CHAPTER III

Phytochemical Study of Euphorbia tuckeyana Results and Discussion

Euphorbia tuckeyana: Results and Discussion

Introduction The phytochemical investigation of Euphorbia tuckeyana methanolic extract has afforded several macrocyclic and polycyclic diterpenes, including three new jatrophane polyesters (Scheme 3.1). In addition, various phenolic compounds have also been isolated and characterized. Some compounds have been isolated in major quantities, which allowed their derivatization through acetylation or methylation reactions. In this chapter, it will be described and discussed the physical and spectroscopic data that have permitted the structural elucidation of all the isolated compounds from E.tuckeyana. Macrocyclic Diterpenes and Related Compounds

Jatrophane-type Tuckeyanol A (64) Tuckeyanol B (63) Euphotuckeyanol (65)

Tigliane-type 4,20-Dideoxy-5-hydroxyphorbol-12,13-diisobutyrate (66) 4,20-Dideoxy-5-hydroxyphorbol-12-benzoate-13-isobutyrate (67)

Other Diterpenes

Ent-abietane lactones

E. tuckeyana

Helioscopinolide Helioscopinolide Helioscopinolide Helioscopinolide

A (69) B (56) D (68) E (55)

3-Acetoxy-helioscopinolide A (70) 3-Acetoxy-helioscopinolide B (57)

Phenolic Com pounds

Flavanoids Naringenin (58)

Naringenin-4’,7-dimethylether (59) Naringenin-7-methylether (60)

Aromadendrin (61)

Neolig nan Dehydrodiconiferyl diacetate (25)

C6C3 Phenols Coniferaldehyde (62)

Scheme 3.1. Isolated compounds from Euphorbia tuckeyana.

147

Euphorbia tuckeyana: Results and Discussion

1. STRUCTURE ELUCIDATION OF DITERPENIC COMPOUNDS 1.1. DITERPENES WITH JATROPHANE SKELETON 1.1.1.

Tuckeyanol

3β,5α,8α-triacetoxy-14β-benzoyloxy-15β-hydroxy-7β-(2-

A,

methylbutanoyloxy)-jatropha-6(17),11E-dien-9-one

20

CH3

BzO HO H3C

12

10 9

2 3

AcO

CH3

13

14 15

1

16

19 11

4

7 5

H AcO

6

8

18

CH3

O OAc

O 17

O

64

CH3 CH3

Compound 64, named tuckeyanol A, was isolated as a colorless oil with [α ]20 D + 32.8. Its HR-LSIMS showed a pseudomolecular ion at m/z 721.32263 [M+Na]+ (calcd. for C38H50O12Na: 721.31999) indicating a molecular formula of C38H50O12 consistent with fourteen degrees of unsaturation. The IR spectrum showed a carbonyl absorption band (1730 cm-1) as well as absorption bands for the hydroxyl group (3590 cm-1) and the aromatic ring (1455 and 756 cm1).

The EIMS displayed fragment peaks due to the loss of CH3COOH and C4H9COOH.

Moreover, a base peak at m/z 105 in the FABMS, also suggested the presence of a benzoyl residue in the molecule. These structural features were confirmed by the 1H NMR spectrum of compound 64, which showed the characteristic signals for one benzoyl group, the presence of a 2-methylbutyrate group and three singlets corresponding to the acetyl residues (Table 3.1). Due to the existence of several broad and overlapped signals in the 1H NMR spectrum of 64 recorded in CDCl3, the solvent was changed to C6D6, which caused a betterseparated NMR peaks, allowing the unambiguous assignments of all the 1H and 13C signals. In addition to the resonances of the ester moieties, the 1H NMR experiment showed signals for four methyl groups (two secondary at δ 0.84 and 1.16, and two tertiary at δ 1.34 and 1.40) and five protons geminal to ester functions, all of them displayed as broad singlets at δ 5.74, 5.80, 6.18, 6.34 and 6.38 (Table 3.1). Moreover, the aforementioned spectrum also revealed the 148

Euphorbia tuckeyana: Results and Discussion

presence of an exocyclic double bond (δ 5.62 and 5.18, br s) and a trans disubstituted double bond (δ 6.01, d, J = 16.0 Hz and δ 6.03 dd, J = 16.0 and 8.0 Hz). The presence of a broad singlet at δ 3.26, without correlation in the HMQC spectrum, confirmed the existence of a hydroxyl group in the molecule.

Table 3.1. NMR data of tuckeyanol A (64), (a CDCl3; b C6C6, 1H 400 MHz, 13C 100.61 MHz; δ in ppm, J in Hz).

Position

1H a

1H b

13C b

DEPT

HMBC b

1α 1β 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17a 17b 18 19 20 15-OH 3-OAc

2.35 m 1.82 m 2.19 m 5.51 br s 2.62 m 5.82 br s — 5.96 br s 5.97 br s — — 5.93 d (16.0) 5.95 m 2.62 m 5.53 br s — 1.0 d (6.8) 5.27 br s 4.94 br s 1.19 s 1.33 s 1.30 d (7.2) 3.21 br s — 2.10 s — 1.82 s — 2.52 dd (13.6; 6.8) 1.20 d (7.4) 1.50 m 1.74 m 0.95 d (7.4) — 2.16 s — — 8.07 d (7.2) 7.59 t (7.2) 7.50 t (7.2)

1.96 m 1.75 m 1.75 m 5.74 br s 2.69 br s 6.18 br s — 6.34 br s 6.38 br s — — 6.01 d (16.0) 6.03 dd (16.0; 8.0) 2.28 m 5.80 br s — 0.84 d (8.0) 5.62 s 5.18 s 1.34 s 1.40 s 1.16 d (8.0) 3.26 br s

47.0

CH2

3, 16

36.2 77.7 48.5 70.8 142.5 66.9 71.9 204.9 49.9 134.8 134.6 38.6 80.3 83.1 14.4 110.9

CH CH CH CH C CH CH C C CH CH CH CH C CH3 CH2

1, 16 5, 16 5, 14 17a, 17b 5, 7, 8, 17a, 17b 5, 17a, 17b — 8, 18, 19 11, 12, 18, 19 12, 18, 19 11, 14, 20 11, 12, 14, 20 12, 20 2, 3, 4, 14 2 —

24.6 25.7 23.5 — 168.5 20.4 168.2 20.0 175.8 40.9 16.5 30.0

CH3 CH3 CH3 — C CH3 C CH3 C CH CH3 CH2

11 11 14 — 3 — 5 — 7 4’ 4’ 2’-Me, 4’

11.5 169.9 20.0 166.3 129.9 130.4 128.9 133.1

CH3 C CH3 C C CH CH CH

2’ 8 — 14, 2’’, 6’’ 3’’, 5’’ 3’’,4’’, 5’’ 2’’,4’’, 6’’ 2’’, 3’’, 5’’, 6’’

5-OAc 7-OMeBu 2’ 2’-Me 3’a 3’b 4’ 8-OAc 14-OBz 1’’ 2’’, 6’’ 3’’, 5’’ 4’’

1.65 s 1.30 s — 2.35 m 1.06 d (8.0) 1.30 m 1.30 m 0.85 t (8.0) — 1.90 s — — 8.41 d (8.0) 7.41 t (8.0) 7.23 t (8.0)

