Pentacyclic triterpenoids from the leaves of Plumeria obtusa

Phytochemistry, Vol. 31, No. 12, pp. 4279 4283, 1992 Printedin Great Britain.

PENTACYCLIC

1X131-9422:92$5.00+0.00 Q 1992 PergamonPress Ltd

TRITERPENOIDS

PLUMERIA SALIMUZZAMAN H.E.J.

FROM

THE LEAVES OF

OBTUSA

SIDDIQUI, BINA SHAHEEN SIDDIQUI, AKHTAR NAEED

and

SABIRA BEGUM

Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan (Received in revised form 24 March 1992)

Key Word Index-Plumeria

ohtusa; Apocynaceae; triterpenoids: 3/I-hydroxy-24-p-E-coumaroyloxyurs-12-en-28oic acid, 3P-hydroxy-27-p-Z-coumaroyloxyurs-12-en-30-oic acid.

Abstract-Nine pentacyclic triterpenoids along with a coumarin have been isolated from the fresh, undried and uncrushed spring leaves of Plumeria obtusa. The new triterpenes obtusin and obtusilic acid have been characterized as the 24-E and 27-Z p-coumaric esters of the novel 3b,24-dihydroxyurs-l2-en-28-oic acid and 3/?,27-dihydroxyurs-12en-30-oic acid, respectively, through chemical and spectral studies while the other eight compounds have been identified as known kaneroside, oleandrin, a-amyrin, neriucoumaric acid, isoneriucoumaric acid, alphitolic acid, oleanonic acid, methyl p-E-coumarate and scopoletin hitherto unreported from this source.

INTRODUCTION

In continuation of studies [l] on the fresh and uncrushed leaves of Plumeria obtusa two new pentacyclic triterpenoids 1 and 2, provisionally named as obtusin and obtusilic acid, have been isolated and their structures elucidated through chemical and spectral studies. Besides these, known constituents kaneroside [2], oleandrin [3, 41, &amyrin [S, 61, neriucoumaric acid, isoneriucoumaric acid [7], alphitolic acid [8, 91, oleanonic acid [lo, 111, methyl p-E-coumarate [ 123 and scopoletin [ 13, 141 have also been isolated and characterized from this plant for the first time.

RESCLTS AND DISCUSSION

Obtusin (1) did not show a molecular ion peak in the EI and FAB mass spectra and the molecular formula &,Hs406 was derived through exact measurement of various mass fragment ions and ’ 3C NMR spectroscopy (broad band, DEPT and heteroCOSY). Compound 1 showed IR absorptions at 35W2600 (br, OH and CO,H), 1738-1680 (acid and ester carbonyls) and l-1380 cm- ’ (four peaks, aromatic ring); while the UV spectrum showed maxima at 203 and 282 nm. The ‘H NMR spectrum in pyridine-d, (Table 1) exhibited two sets of AB doublets at 67.74 (lH, J = 15.6 Hz, H-3’), 6.37 (lH, J= 15.6 Hz, H-2’) and 7.52 (2H, J= 8.5 Hz, H-5’ and H-9’), 7.02 (2H, J= 8.5 Hz, H-6’ and H-8’). The chemical shifts and coupling constants of these doublets indicated the presence of a p-E-coumaroyloxy substituent which was also supported by the 13C NMR spectral values (Table 2) and high resolution mass spectrum which showed significant fragments at m/z 147.0489 (C,H,O,) and 164.0526 (C,H,O,) along with an ion at m/z 454.3460 (C30H,,03) resulting from the loss of p-coumaric acid from the molecular ion (Scheme 1). Besides the p-coumaroyloxy substituent, the ‘H NMR spectrum displayed resonances for an olefinic proton (6 5.45, t, J = 3.4 Hz, H-

