 (CDCI,+few drops of CD,OD, 400 MHz) showed four tertiary methyl singlets at 60.78, 0.81, 0.83 and 1.01. A doublet at 60.82 (J,,,,O = 8.1 Hz) was assigned to the 21secondary methyl group. while the vicinally coupled C-20 methine proton appeared at 62.44 and was further coupled to the C-17~ proton which appeared at b 1.78. A three-proton singlet at 6 1.98 was due to the acetate methyl protons, while a 6H singlet at 62.13 was assigned to the N(Me), protons. A multiplet centred at 64.70 was assigned to the C-6 methine proton geminal to the r-acetoxy group [6], while the neighbouring C-5x proton appeared as a doublet of doublet at 62.50 (J,,, = 11.0 Hz, J,.,,=9.5 Hz). The characteristic [runs-diaxial coupling constant of 11.0 Hz indicated the r orientation of the C-6 acetoxy group. A broad doublet resonating at 63.93 (J28,3Z= 8.7 Hz) was assigned to the C-2fl proton geminal to the hydroxy group. A multiplet centred at 63.82 was assigned to the C-3 proton r to the amide nitrogen, while the NH proton resonated at 66.84 (J NH.3z= 9.5 Hz) as a broadened doublet. This doublet sharpened when the spectrum was run in pure CDCl,. An interesting doublet resonating at 62.96 (J 11,I2p= 2.0 Hz) was assigned to the C-12 proton geminal to the C-9,1 I-epoxide. The J value indicated that the epoxide has p-orientation, because in an r-orientation the C-11/I proton would have afforded a doublet with -3 5 Hz [7]. A doublet of doublet at (r 1.99 :;,r;;.‘;$= i3.8 Hz, J 12z., la = 2.0 Hz) was assigned to C12~ proton while the geminal C-12/I proton appeared at 6 1.75. A broad singlet at 65.56 was due to the C-l olefinic proton while multiplets centred at 67.34 (2H), 7.42 (1H) and 7.71 (2H) were assigned to the five aromatic protons at C-2’, C-6’, C-4’ and C-3’,C-5’ respectively. Two clean AB doublets at (i2.70 and 1.65 (J,9a,,98= 13.5 Hz) were due to the 19a and 19,%methylene protons. The assignments of the resonances to various protons were facilitated by homonuclear 2D J-resolved, COSY-45” and NOESY experiments [S, 93. In the COSY-45” spectrum coupling of C-l 1 H (62.96) was observed with C-19 rH (62.70), C-19 /IH (61.65) C-12aH (61.99) and C-l2/IH (61.75). Similarly C-3aH (63.82) was coupled with C-2BH (b4.00) as well as with the amide NH proton at 66.28. Its three-bond coupling with C-5srH was also observed. The assignment to C6 /3H at 64.70 was confirmed by its COSY-45’ interaction with C-5aH (fi2.50), the latter showing allylic coupling with the C-l vinylic proton at 65.56 and homoallylic
