PHYTOCHEMICAL ANALYSIS, VOL. 4, 19-24 (1993)

Carbon-13 Nuclear Magnetic Resonance Spectra of some Tetracyclic Diterpenoids Isolated from Elaeoselinum Species

Manuel Grande*t, Joaquin R. Morant, Maria Jesus Maciast and Balbino ManchenoS t Departamento de Quimica Organica, Facultad de Ciencias Quimicas, Universidad de Salamanca, Plaza de 10s caidos 1, E-37008 Salamanca, Spain t Departamento de Quimica Orgiinica, Facultad de Ciencias, Universidad de Alicante, Apartado 99, E-03080 Alicante, Spain

The I3C nuclear magnetic resonance (NMR) spectral data for nine ent-kaurenes, 12 ent-beyeranes and four ent-atisene derivatives, isolated from Elaeoselinum species, are discussed. The assignments of the "C signals were based on DEPT sequences, two-dimensional (2D) 6H/K NMR spectra and comparison with known compounds. The assignments of some known ent-kaurenes are revised

Keywords: Umbelliferae; Elaeoselinum; nuclear magnetic resonance; tetracyclic diterpenoids.

INTRODUCTION ~

The most characteristic secondary metabolites found in Umbelliferae (= Apioideae) are coumarins and essen- tial oils (mainly mono- and sesqui-terpenoids), although other less frequent metabolites are also important (Hegnauer, 1973). Diterpenoids are not widety distributed in this family and, as far as we know, the reported metabolites have been isolated only from species of seven genera. Some monocyclic, bicyclic and tricyclic diterpenoids have been found but the most frequent are tetracyclic diterpenoids with kaurane, ati- sane and beyerane skeletons (Grande, 1988), for which structures have been established mainly by spectro- scopic methods.

The I3C nuclear magnetic resonance (NMR) spectra of a number of kaurane diterpenoids have already been reported (Hanson et al., 1976; Wehrli and Nishida, 1977; Von Carstenn-Lichterfelde et al., 1977) and the ability of this technique to discern between C-18 and C-19 hydroxylation has been discussed (Gonzalez et al., 1981). In connection with our phytochemical studies on secondary metabolites isolated from Spanish Elaeoselinurn species, Umbelliferae, (Grande er al., 1986, 1989, 1991a, b) we now report the 13C assign- ments of some new diterpene derivatives with kaurane, atisane and beyerane skeleton4 , based on one-bond and long-range two-dimensional (2D) d H / K spectra of compounds 10, 12, 22 and 36 and also on comparison with the assignments given in literature (Duc et al . , 1981; Gonzalez et al., 1981, Hutchinson et al. , 1984; Mody and Pelletier, 1978; Pinar et al . , 1978; Rodriguez et al., 1978; Von Carstenn-Lichterfelde et al., 1975, 1977; Wahlberg et al . , 1975; Wehrli and Nishida, 1977; Yamasaki et al., 1976). The reported data are also in agreement with the expected substituent effects and can be useful as reference for new assignments.

Author to whom correspondence should be addressed. 8 All these natural compounds belong to the enantiomeric series (ent series). The bold ( p ) and dashed (a) lines shown in the drawings are changed in the names into a and p respectively when the prefix enf is placed before the skeleton name (see Hutchinson et al., 1984).

EXPERIMENTAL

Diterpenoids. We have previously described the isolation of the substances 10, 11, 12, 13, 14 and 15 from Elaeoselinum tenuifolium (Lag.) Lange [ = Distichoselinum tenuifolium (Lag.) Garcia Martin & Silvestre] (Grande et al., 1986, 1991a) and 5 ,6 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 28,29, 31, 34 and 35 from E. asclepium (L.) Bertol subsp. asclepium (Grande et al., 1989, 1991b). Compounds 1,3,4,9,32 and 37

R1 R2 R3 R4 Rs R6

1 CH3 CH3 H H H H 2 CH3 cH20H H H H H 3 CH3 C O O H H H H H 4 CH3 O M e H H H H 5 CH20Ang COOH H H H H 6 CHflAng O M e H H H H 7 CH3 W M e OH H H H 8 a 3 W M e H OH H H 9 c H 3 W M e H H OH H 10 cH3 CH3 H OAng H OH 11 c H 3 CH3 H OH H OH 1 2 c H 3 CH3 H OAng OH OH

E Z Tgl = -COCMe = CHMe Ang = -COCMe= CHMe

Figure 1. Structure of ent-kaur-16-enes 1-12.

