Structure determination and identification

2.3.1.3.1.1 3oc-Hydroxy-tirucaIla-7,24Z-dien-26-oic acid (Cml)

High resolution El mass spectrometry o f Cml showed a [M]^ peak at 456 m/z

corresponding to the molecular formula indicating the presence o f eight

double bond equivalents, five o f which were accounted for one pentacyclic ring nucleus ,

one carboxylic group and two double bonds, confirmed by DEPT spectrum (S-2). The

IR spectrum o f Cml showed absorptions for hydroxyl (3450 cm'^), a,p-unsaturated

carboxylic acid (1690 cm'^) and double bond (1640 cm'^) groups. The and ^^C NMR

spectra (S-1 and S-2) o f Cml showed the presence o f five ter-methyl (Ô 0.79, 0.87, 0.98, 1.04, 1.17), one sec-methyl (ô 0.97, d, 6 Hz) and one vinyhc methyl group (ô 2.15) signals, which are typical o f a pentacyclic triterpene with a side chain. The ^H and ^^C NMR spectra also showed two olefinic protons (ô^ 5.35, ôc 118; 6.06, ôc 142.67), one o f which was deshielded, by the typical P position on an oc,P-unsaturated carboxylic acid (ô^ 170.72) in the side chain o f the tetracyclic triterpene. A significant fi*agment at m/z 316 [M - CgHi302] \ resulting from the cleavage o f the side chain o f the tetracyclic nucleus required the presence o f the a,P-unsaturated COOH at C-26 o f the side chain (Figure 2.3). The geometry relatioship between the C-24/C-25 double bond and the carboxylic acid was identified from the chemical shift o f H-24 (ô 6.06) and its

comparison with methyl angelate (65.97, COOH and H-24 trans) and methyl tiglate (6

6.72, COOH and H-24 cis) (Da Silva et a l, 1990), as well as NOESY spectrum (S-6). The structure o f the side-chain was confirm from COSY-45 (S-3), HMQC (S-4) and

HMBC (S-5) spectra. The COSY-45 spectrum (S-3) showed in^ortant coupling

between the following resonances: H3-2I vs H-20; H-23 vs H-22 and H-24; H-24 vs H3- 26. Futhermore, in the HMBC experiment (S-5) appreciable long-range shift correlations were observed between the following protons and carbons: H3-26 vs C-24 and C-25. Furthermore, appreciable NOEs (S-6) were observed between H-24 and H3-26, and between H-24 and H2-23, thus confirming the geometry o f the double bond located between C-24/C-25.

Placement o f the second double bond at C7/C8 was indicated by fragments at m/z 316, 175, 140, 122 and 96, associated with Retro Diels Alder (RDA) clavage o f ring B, see

Figure 2.3. This was supported by the NMR spectrum which was closely related with that reported for masticadienoic acid (Gewah et a l , 1990; Da Silva et a l , 1990). Thus, indicating a tirucall-7-en instead o f a lanostan-7-en nucleus, this was fiirther

confirmed by COSY-45 spectrum, which showed cross-peaks between H-7 and H2-6.

Furthermore, the NOESY spectrum (S-6) showed strong interaction o f H-7 with Hg-30

and H2-6. = + m/z 315 — ^ m/z 96 H O ' m/z 141 H O ' m/z 316 m/z 140 m/z 175

Figure 2.3 Mass spectral fragmentation o f 3a-hydroxy-tirucalla-7,24Z-dien-26 oic acid

The NMR spectrum further showed a broad singlet (ô^ 3.69; 75.28) attributed to

the proton geminal o f a secondary hydroxyl group, which is placed at C-3 on biogenetic consideration. The coupling constant and chemical shift o f H-3 favoured the a-

orientation (axial) o f the hydroxyl group at C-3. This was confirmed by the COSY-45 experiment o f Cml, which showed in^ortant cross-peaks between the oximethine CHOH (ôjj 3.69), and the two resonances o f the neighbouring protons CH2-2 (ô^ 1.84, d, J =

9.4 Hz and 2.01 m). Furthermore, appreciable NOEs were observed between H-3, H2-2, and H3-29. Finally, HMBC spectrum (S-5) o f Cml showed long-range shift correlations between the following protons and neighbouring carbons: Hg-29 V5 C-3, C- 4, and C-28. Acétylation o f Cm with pyridine-AcjO (Figure 2.4) afforded Cml a, m/z 498

[ M ] + ( C 3 2 H 5 0 O 4 ) .

CO2H

Figure 2.4 Acétylation o f 3a-hydroxy-tirucalla-7,24Z-dien-26 oic acid

The IR spectrum o f the acetylderivate showed absorptions for one acetate group at 1730 and 1210 cm'\ The mass spectrum showed the highest ion peak at m/z 438 [M-AcOH]^. The ^H NMR data (Table 2.2) was similar to that o f Cml except that the oxymethine group was deshielded paramagnetically to high field (ô^ 4.68 brs), confirming the presence o f one secondary hydroxyl group in the molecule. Although, the methyl derivative o f 3 a- hydroxy-tirucalla-7,24Z-dien-26-oic acid has been isolated and its physical properties has been reported (Monaco et a l , 1974), this is the first report o f the isolation and determination o f the physical and spectroscopical data o f 3a-hydroxy-tirucalla-7, 24Z- dien-26-oic acid.

