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Triterpenoids from the fresh fruit coats of Azadirachta indica
Phytochemistry,Vol. 31, No. 12. pp. 4275 4278, 1992 Printed in Great Britain.
0031 9422/92$5.00+ 0.00 Q 1992 Pergamon Press Ltd
TRITERPENOIDS FROM THE FRESH FRUIT COATS OF AZADlRACHTA INDICA BINA
S.
SIDDIQUI,
GHIASUDDI~,
SHAHEEN
FAIZI and SALIMUZZAMANSIDDIQW
H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75274 Pakistan (Received
in revised
form 23 April 1992)
Key Word Index-Azudirachta indica;Meliaceae; neem fruit coats; degraded triterpenoids; 5cl,l3a-androstone and 5a,l3u-17-oxa-androstone derivatives.
Abstract-Three novel degraded triterpenoids, desfurano-~adi~ione [7u-acetoxy-4,4,8-trimethyl-5cY-( lkMe)androsta-1,14-dien-3,16-dione],7a-acetoxy-4,4,8-t~methy1-5a-(13aMe~17-oxa-androsta-1,14-dien-3,16-dio~e and 7cracetoxy-4,4,8-trimethyl-5a-17-oxa-androsta-1,14-dien-3,16-dione which were isolated from the neutral fraction of the ethanolic extract of the fresh, undried uncrushed ripe neem fruit coats of Azadirachta indica are described. Their structures were elu~dated through spectroscopic methods. It is the first report of the isolation of such degraded triterpenoids of this skeleton from any plant source.
INTRODIXX’ION
In view of the enormous therapeutic and pesticidal importance Cl-33 attributed to Azadirachta indica A. Juss, commonly known as neem, widely distributed in Asia and Africa, chemical studies on its various parts have been carried out by various groups of workers [4]. As a result, a host of new constituents has been reported in the literature [4, 53. The present studies on the neutral fraction of the ethanolic extract of the fresh fruit coats have led to the isolation of three novel terpenoids, desfurano-azadiradione [7a-acetoxy-4,4,8-trimethyl-% (13~Me)-androsta-1,14-dien-3,16-dione] (I), 7a-acetoxy4,4,8-trimethyl-5a-(l3~Me)-17-oxa-androsta-l,l4-dien3,16-dione (2) and ?a-acetoxy-4,4,8-trimethyl-5a-17oxa-androsta-1,14-dien-3,16-dione (3) through solvent separation followed by chromatograpbic procedures.
0
0
/
0
&
‘.
“ .oAc
*.
BESL~TSANDDISCIJSSIDN 3
The EI and FD mass spectra of 1 showed a [M]’ at m/z 384 while the HR mass spectrum, [M]” 384.2304, gave the molecular formula as C,, H J2 0,. The presence of five quaternary methyl groups in the ‘HNMR speu trum at 6 1.28 f3H, H-18), 1.22 (3H, H-19), 1.08 (6H, H-28 and H-29) and 1.37 (3H, H-30) indicated its triterpenoidal nature. The spectral data (IR, UV, ‘H and 13CNMR) revealed that 1 has the same tetracyclic skeleton as that of azadiradione [6]_ Thus, the ring Al-en-3-one structure was evident from a pair of AB doublets at 67.14 and 5.88 (J1,2=J,,, = 10.19 Hz)in the ‘HNMR spectrum(TabIe l), signals of C-l, C-2 and C-3 at S 156.9, 126.0 and 204.1 in the 13C NMR spectrum (Table 2) and a fragment at m/z 137.0969 [C,H,,O] in the mass spectrum. H-7 and H-15 were observed at 65.28 (lH, t, J7,6. =J,,68 = 3.04 Hz) and 5.76 (sharp singlet), while two one-proton double doublets related to H-5 and H-9 resonated at 62.20 (J5,6. =2.68, J5,68 =12.16 Hz} and 2.51 (Jgs,,, =6.60, Js,rla = 12.21 Hz), respectively. The acetoxy
methyl was noted at 6 1.94 as a three-proton singlet. The molecular formufa and the 13C NMR spectrum demonstrated that 1 has 24 carbons, 22 of which are part of the skeleton while two are accounted for by the acetyl group, hence 1 is an ~tanortriter~oid. It was supported by the absence of the characteristic signals of a furan ring and H17, present in azadiradione [6] and other meliacins [3]. Two doublets at 62.25 and 2.29 (J= 15.93 Hz) were ascribed to C-17 methylene protons, while the signal due to C-17 was observed at 655.6 (CH,) in the ‘jCNMR spectrum (DEPT). All the assignments noted above were confirmed by hetero-COSY and COSY-45 spectra. In the light of these spectral data the structure of 1 has been deduced as a desfurano-azadiradione [4,4,8-trimethyl-Scr(13aMe)-~drosta-l,l4-dien-7~-~etoxy-3,16-dione], which was substantiated by the high resolution mass spectrum, which showed diagnostic fragments at m/z
4275
Table 1. ‘H NMR spectral data (6, ppm and J/Hz) of triterpenoids l-3 Azadiradione H
[6, 111
1
2
3
1
5
7.11 d J I, z 10.24 5.87 d J 2,f 10.24 2.20 dd
6r
J 5,6;1 2.92 J 5,6p 12.72 1.88 ddd
7.14d J,.,10.19 5.88 d J 2.1 10.19 2.20 dd J 5.6n 2.68 J5,68 12.16 1.87 m
7.11 d J,,,10.20 5.90 d J,,, 10.20 2.19 dd J 5.h 3.33 J 5,~ 12.16 1.82-2.12 m
7.13 d J,,,lO.ZO 5.90 d J 2,, 10.20 2.17 dd J f.6z 2.74 J s,w 13.24 1.88-2.09 m
68
J genr 15.0 J 6a.5 2.92 J 6a.7 2.40 1.92 ddd
2.02
1.82-2.12 m
1.88-2.09 m
7
J QPnI15.0 J 66.5 12.72 J 68.7 3.32 5.30 dd
9
J 7.6.2-40 J 7.68 3.32 2.48 dd
Ila.