0958-0344/93/010019-06 $08.00 0 1993 by John Wiley & Sons, Ltd.

Received 31 July 1992 Accepted (revised) 10 September 1992

20 M. GRANDE ET AL.

,,.s*" RO

13 R = H

14 R = AC

1 5

Figure 2. Structure of ent-kauranes 13-15.

were isolated from the hexane extract of the aerial parts of E. asclepium (L.) Bertol subsp. millefolium (Desf.) Fiori Compound 37 is a new natural product whose structure was established by spectroscopic methods and by hydrolysis to the known 7,15-dihydroxyatis-l6-ene-19-oic acid methyl ester derivative (Rodriguez and Pinar, 1979). Full details on the study of the secondary metabolites from this last plant will be published elsewhere. Compound 36 was isolated some time ago in our laboratory from E. gummiferum (Desf.) Tutin [=Margotia gummifera (Desf.) Lange] (Pascual Teresa et al., 1978), although it was described at the same time by Pinar et al. (1978). The data of the remaining compounds have been included in the tables to modify the original assignments or for comparison purposes: data of 7 and 8 were taken from Yamasaki et al. (1976), of 2 from Gonzilez et al. (1981), of 16 from Von Carstenn-Lichterfelde et al. (1977), of 26,27 and 30 from Duc et al. (1981) and of 33 from Rodriguez et al. (1978).

Spectroscopy. The I3C NMR spectra were measured at 50.3 MHz (Bruker WP 200 SY with ASPECT 2000 computer) in CDCI3 solution with TMS as internal standard. The DEPT program, supplied by Bruker, was used with the following acquisition parameters: AQ = 0.4 s, D1 = 1 s, SI = 16 k, S2 = 6H, D2 = 3.7 ms, PO = 90°, 135". The heteronuclear 2D dH/ dC: NMR experiments were performed using 256 scans accumulation, SI =2k, with delays of D1= 1 s and D 2 = 3.7 ms, chosen to emphasize J values of ca. 135 Hz (one- bond) or with D2= 65 ms, to emphasize values of ca. 7.5 Hz (long-range).

Figure 3. Interactions between C-9 hydroxyl group and adjacent carbon atoms in kaurenes.

16 CH3 H H 17 CH20H H H 18 COOH H H 19 COoMe H H 2 0 CH20H OTgl H 2 1 COOH OTgl H 22 COoMe OTgl H 2 3 COOH OAng H

25 CHO OAng H 2 4 CHO mgl OAC

Figure 4. Structure of ent-beyer-18enes 16-25.

RESULTS AND DISCUSSION

The observed one-bond (JH-c = 135 Hz) and long-range JH-c = 7 Hz) d H / X correlations for 10 and 12 (Table l), combined with DEPT sequences (Bendall and Pegg, 1983; Maudsely and Ernst, 1977), left no doubt about the assignments of carbon resonances for these new kaurenols. The dH/dC correlations displayed by these substances provided assignments for all the carbon

R1 R2 R3

26 CH3 H H 27 CH3 H OH 28 CH20H H OH 2 9 CH20H OH H 30 CH3 =o 31 COoMe =o

Figure 5. Structure of ent-beyeranes 26-31.

I3C NMR SPECTRA OF TETRACYCLIC DITERPENOIDS 21

12 17

~~

32 CH3 H H 33 CH2OH H H 34 COOH H H 35 O O M e H H 36 O O M e OAng H 37 O O M e OTgl OAc

Figure 6. Structure of ent-atis-16-enes 32-37.

signals except those for C-2 and C-12, but their chemi- cal shift are easily assigned because these carbon atoms absorb at quite different fields. Comparison with other kaurane derivatives and using the usual techniques based on the additivity of increments of shielding effects, led us to propose the assignments given in Table 2 for kaurenes 1-15.