2.3.1.3.1.2 3-Oxo-tinicalla-7,24Z-dien-26-oic acid (Cm2)

The conpound Cm2 showed a molecular ion at m/z 454 [M]^ (C3 0 H^g O3). Its IR

spectrum showed a peak at 1710 cm'^ o f ketone group. Conparison o f ^H and C NMR spectra (S-7 and S-8) o f Cm2 with 3 a-hydroxy-tirucalla-7,24Z-dien-26-oic, clearly.

indicated that the hydroxyl group at C-3 was replaced by a keto group in Cm2. Consistent with this proposal was the presence o f two protons [ô^ 2.5 (td, J = 14,3 Hz) and Ôjj 2.76 (dt, J = 14,5 Hz)] a to the carbonyl instead o f a hydroxyl group, and the signal o f C-2 was shifted about 7 ppm downfield to 6^ 34.96 owing to the presence o f a carbonyl (ô^ 217) at C-3. Oxidation o f 3a-hydroxy-tirucalla-7, 24Z-dien-26-oic acid with CrOg-pyridine yielded 3-oxo-tirucalla-7,24(Z)-dien-26-oic acid (Figure 2.5), which had identical spectroscopic data to 3-oxo-tirucalla-7,24Z-dien-26 oic acid (Konno et al,

1981).

HO'

Figure 2.5 Chemical conversion o f 3a-hydroxy-tirucalla-7,24Z-dien-26 oic acid to 3-oxo-tirucalla-7,24Z-dien-26 oic acid.

2.3.1.3.1.3 Epi-oleanolic acid

The FABMS (MNOBA + Na matrix) and EIMS o f epi-oleanohc acid revealed a [M]"^, m/z 456 (C3 0 H48O3), and fragments (m/z 249, 208, 204, and 190) typical o f a RDA

fragmentation o f an olean-12-ene. The ^H and ^^C NMR spectra (S-9 and S-10) exhibited signals due to seven tertiary methyl groups (ô 0.74-1.14), and three isolated hydrogens (ô 2.81, ô 3.41 and ô 5.27) o f H-18, H-3 and H-12. The signal o f H-18 (ôg 2.81, dd, J = 14, 4 Hz), permitted the identification o f an oleanen skeleton bearing a carboxyl (ôc 184.29) group at C-28; the presence o f H-3 as a broad singlet signal (ôjj 3.41; ôc 76.62) indicative o f the a-configuration o f the hydroxyl group; and the chemical shifts o f C-12/C-13 , and the olefinic proton on H-12 (ôjj 5.27, t, J = 3.3 Hz;

Ô C 123, 144), indicated an unsaturated oleaneno. 2D NMR experiments such as

epi-oleanolic acid. COSY-45 spectrum showed cross-peaks between the following

protons: H-3 vs H-2; H-18 H-19; and H-12 v.s H -11. NOESY interactions could

be observed: H-3 V5 23-Hg, 24-Hg; H-12 H-11, H-18, H-26; and H-18 3O-H3,

26-H3. The spectroscopical data o f epi-oleanohc (3 a-hydroxy-12-oleanen-28-oic acid) acid was in agreement with hterature (Chen et a l , 1983; Ikuta & Itokawa, 1988).

2.3.1.3.1.4 Friedelin, maytensifolin B and 3P-hydroxyMedelan-16-one

The identification o f friedelin (Gunatilaka et a l, 1983), maytensifolin B (Nosaki et a l ,

1986) and 3p-hydroxyffiedehn-16-one (Castaneda et a l , 1992) was carried out by comparison o f their spectral data with hterature. Conparative analysis o f ^H NMR spectra (S-13, S-14 and S-15) o f three terpenoids indicated that they were closely related fiiedelan-type triterpenes. The three triterpenes showed seven tertiary (ô 0.71 -1 .5 4 ), and one secondary (60.87 - 0.96, d, J= 5.3 Hz) methyl groups, however, some differences could be observed. In the case o f fnedehn one methylene group a to a keto group (6

1.68, dd, J = 14, 5.7 Hz, H-2) was observed; maytensifolin B displayed two methylene groups and each has an a keto group (6 1.68 and 2.35 each IH, dd, J= 14, 5.7 Hz, H-2 and 6 2.08 and 2.41 each IH, d, J = 19 Hz, H-15), and 3 P-hydroxy-friedelan-16-one revealed one methylene group (2.41 each IH, d, J=19 Hz, H-15) a to a keto group, and one methyne bearing an hydroxyl group (6^ 3.74) at C-3.