coupling with C-2 /IH at 6 3.98. The C-20 methine proton (62.44) showed a cross-peak with C-17 rH at 61.72. The upfield value of C-17xH suggested the lack of any electron withdrawing substituent at C-16. On irradiation at 63.98 (H-2/?). NOE was observed at H-32 (63.82) and at the C-l olefinic proton at S5.56. On the other hand when irradiation was carried out at 65.56, NOES were seen at 63.98 (H-2/$, 3.82 (H-3x) and 62.70 (H-19x). These results unambiguously indicate that the H-28 and H-3x are close in space. while the H-l olehnic proton is also in close proximity. The a-stereochemistry of the acetate group at C-6 can be deduced from the NOE results, as follows: on irradiation of the C-68 proton (fi4.70), NOES were seen at H-19/1 proton (62.70) and at the H-7% and /I protons (6 1.95 and 1.54). Moreover, when H-5% (62.50) is saturated no NOE was observed at 64.70 (H-6/I). This finding is consistent with the r-stereochemistry of the C-6 acetate group. The observed NOES are shown in Table 1. DEPT experiments established the multiplicity of the 13C signals (Table 2). Further insight into the structure of 1 was provided by 2D one-bond ‘H-“C correlation (HETCOR) experiment (Table 2) which permitted the assignment ofall the protonated carbons in the 13C NMR spectrum of 1. The presence of an epoxide function could be deduced from the signal at 362.48 which is consistent with a quaternary carbon of an oxirane system and assignable to C-9; the other carbon of the oxirane system resonated at 663.36 and was assigned to C-l 1. The high resolution mass spectrum of 1 showed the molecular ion at ml: 578.3721 corresponding to the molecular formula C35H50N205. indicating the presence of 12 double bond equivalents in the molecule. The [M] + was further confirmed by FAB and FD techniques. A peak at m/z 563.3480 was due to the loss of a methyl group from the molecular ion while a large peak at m/z 105.0331 corresponded to the benzoyl cation. Compound 1 showed a base peak at m,/z 72.0813 which arose by cleavage of the nitrogen-containing ring D side chain [lo]. A peak at m/z 175.1 167 was the result of a retroDiels-Alder cleavage of ring A. On the basis of the above spectroscopic studies. structure 1 was assigned to ( -)buxoxybenzamine.
Table 1. Connectivities established by NOE difference spectra ___--___ Proton irradiated
Chemical shift (d)
NOE connectivity (s)
“4, NOE
H-l
5.56
H-2/1
3.98
H-51 H-60
2.50 4.70
H-20 H-8/I
2.36 2.00
H-2p (3.98) H-3x (3.82) H-19/I(1.65) H-3x (3.82) H-l (5.56) H-31 (3.82) H-198(2.70) H-7/J(l.9.5) H-16/$(1.35) H-19/j (1.65) H-68 (4.70) 30-CH, (1.01) H-7/?(1.95)
10.0 6.0 7.5 7.0 5.0 5.0 8.0 5.0 1.8 3.0 5.0 6.0 3.0
Short Reports
Table
2. 13C NMR,
multiplicities (DEPT) nectivities (HETCOR)
C
(6°C)
DEPT
1 2 3 4 5 6 7 8 9 10 11 12
134.09 67.48 61.13 38.52 53.35 77.89 35.54 41.07 62.48 131.78 63.36 35.11
CH CH CH C CH CH CH, CH C c CH CH,
13 14 15
42.94 38.65 26.8
C C CH,
16 17 18 19
32.41 49.91 16.63 44.41
CH, CH Me CH,
61.51 9.70 17.29 27.22 17.01 134.41 128.33 126.95 131.42 126.95 128.33 39.56 21.61 169.98 169.76
CH Me Me Me Me c CH CH CH CH CH Me Me C C
20 21 30 31 32 1’ 2 3’ 4 5’ 6 -N (Me), O-XX& &Me<0 3-HN-CO-Ar
‘H-i3C (6iH)
and
iH-13C
con-
connectivities