12), two methylene protons (64.52, d J= 12.1 Hz, H-24a and 64.35, d, J= 12.1 Hz, H-24b), two characteristic methine protons at 63.33 (dd, J=9.6 and 4.4 Hz, H-3cr) and 62.55 (d, J = 11.4 Hz, H-18), four methyls located on tertiary carbons (6 1.08, 1.02,0.98 and 0.92 each 3H s) and two secondary methyls (6 1.05, J=6.0 Hz and 0.93, J =5.6 Hz each 3H doublet). Compound 1 formed the dimethyl derivative la (6OMe 3.57, 3.75, 2 x s) on with diazomethane confirming the reaction p-coumaroyloxy and carboxyl functions. Appearance of H-18 as a doublet (62.55 in the case of 1, pyridine-d, and 62.23 in the methyl ester la, CDCl,) and the characteristic retro-Diels-Alder fragmentation [15] of 1 (m/z 248.1783, C16HZ402 and 206.1664, C,,H,,O) along with the characteristic 13C NMR chemical shifts of C-12 and C-13 at 6126.5 and 138.1, respectively, suggested [16] that 1 belongs to the A12-ol-amyrin series of triterpenoids with the carboxylic group at C-17 and the hydroxyl and ester groups in the A/B rings. The relative intensities of the fragments at m/z 248 (100%) and 203 (48%) further supported the C-17 position of the carboxyl function [15]. One of the hydroxyl groups indicated by the ‘H NMR spectrum was placed at C-3 on biogenetic grounds. Its fl-disposition was evident from the chemical shift and coupling constants of H-3 [17]. The appearance of the methylene AB doublets at 64.52 and 4.35 permitted the placement of the ester function at C-24 since in case of its equatorial orientation (C-23) these would have appeared at 6 N 3.8 [18, 193. Thus the structure of obtusin (1) is 3/Ghydroxy-24-p-E-coumaroyloxyurs-12-en-28-oic acid; the corresponding free alcohol does not appear to have been isolated earlier. Obtusilic acid (2) showed IR absorptions at 3630-2600 (br, OH and CO,H), 17.50-1690 (ester and acid carbonyls) and 165&1560 cn-’ (aliphatic and aromatic C=C) and UV absorptions at 226 and 313.6 nm. It yielded a diacetyl derivative 2a (6OAc 2.28 s and 2.01 s, Table 1) on reaction with .acetic anhydride in pyridine which afforded the diacetylmonomethyl derivative 2b on treatment with

4279

S. SIDDIQUI er

4280

al.

Table 1. *H NMR spectral data (&,&pmand f/Hz) of triterpenes 1, la, 2, 2a and 2b H 3c(

12 18 19 20 24a 24b 27a 27b Me

2 3 5’ 6 8 9 OMe ArOMe ArOAc OAc

1

la

2

?‘a

2b

3.05 dd (11.3)

4.15

4.16 dd (11.3)

3.33 dd

3.11 dd

(9.6) (4.41 5.45 f (3.4) 2.55 d (11.4)

(9.5) (4.6) 5.26 t (3.6) 2.23 d (11.6)

4.52 d (12.1) 4.35 d {12.1}

4.38 d (12.2) 4.30 d (12.2)

0.92 s 0.93 d

0.93 s 0.97 d

(5.6)

0.98 s 1.02 s 1.05d

(5.8) 0.97 s 1.03 s 1.07 d

(6.0) 1.08 6.37 7.74 7.52 7.02 7.02 7.52 _. -

(6.0) 1.12 s 6.18 d 7.50 d 7.09 d 6.82 d 6.82 d 7.09 d 3.57 s 3.75 s

s d d d d d d

(15.6) (15.6) (8.5) (8.5) (8.5) (8.5)

(4.5) 5.51 t (3.5) 2.29 d (11.7) 1.22 In 1.90 M -4.41 4.12 0.75 0.82 0.90

d (12.7) d (12.7) s s d

(6.3) 0.92 s 0.93 s 0.94 s 5.71 d 6.84 d 7.62 d 6.75 d 6.75 d 7.62 d

(15.9) (15.9)

(8.6) (8.6) (8.6) (8.6)

(12.6) (12.6) (8.8) (8.8) (8.8) (8.8)

dd (11.2) (4.5) 5.52 1(3.5) 2.30 d (11.6)

(4.4) 5.51 t (3.4) 2.30 d (11.6) -.