J 9.1LZ 4.50 J 9.1 ,a 9.60 1.85 dddd
2
5.28
5.28 r
J
J,,,,=J,.6p
t ~,cia=J,.w 3.04
2.51 dd J 9.1 ,a 6.60
5.19 dd 3.03
57.6. 1.71 J ,,ap 3.74 2.31 m
JY.,,~ 12.21 1.83 m
2.41 dd J 9.1 Ia 5.56 J 9.1 i/3 12.58 1.82-2.12 m
118
J #em15.0 J 11a.y 4.50 J 11a.12a 2.90 J Ite,,zg 2.90 2.08 dddd
2.11 m
1.82-2.12 m
1.88-2.09 m
12a
J 9Pnl15.0 J 1,g.y 9.60 J ita.xzz 8.0 J ,,@.I28 4.0 1.84 ddd
1.83 m
1.82-2.12 m
1.88-2.09 M
I2 @
J jJ” 15.0 J ilx.llo 3.20 J lZa.lIS 8.0 2.05 ddd
2.01 m
1.82-2.12 m
1X8-2.09 m
15 17 17a
J BEIn15.0 J 1z/$.L*a 2.90 J *xi. 11s 4.0 5.86 s 3.41 br s ~-.
5.76 s
5.71 s ..
5.33 s -
17b 18
--. J spm15.93 1.02 s
19
2.25 d J Qe~15.93 2.29 d
1.88-2.09 m
._
1.28 s
1.62 s
1.60 s
1.24 s
1.22 s
1.19 s
1.18 s
21
7.46 ddd
_..
22
J P.I.ZS 1.84 Jz,.,, 0.80 J 2i.t~ 0.80 6.25 dd
23
J 22.23 1.84 522.21 0.80 7.41 t
28 29 30 OAc
J 23.22 =-I,,,,, 1.08 .Y 1.09 s 1.33 s 1.93 s
--_
-
.__
-_
1.08 s 1.09 s 1.34 s 1.98 s
1.06 s 1.10 s 1.31 s 2.06 s
1.84 1.08 s 1.08 s 1.37 s 1.94 s
Triterpenoids from Azadirachta in&a
4211
Table 2. r3C NMR spectral data of compounds l-3
c 1
2 3 4 5 6 7 8 9 10 11 12 13
Azadiradione Cll, 121
1
2
3
C
Azadiradione Cll, 121
157.4 125.8 203.8 44.0 46.0 23.3 73.8 44.5 38.1 39.9 15.7 30.2 41.9
156.9 126.0 204.1 43.9 46.2 23.5 14.4 44.1 37.9 39.9 15.7 30.8 44.7
156.2 126.2 203.8 44.2 466.4 23.6 73.3 44.6 38.9 39.5 15.1 29.9 86.4
156.7 126.5 204.0 44.0 47.3 22.7 73.3 44.3 38.3 40.0 18.4 31.9 87.2
14 15 16 17 18 19 20 21 22 23 28 29 30 WOMe OCOhje
192.2 123.2 204.8 60.6 26.9 18.9 118.4 141.5 111.1 142.6 26.3 21.2 26.1 169.4 20.8
1
2
3
195.1 124.6 207.4 55.1 29.4 19.0
183.7 113.6 172.0
* 111.7 169.0
27.2 19.0
27.5 19.5
27.0 21.3 26.5 169.4 20.9
27.0 21.3 26.9 169.4 21.2
27.5 21.2 26.9 168.5 20.3
*Weak signal.