149

Euphorbia tuckeyana: Results and Discussion

Apart from the ester signals, the combined analysis of the

13C

and DEPT spectra

showed the presence of four quaternary carbons (a ketone group at δC 204.9, an olefinic carbon at δC 142.5, an oxygenated carbon at δC 83.1 and one sp3 carbon at δC 49.9), ten methines (two sp2 at δC 134.8 and 134.6, and five oxymethines at δC 66.9, 70.8, 71.9, 77.7 and 80.3), two methylenes (one sp2 at 110.9) and four methyl groups (Table 3.1). The diterpenic nature of 64 was confirmed by the combined analysis of the HMQC and 1H-1H COSY spectra, which allowed the establishment of two correlated spin systems: –CH2–CH(CH3)– CH(OR)–CH(R)–CH(OR)–C(=CH2)–CH(OR)– (A) and –CH=CH–CH(CH3)– (B), (Figure 3.1). The connection of these two partial structures was deduced from the 2JC-H and 3JC-H correlations observed in the HMBC spectrum, between the quaternary carbons and the protons of the A and B spin systems (Table 3.1 and Figure 3.1). In particular, the long-range correlations between C-15 (δC 83.1) and H-2 (δ 1.75 m), H-3 (δ 5.74 br s) and H-4 (δ 2.69 br s), revealed the methyl-substituted cyclopentane ring A, present in all jatrophane diterpenes (Evans and Taylor, 1983). Moreover, the long-range correlations between the olefinic carbon C-6 (δC 142.5) and the protons at δ 6.18 (H-5), 6.34 (H-7), and 6.38 (H-8), and the cross-peaks between the carbonyl signal at δC 204.9, the proton signal at δ 6.38 (H-8) and the methyl singlets at δ 1.34 and 1.40 (CH3-18 and CH3-19), indicated that the ketone group must be situated at C-9, a fact that was corroborated by the downfield shift of C-10 (δC 49.9), (Hohmann et al, 2003 a). Finally, the connection between fragments A and B was allowed by the observation of 2JC-H and 3JC-H correlations between the quaternary carbon at δC 49.9 (C-10) and the olefinic protons H-11 and H-12 (6.01 d; 6.03 dd), together with the existence of crosspeaks between the carbon at δC 134.6 (C-12) and the oxymethine proton at δ 5.80 (H-14). The positions of all the ester groups were also established by the long-range correlations between the carbonyl groups and the corresponding oxymethine protons. In this way, the correlation of the carbonyl signal at δC 166.3 with the proton signal at δ 5.80 (H-14), indicated the presence of the benzoyl group at C-14; the cross-peaks of the carbonyl group at δC 175.8 and the protons at δ 2.35 (H-2’, MeBu) and 6.34 (H-7) located the 2-methylbutyrate residue at C-7. Similarly, the three acetyl esters could be undoubtedly assigned to carbons C-3, C-5 and C-8 due to the observed long range correlations between the carbonyl carbons of the ester residues (δC 168.5, 168.2 and 169.9) with the oxygenated methines at δ 5.74, 6.18 and 6.38, respectively. Because all ester carbonyl groups could be correlated with oxymethine protons, the position of the free hydroxyl group was assigned to C-15, a conclusion that was corroborated by the high-field resonance of this carbon (δC 83.1) when compared with C-15 acyl derivatives (Hohmann et al, 2003 a).

150

Euphorbia tuckeyana: Results and Discussion

CH3

BzO H H H

.

H HO

H H3 C

H AcO

H

.

H AcO

. . . H

H

H OR H

CH3 CH3 O

OAc

Figure 3.1. 1H-spin systems (A-B) of compounds 63 and 64 assigned by the HMQC and COSY experiments (▬) and their connection by the main heteronuclear 2JC-H and 3JC-H correlations displayed in the HMBC spectrum (→).

The relative configuration of compound 64 was determined through a NOESY experiment (Table 3.2) and by comparison of the coupling constant values with those reported on the literature, assuming a trans-ring junction and an α orientation for H-4, similarly to other jatrophane diterpenes isolated to date whose stereochemistry have been established by X-ray analysis (Hohmann et al, 1997; Liu et al, 2001; Li et al, 2003).

Table 3.2. NOE data for compounds 63 – 65.

Position

63, 64

65

1α 1β 2 3 4 5 7 8 9 11 12 13 14 16 17a 17b 18 19 20 15-OH 14-OBz 2’, 6’ 3, 5’ 4’

1β, 13, 14 1α, 15-OH 3, 4, 16 2, 4 2, 3, 5, 7, 11, 13 4, 8, 2’-Bz, 6’-Bz 4, 11 5, 19, 2’-Bz, 6’-Bz — 4, 7 19, 20, 2’-Bz, 6’-Bz 1α, 4, 11, 14 1α, 13, 20 2 17b 17a 11 8, 12 12, 14 1β

1β, 13 1α, 16 — 4 3, 7, 13 16, 17a 4, 11 17b 9, 19 8, 18, 19 7, 12, 13, 18 11, 19, 20 1α, 4, 11, 20 — 1β, 5 5, 17b 7, 17a 9, 11, 19 8, 9, 12, 18 12, 13 — — — — —

3, 5’, 5, 8, 12 2’, 4’, 6’ 3, 5’

151

Euphorbia tuckeyana: Results and Discussion

In this way, the NOESY cross-peaks between H-4/H-2, H-4/H-3 and H-4/H-7 established the α position of these protons. Strong NOE enhancements were also observed between H-4/H-13 indicating, unambiguously, the β configuration of CH3-20. The NOE interactions detected between CH3-20/H-12, H-12/CH3-19 and CH3-19/H-8 indicated that these protons are β-located. On the other hand, a strong NOESY correlation between H-8/H5 provided evidence for the β orientation of these protons, a fact that is corroborated by the coupling constant J7,8 (0 Hz), (Corea et al, 2005

b

and 2003 a). The existence of strong NOE

enhancements between the signals of H-5 and H-8 with the ortho-benzoyl protons (δ 8.41 d) dictated the β configuration of the aromatic ester at C-14. It should be noticed that the configuration at C-14 could not be confirmed from the analysis of the coupling constant between H-13 and H-14, since the constants J13α,14α and J13α,14β were found to be 0 Hz in both cases and therefore could be compatible with either configuration of this carbon (Marco et al, 1998; Jakupovic et al, 1998 a). Finally, the existence of a NOESY cross-peaks between H-13/H1α, H-1β/OH-15 (δ 3.26) and the absence of effects between OH-15/H-4, confirmed the translinked cyclopentane ring. As extensively reported in the literature by several authors, the majority of Δ6(17), Δ11 jatrophane diterpenes isolated from Euphorbia species are characterized by a particular coupling constant pattern because all coupling constants are almost zero Hz, suggesting an orthogonal relationship between all the protons of the macrocycle (Corea et al, 2004; Jakupovic et al, 1998

a,b;

Marco et al, 1998). In this type of compounds, the macrocyclic ring

can adopt two predominant conformations (endo and exo-type), which are dependent of the spatial orientation of the exomethylene group with respect to the mean plane of the macrocycle (Jakupovic et al, 1998 a,b; Appendino et al, 1998; Marco et al, 1998). Therefore, in the exo-type conformation, the exomethylene group is parallel to the mean plane of the molecule, H-4 and H-5 are almost orthogonal and the coupling constant between them is small (J4,5 = 0 - 2 Hz). Furthermore, this conformation is also characterized by the existence of diagnostic NOESY cross-peaks between H-4/H-5 and H-5/H-8 due to the close proximity of H-5 and H-8. On the other hand, the endo-type conformation is characterized by a large coupling constant between H-4 and H-5 (J4,5 = 9 - 11 Hz), showing that these protons have an antiperiplanar orientation, and by the existence of a diagnostic NOESY cross-peak between H-5 and H-17, due to the perpendicular orientation of the exomethylene group with respect to the mean plane of the macrocycle (Jakupovic et al, 1998 a,b; Appendino et al, 1998; Marco et al, 1998). Recently, in order to investigate the conformations adopted by Δ6(17), Δ11 jatrophane diterpenes, some molecular mechanic and dynamic calculations were performed and the 152

Euphorbia tuckeyana: Results and Discussion

results were in agreement with the data obtained by the NMR experiments (Corea et al, 2005a). Considering that J4,5 = 0 Hz, the presence of NOESY correlations between H-4/H-5 and H-5/H-8, together with the absence of NOESY effects between H-5 and the exomethylene protons, it can be concluded that tuckeyanol A (64) adopts preferentially the exo-type conformation. All the above data are in agreement with structure 64, corresponding to a new highly acylated jatrophane diterpene, named as tuckeyanol A.