-

.4.47 4.14 0.73 0.84 0.87

d (12.9) d (12.91 s s d

4.46 d (12.9) 4.13 d (12.9) 0.72 s 0.84 s 0.89 d

(6.6) 0.88 s 0.93 s 0.95 s

(6.4) 0.89 s 0.92 s 0.95 s

5.80 d (12.8) 6.85 d (12.8) 7.72 d (8.6)

5.86 6.85 7.73 6.84

d (12.5) d (12.5) d (8.5) d (8.5) 6.84 d (8.5) 7.73 d (8.5)

6.84 d (8.6) 6.84 d (8.6) 7.72 d (8.6)

3.61 s 2.28 s 2.03 s

2.28 s 2.01 s

-

30 :

&z 187.1476 t

- Hz0 133.1019

ml% 2OS.1620

1

- Ws

203.1790

?nfz 454.3460

-HI

I+&

ml2 164.0526

1

R H (mass fragmentscorrespoadto compound 1)

la

Me

Scheme 1.

diazomethane showing the presence of two hydroxyl groups-one aromatic (SOAc 2.28s)and the other aliphatic (6OAc 2.03s), and a carboxyl(60Me 3.61s,Table 1) function in the molecule. The ‘H NMR spectrum (Table 1) of 2 exhibited resonances for two sets of AB doublets at 66.84 (lH, J= 12.6 Hz, H-3’) and 65.71 (IH, J = 12.6 Hz, H-2’) and 67.62 (2H, J = 8.8 Hz, H-S and H-9’)

and 6 6.75(2H,J = 8.8Hz,H-6'and H-8’) attributable to the p-Z-coumaroyloxy substituent, which was supported by the r3C NMR chemical shifts (Table 2) and the HR mass spectral fragments at m/z 164.0446 (&H,O,) and 147.0438 (C&H,O& Furthermore, the ‘H NMR spectrum of 2 showed signals of six methyl protons, five as singlets (60.75, 0.82, 0.92, 0.93 and 0.94) and one as a

Triterpenoids from Plumeria Table 2. 13C NMR spectral data (djppm) of triterpenes 1 and 2 C

2 3 4 6 I

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1’ 2 3 4 5 6 7 8 9

1

2

38.7 21.2 78.1 38.8 55.3 18.3 32.7 39.5 47.7 37.1 23.6 126.5 138.1 42.1 28.1 23.6 48.6 52.5 38.9 38.6 30.3 36.8 28.2 68.3 15.7 16.9 23.7 180.6 16.8 21.2 167.2 115.4 144.6 132.7 129.9 116.2 160.7 116.2 129.9

38.1 27.8 79.4 38.3 56.6 19.4 34.8 41.5 49.1 39.8 24.6 131.6 134.6 46.8 25.4 24.6 30.8 53.9 40.5 39.9 31.4 41.5 28.7 16.3 16.4 18.9 66.1 21.6 18.1 182.0 168.3 116.8 144.4 127.6 133.5 115.9 160.1 115.9 133.5

doublet (60.90, J =6.3 Hz); an isolated olefinic proton (65.51 t, J= 3.5 Hz, H-12), two methylene protons (64.41 d, J= 12.7 Hz, H-27a and 64.12 d, J= 12.7 Hz, H-27b) and two methine protons (63.05 dd, J= 11.3 and 4.5 Hz, H-3cr and 62.29 d, J = 11.7 Hz, H-18). These data indicate that 2 belongs to the m-sane series of pentacyclic triterpenes. The aliphatic hydroxyl group was placed at C-3 on the biogenetic grounds and its /?-disposition was decided on the basis of the 6 value and coupling constants of H-3 [17-J. The downfield chemical shifts of H-12 (65.51), C-12 (6131.6) and C-14 (646.8) and upfield shift of C-13 (6134.6; Table 2) [S, 20, 211 as compared to the values of these nuclei in ursolic acid suggested a substituent at C-27, whereas only one doublet of the methyl protons (H-29/H-30) indicated the location of the other substituent at C-19/C-20. Since the carbinylic methylene protons appeared as two AB doublets, the p-Z-coumaroyloxy group was placed at C-27. The carboxyl group was placed