5.56 3.98 3.82 2.50 4.70 (61) 1.54 (H-7a), 2.00
1.95 (H-7B)
2.96 1.98 (H-12a) 1.75 (H-12b)
1.80 (H-15a) 1.44 (H-15/1) 1.38(H-16a),(l.35)(H-16p) 1.72 0.78 2.70 (H-19a) 1.65 (H-198) 2.44 0.82 0.83 1.01 0.81 7.34 7.71 1.42 7.71 7.34 2.13 -
EXPERIMENTAL
The leaves of B. papillosa (dry wt 50 kg) were collected from the northern regions of Pakistan. The EtOH extracts of the airdried leaves was evapd to a gum. The total alkaloids were
685
obtained by extraction into 10% HOAc. Partial separation of the alkaloids was achieved by extraction with CHCI, at different pH values. The fraction obtained at pH 8.5 (20 g) was loaded on a silica gel column (1.0 kg). Elution with 96% CHCI,4% MeOH afforded fraction A (1.0 g). Fraction A was further purified by prep. TLC (silica gel) in C,H,-CHCI,-MeOH (20:20:3) to afford (-)-buxoxybenzamine as an amorphous solid (15 mg), [a];‘= -22” (MeOH); UV 1”,$” nm: 224; IR v!,!,f; cm-‘, 3400 (NH and OH); 1730 (ester carbonyl), 1640 (amide carbonyl) and 1540 (C=C); ‘H NMR (CDCI,+few drops of CD,OD, 400 MHz) 6: 0.78(38, s, Me), 0.81 (3H, s, Me), 0.82 (3H, d, J,,,,, =8.1 Hz, 21-Me), 0.83 (3H, s, Me), 1.01 (3H, s, Me), 1.98 (3H, s, OAc Me), 1.65 (lH, d, 5198,,9.=13.88 Hz, H-19&, 1.75 (lH, m, H-12a), 1.72 (lH, m, H-17), 1.99 (lH, m, H-12/3), 2.13 (6H, s, N(CH,),),2.44(1H,dd,J,,,,,=8.1 Hz,J,,,,,= 10.13 Hz,H-20), 2.50 (lH, d, J,,,,B=ll.O Hz, .I,,.,,=95 Hz, H-5), 2.70 (lH, d, J 16n.198=13.85 Hz H-19a), 2.96 (lH, d, J,,,~,z8=2.0 Hz, C-11) 3.82 (lH, br d, J 3=,aa=8.7 Hz,C-3a), 3.98(1H,d,J,,*,,=8.7 Hz, C-2~),4.70(1H,m, H-6/3), 5.56,(1H, br s, H-l), 6.28(1H,d,.lNH.a. =9.5 Hz, NH), 7.34-7.71 (5H, m, Ar-H); MS m/z (formula rel. int.,): 578.3721 (C,,H,,N,O,, calcd. 578.3729, (2)) 563.3480 (C 34H 47N 2O,,calcd, 563.3475,(4)), 175.1167(C,,H,,NO,calcd. 175.1259, (6)), 119.1013 (C,H,NO, calcd. 119.1102, (5)), 105.0331 (C,H,O, calcd. 105.0340, (lo)), 72.0813 (C,H,ON, calcd. 72.0810, (loo)). REFERENCES
1. A.-ur-Rahman, Alam, M., Ahmad, G. and Choudhary, M. I. (1988) Heterocycles 27, 89. 2. A.-ur-Rahman, Choudhary, M. I. and Nisa, M. (1985) Hetero&es, 23, 1951. M. I., A.-ur-Rahman, Freyer, A. J. and Shamma, 3. Choudhary, M. (1987) J. Nat. Prod. 50, 84. Alam, M. and Choudhary, M. I. (1988) 4. A.-ur-Rahman, Phytochemistry 27, 3342. Alam, M. and Choudhary, M. I. (1988) J. 5. A.-ur-Rahman, Nat. Prod. 51, 309. 6. Kupchan, S. M., Kennedy, R. M., Schleigh, W. R. and Ohta, G. (1967) Tetrahedron 23,4563. 7. Bhacca, N. S. and Williams, D. H. (1966) Application of NMR Spectroscopy in Organic Chemistry, p. 101. HoldenDay, San Francisco. (1986) Nuclear Magnetic Resonance Spectro8. A.-ur-Rahman scopy, p. 202. Springer, New York. (1989) One and Two-Dimensional NMR Spec9. A.-ur-Rahman troscopy, p. 359. Elsevier, Amsterdam. 10. Wailer, G. R. and Dermer, 0. C. (1980) Biochemical Applications of Mass Spectrometry, p. 783. Wiley Interscience, New York.