1.1.2.

Tuckeyanol

B,

3β,5α,8α-triacetoxy-14β-benzoyloxy-15β-hydroxy-7β-(2-

methylpropanoyloxy)jatropha-6(17),11E-dien-9-one

20

CH3

BzO HO H3C

12

10 9

2 3

AcO

CH3

13

14 15

1

16

19 11

4

7 5

H AcO

6

8

O O

CH3

O OAc CH3

17

63

18

CH3

Tuckeyanol B (63) was obtained as a colorless oil with [α ]20 D + 26.3. Its molecular formula (C37H48O12) was determined by HR-LSIMS, which showed a pseudomolecular ion at m/z 707.3021 [M + Na]+ (calcd for C37H49O12Na: 707.3043). This compound showed IR, 1H and 13C

NMR data closely similar to those of tuckeyanol A (64), (Table 3.3). As for that

compound, the 1H NMR spectrum also indicated the presence of two double bonds (an exocyclic and a trans-substituted one) and the same number of protons geminal to oxygenated carbons (all signals displayed as broad singlets at δ 5.74, 5.82, 6.19, 6.35 and 6.40), as well as four methyl groups (two secondary at δ 0.83 and 1.19 and two tertiary at δ 1.35 and 1.41). Accordingly to the spectroscopic data and taking into account the molecular formula, the only difference is the presence of an isobutyrate group (δ 2.47 m, δ 1.12 d, J = 6.8 Hz and δ 1.05 d, J = 6.8 Hz; δC 176.3, 33.9, 18.8 and 18.6) instead of the 2-methylbutyrate ester.

153

Euphorbia tuckeyana: Results and Discussion

The location at C-7 was determined by the long-range correlation between the carbonyl group at δC 176.3 and the oxymethine proton at δ 6.35 (H-7). The structure of tuckeyanol B (63) was confirmed by extensive 2D NMR experiments that allowed the unambiguous assignment of all

1H

and

13C

signals. The relative

configuration of compound 63 was deduced considering the coupling constant pattern and the NOESY spectrum (Table 3.2). The stereochemistry of all tetrahedral stereocenters was found to be identical to that of tuckeyanol A (64).

Table 3.3. NMR data of tuckeyanol B (63), (a CDCl3; b C6C6, 1H 400 MHz, 13C 100.61 MHz; δ in ppm, J in Hz).

Position

1H a

1H b

13C b

DEPTb

1α 1β 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17a 17b 18 19 20 15-OH 3-OAc

2.36 m 1.82 m 2.16 m 5.50 br s 2.58 m 5.80 br s — 5.94 br s 5.95 br s — — 5.93 d (16.0) 5.91 m 2.58 m 5.51 br s — 0.99 d (6.4) 5.25 s 4.92 s 1.16 s 1.33 s 1.28 d (7.2) 3.19 br s — 2.08 s — 1.82 s — 2.67 dd (14.0; 6.8) 1.19 d (6.8) 1.18 d (6.8) — 2.14 s — — 8.05 d (7.2) 7.57 t (7.2) 7.49 t (7.2)

1.98 m 1.75 m 1.75 m 5.74 br s 2.68 br s 6.19 br s — 6.35 br s 6.40 br s — — 6.02 d (16.0) 6.03 dd (16.0, 8.0) 2.29 m 5.82 br s — 0.83 d (6.0) 5.63 s 5.20 s 1.35 s 1.41 s 1.19 d (6.8) 3.20 br s — 1.66 s — 1.32 s — 2.47 m 1.05 d (6.8) 1.12 d (6.8) — 1.92 s — — 8.42 d (8.0) 7.42 d (8.0) 7.24 d (8.0)

47.0

CH2

36.2 77.7 48.5 70.8 142.2 67.0 71.9 205.0 50.0 134.9 134.6 38.6 80.6 83.1 14.4 111.0

CH CH CH CH C CH CH C C CH CH CH CH C CH3 CH2

24.6 25.7 23.5 — 168.5 20.3 168.2 20.5 176.3 33.9 18.6 18.8 169.9 20.1 166.3 129.9 130.4 128.9 133.1

CH3 CH3 CH3 — C CH3 C CH3 C CH CH3 CH3 C CH3 C C CH CH CH

5-OAc 7-OiBu 2’ 3’a 3’b 8-OAc 14-OBz 1’’ 2’’, 6’’ 3’’, 5’’ 4’’

154

Euphorbia tuckeyana: Results and Discussion

1.1.3.

Euphotuckeyanol,

3β,5α,8α,9α,15β-pentacetoxy-2α,7β-dibenzoyloxyjatropha-

6(17),11E-dien-14-one

20

CH3

O BzO 16

1 2

H3C AcO

3

19

11

AcO

14

12

10

15

9

4

7 5

H AcO

CH3

13

8

6

18

CH3 OAc

OBz OAc 17

65

Compound 65, named euphotuckeyanol, was isolated as a colorless oil with [α ]20 D – 42.8. Its molecular formula was determined as C44H50O15 by HR-LSIMS, which showed a pseudomolecular ion at m/z 841.3059 (calcd for C44H50O15Na: 841.3047). The IR spectra of compound 65 showed absorption bands corresponding to a ketone (1723 cm-1) and ester functions (1745, 1274 and 1229 cm-1), as well as bands characteristic of the aromatic ring (1446 and 756 cm-1). The FABMS displayed a base-peak at m/z 105 [C6H5CO]+ and ions corresponding to the loss of AcOH and C6H5COOH, suggesting the presence of acetyl and benzoyl esters in the molecule. These structural features were confirmed through the analysis of the 1H and

13C

NMR spectra of compound 65, which indicated the presence of

two benzoyl and five acetyl residues (Table 3.4). Moreover, the 1H NMR spectrum also showed signals for one secondary (δ 1.16 d, J = 6.8 Hz) and three tertiary methyl groups (δ 0.67, 1.17 and 1.75), and five oxymethine protons, two of them displayed as doublets at δ 5.88 (J = 9.3 Hz) and δ 5.90 (J = 4.0 Hz), and the remaining three signals displayed as broad singlets at δ 4.85, 5.15 and 6.23. Additionally, the presence of an exocyclic double bond indicated by two broad singlets at δ 5.16 and 5.38 and a trans-disubstituted double bond (δ 5.24 d, J = 16.0 Hz; δ 5.44 dd, J = 16.0 and 9.6 Hz), was also observed. Besides the signals assigned to the ester groups, the combined analysis of the 13C and DEPT spectra of compound 65, showed signals corresponding to four methyls, two methylenes (one sp2 at δC 122.9), nine methines (two sp2 at δC 130.4 and 134.7, and five oxymethines at δC 66.4, 70.1, 71.4, 79.1 and 81.9) and, five quaternary carbons (a ketone group at δC 204.1, two oxygenated carbons at δC 88.3 and 90.5 and an olefinic carbon at δC 134.9). On the basis of the twenty degrees of unsaturation deduced by the molecular formula 155

Euphorbia tuckeyana: Results and Discussion

C44H50O15 a polyacylated-jatrophane diterpene structure was proposed for euphotuckeyanol (65).

Table 3.4. NMR data of euphotuckeyanol (65), (CDCl3; 1H 400 MHz, 13C 100.61 MHz; δ in ppm, J in Hz).