We would like to call attention to the data shown in Table 2 for C-1 and C-7 in compound 1, which are in agreement with those proposed for ent-kaurene (Yama- saki et af., 1976) and related compounds (Grande et al., 1989; Hutchinson et af., 1984; Wahlberg et af., 1975), but are reversed with respect to those reported by Hanson et al. (1976) and Gonzalez et al. (1981). The assignments in which the C-1 absorbs at higher field than C-7 seems to be more consistent than the opposite if the effects of C-18 and C-3 hydroxylation (com- pounds 5, 6, 10-15) are considered. In order to decide between both assignments, the C-15 oxygenated com- pounds 7 and 8 are more suitable substances because in these compounds the C-l5p substituent should produce a strong y-gauche effect on C-7 whereas C-1 should

remain almost unaffected; we therefore assign the 40.5 signal to C-1 (A650 .2 ppm) and the 41.3 signal to C-7 (A6 ca. 5 ppm). The three-bond correlation observed between C-1 and H-20 signals in 10 (Table 1) confirmed the proposed assignments for C-1 and C-7.

Compounds 9 and 12 have an axial hydroxyl group at C-9 which causes outstanding shielding effects on C-1, C-5, C-7 and C-15 (y-gauche effect) and deshielding effects on the contiguous carbon atoms C-10, C-8 and C-11 as well as less intense deshielding in the anti carbon atoms C-20, C-14 and C-12 (see Fig. 3). However, the C-14 signal in compound 12 is shielded with respect to 10, but this can be explained by the steric interaction of the 1,3-syn-diaxial substituents on C-9 and C-15 which induces a conformational change of ring B (Hutchinson et af., 1984). These effects are also present in the data displayed by other C-9 hydroxy- lated kaurenoids (Hutchinson et af., 1984; Yamasaki el al., 1976); however, they have not €or instance been considered in the assignments given for 9 by Hutchinson et a f . (1984), so the assignments of this substance should be revised as we propose in Table 2.

The values of the chemical shifts for the carbon atoms of the rings A and B in the beyerane diterpenes (Tables 3 and 4), could easily be assigned by compari- son with those of kaurane derivatives 1-15 due to the close structural similarities of both A/B ring systems. The C-11 to C-17 shifts were initially assigned by comparison with those previously described in the liter- ature (Von Carstenn-Lichterfelde et al., 1977; Wahlberg et af., 1975) and a good correlation between the calculated and observed values were obtained for all beyerane derivatives 16-31. These assignments are also in agreement with those deduced from the 6 H / W spectra of 22. The 2D data provided assignments for all carbon signals except that of C-11 which was thus assigned by elimination.

The chemical shifts for the atisane diterpenoids (Table 5) have been obtained by comparison with the 13C NMR shifts of gummiferolic acid methyl ester 36, whose assignments have been confirmed by one-bond and long-range 2D 6H/6C correlations (Table 1). The observed differences between the gummiferolic ester 13C shifts and those of the compounds 32-35 (Table 5) can be easily explained by the shielding effects of the C-7 axial angeloyloxy group which produces strong upfield shifts on C-15, C-9 and C-5 carbon atoms due to a y-gauche effect. In this case, we did not observe in the

~~

Table 1. Long-range 6H/W correlations for compounds 10, 12, 22 and 36 (C) 7 3 4 5 8 9 10 I ? 12 13 15 16 17 18

10 H 20 18 18 18 20 20 14 14 19 19 19 7 15 15 5

9 9 17 17 3 12 n 18 18 14 14 19

19 19 15 15 15 20 20 20 20 17 17

22a H 20 18 18 20 16 20 20 17 17 17 3 3 6 16 14 14

2 2 7 2 7/9

20 20 15 20 15 15

1 6 117 5 5

36 H 18 18 18 14 17

5 9 a Other correlations for 22: C-2 and H-3; C-6 and H-5; C-7' and H-14.