122.0779 (CsH,,O; fragment a), 342.2207 (C,,H,OO,; fragment b), 324.2091 (CZZH2s02; [M-HOAc]+), 3O9.1810 (C2iH,,02; [M-HOAc-Me]), 274.1554 (C,,H,,O,;fragment c), 215.1478 (C,,H,,O; fragment d), 189.1281 (C,,H,,O; fragment e) and 163.1125 (C 1r H r5 0; fragment f ). The configuration of various centres of 1 has been established through 2D NOE (NOESY) experiment, which showed spatial connectivities of H-l with H-2 and H-19; H-9 with H-18 and H-5; H7 with H-30; and H-l.5 with H-7’and H-30. It is noteworthy that 1 is the first octanortriterpenoid hearing this nucleus ever isolated from any plant source, while there are a few reports of isoiation of pentanor- [7], hexanor- [S], and heptanortriterpenoids [9] possessing this nucleus. In dammarane triterpenoids, an octanortriterpenoid has heen reported earlier [lo]. 7~-A~toxy-4,4,8-trimethyl-5u~ 13crMe)-17-oxa-androsta-1,14-dien-3,16-dione (2) gave a CM]’ at m/z 386 (EI and FD mass spectra). The HR mass spectrum gave its molecular formula as Cz3H,,0, C386.21131. The mass and ‘HNMR spectra of 2 showed that it has the same composition of the A-C rings and C-15, C-16 as that of azadiradione [6] and 1. Thus in the 1H NMR spectrum of 2 (Table 1) two AB doublets at S7.11 and 5.90 (J1,2 =.i 2,i = 10.20 Hz) for H-l and H-2, respectively, the signals of C-l, C-2 and C-3 at 6 156.2, 126.2 and 203.8 in the 13CNMR spectrum and a fragment at m/z 137.0967 [C9Hi30, fragment a] in the mass spectrum showed the presence of a ring A I-en-3-one system. Furthermore, a sharp singlet at 65.71 (lH), two one-proton double doubletsat~2.19(~~,~~=3.33,~~.~~=12.16Hz)and2.41 (J 9,11a=5.%J~.,Is = 12.58 Hz), a one-proton triplet at = 3.03 Hz) and a three-proton singlet 65.28 (57 ea= JT,.SB at 61.98 displayed the presence of H-15, H-5, H-9, H-7 and C-7 acetoxy methyl signals, respectively, as in azadiradione [6]. The ‘HNMR spectrum further showed five quaternary methyl singlets at 61.62 (H-18), 1.19 (H-19), 1.08 (H-28), 1.09 (H-29) and 1.34 (H-30). The molecular formula indicated that 2 has one carbon less than 1 and has five oxygen atoms, instead of four in 1. As four oxygen atoms were accounted for by the ring A I-en-3-one
system, the acetoxy group and the C-16 carbonyl function, the remaining oxygen atom was assigned as part of a lactone system in ring D (IR Y,,: 1755-1720, cm-‘), since no further unsaturation and/or carbinylic proton was indicated by the mass and ‘H NMR spectra. This was supported by the downfield shifts of H-18 as compared to that of 1 and azadiradione [ 111, and appearance of a quaternary carbon at 686.4 in the i3CNMR spectrum. The structure was corroborated by the high resolution mass spectrum which showed fragments at m/z 327.1923 343.1919 (C,,H,,O,; fragment b), (C2rH,,0J; [M-OAc] +), 326.1884 (C,,H,,O,; [M -HOAc]+), 233.1559 (CiSH,,02; fragment c) and 230.1683 (C,,H,,O; fragment d). The stereochemistry of various centres of 2 was also established through a 2D NOE (NOESY) experiment which showed the connectivities of H-5 with H-28; H-9 with H-18; H-7 with H-30; H15 with H-7 and H-30; H-30 with H-19; and H-29 with H19. The spectral data (IR, W, mass, 1H and 13C NMR) of 3 showed its close similarity with 2. However, a marked difference in the chemical shift of H-15 was noted which appeared at 65.33 in 3 as against 65.76 and 5.71 in 1 and 2, respectively. That this was due to a difference in configuration at C-13 was revealed by NOESY interactions of H-19 with H-29 and H-30; and H-18 with H-30 which demonstrated that in 3, the C-13 methyl is /Ioriented. The exact assignment of carbons particularly those of the methyl signals are based on HMQC spectroscopy. In the light of these spectral data the structure of 3 is 7cr-acetoxy-4,4,8-trimethyl-5cr-17-oxa-androsta-1,14dien-3,16-dione. EXPERIMENTAL
Mps: uncorr. IR: CHCI,; UV: in MeOH. Optical rotations: 27” in CHCI,. ’ H NMR spectra: in CDCS, at 400 MHz; ‘“CNMR spectra: (broad band and DEPT): in CDC13 at 75 MHz. Chemical shifts are recorded in ppm (6) and coupling constants(J) are in Hz. Exact assignments of *H NMR chemical shifts of l-3 were made through double resonance experiments
4278
B. S. SIDDIQUIet al.