Position

1H

13C

DEPT

COSY

HMBC

1α 1β 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17a 17b 18 19 20 2-OBz 1’ 2’, 6’ 3’, 5’ 4’ 3-OAc

3.76 d (17.6) 2.70 d (17.6) — 5.90 d (4.0) 3.93 br dd (9.3; 4.0) 5.88 d (9.3) — 6.23 br s 5.15 br s 4.85 br s — 5.24 d (16.0) 5.44 dd (16.0; 9.6) 3.35 ddd (6.8; 6.4; 2.8) — — 1.75 s 5.38 br s 5.16 br s 0.67 s 1.17 s 1.16 d (6.8)

46.8

CH2

3, 4, 16

88.3 79.1 46.2 71.4 134.9 66.4 70.1 81.9 39.8 134.7 130.4 43.5 204.1 90.5 18.8

C CH CH CH C CH CH CH C CH CH CH C C CH3

1β 1α — 4 3, 5 4 — 8 7 — — 12 11, 13 12, Me-20 — — —

1α, 16 1α, 16 1α 17a — 17a, 9 9 18, 19 9, 11, 12 9, 18, 19 12, 20 11, 12, 20 1α, 1β, 12, 20 1β, 3, 5 1β

122.9

CH2

—

7

25.0 23.5 19.8 165.3 130.8 130.2 128.8 133.4 169.5 20.8 169.3 20.5 165.2 130.7 129.8 128.4 133.3 170.3 21.1 169.9 20.3 169.5 21.3

CH3 CH3 CH3 C C CH CH CH C CH3 C CH3 C C CH CH CH C CH3 C CH3 C CH3

— — 13 — — 3’, 5’ 2’, 4’, 6’ 3’, 5’ — — — — — — 3’’, 5’’ 2’’, 4’’, 6’’ 3’’, 5’’ — — — — — —

19 9, 11, 18 12, 13 — 2’, 3’, 5’, 6’ — — — 3

— 8.51 d (7.6) 7.62 t (7.6) 7.50 t (7.6) 2.02 s

5-OAc 1.32 s 7-OBz 1’’ 2’’, 6’’ 3’’, 5’’ 4’’ 8-OAc

— 8.17 d (7.6) 7.62 t (7.6) 7.50 t (7.6) 2.16 s

9-OAc 1.55 s 15-OAc 2.20 s

156

5 7 2’’, 3’’, 5’’, 6’’ — — — 8 9 — — —

Euphorbia tuckeyana: Results and Discussion

The HMQC and 1H-1H COSY spectra defined four structural fragments with correlated protons: –CH2– (A), –CH(OR)–CH(R)–CH(OR)– (B), –CH(OR)–CH(OR)– (C) and –CH=CH– CH(CH3)– (D), (Figure 3.2). The linkage of these fragments was done through the heteronuclear 2JC-H and 3JC-H connectivities displayed in the HMBC spectrum of compound 65. Accordingly, the observed correlations of C-5/H17a, C-7/H-17a and C-7/H-9 allowed the connection of the fragments B and C with the exocyclic double bond. The cross-peaks between C-9/CH3-18, C-9/CH3-19 and the quaternary carbon C-10/H-11 and C-10/H-12 established the linkage of the fragments C and D. The long-range correlations observed for the carbonyl group at δC 204.1 with H-12, CH3-20 and the diastereotopic methylene protons H-1, located the ketone group at C-14. Finally, The HMBC cross-peaks between C-15/H-1β, C-15/H-3 and C-15/H-5 as well as the 3JC-H correlations of C-1/H-3, C-1/H-4 and C-1/CH316, allowed the connection of the remaining fragments and the establishment of the cyclopentane ring A. The HMBC experiment also allowed the establishment of the acylation pattern. The correlation of the carbonyl signal at δC 165.2 with the proton signal at δ 6.23 indicated the presence of one benzoyl group at C-7. In a similar way, the long range correlations of the carbonyl groups at δC 169.9, 169.5, 169.3 and 170.3 with the oxymethine protons H-3 (δ 5.90), H-5 (δ 5.88), H-8 (δ 5.15) and H-9 (δ 4.85) pointed out the presence of the acetyl groups at these carbons. The positions of the remaining acetyl and benzoyl esters could not be assigned through the analysis of 3JC-H correlations and these groups must be situated in quaternary carbons (C-2 and C-15). The presence of the aromatic ester at C-2 was suggested by the downfield shift of CH3-16 (δ 1.75 s) when compared with the chemical shift of the same methyl group (δ 1.58) in jatrophane derivatives acetylated at this position (Liu and Tan, 2001). The relative stereochemistry and the preferred conformation of euphotuckeyanol (65) were deduced from the NOESY spectrum (Table 3.2) and from the analysis of the coupling constant values. Starting from the α configuration of H-4 (Corea et al, 2005 b; Liu et al, 2001; Li et al, 2003), the strong NOE interactions between H-4 and H-3, H-7 and H-13, indicated the α orientation of these three protons, as well as the β configuration of CH3-20. Furthermore, NOESY correlations between H-13/H-1α, H-13/H-11 and H-11/CH3-18, established their α configuration, while the cross-peaks between CH3-20/H-12, and H-12/CH3-19 indicated the position of these methyl groups above the plane of the macrocycle. The NOESY cross-peaks observed

between

CH3-19/H-9,

CH3-19/H-8

and

H-8/H-9

indicated

the

same

β configuration of these two protons. Finally, the β orientation of CH3-16 and H-5 was

157

Euphorbia tuckeyana: Results and Discussion

deduced from the nuclear Overhauser enhanced signals observed between CH3-16/H-1β and CH3-16/H-5. In contrast to tuckeyanol A (64) and B (63), the presence of a larger J4,5 value (9.3 Hz) and the existence of a NOE cross-peak between H-5 and H-17a showed that compound 65 had a strong preference to remain in the endo-type conformation, where the exomethylene group is perpendicular to the mean plane of the molecule (Jakupovic et al, 1998

a,b;

Marco et

al, 1998). All the above data are in agreement with structure 65, which corresponds to a new polyacylated jatrophane diterpene that was named euphotuckeyanol.

H BzO H3C AcO

..

O H AcO

.

H CH3

H H

H

AcO H

H

H

.

.

CH3 CH3 OAc

H H H OBzOAc H

Figure 3.2. 1H-spin systems (A-B) of compound 65 assigned by the HMQC and COSY experiments (▬) and their connection by the main heteronuclear 2JC-H and 3JC-H correlations displayed in the HMBC spectrum (→).

Jatrophane diterpenes are found exclusively in the Euphorbiaceae family, mainly as polyesters. Although the compounds isolated from Euphorbia tuckeyana have a jatrophane core similar to some diterpenes previously isolated from other Euphorbia species (Corea et al, 2004 b; Marco et al, 1998; Liu et al, 2001), they have a different and unique esterification pattern. This feature contributes to expand the data base of this class of compounds, which are very promising molecules from the biological point of view.

158

Euphorbia tuckeyana: Results and Discussion

1.2. DITERPENES WITH TIGLIANE SKELETON 1.2.1. 4,20-Dideoxy-5-hydroxyphorbol-12,13-diisobutyrate

3'

CH3

3''

2'

H3C

O

1'

3'

18

H

19

H3C

1

11

9

2

13

O

H H

7 5

H

16

CH3

H

8

4

CH3

14

10 3

3''

17

15

OH

H

CH3

O

12

H3C

2'' 1''

H

O

CH3

O

H

6

OH

CH3 20

66

Compound 66 was obtained as a colourless oil with

[α ]20D

+ 45.0. It gave a

pseudomolecular ion at m/z 511 [M + Na]+ corresponding to the molecular formula C28H40O7, from which nine degrees of unsaturation were deduced. Its IR spectrum showed the existence of a hydroxyl function (3420 cm-1), ester carbonyl groups (1720 and 1715 cm-1) and an α,β-unsaturated ketone carbonyl (1690 cm-1). In the EIMS, the ions at m/z 400 [M – C4H8O2]+, 312 [M – 2 × C4H8O2]+ and 71 [C3H7CO]+ pointed to the existence of a butanoyl residue in the molecule. This feature was also supported by the 1H and