19 18 5 3

18

5

OMe 3f5 18

OMe 5

20

5 9

5 1

1 9 5

N

N

Tab

le 2

. ''C

NMR sp

ectr

al s

igna

ls o

f enf

-kau

r-16

-ene

s 1-1

2 an

d en

t-ka

uran

es 13

-15

(in p

pm fr

om T

MSY

C

arbo

n l

b

1

1 40

.5

40.5

2

18.9

* 18

.7'

3 42

.2

42.1

4

33.3

33

.3

5 56

.2**

56

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6

20.4

20

.3

7 41

.3

41.3

8

44.3

44

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9 56

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56

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10

39

.5

11

18.3

* 12

33

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13

44.3

14

40

.0

15

49.4

16

15

5.8

17

103.

4 18

33

.7

19

21.7

20

17

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1'

-

2'

-

3'

-

4'

-

5'

-

CO

O&

-

39.4

18

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33.3

44

.0

39.9

49

.2

156.

0 10

2.8

33.7

21

.7

17.6

-

-

-

-

-

-

2b

40.5

18

.3

35.6

38

.7

56.8

20

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41.6

44

.2

56.2

39

.2

18.2

33

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44.0

39

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49.1

15

5.8

103.

0 27

.1

65.4

18

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-

-

-

-

-

-

3

40.7

19

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37.8

43

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57.1

21

.8

41.3

44

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55.1

39

.7

18.4

33

.1

43.9

39

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49.0

15

5.9

103.

0 29

.0

184.

6 15

.6

-

-

-

-

-

-

4b

40.8

19

.5

38.2

43

.9

56.9

22

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41.4

44

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55.1

39

.6

18.6

33

.3

44.2

39

.8

49.1

15

5.7

103.

6 28

.6

177.

5 15

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51.1

-

-

-

-

-

4

40.9

19

.2

38.1

43

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57.2

21

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41.4

44

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55.2

39

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18.4

33

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43.9

39

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49.0

15

5.8

103.

1 28

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178.

0 15

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51.1

-

-

-

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-

5

40.2

*d

18.4

32

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47

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52.4

21

.8

40.8

44

.0

55.3

39

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18.4

33

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43

.8

39.5

* 48

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155.

4 10

3.2

72.0

18

1.7

15.6

167.

5 12

7.6

138.

6 15

.7

20.4

-

6 40

.1 *

18.5

32

.9**

47

.8

52.3

21

.8

40.8

43

.8

55.2

39

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33

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43

.8

39.6

* 48

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155.

5 10

3.1

71.9

17

5.1

15.4

51

.4

167.

5 12

7.6

138.

4 15

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20.5

7b

40.9

19

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38.3

44

.0

57.1

21

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36.6

' 48

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54.0

39

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18.7

33

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36

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82.6

16

1.1

107.

8 28

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177.

7 15

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51.1

-

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-

7 40

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19.2

38

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43.9

57

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21.1

36

.3*

47.8

53

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39.7

18

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32.6

42

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35.3

* 82

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160.

3 10

8.2

28.8

17

8.0

15.7

51

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-

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-

8b

40.9

19

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38.3

44

.0

56.7

22

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36.8

46

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46.0

39

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33

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39

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82.2

15

9.6

104.

4 28

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177.

7 15

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51.1

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40.7

19

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38.1

43

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21

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36.3

45

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39

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33

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38

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82.5

15

8.4

104.

7 28

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178.

0 15

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51.1

-

-

-

-

-

9

32.2

19

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37.8

43

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50.1

21

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49

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77.3

44

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29.6

34

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40

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43.8

15

5.2

103.

1 28

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178.

2 17

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51.1

-

-

-

-

-

10

39.0

27

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78.9

38

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54.9

19

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36.5

46

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48.2

38

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17.8

33

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40.8

38

.8

81.2

15

3.9

106.

3 28

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15.6

17

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168.

1 12

8.1

138.