4281

obtusa

at C-20 in the light of the interactions

observed in COSY45 spectrum which showed very clear connectivities between H-18 and H-19; H-19 and H-20 and H-19 and H29 apart from other spin systems. Additional evidence was obtained from attempts to form a lactone of 2 on its reaction with Br,-acetic acid [22] which, however, failed to give any y-lactone thus excluding the possibility of the carboxyl group at C-19. It may also be noted that C-17 of 2 appeared at upfield (630.8) as compared to that of ursolic acid (647.9) since in the former the carboxyl group is not at C-17. Moreover, the chemical shift of this carbon is comparable with that of cr-amyrin [S]. Thus the structure of 2 is 3/?-hydroxy-27-p-Z-coumaroyloxyurs12-en-30-oic acid, which was substantiated by various fragments in the EI mass spectrum at m/z 246.1633 (C,,H,,O,), 207.1809 (C,,H,,O), 201.1620 (C,,H,,) and 189.1597 (C,,H,,) (Scheme 2) and 13CNMR spectral data (Table 2); the corresponding free alcohol does not appear to have been isolated earlier. Kaneroside [2], oleandrin [3, 41, cl-amyrin [S, 63, neriucoumaric acid, isoneriucoumaric acid [7], alphitolic acid [8,9], oleanonic acid [lo, 111, methyl p-E-coumarate [12] and scopoletin [13, 143 were identified through comparison of their spectral data with the published data.

EXPERIMENTAL

NMR spectra of 1 (pyridine-d,): 300 MHz for ‘H and 75 MHz for r3C, 2 (CD,OD): 400 MHz for ‘H and 100 MHz for “C. The 13C NMR spectral assignments (Table 2) were made partly through a comparison of the chemical shifts with the published data for similar compounds [S, 18-211 and partly through the appearance of signals in DEPT spectrum. The r3C NMR assignments of 1 have also been supported through the appearance of signals in heteroCOSY spectrum. The plant was identified by Professor S. I. Ah (Department of Botany, University of Karachi) and a voucher specimen (No. 9317 KUH) is deposited in the Herbarium. The methanolic extract of fresh, uncrushed and undried leaves (12 kg) collected from the Karachi region in April (1987) was coned under red. pres. and shaken with EtOAc and H,O. The EtOAc phase was treated with 4% aq. soln of Na,CO, to separate the acidic from the neutral fr. The EtOAc layer was washed with water, dried (Na,SOJ and the residue left on removal of the solvent was divided into petrol-soluble and petrol-insoluble portions and the petrol-insoluble fr. was SUCC~Ssively treated with petrol- EtOAc (7:3) and petrol-EtOAc (1:l). The petrol-EtOAc (7:3)-soluble fraction (6 9) was subjected to flash CC (petrol, petrol-EtOAc in order of increasing polarity). Various frs were combined on the basis of TLC. Fractions eluted with petrol-EtOAc (7:3) afforded pure kaneroside and oleandrin in the order of polarity. Fraction (3.5 g) obtained on removal of the solvent from the petrol-EtOAc (1: 1)-insoluble fraction was subjected to FCC (CHCl,, CHCl,-MeOH in the order of increasing polarity). Different frs were combined on the basis of TLC. Ten frs were ultimately obtained of which fr. 1, eluted with CHCl,, gave pure a-amyrin. Fr. 2 obtained on elution with CHCl,-MeOH (19:l) showed 3 spots on TLC and was rechromatographed on FCC (petrol-EtOAc, 6:4). Three pure compounds were ultimately obtained and characterized as neriucoumaric acid, isoneriucoumaric acid and alphitohc acid in the order of polarity. Fr. 3 which was CHCI,-MeOH (19:l) eluate of FCC, afforded pure obtusin (1). Frs 9 and 10 eluted with CHCl,-MeOH (47:3) gave pure oleanonic acid. Petrol-EtOAc

s. SIDDIQI;Ief al.