13C

spectra of

compound 66 (Table 3.5), which showed signals for two iso-butanoyl groups (δ 1.14 m, 12 H; 2.56 m, 2 H; and δC 179.2, 176.5, 34.2, 34.1, 19.0, 18.8, 2 × 18.5). In addition, the 1H NMR spectrum also showed signals for two tertiary methyl groups displayed as singlets at δ 1.16 and 1.20, one secondary methyl (δ 0.90 d, J = 6.0 Hz) and two vinylic methyl groups (δ 1.74 and 1.84 br s). Moreover, two oxymethine protons (δ 4.82 br s; δ 5.36 d, J = 9.6 Hz) and two olefinic protons at δ 5.29 (br s) and δ 7.67 (s) could also be observed. A broad singlet at δ 5.67 without correlation in the HMQC spectrum is in agreement with the existence of the hydroxyl group in the molecule. Apart from the esters signals the combined analysis of 13C and DEPT spectra (Table 3.5) revealed five methyl groups, nine methines (two oxygenated at δC 71.0 and 76.6, and two olefinic at 127.3 and 162.9) and six quaternary carbons (a carbonyl group at δC 208.6, two 159

Euphorbia tuckeyana: Results and Discussion

olefinic carbons at 138.2 and 140.5, and one oxygen bearing carbon at δC 78.2). The presence of a pentacyclic α,β-unsaturated ketone was supported by the downfield vinylic proton resonance at δ 7.67 and the chemical shift value of the carbonyl signal at δC 208.6, evidencing mesomeric effects due to a conjugated system. From the spectroscopic data, a tigliane-type scaffold was proposed for compound 66. These structural features were confirmed by twodimensional NMR experiments (COSY, HMQC and HMBC, Figure 3.3) which allowed the unambiguous assignment of all 1H and 13C resonances.

Table 3.5. NMR data of 4,20-dideoxy-5-hydroxyphorbol-12,13-diisobutyrate (66) and 4,20-dideoxy-5hydroxyphorbol-12-benzoate-13-isobutyrate (67), (1H 400MHz; 13C 100.61 MHz; CDCl3, δ in ppm, J in Hz).

66 Position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

67

1H

13C

DEPT

1H

13C

DEPT

7.67 s ⎯ ⎯ 2.56 m 4.82 br s ⎯ 5.29 br s 2.25 br t ⎯ 3.50 br t (1.8) 1.49 m 5.36 d (9.6) ⎯ 0.99 d (5.6) ⎯ 1.20 s 1.16 s 0.90 d (6.0) 1.74 br s 1.84 s

162.9 138.2 208.6 51.3 71.0 140.5 127.3 42.3 78.2 51.4 42.7 76.6 65.0 36.4 25.9 23.7 16.9 15.2 10.1 21.7

CH C C CH CH C CH CH C CH CH CH C CH C CH3 CH3 CH3 CH3 CH3

7.72 s ⎯ ⎯ 2.64 m 4.88 br s ⎯ 5.35 d (4.8) 2.37 br t ⎯ 3.56 br s 1.70 m 5.65 d (9.6) ⎯ 1.09 d (5.6) ⎯ 1.24 s 1.33 s 0.99 d (6.4) 1.77 d (0.8) 1.89 s

162.8 138.3 208.6 51.4 71.1 140.6 127.3 42.4 78.4 51.5 43.1 77.7 65.1 36.8 26.2 23.7 17.1 15.4 10.2 21.7

CH C C CH CH C CH CH C CH CH CH C CH C CH3 CH3 CH3 CH3 CH3

Other signals: for 66: δ 1.14 m (CH3-3’, CH3-3’’, CH3-4’, CH3-4’’), 2.56 m (H-2’ and H-2’’), 5.67 br s (OH); 176.5 (C-1’), 34.2 (C-2’), 19.0 (C-3’), 18.5 (C-4’), 179.2 (C-1’’), 34.1 (C-2’’), 18.8 (C-3’’), 18.5 (C-4’’); for 67: δ 1.20 d (J = 7.0 Hz, CH3-4’’), 1.22 d (J = 7.0 Hz, CH3-3’’), 2.64 m (H-2’’), 5.85 br s (OH), 7.48 t (J = 7.2, H-3’ and H-5’), 7.61 t (J = 7.2 Hz, H-4’), 8.03 d (J = 7.2 Hz, H-2’ and H-6’); 166.0 (COBz) 129.9 (C-1’), 129.7 (C-2’ and C-6’), 128.5 (C-3’ and C-5’), 133.2 (C-4’), 179.4 (C-1’’), 34.2 (C-2’’), 18.6 (C-3’’), 18.6 (C-4’’).

From the analysis of HMQC and 1H-1H COSY spectra, two correlated spins systems were deduced and their connection was done by the heteronuclear 2J and 3J correlations displayed in the HMBC spectrum, which also allowed the establishment of the acylation pattern (Figure 3.3). The correlation of the carbonyl signal at δC 176.5 with the proton signal at δ 5.36 indicated the presence of one iso-butanoyl group at C-12. The position of the other ester residue could not be established by the analysis of HMBC correlations, thus this group

160

Euphorbia tuckeyana: Results and Discussion

must be located in a quaternary carbon. By comparison of all spectroscopic data with those reported in the literature for similar phorbol derivatives, the remaining iso-butanoyl ester was located at C-13 and compound 66 was identified as 4,20-dideoxy-5-hydroxy-12,13 diisobutyrate (Dagang et al, 1994; Marco et al, 1999; Appendino et al, 1998).

H3C H H

H3C

O

.

.

H OR

RO H

.

OH

CH3 CH3 H

H

H H H

CH3

OH

Figure 3.3. 1H-spin systems of compound 66 assigned by the HMQC and COSY NMR (▬) and their connection by the main 2JC-H and 3JC-H correlations displayed in the HMBC spectrum (→).

1.2.2. 4,20-Dideoxy-5-hydroxyphorbol-12-benzoate-13-isobutyrate

5' 6' 3''

4'

O

1'

3'

2'

18

H

19

H3C

1

11

H 9

2

13

O

16

CH3

H

6

OH

H

H

H 7

5

CH3

14

8

4

H

3''

17

15

OH

10 3

CH3

O

12

H3 C

2'' 1''

H

O

CH3

O

CH3 20

67

Compound 67 was obtained as a colourless oil with [α ]20 D + 38.1. Its FABMS showed a pseudomolecular ion at m/z 545 [M + Na]+ and ions at m/z 401 [M + H – C6H5CO2H]+, 313 [M + H – C4H8O2 – C6H5CO2H]+, 105 [C6H5CO]+ and 71 [C3H7CO]+, suggesting the presence of a 161

Euphorbia tuckeyana: Results and Discussion

benzoyl and a butanoyl group in the molecule. The IR spectrum displayed absorption bands for hydroxyl (3399 cm-1) and carbonyl (1714 cm-1) groups, as well as for an aromatic ring (1630 and 711 cm-1). The 1H and

13C

NMR spectra of compound 67 (Table 3.5) closely resembled

those recorded for 4,20-dideoxy-5-hydroxy-12,13-diisobutyrate (66). In fact, the most remarkable difference was the presence of signals for the benzoyl group (δ 7.48 t, J = 7.2 Hz; 7.61 t, J = 7.2 Hz; 8.03 d, J = 7.2 Hz; and δC 166.0, 129.9, 129.7, 128.5 and 133.2), while one of the iso-butyrate groups was found to be absent. Taking into account the NMR data and the molecular formula C31H38O7, compound 67 was identified as 4,20-dideoxy-5-hydroxyphorbol12-benzoate-13-isobutyrate. The structure was confirmed by two-dimensional NMR experiments (COSY, HMQC and HMBC), which provided evidence for the unambiguous assignment of all the proton and carbon resonances. The HMBC experiment also allowed the location of the benzoyl ester at C-12 due to the presence of a long range correlation between the carbonyl carbon at δC 166.0 and the oxymethine proton at δ 5.65 (H-12). All the spectroscopic data were in agreement to those described in the literature for tigliane-type diterpenes (Dagang et al, 1994; Marco et al, 1999; Appendino et al, 1998).

