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The EIMS and Laser MS o f quercetin exhibited a molecular ion at m/z 302, which

corresponds to the molecular formula H^o O7, confirmed by DEPT spectrum (S-31).

The ^H NMR spectrum (E-30) showed the presence o f an AB system at ô 6.19 (H-8) and 6.41 (H-6) (each d, J = 1.8 Hz) o f a 5,7-disubstituted ring A, an ABX system at ô 6.89 (d, J = 8.4 Hz, H-5'), 7.54 (dd, J = 8.4, 2.1 Hz, H-6') and 7.68 (d, J = 2 Hz, H-2'), o f a 3',4'-disubstituted ring B, and a chelated hydroxyl group at C-5 (ô^ 12.49,

29 OH 29 128 _{OH 128} Galphimine C Galphimine D 29 OCOCH3 ■'OCOCH3 23 uCOCHs Galphimine E OH OH Glaucamine 2^''i.,,20 21' 'i,.,20 H O ' Sitosteryl-3-O-p-D-glucopyianoside Stigmasterol 26 Quercetin

o f ring C, indicated the presence o f two phenolic groups in ring A and two phenohc groups in ring B (Figure 2.7). Full assignation o f protons and carbons was confirmed with HMQC spectrum The physical and spectroscopical data o f the flavonol quercetin was in agreement with those in literature (Pakulski et a l , 1996; Markham & Chari, 1982).

•OH

M/Z 302 [M]+ m /z 137

m/z 153

Figure 2.7 Mass spectral fragmentation o f quercetin

2.3.2.3.1.2 Galphimine C

High-resolution FAB-mass spectrometry o f galphimine C, showed a molecular ion at m/z 616 [M]^ (C34 H48 Oio ). The loss o f a molecule o f acetic acid led to the fragment, m/z 556 [C32 H44 Og, M"^- CH3CO2 + H], generated from the molecular ion, through a McLafferty rearrangement in the ring A o f galphimine C (Figure 2.6). The IR spectrum

showed the presence o f hydroxyl (3500 cm’') and ester (1760-1720 crn^). The NMR

spectrum (S-32) exhibited the presence of four methyl groups [ô 1.09 (s), 1.17 (d, J = 6.4 Hz), 1.31 (s), and 1.34 (s)], two acetoxyl groups [ô 1.99 (s) and 2.07 (s)], a methoxyl group [Ô 3,49 (s)], two methine signals o f an epoxide function [ô 3.55 (d, J = 4 Hz) and 3.56 (dd, J = 4, 1.26 Hz)], an exo-methylene [ô 4.46 and 4.49, (each IH, AB, d, J = 2 Hz)], a methylene [ô 3.63 and 5.07, (each IH, AB, d, J = 12.55 Hz)] and a methine [ô 5.16 (brt, J = 7.96 Hz)] bearing each one acetoxyl group.

^H and ^^C NMR assignments o f galphimine C (Tables 2.4) were greatly facihtated by

HMQC spectrum (S-35). Long-range C-H correlations were observed in COLOC

spectrum (S-36) with the following protons and carbons: H-1 (ô^ 3.55) and H-2 (ô^ 3.58) v.y C-3 (ô^ 168.69); H-1 (ôg 3.55) and H-10 (ô^ 1.63) V5 C-2 (ô^ 56.80), which were utilized to confirm the epoxide function at C-1, C-2. The lactone system was

confirmed by NMR spectrum (S-33) through the signals at ô 52.73 (C-1), 56.38 (C- 2) and 168.57 (C-3) and its localization in the heptacycle ring A, was done in accordance with the chemical shifts o f carbon atoms: ô 76.31 (C-4), 38.63 (C-5), 53.85 (C-10), 13.04 (C-23), and the conparison with a model structure (Abreu et al., 1990). The peak at m/z 212,196,152, and 137 in the mass spectrum (Figure 2.8), suggested the presence o f methylene bearing an acetoxyl group at C-5. Long-range C-H correlations in COLOC spectrum (S-36) enabled the determination o f the following carbon sequence: C-9 -» C-

8 - C-25; C-26 -> C-14; C-5 - C-10; C-8 - C-14 - C-26 and C-9 - C-10 - C-11. On the other hand, the carbon sequence C-6 - C-7 - C-8; C-15 - C-16; C-11 - C-12; C-22 - C-21 - C-20 - C-29 and C-19 - C-20 - C-29, was disclosed by 'H-'H COSY correlations (S-34) arising fi'omthe geminal and vicinal sping coupling constants between involucrated protons. Further, the localization o f the acetoxyl groups at C-7 and C-24, the hydroxyl group at C-18, the epoxide at C-1-C-2, and the methoxyl group at C-13 was made possible by H and ^^C chemical shifts, multiplicity o f the signals observed in the 2D NMR spectra (COSY-45, HMQC, HMBC, COLOC).