4282

30

m/z 189.1597

~0~“”

I - Hz0 m/r 207.1809

-

m/z 147.0438

2 2a 2b

R’ H AC AC

R2 (mass fragments correspond to compound 2) H Me Scheme 2.

(l:l)-insoluble fraction (3 g) was subjected to VLC (CHCl,, CHCI,-MeOH in order of increasing polarity). The eluates were combined on the basis of TLC affording 4 frs of which frs 1-3 had ursolic acid as the main constituent. On recrystallization from MeOH-C,H, (1:l) pure ursolic acid (2 g) was ultimately obtained. Fr. 4 and the mother liquor of ursohc acid showed more or less similar spots on TLC and were, therefore, combined giving 1 g of a residue. This fraction was subjected to FCC (CHCl,-MeOH in order of increasing polarity). Various frs obtained were added together on the basis of their TLC. As a result pure methyl p-E-coumarate (CHCl,-MeOH, 19:l) and obtusilic acid (2) (CHCl,-MeOH, 17:3) were obtained. The 4% aq. Na,CO, phase referred to at the outset was acid&d with 30% HCl and extracted out with EtOAc after addition of NaCl. The EtOAc phase was washed, dried (Na,SO,) and charcoaled. The charcoal bed was successively eluted with EtOAc and MeOHC,H, (1:l). The residue obtained on removal of the solvent from the combined EtOAc filtrate and eluate was divided into petrol-soluble and petrol-insoluble frs. The latter fr. was combined with the petrol- insoluble fr. obtained from the residue of the MeGH-C&H, (1: 1)eluate of the charcoal. The combined petrol-insoluble fr. (8 g) was subjected to VLC in order of (silica gel, Merck, GF2s4; CHCI,, CHCl,-MeOH increasing polarity). The eluates were added together on the basis of TLC and afforded a pure fr. containing scopoletin. Obtusin (1). Obtusin (28 mg) was obtained as irregular plates (MeOH), mp 198-199”. UV 1:::” nm: 203 and 296. IR *z cm-‘: 3500-2600,1738-1680 and 160&1380 cm-‘. HRMS m/z (rel. int.): 454.3460 [M - p-coumaric acidJ + (C 30H 460 3, 17) 439.3258 (C,,H,,O, 14), 436.3335 (C 30H 440 *, 8) 421.3132

Methylation of obtusin. Compound 1 (10 mg) was taken in MeOH, treated with an ethereal solution of CH,N, and kept at room temp. overnight. Usual work-up gave lla as needles (MeOH), mp 120-121”. EIMS m/z (rel. int.): 468 [M-p-coumaric acid] + (18) 409 (lo), 391 (1) 262 (80), 249 (34), 205 (20), 203 (lOO), 189(40),178(3), 161(12)and 130(16). ‘HNMR(4OOMHz, CDCl,): Table 1. Obtusilic acid (2). Compound 2 (17 mg) was obtained as needles (CHCl,-MeOH, 1: l), mp 29s 291”. UV ;(~~~r’ nm: 226 and 313.6. IR vzr$(IcI1cm--‘: 363@2600 br (OH and CO,H), 1750-1660 (ester and acid carbonyls) and 1660-1560 (aliphatic and aromatic C=C). HRMS m/z (ret int.): 454.3461 [M-pcoumaric acid]+ (CJOH4603, 84) 441.3397 (C,,H,,O,, 20), 393.3162 (C,,H,,O, 3). 300.2136 (&,H2sOZ, 4). 255.2093 (C,,H,,, 8) 246.1633 (C,,H,,O*, 16) 207.1809 (C,,H,,O, 60) 201.1620 (C,sHZ1, 5) 189.1597 (C,,H2,, 20) 164.0446 (C,H,O,, 94) and 147.0438 (C,H,OI, 100). ‘H NMR (400 MHz, CD,OD): Table 1. *jC NMR (100 MHz, CD,OD): Table 2. Acetylation of’ compound 2. Compound 2 was dissolved in pyridine (4 ml) and Ac,O (1 ml) added. The reaction mixture was left at room temp. overnight and worked-up in the usual manner affording the diacetate derivative 2~ as a gummy residue. EIMS m/z (rel. int.): 496 [M -p-acetylcoumaric acid] ’ (14), 483 (20) 435 (6) 300 (20) 255 (35) 249 (30) 246 (32), 206 (60), 201 (17) and 189 (40). ‘H NMR (400 MHz, CD,OD): Table 1. Methyl&on of 2a. Compound 2b was obtained on reaction of 2s with CH,N, (in excess) in Et,0 at room temp. overnight and usual work-up. EIMS m/z (rel. int.): 510 [M -p-acetylcoumaric acidJ+ (6), 497 (20) 464 (14) 450 (lo), 260 (45), 201 (35) and 189 (100). ‘H NMR (4.00 MHz, CD,OD): Table 1.