1.3. Ent-ABIETANE LACTONES 1.3.1. Helioscopinolide A

O O

H 11

19

16 15

12

9

14

1

RO

2

10

3

5

H

4

H

20

13

8

H

7

6

H 17

18

69 R = H 70 R = Ac

Helioscopinolide A (69) was obtained as white crystals of m.p. 206 – 208 ºC. Its EIMS showed a molecular ion at m/z 316, consistent with the molecular formula C20H28O3 (seven degrees of unsaturation) and an ion at m/z 298 due to the loss of water by the molecular ion.

162

Euphorbia tuckeyana: Results and Discussion

The IR spectrum confirmed the presence of a hydroxyl group (3483 cm-1), and showed in addition, the absorption bands for a lactone carbonyl group at 1732 cm-1. The 1H NMR spectrum (Table 3.6) exhibited signals for three tertiary methyl groups displayed as singlets at δ 0.80, 0.95 and 1.0 and one vinylic methyl at δ 1.80 (d, J = 1.2 Hz). Moreover, the aforementioned spectrum also showed signals for an isolated olefinic proton (δ 6.25, s) and two protons on carbon bearing oxygen atoms (δ 3.25, dd, J = 11.6 and 4.0 Hz, and δ 4.84, dd, J = 13.6 and 5.2 Hz).

Table 3.6. 1H NMR data of helioscopinolides A (69), B (56), D (68), E (55), 3-acetoxy-helioscopinolide B (57) and 3acetoxy-helioscopinolide A (70) (400 MHz, CDCl3, δ in ppm, J in Hz).

Position

55

56

57

68

3

⎯

3.49 t (2.8)

4.71 t (2.8)

⎯

9

2.27 d (8.4) 4.89 dd (13.2; 5.2) 6.34 s 1.14 s 1.07 s 1.10 s 1.85 d (0.8) ⎯

2.38 d (8.4) 4.89 dd (13.2; 4.8) 6.28 s 1.00 s 0.89 s 0.96 s 1.84 d (1.2) ⎯

2.32 d (8.4) 4.89 dd (13.2; 4.8) 6.30 s 0.97 s 0.91 s 0.95 s 1.85 s 2.09 s

⎯ 4.90 dd (12.0; 4.8) 6.42 d (1.6) 1.16 s 1.08 s 1.10 s 1.88 d (1.8) ⎯

12 14 17 18 19 20 CH3CO

The

13C

69

70

3.25 dd (11.6; 4.0) 2.14 d (8.4) 4.84 dd (13.6; 5.2) 6.25 s 1.00 s 0.80 s 0.95 s 1.80 d (1.2) ⎯

4.55 dd (12.0; 4.4) 2.19 d (8.4) 4.89 dd (13.2; 4.8) 6.30 s 0.97 s 0.91 s 0.95 s 1.85 s 2.09 s

NMR spectrum (Table 3.7) revealed the presence of twenty signals

discriminated by a DEPT experiment as four methyl groups (one vinylic at δC 8.2), five methylenes, five methines (two oxygenated at δC 76.0 and 78.6, and one olefinic at δC 114.2) and six quaternary carbons (a conjugated lactone carbonyl at δC 175.3 and three olefinic carbons at δC 116.5, 151.3 and 156.1). All the previous data suggested that compound 69 was an ent-abietane diterpenic lactone and was identified as helioscopinolide A, through comparison of the spectroscopic data with those described in the literature (Borghi et al, 1991).

163

Euphorbia tuckeyana: Results and Discussion

Table 3.7. 13C NMR and DEPT data of helioscopinolides B (56) and A (69), (100.61 MHz, CDCl3, δ in ppm).

56

69

Position

13C

DEPT

13C

DEPT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

32.1 25.7 75.6 37.8 48.3 23.4 37.1 152.1 51.6 41.3 27.5 76.0 156.1 114.1 116.3 175.3 28.7 22.2 16.7 8.2

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C CH C C CH3 CH3 CH3 CH3

37.4 27.5 78.6 39.0 54.4 23.4 37.1 151.3 51.6 41.3 27.5 76.0 156.1 114.2 116.5 175.3 28.7 15.5 16.7 8.2

CH2 CH2 CH C CH CH2 CH2 C CH C CH2 CH C CH C C CH3 CH3 CH3 CH3

Treatment of helioscopinolide A (69) with acetic anhydride and pyridine at room temperature yielded 3β-acetoxy-helioscopinolide A (70). The IR spectrum exhibited the presence of an additional absorption band for the ester carbonyl group (1742 - 1729 cm-1), and the absence of the hydroxyl absorption band. The presence of the acetyl group was confirmed by the MS of the derivative which gave a molecular ion at m/z 358. The 1H NMR spectrum (Table 3.6) of compound 70 showed that acetylation had taken place due to downfield chemical shift of H-3 (δ 4.55, dd, J = 12.0 and 4.4 Hz).

164

Euphorbia tuckeyana: Results and Discussion

1.3.2. Helioscopinolide B

O O

H 11

19

13 14

1

RO

10

3

5

H

4

H

20

15

12

9 2

16

8

H

7

6

H 17

18

56 R = H 57 R = Ac

Compound 56 was isolated as white crystals of m.p. 185 – 186 ºC. Its MS, IR, 1H and 13C NMR data were very similar to those obtained for helioscopinolide A (69), except for the signal of H-3, which appeared in compound 56 as a triplet with a downfield chemical shift (δ 3.49). This difference could be rationalized in terms of the different configuration at C-3 (Figure 3.4). In compound 56, H-3 is in an equatorial position as can be deduced by its coupling constant values and splitting pattern (t, J3e,2a ≈ J3e,2e ≈ 2.8 Hz), while in compound 69, H-3 has an axial orientation, being displayed as a double doublet (J3a,2a = 11.6 Hz and J3a,2e = 4.0 Hz). By comparison of all the spectroscopic data to those reported in the literature, compound 56 was identified as helioscopinolide B (Perellino et al, 1996).

H 3

HO

OH H

H

3

2

H H 69 J3a,2a = 11.6 Hz J3a,2e = 4.0 Hz

2

H 56 J3e,2a = J3e,2e = 2.8 Hz

Figure 3.4. Coupling constant values of H-3 NMR signals in helioscopinolides A (69) and B (56).

165

Euphorbia tuckeyana: Results and Discussion

Treatment of helioscopinolide B (56) with acetic anhydride and pyridine at room temperature yielded 3β-acetoxy-helioscopinolide B (57). The IR spectrum exhibited the presence of an additional absorption band for the ester carbonyl group (1742 - 1729 cm-1), and the absence of the hydroxyl absorption band. The presence of the acetyl group was confirmed by the MS of the derivative which gave a molecular ion peak at m/z 358. The 1H NMR spectrum (Table 3.6) of compound 57 showed that acetylation had taken place due to downfield chemical shift of H-3 (δ 4.55, br s).