and 2D studies (NOESY, COSY-45, hetero-COSY and HMQC). i3CNMR spectral assignments have been made partly through DEPT and hetero-COSY (for compounds 1 and 2) and HMQC (for 3) spectra and partly through a comparison of chemical shifts with those reported for azadiradione [ll, 123. Vacuum liquid [13, 141 and Rash CC [IS] were done with E. Merck Kieselpl GFzs4 and E. Merck 9385 silica gel, respectively. The purity of samples was checked on silica gel GF,,, coated glass plates, which were examined under the UV Iight, and with I, vapours. HPLC (Shimadzu) was performed in the reverse phase mode (Cis, Techspher, 50 DS, 30 cm x 10 mrm mobile phase 70% MeOH-H,O (v/v) + few drops of HOAc; loop 20 ~1,flow rate 3 ml _ ’ ). fsdution ofcompounds1-3. The thi~kish residue obtained on removal of the solvent from the combined ethanohc extracts of fresh, undried, uncrushed ripe neem fruit coats (20 kg) in uacuo, was partitioned between EtOAc and H,O. The EtOAc layer was extracted with 4% aq. Na,CO, soln to separate the acidic from the neutral fr. The EtOAc phase was washed, dried (Na,SO,) and evapd in vacua. The residue from the neutral fraction was successively treated with petrol and Et,O. Et,0 soluble portion was treated with 90% MeOH after addition of petrol. The 90% MeOH phase was extracted with EtOAc after addition of saline H,O. The EtOAc phase was washed, dried (Nar,SO,) and freed of the solvent affording 200 g of neutral gummy residue, 180 g of which was subjected to vacuum liquid chromatography [13,14] (petrol, petrol-EtOAc in order of increasing polarity). The petrol-EtOAc (1:4,1:9), EtOAc and EtOAc-MeOH (9: 1,4: 1, 7 : 3 and 2 : 3) eluates were combined together on the basis of TLC to give fraction E (3Og) which was again subjected to VLC (CHCI,-MeOH in order of increasing polarity). The CHCl,-MeOH (99: 1) eluates furnished various frs. As frs 5 and 6 showed the same spots on TLC, they were combined together affording fr. G56 (5.2 g), which was again subjected to flash CC [lS] (petrol, petrol-EtOAc in order of increasing polarity). The petrol-EtOAc (31:9) eluate (0.253 g), showed a single spot on TLC. However, its ’ H NMR spectrum showed that it was a mixture of at least 4 compounds which were sepd by reversed phase HPLC to afford 3 new constituents; namely desfuranoaxadiradione [7a-acetoxy-4,4,8-trimethyl-5a-( 13aMe)-androstal,l~dien-3,l~dione, l] flowers of needles, 8.5mg, mp 226- 231”) 7a-acetoxy-4,4,8-trimethyl-Sa-(13aMe)-l7-oxa-androsta-l,l4dien-3,16-dione (2) (amorphous powder, 57.2 mg) and 7aacetoxy-4,4,8-trimethyl-5a-17-oxa-androsta-l.l4-dien-3,16dione (3) (amorphous powder, 35 mg). Des~ra~o-az~diradio~e
(7a-acetoxy-4,4,8-
tr~methy~-5a-
(13aMe)-androsta-1,14-dien-3,16&one) (1). [a];’ -51.7” (CHCI,). FDMS m/z 384; HRMS m/z (rel. int.): [M]’ 384.2304 [talc. for C,,H,,O,, 384.23001 (lOO), 369.2077 [C,,H,,O,] (5), 342.2207 (CaaH,,O,; fragment bf (19), 324.2091 [C,,H,,O,] (72X 309.1810 (C,,H,,O,) (171, 296.2090 (C,,H2,0)(8),274.1S54(C,,H,,O,;fragmentc)(9),255.169O (C,,H,,0)(14),215.1478(C,,H,,0;fragmentd)(6~201.1335 [C,,H,,O](l2), 189.1281 (C,,H,,O,fragmente)(7), 163.1125 (CriHts0; fragment f) (17), 122.0779 (CsH,,O; fragment a) (81, 121.0697 [C,H,O] f26), and 60.0169 [C,H,O,] (6). ‘H (Table 1) and i3CNMR (Table 2). UV $$‘” nm: 235.2; IR v~~~‘3crn-‘, 1725-1710, 1620 and 1380.
7a-Acetoxy-4,4,8-trimethyl-5a~13aMe)-l7-oxa-androsta-l,14dien-3,16-dione (2). [a]P+66.7” (CHCI,). FDMS m/z 386: HRMS m/z (rel. int.): [M]’ 386.2113 [talc. for C2sHa00s.
386.20931 (57), 344.1998 [C&H,,O,] (18), 343.1919 (C2,Hs70,; fragment b) (2l), 326.1884 [C,,H,,O,] (lOO), 239.1306 [C13H1904] (27), 233.1559 (C,,H,,Or; fragment c) (15), 230.1683 (C,,H,,O, fragment d) (12) and 137.0967 (C~H,~~fra~enta)(l3). ‘H(Tabiel)and t3CNMR(Table 2). IR vFz’3 cm -I: 1755-1720, 1665-1650, 1595, and 1365. 7a-Acetoxy-4,4,8-trimethyl-5a-l7-oxa-androsta-l,l4-dien-
3,16-dione (3). [a];‘+ SO”(CHCI,). FDMS m/z 386: HRMS m/z (rel. int.): [M]’ 386.2114 [talc. for C 23H 300 51386.20931 (36), 327.1941 [C,,H,,O,] (25), 326.1883 [C2iHZ603] (lOOk 258.1275 [CLbH1s03] (13), 232.1488 [C,,H,,O,] (19), 214.1391 [C,,H,sO] (14), 121.0641 [C,H,O] (S), and 73.0307 [C,H,O,] (15). ‘H (Table 1) and 13CNMR (Table 2); UV l.:z$ nm: 227.6 nm >IR ,,cnCI,cm- 1.. 1740-1710, !660-1630, 1595 and 1365. ltl*x Acknowledgement-The Pakistan Agricultural Research Council is gratefully acknowledged for the provision of a research grant during the course of these studies.
REFERENCES 1. Dymock, W., Warden, C. J. H. and Hooper, D. (1890) Phurmucogruphiu Indica Vol. 1, p. 322 The Institute of
2. 3.
4. 5. 6.
Health and Tibbi Research, republished under the auspices of Hamdard National Foundation, Pakistan. Chopra, R. N., Nayar, S. L. and Chopra, I. C. (1956) Glossary of lndiun ~ed~~~~ Plants, p. 31. C.S.I.R. New Delhi, India. S&mutterer, H. and Ascher, K. R. S. (eds) (1988) Natural Pesticides from Neem Tree and other Tropical Plants. Proceedings of the 3rd International Neem Conference, Kenya, Africa July 1986, Deutsche Gesellschaft fiir Technische Zusammenarbeit, Eschborn, Federal Republic of Germany. Siddiqui, S., Siddiqui, B. S., Faizi, S. and Mahmood, T. (1988) J. Nat. Prod. 51, 30. Ara, I., Siddiqui, B. S., Faizi, S. and Siddiqui, S. (1989) .J. Chem. Sot., Perkin Trans f 343. Lavie, I)., Levy, E. C. and Jain, M. K. (1971) Tetrahedron 27, 3927.