1 15

.8

20.8

-

11

38.9

27

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79.0

38

.9

54.6

19

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36.5

45

.7

46.5

38

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18.2

33

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40.2

38

.8

82.4

15

8.6

104.

8 28

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15.5

17

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-

-

-

-

-

-

12

34.7

27

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78.4

38

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46.2

19

.8

30.8

50

.0

78.9

44

.9

29.7

35

.5

39.3

36

.5

84.2

15

2.2

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9 28

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15.6

19

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168.

4 12

6.8

139.

9 15

.9

20.7

-

13

39.0

* 38

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27.2

23

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78.7

80

.8

38.9

37

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54.7

54

.9

19.4

19

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35.9

36

.1

47.4

47

.6

47.7

47

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38.7

38

.7

19.0

19

.1

29.0

29

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39.0

39

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38.9

* 38

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81.6

51

.7

68.6

66

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48.0

48

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28.3

28

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15.5

16

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17.7

17

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166.

7 16

6.6

127.

2 12

7.2

139.

3 13

9.3

15.7

15

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20.5

20

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-

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37.9

27

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78.8

38

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54.4

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35.0

34

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202.

3

B 24

.8

id

0 5 l2 h

10.1

b

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17

.8

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Y

28.2

!-

a D

eter

min

ed in

CD

Cl3

exce

pt w

here

sta

ted.

In

pyr

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. A

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te s

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, 17

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Me,

21.

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*,*I

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sign

men

ts m

ay b

e in

terc

hang

ed.

I3C NMR SPECTRA OF TETRACYCLIC DITERPENOIDS 23

Table 3. 13C NMR spectral signals of ent-beyer-15-enes 16-25 (in ppm from TMS)" Carbon 16 17 18 19 20 2lb 22 Wb

1 39.3 39.3 39.6 39.6 39.4 40.4 39.6 40.5 2 18.7 18.3 19.3 19.3 18.3 20.0 19.2 20.1 3 42.2 35.6 38.0 38.2 35.6 38.6 38.0 38.8 4 33.3 38.5 43.9 43.6 38.4 44.0 43.8 44.1 5 56.1 56.9 57.2 57.1 56.1 56.9 56.3 56.9 6 20.3*d 20.4' 21.6 21.6 19.7' 21.5 20.7 21.6 7 37.4 37.7 37.7 37.7 31.6** 32.3 31.4 32.4 8 49.1 49.1 49.2 49.1 53.2 54.0 53.3 54.0 9 53.0 53.0 52.4 52.3 52.9 53.0 52.1 53.1

10 37.4 37.3 37.7 37.7 37.3 38.8 37.7 38.7 11 20.5* 20.2' 20.5 20.4 19.4* 20.5 19.7 20.5 12 33.7 33.2 33.2 33.2 32.6** 33.1 32.4 33.1 13 43.6 43.7 43.7 43.6 48.3 48.9 48.2 48.9 14 61.3 61.2 61.1 61.1 94.4 94.9 94.3 94.8 15 135.2 135.1 134.8 134.7 132.4 132.9 132.0 133.1 16 136.1 136.5 136.5 136.4 133.5 134.1 133.5 134.2 17 25.0 24.9 24.9 24.8 19.2 19.6 19.2 19.7 18 33.8 27.0 29.1 28.9 27.0 29.4 28.8 29.5 19 22.0 65.6 184.3 177.9 65.4 179.1 177.9 179.0 20 15.1 15.8 13.8 13.6 16.0 14.6 13.9 14.6

1' - - - - 168.2 168.1 168.3 168.2 2' - - - - 128.9 129.6 128.8 129.5 3' - - - - 136.4 136.9 136.6 137.4 4' - - - - 14.2 14.2 14.2 15.9 5' - - - - 12.0 12.2 12.0 20.9

51.1 - - 51.0 - COOrv&- - - -

a Determined in CDCI, except where stated. In acetone-d,. Acetate: CO, 170.4; Me, 21.0. *,** These assignments may be interchanged.