1.3.3. Helioscopinolide D

O O

H 11

19

O 17

10 5

14 8

OH7

4

20

13

9

3

15

12

1 2

16

H

6

18

68

Compound 68, identified as helioscopinolide D, was obtained as white crystals of m.p. 248 – 250 ºC. Its molecular formula C20H26O4 was deduced by the EIMS, which showed, in addition to a molecular ion at m/z 330, an ion at m/z 312 due to the loss of water. The IR and NMR spectra of compound 68 closely resembled those of helioscopinolides A and B, suggesting a similar ent-abietanolide structure. In fact, the 1H NMR spectrum (Table 3.6) also showed the presence of signals corresponding to three tertiary (δ 1.08, 1.10 and 1.16) and one vinylic (δ 1.80 d, J = 1.8 Hz) methyl groups as well as an olefinic signal displayed as a doublet at δ 6.42 (J = 1.6 Hz). However, the presence of the H-3 and the H-9 signals was not observed. The

13C

and DEPT NMR spectra (Table 3.8) of compound 68 showed twenty

carbon resonances corresponding to four methyls (one vinylic at δC 8.4), five methylenes, three methines (one oxymethine at δC 76.9 and one sp2 at δC 115.8) and eight quaternary carbons (three olefinic carbons at δC 118.0, 152.3 and 154.7, an α,β-unsaturated lactone carbonyl at δC 174.9 and a ketone carbonyl at δC 216.6). Comparing the 13C NMR spectrum of

166

Euphorbia tuckeyana: Results and Discussion

this compound with those of helioscopinolides A (69) and B (56), the most evident differences were the replacement of the C-3 oxymethine signal by a ketone signal at δC 216.6 and the presence of a tertiary oxygenated carbon at δC 76.9, along with changes of the chemical shifts of the corresponding neighbouring carbons. From the analysis of the spectroscopic data, it could be concluded that compound 68 has a carbonyl group instead of a hydroxyl group at C-3 and an additional hydroxyl function at C-9, which was in agreement with the data described in the literature for helioscopinolide D (Borghi et al, 1991).

1.3.4. Helioscopinolide E

O O

H 11

19

O 17

3

5

H

4

20

13

9 10

15

12 14

1 2

16

8

H

7

6

18

55

Helioscopinolide E (55) was obtained as white crystals of m.p. 180 – 183 ºC. The EIMS showed a molecular ion at m/z 314 that was consistent with the molecular formula C20H26O3. Its IR spectrum displayed absorption bands for an α,β-unsaturated γ-lactone and a ketone carbonyl group at 1751 and 1701 cm-1, respectively. Moreover the absence of the absorption band for the hydroxyl group was also observed. Comparing the 1H and 13C NMR data (Tables 3.6 and 3.8) of compound 55 with those of helioscopinolide D (68), and taking into account its molecular formula, it could be concluded that both compounds showed the same general structure but differ on the substitution of C-9. For compound 55, this tertiary carbon resonates at δC 50.7 instead of the downfield chemical shift of δC 76.9 observed for helioscopinolide D (68), which indicated that helioscopinolide E (55) is a 9-deoxy derivative of helioscopinolide D (68). The identification of helioscopinolide E was confirmed through comparison of its spectroscopic data to those reported in the literature (Borghi et al, 1991).

167

Euphorbia tuckeyana: Results and Discussion

Table 3.8. 13C NMR and DEPT data of helioscopinolides E (55) and D (68), (100.61 MHz, CDCl3, δ in ppm).

55

168

68

Position

13C

DEPT

13C

DEPT

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

37.4 34.4 215.5 47.5 54.8 24.6 36.6 150.2 50.7 40.9 27.8 75.6 155.6 114.7 117.1 175.0 26.5 21.8 16.2 8.3

CH2 CH2 C C CH CH2 CH2 C CH C CH2 CH C CH C C CH3 CH3 CH3 CH3

30.6 34.3 216.6 47.2 46. 1 24.1 32.2 152.3 76.9 43.9 40.0 76.9 154.7 115.8 118.0 174.9 27.1 21.7 17.8 8.4

CH2 CH2 C C CH CH2 CH2 C C C CH2 CH C CH C C CH3 CH3 CH3 CH3

Euphorbia tuckeyana: Results and Discussion

2. STRUCTURE ELUCIDATION OF PHENOLIC COMPOUNDS 2.1. FLAVANOIDES 2.1.1. Naringenin

3' 4'

2' 8

RO

9

7

O 2

6

5'

1' 6'

3

10

4

5

OH

OR1

O

58 R = R1 = H 59 R = R1 = CH3 60 R = CH3; R1 = H

Compound 58 was obtained as an amorphous white solid, [α ]20 D – 23.2, and identified as naringenin on the basis of its spectroscopic data. The FABMS showed pseudomolecular ions at m/z 295 [M + Na]+ and 273 [M + H]+, compatible with the molecular formula C15H12O5. The UV spectrum (MeOH) displayed an intense band II absorption (λmax 289 nm) and a small inflection representing band I (λmax 326 nm), very characteristic of the flavanones and dehydroflavonols (Table 3.9). These bands are the result of the lack of conjugation between A and B rings and distinguishes these compounds from the corresponding flavones, flavonols and chalcones (Mabry et al, 1970; Grayer, 1989). In the 1H NMR spectrum (Table 3.10), the flavanone skeleton of compound 58 was indicated by the presence of three double doublets at δ 5.29 (J = 12.8 and 2.8 Hz, H-2), 3.08 (J = 17.2 and 12.8 Hz, H-3ax), and 2.66 (J = 17.2 and 2.8 Hz, H-3eq). The 5,7-substitution pattern of the A-ring was deduced from the observation of aromatic singlets at δ 5.87 (2 H, H-6 and H-8).

169

Euphorbia tuckeyana: Results and Discussion

Table 3.9. UV spectra (band II) of naringenin (58), naringenin-4’,7-dimethylether (59), naringenin-7methylether (60) and aromadendrin (61), in neutral conditions (MeOH) and in the presence of some shift reagents (λmax, nm).

58 Band II

Reagents

λmax (nm)

MeOH NaOMe NaOAc NaOAc + H3BO3 AlCl3 AlCl3 + HCl

59 Δλmax

Band II λmax (nm)

60 Δλmax

Band II λmax (nm)

61 Δλmax

Band II λmax (nm)

Δλmax

289 325 325

⎯ + 36 + 36

287 ⎯ 287

⎯ ⎯ 0

286 ⎯ 286

⎯ ⎯ 0

291 327 325

⎯ + 36 + 34

288

–1

288

+1

286

0

288

–3

311

+ 22

311

+ 24

310

+ 24

315

+ 24

311

+ 22

310

+ 23

310

+ 24

315

+ 24

The existence of free hydroxyl groups at those positions was confirmed by the UV experiments that were performed in the presence of various shift reagents (Mabry et al, 1970; Grayer 1989). In this way, the bathochromic shift observed at band II with the addition of NaOMe (Δλmax = + 36 nm) and NaOAc (Δλmax = + 36 nm) indicated that not only the A ring was hydroxylated, but also that there was a free hydroxyl group at C-7. Furthermore, the absence of a bathochromic shift with the addition of NaOAc + H3BO3 corroborated that the two hydroxyl groups in ring A were not in ortho positions. Finally, the presence of a free hydroxyl group at C-5 was evident by the addition of AlCl3 followed by HCl, which caused a consistent bathochromic shift of band II (Δλmax = + 22 nm), (Mabry et al, 1970). The para-substitution pattern of ring B was pointed out in the 1H NMR spectrum, by the existence of a typical pair of doublets at δ 7.29 (2 H, J = 8.8 Hz, H-2’ and H-6’) and 6.80 (2 H, J = 8.8 Hz, H-3’ and H-5’); the doublet for the C-3’ and C-5’ protons (which are shielded by the C-4’ oxygen substituent) appeared upfield from the C-2’ and C-6’ protons, as expected (Mabry et al, 1970). The

13C

NMR spectrum (Table 3.10) showed fifteen carbon resonances and together

with the above statements corroborated the proposed structure for compound 58 that was identified as naringenin (Prescott et al, 2002; Ibrahim et al, 2003; Kim et al, 2005).

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Euphorbia tuckeyana: Results and Discussion

Table 3.10. NMR data of naringenin (58), (1H 400MHz; MHz; MeOD; δ in ppm, J in Hz).