OH 24 o m/z 196 ► O O m/z 137 Galphimine C

Figure 2.8 Mass spectral fi'agmentation o f galphimine C

The spectral analysis pointed to a nor-fiiedelane type skeleton foi^galphimine C. With the constitution estabhsed, all that was left was to determine the configuration and conformation o f the molecule, this was achieved by a NOESY spectrum (S-37). The analysis o f results indicated that the position and orientation o f 25, 26 and 28-CH3 is consistent only with rings C and D in a chair-trans-chair arrangement with the three

methyl groups and the hydroxyl group at C-18 all axial and P-orientated. Furthermore, the hydrogens on C-10, C-8 are all axial and oc-orientated, whereas H-1 and H-2 are

pseudo equatorial and a-orientated. The a-axial carbomethoxy group at C-13 is

consistent only with a cis D/E ring junction. The acetylated methine at C-7 is ct- orientated, because cross-peaks were observed between 25- and 26-methyl-signals with the p-hydrogen at C-7. The B/C junction is trans, and ring B is in a twist-boat conformation. The coupling constants in ring B (Jgy = 16.6, 5.6 and J^g = 7.8 Hz) confirm gauche dihedral angles for each pair o f protons. Finally, the two 4-8-10 and 8- 10-13 sequences must place these protons on the a-face o f the molecule, which completes the assignment o f the relative configuration as shown in spectrum (S-37). On the basis o f the above information, galphimine C was identified as the novel confound 7a,24- diacetoxy-13 a-carbomethoxy- 1 p ,2 P-epoxy-18 p-hydroxy-27,3 O-bisnor-3,4-secofnedela- 20(29)-en-3,4R-ohde.

2.3.2.3.1.3 Galphimine D

The molecular formula o f galphimine D, was determined to be C^g Hgg 0^2, [M + H ]\ m/z 674 by HRFABMS. ^H and ^^C NMR spectra (S-38 and S-39) closely resembled those o f the nor-triterpenoid galphimine C, except for the presence o f additional signals o f acetoxyl group [6^ 2.05, s, CH3CO2 ; ôc 21.23,170.20] and the methine bearing this group [ôn 5.56, dd, J = 12.3, 7 Hz; ôc 70.94]. The location o f the acetoxyl group at C-15 in the moiety was estabhshed by the analysis o f the cross-peaks observed between H-15 and H-16 in the COSY-45 spectrum (S-40) o f galphimine D. The assumption was

confirmed by analysis o f HMQC (S-41), HMBC and COLOC (S-42) experiments. ^H

- ^^C long range observed in COLOC and HMBC experiments showed as inportant features, a correlation o f the signal o f C-18 - C-28 C-19; C-26 ^ C-10; C-5 ^ C-23 ^ C-10 -* C-1. Finally, the stereochemistry of acetoxyl group at C-15 in galphimine D was

determined by a NOESY spectrum (S-43). NOE’s were observed between H-8 and H-

11, thus H-15 is a and axial and 15-OAc must be P ecuatorial. On the basis o f the above information galphimine D was identified as the novel confound 7a,15p,24-triacetoxy- 13a-carbomethoxy-lp,2p-epoxy-18P-hydroxy-27,30-bisnor-3,4-secofriedela-20(29)-en- 3,4R-ohde.

2.3.2.3.1.4 Galphimine £

Thé molecular formula o f galphimine E was determined to be [M + H]^, m/z

733 by HRFABMS, and was consistent with an additional acetoxyl group in comparison

with glaucamine D. This was corroborated by ’H and NMR experiments (S-44 and

S-45) comparison o f galphimine E, with those o f galphimine D. The NMR data o f galphimine E was very similar to galphimine D, except for the presence o f an additional acetoxyl group signal (6^ 1.98, s, CH3CO2; ôc 22.64, 171.49) and the methine bearing this group (ôjj 5.6, dd, J = 12.7, 4; ôc 76.32). The position o f the additional acetoxyl group at C-12 was determined by the analysis o f COSY-45 (S-46), which showed cross­ peaks between the protons H -11 (ô^ 0.97, dd, J = 12.6, 4 Hz and 2.98, t, J = 12.6 Hz;

Ô C 35.26) and H-12 (ô^ 5.6, dd, J = 12.7, 4; ôc 76.32). The above features were

confirmed with HMQC (S-47), HMBC and COLOC (S-48) experiments. The ^H - ^^C long range correlations, observed in COLOC experiment showed the following sequence o f carbons: C-18 - C-28 - C-19; C-26 - C-10; C-5 - C-23 - C-10 - C-1. Furthermore, the NOESY spectrum (S-49) o f galphimine E, showed the interactions between the following protons: H-12 / H -11 / 25-H^ / 26-Hg, this allowed to conclude that the proton bearing the acetoxyl group at C-12 was P and axial and the acetoxyl group must be ecuatorial and a orientated. On the basis o f the above information galphimine E was identified as the novel conpound 7a, 15 p, 12a,24-tetra-acetoxy-13 a-carbomethoxy-

1 p,2P-epoxy-18 P-hydroxy-27,3 O-bisnor-3,4-secofiiedela-20(29)-en-3,4R-ohde.