7. Kraus, W. and Cramer, R. (1981) Chem. Ber. 1112375. 8. Siddiqui, S., Mahmood, T., Faizi, S. and Siddiqui, B. S. (1987) .I. Chem. SW. Perkin Trans I 1429. 9. Tchouankeu, J. C., Tsamo, E., Sondengam, B. L., Connolly, J. D. and Rycroft, D. S. (1990) Tetrahedron Letters 31,4505. 10. Provan, G. J. and Waterman, P. G. (1986) Phytochemistry 25,917.
11. Siddiqui, S., Siddiqui, B. S., Ghiasuddin and Faizi, S. (1990) Proc. Pakistan Acad. Sci. 27, 333. 12. Kraus, W. and Cramer, R. (1978) Tetrahedron Letters 2395. 13. Pelletier, S. W., Chokhi, H. P. and Desai, H. K. ( 1986) J. Nat. Prod. 49,892.
14. Coil, J. C. and Bowden, B. F. (1986) J. Nat. Prod. 49,934. 15. Still, W. C., Kahn, M. and Mitra, A. (1978) J. Org. Chem. 43, 2923.
0031 9422/92$5.00+ 0.00 Q 1992 Pergamon Press Ltd
TRITERPENOIDS FROM THE FRESH FRUIT COATS OF AZADlRACHTA INDICA BINA
S.
SIDDIQUI,
GHIASUDDI~,
SHAHEEN
FAIZI and SALIMUZZAMANSIDDIQW
H.E.J. Research Institute of Chemistry, University of Karachi, Karachi 75274 Pakistan (Received
in revised
form 23 April 1992)
Key Word Index-Azudirachta indica;Meliaceae; neem fruit coats; degraded triterpenoids; 5cl,l3a-androstone and 5a,l3u-17-oxa-androstone derivatives.
Abstract-Three novel degraded triterpenoids, desfurano-~adi~ione [7u-acetoxy-4,4,8-trimethyl-5cY-( lkMe)androsta-1,14-dien-3,16-dione],7a-acetoxy-4,4,8-t~methy1-5a-(13aMe~17-oxa-androsta-1,14-dien-3,16-dio~e and 7cracetoxy-4,4,8-trimethyl-5a-17-oxa-androsta-1,14-dien-3,16-dione which were isolated from the neutral fraction of the ethanolic extract of the fresh, undried uncrushed ripe neem fruit coats of Azadirachta indica are described. Their structures were elu~dated through spectroscopic methods. It is the first report of the isolation of such degraded triterpenoids of this skeleton from any plant source.
INTRODIXX’ION
In view of the enormous therapeutic and pesticidal importance Cl-33 attributed to Azadirachta indica A. Juss, commonly known as neem, widely distributed in Asia and Africa, chemical studies on its various parts have been carried out by various groups of workers [4]. As a result, a host of new constituents has been reported in the literature [4, 53. The present studies on the neutral fraction of the ethanolic extract of the fresh fruit coats have led to the isolation of three novel terpenoids, desfurano-azadiradione [7a-acetoxy-4,4,8-trimethyl-% (13~Me)-androsta-1,14-dien-3,16-dione] (I), 7a-acetoxy4,4,8-trimethyl-5a-(l3~Me)-17-oxa-androsta-l,l4-dien3,16-dione (2) and ?a-acetoxy-4,4,8-trimethyl-5a-17oxa-androsta-1,14-dien-3,16-dione (3) through solvent separation followed by chromatograpbic procedures.
0
0
/
0
&
‘.
“ .oAc
*.
BESL~TSANDDISCIJSSIDN 3
The EI and FD mass spectra of 1 showed a [M]’ at m/z 384 while the HR mass spectrum, [M]” 384.2304, gave the molecular formula as C,, H J2 0,. The presence of five quaternary methyl groups in the ‘HNMR speu trum at 6 1.28 f3H, H-18), 1.22 (3H, H-19), 1.08 (6H, H-28 and H-29) and 1.37 (3H, H-30) indicated its triterpenoidal nature. The spectral data (IR, UV, ‘H and 13CNMR) revealed that 1 has the same tetracyclic skeleton as that of azadiradione [6]_ Thus, the ring Al-en-3-one structure was evident from a pair of AB doublets at 67.14 and 5.88 (J1,2=J,,, = 10.19 Hz)in the ‘HNMR spectrum(TabIe l), signals of C-l, C-2 and C-3 at S 156.9, 126.0 and 204.1 in the 13C NMR spectrum (Table 2) and a fragment at m/z 137.0969 [C,H,,O] in the mass spectrum. H-7 and H-15 were observed at 65.28 (lH, t, J7,6. =J,,68 = 3.04 Hz) and 5.76 (sharp singlet), while two one-proton double doublets related to H-5 and H-9 resonated at 62.20 (J5,6. =2.68, J5,68 =12.16 Hz} and 2.51 (Jgs,,, =6.60, Js,rla = 12.21 Hz), respectively. The acetoxy