100.61

58

Position 2 3ax 3eq 4 5 6 7 8 9 10 1’ 2’ 3’ 4’ 5’ 6’ 4’-OCH3 7-OCH3

13C

1H

13C

5.29 dd (12.8; 2.8) 3.08 dd (17.2; 12.8) 2.66 dd (17.2; 2.8) ⎯ ⎯ 5.87 s

80.4 44.0

5.87 s ⎯ ⎯ ⎯ 7.29 d (8.8) 6.80 d (8.8) ⎯ 6.80 d (8.8) 7.29 d (8.8) ⎯ ⎯

197.7 163.7 97.0 168.3 96.2 164.8 103.3 131.1 129.0 116.3 159.0 116.3 129.0 ⎯ ⎯

Naringenin (58) was methylated with diazomethane to afford two derivatives: naringenin-4’,7-dimethylether (59) and naringenin-7-methylether (60), also known as sakuranetin. The EIMS of the dimethylether derivative 59 displayed a molecular ion at m/z 300 [M]+ compatible with the molecular formula C17H16O5. The presence of two methoxy groups was indicated in the 1H and 13C NMR spectra (Table 3.11) by the presence of two singlets at δ 3.98 (δC 55.4) and 3.83 (δC 55.7). The location of these methoxy groups was achieved by UV experiments (Table 3.9). The absence of a band II bathochromic shift with the addition of NaOAc (a weaker base that ionizes only the more acidic hydroxyl groups) and the existence of a bathochromic shift (Δλmax = + 22 nm) with AlCl3 that did not disappeared with the addition of HCl (due to the formation of acid stable complexes between 5-OH and the carbonyl group) allowed the unambiguous assignment of the methoxy groups to C- 7 and C4’ (Mabry et al, 1970; Seidel et al, 2000).

171

Euphorbia tuckeyana: Results and Discussion

Table 3.11. NMR data of naringenin-4’,7-dimethylether (59) and naringenin-7-methylether (60), (1H 400MHz; 13C 100.61 MHz; CDCl3; δ in ppm, J in Hz).

59

Position

2 3ax 3eq 4 5 6 7 8 9 10 1’ 2’ 3’ 4’ 5’ 6’ 4’-OCH3 7-OCH3

60

1H

13C

1H

13C

5.39 dd (12.8; 3.0) 3.13 dd (17.2; 12.8) 2.81 dd (17.2; 2.8) ⎯ ⎯ 6.09 d (2.2) ⎯ 6.06 d (2.2) ⎯ ⎯ ⎯ 7.41 d (8.8) 6.98 d (8.4)

79.0 43.2

5.36 dd (12.8; 2.8) 3.11 dd (17.2; 12.8) 2.80 dd (17.2; 2.8) ⎯ ⎯ 6.02 d (2.0) ⎯ 6.06 d (2.0) ⎯ ⎯ ⎯ 7.33 d (8.8) 6.90 d (8.4) ⎯ 6.90 d (8.4) 7.33 d (8.8) ⎯ 3.82 s

79.0 43.1

6.98 d (8.4) 7.41 d (8.8) 3.98 s 3.83 s

196.1 162.9 95.1 168.0 94.2 164.1 103.2 130.4 127.8 114.2 160.0 114.2 127.8 55.4 55.7

196.3 162.9 95.1 168.1 94.3 164.1 103.1 130.3 128.0 115.7 156.4 115.7 128.0 ⎯ 55.7

Compound 60 was identified as naringenin-7-methylether on the basis of the comparison of its spectroscopic features to those of compounds 58 and 61. The NMR spectra (Table 3.11) showed one signal corresponding to a methoxy group at δ 3.82 and δC 55.4. The EIMS spectrum exhibited a molecular ion at m/z 286 that suggested the molecular formula C16H14O5. The UV spectra showed no bathochromic shift with the addition of NaOAc indicating that the methoxy group was located at C-7 (Table 3.9). This was also corroborated by the observation of a NOE correlation between the methoxy singlet at δ 3.82 and the aromatic proton signals at δ 6.09 and 6.06 (H-6 and H-8), in the NOESY spectrum. All the spectroscopic data were in agreement to those described in the literature for that compound (Liu et al, 1992; Ibrahim et al, 2003).

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Euphorbia tuckeyana: Results and Discussion

2.1.2. Aromadendrin

3' 4'

2' 8

HO

9

7

O

5'

1' 2

OH

6'

3

6

10 5

OH

4

OH

O

61

Compound 61 was isolated as yellow crystals of m.p. 230 - 232 ºC, [α ]20 D + 20.0 and was identified as the dehydroflavonol aromadendrin. The FABMS showed a pseudomolecular ion at m/z 307 [M + K]+. Its IR, UV and NMR spectra (Tables 3.9 and 3.12) were very similar to those of naringenin (58) indicating a flavanoid structure for compound 61.

Table 3.12. NMR data of aromadendrin (61), (1H 400MHz; 100.61 MHz; MeOD; δ in ppm, J in Hz).

13C

61 Position 2 3 4 5 6 7 8 9 10 1’ 2’ 3’ 4’ 5’ 6’

1H

13C

4.99 d (11.6) 4.55 d (11.6)

84.9 73.6 198.4 165.2 97. 3 168.7 96.3 164.5 101.8 129.3 130.3 116.1 159.2 116.1 130.3

5.94 d (2.4) 5.90 d (2.0)

7.37 d (8.8) 6.85 d (8.8) 6.85 d (8.8) 7.37 d (8.8)

When comparing to narigenin (58), the most remarkable differences in the 1H NMR spectra of both compounds were the presence of an extra oxymethine signal at δ 4.55 (d, J = 11.6 Hz) that replaced the pair of double doublets (δ 3.08 and 2.66) corresponding to the methylene protons of naringenin (58). The axial position of H-3 was deduced by the large

173

Euphorbia tuckeyana: Results and Discussion

value of the coupling constant (J2,3 = 11.6 Hz), (Prescott et al, 2002). The

13

C NMR spectrum

exhibited fifteen carbon resonances that in combination with the FABMS data (m/z 307 [M + K]+) corroborated the proposed molecular structure. As expected, the existence of an extra hydroxyl function at C-3 lead to the downfield shift of this carbon atom (δC 73.6, ΔδC = + 29.6), as well as of C-2 and C-4 (β-carbons, ΔδC = + 4.5 and + 0.7, respectively). The identification of compound 61 as aromadendrin was confirmed by comparison of its spectroscopic data to those referred in the literature (Prescott et al, 2002).

2.2. OTHER PHENOLIC COMPOUNDS: coniferaldehyde

H

CHO 1

9 8

HO

4

3

2

H

5 7

6

OMe

62

Compound 62 was obtained as yellow oil. The IR spectrum showed absorption bands for hydroxyl (3480 cm-1), carbonyl (1660 cm-1) and aromatic groups (807, 704 cm-1). The EIMS exhibited a molecular ion at m/z 178. The 1H NMR (Table 3.13) disclosed three aromatic proton signals (δ 6.98, d, J = 8.0 Hz; 7.09, d, J = 1.6 Hz; 7.14, dd, J = 8.4 and 2.0 Hz), a doublet corresponding to an aldehyde proton at δ 9.67 (J = 8.0 Hz), and a metoxy signal at δ 3.97. Moreover, two olefinic proton signals at δ 6.61 (dd, J = 16.0 and 7.6 Hz) and 7.45 (d, J = 16.0 Hz) showed the presence of a trans disubstituted double bond. Thus, on the basis of its spectroscopic features, compound 62 was identified as coniferaldehyde (Etse et al, 1988).

Table 3.13. 1H NMR data of coniferaldehyde (62), (1H 400 MHz, CDCl3; δ in ppm, J in Hz).

Position 1 2 3 4 5 6

174

1H

9.67 d (8.0) 6.61 dd (16.0; 7.6) 7.45 d (16.0) ⎯ 7.09 d (1.6) ⎯

Position 7 8 9 6-OCH3 7-OH

1H

⎯ 6.98 d (8.0) 7.14 dd (8.4; 2.0) 3.97 s 6.07 br s