2.3.2.3.1.5 Glaucamine

The molecular formula o f glaucamine was ascertained to be C34 H52 Oio [M + H]^, m/z 621 by high resolution FAB-mass spectrometry. The IR spectrum o f glaucamine showed as main features the presence o f hydroxyl (3500 cm'^) and ester (1720 cm'^). The NMR data (Tables 2.4) o f glaucamine resembles those o f galphimine C-E, except for the absence o f the lactone moiety in ring A. Analysis o f the ^H and ^^C NMR spectra (S-50 and S-51) o f glaucamine revealed signals for four methyl groups [1.02 (s), 1.18 (s), 1.27 (s), 1.35 (d, J = 7.16 Hz)], two acetoxyl groups [ô 2.04 (s) and 2.11 (s)], one methoxyl [Ôjj 3.65 (s); Ô C 51.37,174.87] group, two secondary hydroxyl groups [ô 3.31, d, J = 8.36

144.07], one methylene bearing one acetoxy group 4.5, 4.73 (each, d, J = 12.5 Hz);

Ô C 65.11], and one methine bearing one acetoxy group [ô^ 4.93, brd, J = 2.2 Hz; ôc

74.75]. Further, the presence o f one tertiary hydroxyl group (ôc 79.87) and the absence o f the lactone moiety was revealed by DEPT and HMQC spectra (S -51 and S-53). The above spectral analysis pointed to a basic skeleton o f a nor-friedelane type for glaucamine. Localization o f acetoxyl groups at C-3 and C-24, the secondary hydroxyl groups at C-6 and C-7, and the methoxyl group at C-22 was made possible by H and ^^C chemical shifts, multiplicity o f the signals, ^H - ^H 2D homonuclear, and ^H - ^^C 2D

heteronuclear correlations. The long-range C-H correlation observed in COLOC

spectrum (S-54) enabled determination o f the following carbon sequence: C-3 - C-4 -» C-5; C-19 - C -20- C-21; - C-16 - C-14; C-17 - C-18 - C-22 - C-28; C-25 - C-9 - C-10 - C-8. Further the carbon sequence C-1 - C-2 - C-3; C-6 - C-7 - C-8; C-11 - C-12; C-15 - C-16; and C-20 - C-21 - C-22, was disclosed by ^H - ^H 2D homonuclear COSY-45 (S-52) correlations arising from the geminal and vicinal spin coupling constants between involucrated protons. The stereochemistry of glaucamine was determined by the coupling constants observed between the protons and confirmed by the NOESY spectrum

(S-55). Cross-peaks were observed between: H-3 and H-2, H-4, H-23; H-29 and

H-19, H-21; H-24 and H-25, H-7; H-7 and H-26, H-

25, H-24; i H-28 and H-22; and H-22 and H-21. On the basis o f the above

information glaucamine was identified as the novel confound 3p,24-diacetoxy- 6p,7o£, 13 a, 18P-tetra-hydroxy-27,30-bisnor-fnedela-20(29)-en-22a-carboxylate.

It is interesting to note that galphimine C, galphimine D and galphimine E are naturally occurring 3,4-seco(3 ^ 4 lactone)-27,30-bisnor-triterpenoid o f the ffiedelane series with a carbomethoxy group at C-13. The biosynthesis o f galphimine C, galphimine D and galphimine E has not yet been investigated, however might involved a ring A cleavage thr ough an enzymatically controlled Bayer-Villiger reaction as proposed by Baas (1985).

H O — H C O O H H O H _{O H} OH G laucam ine G alp h im in e C -E O H O H H

Figure 2.9 Putative Biosynthetic pathway o f galphimine C, galphimine D, galphimine E and glaucamine

The biosynthesis o f glaucamine has not yet been studied. However, it can be suggested that the secondary biomodification o f the E ring might include elimination o f the Me-20 by oxidative déméthylation (as formic acid or decarboxylation) and méthylation or formylation at C-22 (Torssell, 1983). Alternatively, this biotransformation might involve a ring E cleavage and induce biogenetic cyclization through the reactions suggessted in Figure 2.9. The other biosynthetic modifications observed in galphimine C - E and glaucamine (hydroxylation, olefinic groups, oxidation o f the alcohol function to a carboxyl group, acétylation, méthylation and epoxidation) are common processes o f secondary metabolism (Torssell, 1983).

2.3.2.3.1.6 Stigmasterol

The molecular formula o f stigmasterol (Qp H^g O) was deduced from EIMS, and DEPT

spectrum (S-57). The and NMR spectra (S-56 and S-57) displayed signals typical

o f a tetracychc terpenoid with six methyl groups [ô 0.68 (s), 0.81 (s), 0.83, 0.84 (s), 0.92 (d, J = 5 Hz), 1.00 (s)]; one methine bearing an hydroxyl group 3.52, m; ôc 72.22]; and one trisubstituted olefinic proton [ô^ 5.35 (d, J = 5.12 Hz); ôc 122.15, 139]. The physical and spectroscopic data of stigmasterol (3p,22E-stigmasterol-5,22-dien-3-ol) was in agreement with literature (Rubinstein et a l, 1976).