(C,,H,,O,, 1 l), 408.3392 (C,,H,,O, 16), 394.3215 (C,,H,,O, 8) 393.3163 (C,,H,,O, 20) 300.2112 (C,,H,,O,, 14) 285.1898 (C19H2502, 31), 249.1847 (C,,H,,O,, 91), 248.1783 (C,BH2402, 48), 206.1664 (C,,H,,O, 36) 203.1790 (C,,H,,, 100). 164.0526 (C9H,0J,45), 147.0484(C,H,O, 17)and 133.1019(C,,H,,,46). HeteroCOSY. Clear interactions were observed between the following “C and ‘H nuclei. The upfield region was not very well resolved. C-3 (678.1)/H-3 (63.33) C-12 (6 126.5), H-12 (65.45) C-24 (668.3)/H-24a (64.52) H-24b (64.35), C-2’ (6 115.4)/H-2’ (66.37), C-3’ (b 144.6)/H-3’ (67.74), C-5’, C-9’ (6 129.9)/H-5’, H-9’ (67.52) C-6, C-8’ (6 116.2)/H-6’, H-8’ (67.02). ‘H NMR (300 MHz, pyridine-d,): Table 1. 13C NMR (75 MHz, pyridine-d,): Table 2.

1. Siddiqui, S., Siddiqui, B. S., Begum, S. and Naeed, A. (1990) Phytochemistry 29, 3615. 2. Siddiqui, S., Hafeez, F., Begum, S. and Siddiqui, B. S. (1987) Phytochemisbry 26, 237. 3. Straub, W. (1918) Arch. Exp. Parth. Pharm. 82, 327. 4. Omura, S., Neszmelyi, A., Sangare, M. and Lukas, G. (1975) Tetrahedron Letters 2939. 5. Kang, S. S. (1987) Kor. J. Pharmacogn. 18, 151.

REFERENCES

Triterpenoids from Plumeria obtusa 6. Spring, F. S. and Vickerstoff, T. (1937) J. Chem. Sot. 249. 7. Siddiqui, S., Siddiqui, B. S., Hafeez, F. and &gum, S. (1987) Planta Med. 53,424. 8. Guise, G. B., Ritchie, E. and Taylor, W. C. (1962) Aust. J. Chem. 15,314. 9. Cheung, H. T. and Feng, M. C. (1968) J. Chem. Sot. 1047. 10. Tursch, B., Savoir, R., Ottinger, R. and Chiurdoglu, G. (1967) Tetrahedron Letters 539. 11. Mladenovic (1953) Acta Pharm. Yugoslao. 3, 1. 12. Buckingham. J. (chief ed.) (1987) Dictionary of Organic Compounds, Fifth Edn. fifth Suppl. Chapman & Hall. London. 13. Head, F. S. H. and Robertson, A. (1931) J. Chem. Sot. 1241. 14. Siddiqui, S, Mahmood, T., Siddiqui, B. S. and Faizi, S. (1988) Planta Med. 457.

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