methyl was noted at 6 1.94 as a three-proton singlet. The molecular formufa and the 13C NMR spectrum demonstrated that 1 has 24 carbons, 22 of which are part of the skeleton while two are accounted for by the acetyl group, hence 1 is an ~tanortriter~oid. It was supported by the absence of the characteristic signals of a furan ring and H17, present in azadiradione [6] and other meliacins [3]. Two doublets at 62.25 and 2.29 (J= 15.93 Hz) were ascribed to C-17 methylene protons, while the signal due to C-17 was observed at 655.6 (CH,) in the ‘jCNMR spectrum (DEPT). All the assignments noted above were confirmed by hetero-COSY and COSY-45 spectra. In the light of these spectral data the structure of 1 has been deduced as a desfurano-azadiradione [4,4,8-trimethyl-Scr(13aMe)-~drosta-l,l4-dien-7~-~etoxy-3,16-dione], which was substantiated by the high resolution mass spectrum, which showed diagnostic fragments at m/z
4275
Table 1. ‘H NMR spectral data (6, ppm and J/Hz) of triterpenoids l-3 Azadiradione H
[6, 111
1
2
3
1
5
7.11 d J I, z 10.24 5.87 d J 2,f 10.24 2.20 dd
6r
J 5,6;1 2.92 J 5,6p 12.72 1.88 ddd
7.14d J,.,10.19 5.88 d J 2.1 10.19 2.20 dd J 5.6n 2.68 J5,68 12.16 1.87 m
7.11 d J,,,10.20 5.90 d J,,, 10.20 2.19 dd J 5.h 3.33 J 5,~ 12.16 1.82-2.12 m
7.13 d J,,,lO.ZO 5.90 d J 2,, 10.20 2.17 dd J f.6z 2.74 J s,w 13.24 1.88-2.09 m
68
J genr 15.0 J 6a.5 2.92 J 6a.7 2.40 1.92 ddd
2.02
1.82-2.12 m
1.88-2.09 m
7
J QPnI15.0 J 66.5 12.72 J 68.7 3.32 5.30 dd
9
J 7.6.2-40 J 7.68 3.32 2.48 dd
Ila.
J 9.1LZ 4.50 J 9.1 ,a 9.60 1.85 dddd
2
5.28
5.28 r
J
J,,,,=J,.6p
t ~,cia=J,.w 3.04
2.51 dd J 9.1 ,a 6.60
5.19 dd 3.03
57.6. 1.71 J ,,ap 3.74 2.31 m
JY.,,~ 12.21 1.83 m
2.41 dd J 9.1 Ia 5.56 J 9.1 i/3 12.58 1.82-2.12 m
118
J #em15.0 J 11a.y 4.50 J 11a.12a 2.90 J Ite,,zg 2.90 2.08 dddd
2.11 m
1.82-2.12 m
1.88-2.09 m
12a
J 9Pnl15.0 J 1,g.y 9.60 J ita.xzz 8.0 J ,,@.I28 4.0 1.84 ddd
1.83 m
1.82-2.12 m
1.88-2.09 M
I2 @
J jJ” 15.0 J ilx.llo 3.20 J lZa.lIS 8.0 2.05 ddd
2.01 m
1.82-2.12 m
1X8-2.09 m
15 17 17a
J BEIn15.0 J 1z/$.L*a 2.90 J *xi. 11s 4.0 5.86 s 3.41 br s ~-.
5.76 s
5.71 s ..
5.33 s -
17b 18
--. J spm15.93 1.02 s
19
2.25 d J Qe~15.93 2.29 d
1.88-2.09 m
._
1.28 s
1.62 s
1.60 s
1.24 s
1.22 s
1.19 s
1.18 s
21
7.46 ddd
_..
22
J P.I.ZS 1.84 Jz,.,, 0.80 J 2i.t~ 0.80 6.25 dd
23
J 22.23 1.84 522.21 0.80 7.41 t
28 29 30 OAc
J 23.22 =-I,,,,, 1.08 .Y 1.09 s 1.33 s 1.93 s
--_
-
.__
-_
1.08 s 1.09 s 1.34 s 1.98 s
1.06 s 1.10 s 1.31 s 2.06 s
1.84 1.08 s 1.08 s 1.37 s 1.94 s
Triterpenoids from Azadirachta in&a
4211
Table 2. r3C NMR spectral data of compounds l-3
c 1
2 3 4 5 6 7 8 9 10 11 12 13
Azadiradione Cll, 121
1
2
3
C
Azadiradione Cll, 121
157.4 125.8 203.8 44.0 46.0 23.3 73.8 44.5 38.1 39.9 15.7 30.2 41.9
156.9 126.0 204.1 43.9 46.2 23.5 14.4 44.1 37.9 39.9 15.7 30.8 44.7
156.2 126.2 203.8 44.2 466.4 23.6 73.3 44.6 38.9 39.5 15.1 29.9 86.4
156.7 126.5 204.0 44.0 47.3 22.7 73.3 44.3 38.3 40.0 18.4 31.9 87.2
14 15 16 17 18 19 20 21 22 23 28 29 30 WOMe OCOhje
192.2 123.2 204.8 60.6 26.9 18.9 118.4 141.5 111.1 142.6 26.3 21.2 26.1 169.4 20.8
1
2
3
195.1 124.6 207.4 55.1 29.4 19.0
183.7 113.6 172.0
* 111.7 169.0
27.2 19.0
27.5 19.5
27.0 21.3 26.5 169.4 20.9
27.0 21.3 26.9 169.4 21.2
27.5 21.2 26.9 168.5 20.3
*Weak signal.
122.0779 (CsH,,O; fragment a), 342.2207 (C,,H,OO,; fragment b), 324.2091 (CZZH2s02; [M-HOAc]+), 3O9.1810 (C2iH,,02; [M-HOAc-Me]), 274.1554 (C,,H,,O,;fragment c), 215.1478 (C,,H,,O; fragment d), 189.1281 (C,,H,,O; fragment e) and 163.1125 (C 1r H r5 0; fragment f ). The configuration of various centres of 1 has been established through 2D NOE (NOESY) experiment, which showed spatial connectivities of H-l with H-2 and H-19; H-9 with H-18 and H-5; H7 with H-30; and H-l.5 with H-7’and H-30. It is noteworthy that 1 is the first octanortriterpenoid hearing this nucleus ever isolated from any plant source, while there are a few reports of isoiation of pentanor- [7], hexanor- [S], and heptanortriterpenoids [9] possessing this nucleus. In dammarane triterpenoids, an octanortriterpenoid has heen reported earlier [lo]. 7~-A~toxy-4,4,8-trimethyl-5u~ 13crMe)-17-oxa-androsta-1,14-dien-3,16-dione (2) gave a CM]’ at m/z 386 (EI and FD mass spectra). The HR mass spectrum gave its molecular formula as Cz3H,,0, C386.21131. The mass and ‘HNMR spectra of 2 showed that it has the same composition of the A-C rings and C-15, C-16 as that of azadiradione [6] and 1. Thus in the 1H NMR spectrum of 2 (Table 1) two AB doublets at S7.11 and 5.90 (J1,2 =.i 2,i = 10.20 Hz) for H-l and H-2, respectively, the signals of C-l, C-2 and C-3 at 6 156.2, 126.2 and 203.8 in the 13CNMR spectrum and a fragment at m/z 137.0967 [C9Hi30, fragment a] in the mass spectrum showed the presence of a ring A I-en-3-one system. Furthermore, a sharp singlet at 65.71 (lH), two one-proton double doubletsat~2.19(~~,~~=3.33,~~.~~=12.16Hz)and2.41 (J 9,11a=5.%J~.,Is = 12.58 Hz), a one-proton triplet at = 3.03 Hz) and a three-proton singlet 65.28 (57 ea= JT,.SB at 61.98 displayed the presence of H-15, H-5, H-9, H-7 and C-7 acetoxy methyl signals, respectively, as in azadiradione [6]. The ‘HNMR spectrum further showed five quaternary methyl singlets at 61.62 (H-18), 1.19 (H-19), 1.08 (H-28), 1.09 (H-29) and 1.34 (H-30). The molecular formula indicated that 2 has one carbon less than 1 and has five oxygen atoms, instead of four in 1. As four oxygen atoms were accounted for by the ring A I-en-3-one
system, the acetoxy group and the C-16 carbonyl function, the remaining oxygen atom was assigned as part of a lactone system in ring D (IR Y,,: 1755-1720, cm-‘), since no further unsaturation and/or carbinylic proton was indicated by the mass and ‘H NMR spectra. This was supported by the downfield shifts of H-18 as compared to that of 1 and azadiradione [ 111, and appearance of a quaternary carbon at 686.4 in the i3CNMR spectrum. The structure was corroborated by the high resolution mass spectrum which showed fragments at m/z 327.1923 343.1919 (C,,H,,O,; fragment b), (C2rH,,0J; [M-OAc] +), 326.1884 (C,,H,,O,; [M -HOAc]+), 233.1559 (CiSH,,02; fragment c) and 230.1683 (C,,H,,O; fragment d). The stereochemistry of various centres of 2 was also established through a 2D NOE (NOESY) experiment which showed the connectivities of H-5 with H-28; H-9 with H-18; H-7 with H-30; H15 with H-7 and H-30; H-30 with H-19; and H-29 with H19. The spectral data (IR, W, mass, 1H and 13C NMR) of 3 showed its close similarity with 2. However, a marked difference in the chemical shift of H-15 was noted which appeared at 65.33 in 3 as against 65.76 and 5.71 in 1 and 2, respectively. That this was due to a difference in configuration at C-13 was revealed by NOESY interactions of H-19 with H-29 and H-30; and H-18 with H-30 which demonstrated that in 3, the C-13 methyl is /Ioriented. The exact assignment of carbons particularly those of the methyl signals are based on HMQC spectroscopy. In the light of these spectral data the structure of 3 is 7cr-acetoxy-4,4,8-trimethyl-5cr-17-oxa-androsta-1,14dien-3,16-dione. EXPERIMENTAL
Mps: uncorr. IR: CHCI,; UV: in MeOH. Optical rotations: 27” in CHCI,. ’ H NMR spectra: in CDCS, at 400 MHz; ‘“CNMR spectra: (broad band and DEPT): in CDC13 at 75 MHz. Chemical shifts are recorded in ppm (6) and coupling constants(J) are in Hz. Exact assignments of *H NMR chemical shifts of l-3 were made through double resonance experiments
4278
B. S. SIDDIQUIet al.
and 2D studies (NOESY, COSY-45, hetero-COSY and HMQC). i3CNMR spectral assignments have been made partly through DEPT and hetero-COSY (for compounds 1 and 2) and HMQC (for 3) spectra and partly through a comparison of chemical shifts with those reported for azadiradione [ll, 123. Vacuum liquid [13, 141 and Rash CC [IS] were done with E. Merck Kieselpl GFzs4 and E. Merck 9385 silica gel, respectively. The purity of samples was checked on silica gel GF,,, coated glass plates, which were examined under the UV Iight, and with I, vapours. HPLC (Shimadzu) was performed in the reverse phase mode (Cis, Techspher, 50 DS, 30 cm x 10 mrm mobile phase 70% MeOH-H,O (v/v) + few drops of HOAc; loop 20 ~1,flow rate 3 ml _ ’ ). fsdution ofcompounds1-3. The thi~kish residue obtained on removal of the solvent from the combined ethanohc extracts of fresh, undried, uncrushed ripe neem fruit coats (20 kg) in uacuo, was partitioned between EtOAc and H,O. The EtOAc layer was extracted with 4% aq. Na,CO, soln to separate the acidic from the neutral fr. The EtOAc phase was washed, dried (Na,SO,) and evapd in vacua. The residue from the neutral fraction was successively treated with petrol and Et,O. Et,0 soluble portion was treated with 90% MeOH after addition of petrol. The 90% MeOH phase was extracted with EtOAc after addition of saline H,O. The EtOAc phase was washed, dried (Nar,SO,) and freed of the solvent affording 200 g of neutral gummy residue, 180 g of which was subjected to vacuum liquid chromatography [13,14] (petrol, petrol-EtOAc in order of increasing polarity). The petrol-EtOAc (1:4,1:9), EtOAc and EtOAc-MeOH (9: 1,4: 1, 7 : 3 and 2 : 3) eluates were combined together on the basis of TLC to give fraction E (3Og) which was again subjected to VLC (CHCI,-MeOH in order of increasing polarity). The CHCl,-MeOH (99: 1) eluates furnished various frs. As frs 5 and 6 showed the same spots on TLC, they were combined together affording fr. G56 (5.2 g), which was again subjected to flash CC [lS] (petrol, petrol-EtOAc in order of increasing polarity). The petrol-EtOAc (31:9) eluate (0.253 g), showed a single spot on TLC. However, its ’ H NMR spectrum showed that it was a mixture of at least 4 compounds which were sepd by reversed phase HPLC to afford 3 new constituents; namely desfuranoaxadiradione [7a-acetoxy-4,4,8-trimethyl-5a-( 13aMe)-androstal,l~dien-3,l~dione, l] flowers of needles, 8.5mg, mp 226- 231”) 7a-acetoxy-4,4,8-trimethyl-Sa-(13aMe)-l7-oxa-androsta-l,l4dien-3,16-dione (2) (amorphous powder, 57.2 mg) and 7aacetoxy-4,4,8-trimethyl-5a-17-oxa-androsta-l.l4-dien-3,16dione (3) (amorphous powder, 35 mg). Des~ra~o-az~diradio~e
(7a-acetoxy-4,4,8-
tr~methy~-5a-
(13aMe)-androsta-1,14-dien-3,16&one) (1). [a];’ -51.7” (CHCI,). FDMS m/z 384; HRMS m/z (rel. int.): [M]’ 384.2304 [talc. for C,,H,,O,, 384.23001 (lOO), 369.2077 [C,,H,,O,] (5), 342.2207 (CaaH,,O,; fragment bf (19), 324.2091 [C,,H,,O,] (72X 309.1810 (C,,H,,O,) (171, 296.2090 (C,,H2,0)(8),274.1S54(C,,H,,O,;fragmentc)(9),255.169O (C,,H,,0)(14),215.1478(C,,H,,0;fragmentd)(6~201.1335 [C,,H,,O](l2), 189.1281 (C,,H,,O,fragmente)(7), 163.1125 (CriHts0; fragment f) (17), 122.0779 (CsH,,O; fragment a) (81, 121.0697 [C,H,O] f26), and 60.0169 [C,H,O,] (6). ‘H (Table 1) and i3CNMR (Table 2). UV $$‘” nm: 235.2; IR v~~~‘3crn-‘, 1725-1710, 1620 and 1380.
7a-Acetoxy-4,4,8-trimethyl-5a~13aMe)-l7-oxa-androsta-l,14dien-3,16-dione (2). [a]P+66.7” (CHCI,). FDMS m/z 386: HRMS m/z (rel. int.): [M]’ 386.2113 [talc. for C2sHa00s.
386.20931 (57), 344.1998 [C&H,,O,] (18), 343.1919 (C2,Hs70,; fragment b) (2l), 326.1884 [C,,H,,O,] (lOO), 239.1306 [C13H1904] (27), 233.1559 (C,,H,,Or; fragment c) (15), 230.1683 (C,,H,,O, fragment d) (12) and 137.0967 (C~H,~~fra~enta)(l3). ‘H(Tabiel)and t3CNMR(Table 2). IR vFz’3 cm -I: 1755-1720, 1665-1650, 1595, and 1365. 7a-Acetoxy-4,4,8-trimethyl-5a-l7-oxa-androsta-l,l4-dien-
3,16-dione (3). [a];‘+ SO”(CHCI,). FDMS m/z 386: HRMS m/z (rel. int.): [M]’ 386.2114 [talc. for C 23H 300 51386.20931 (36), 327.1941 [C,,H,,O,] (25), 326.1883 [C2iHZ603] (lOOk 258.1275 [CLbH1s03] (13), 232.1488 [C,,H,,O,] (19), 214.1391 [C,,H,sO] (14), 121.0641 [C,H,O] (S), and 73.0307 [C,H,O,] (15). ‘H (Table 1) and 13CNMR (Table 2); UV l.:z$ nm: 227.6 nm >IR ,,cnCI,cm- 1.. 1740-1710, !660-1630, 1595 and 1365. ltl*x Acknowledgement-The Pakistan Agricultural Research Council is gratefully acknowledged for the provision of a research grant during the course of these studies.
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