24'

36.6 24.1 78.6 51.8 56.9 20.2 31 .O 52.8 51.3 37.0 19.3 32.3 48.3 94.1

131.8 134.1 19.2 20.2

204.4 16.3

168.3 128.8 136.9 14.3 12.1

-

25 38.9 18.4 34.2 48.2 56.0 19.9 31.4 52.9 51.6 37.6 18.8 32.3 48.2 94.1

131.9 133.9 19.3 24.4

205.6 14.8

168.3 128.3 137.2 15.8 20.6

-

2D-NMR spectra of compound 36 any correlations for C-2, C-6, C-7, C-11, C-13 and C-14.

Compound 37, a new natural product, showed one more ester group than 36 and this new ester was placed in C-15/3 according to the observed shielding effects on the methine carbon C-9 as compared with 36 and to the

Table 4. I3C NMR spectral signals of ent-beyeranes 26-31 (in ppm from TMS)"

Carbon 26 27 28b 29b 30 31

1 39.7 39.7 40.3 40.4 40.0 40.3 2 18.5 18.5 18.7 18.7 18.3 19.0 3 42.0**' 42.0 36.2 36.1 41.8 37.9 4 33.1 33.2 39.2 39.2 33.1 43.8 5 56.5 56.0 57.0 57.1'" 55.1 56.0 6 20.4' 19.2 20.2' 20.3' 18.6 20.2 7 41.2'' 38.5 39.5 37.2 32.2 32.6 8 44.9 45.3 45.9 49.5 51.2 51.1 9 56.9 46.8 47.1 56.7*" 60.2 59.3

10 37.6 37.3 37.6 38.2 38.9 39.1 11 20.2' 19.9 20.6' 20.4' 19.1 19.5 12 40.0" 32.4 32.9 39.2 42.2 42.1 13 39.2 40.1 40.6 44.1 46.8 46.8 14 57.7 83.8 83.3 91.4 222.7 222.7 15 37.6 31.9 32.6 31.7 30.3 30.3 16 33.6 29.6 30.0 30.0 27.8 27.4 17 27.1 25.1 25.7 22.6 19.6 19.5 18 33.7 33.6 28.0 28.1 33.6 28.9 19 21.9 22.0 64.4 64.4 21.3 177.8 20 15.1 15.5 16.3 16.3 15.4 13.7

- 51.1 coom - - - - a Determined in CDCI, except where stated.

In pyridine-d,. *,I* These assignments may be interchanged.

fact that the chemical shifts of the methine carbon atoms C-5 and C-12 are nearly the same in both substances. The long-range 2D 6W6C of 37 (D2 = 70 ms

Tables. 13C NMR spectral signals of ent-atis-1Cenes 32-37

Carbon 32 33 34 35 36b 37c

1 39.6*d 39.9' 39.7 39.7 39.3 39.5 2 18.2 17.8 18.8 18.8 18.3 18.3 3 42.3 35.8 38.0 38.2 37.7 37.8 4 33.1 37.6 43.9 43.8 42.7 42.8 5 56.4 57.0 57.3 57.2 49.0 49.1 6 18.8 19.0 20.3 20.3 25.0 25.6 7 39.5* 39.5, 39.7 39.7 75.1 74.3 8 33.5 33.5 33.6 33.4 36.3 38,6 8 52.9 53.0 52.3 52.1 147.2 39.5

10 37.8 38.4 38.4 38.2 37.5 37.4

112 36.6 36.5 36.6 36.6 35.8 35.1

14 27.4'' 27.3" 28.4' 28.3' 26.4' 25.9" 16 48.4 48.1 68.2 48.2 41,2 74.7 16 152.7 162.5 152.7 162.7 160.4 161.3

18 33.7 26.8 29.0 28.7 27.9 28.1 19 2117 66.6 1177.7 177.7 176.6 107.0

- 51,O 50.5 50.7

(in ppm from TMSY

i n ~ 8 . 7 28.6*+ 27.2 27.2 27 .1~ 26.2+

n3 28.7** 28.8*+ 28.8' 2 8 . ~ 27.4' 2 6 . 1 ~

17 104.2 104.3 104.6 104.5 104.9 1ni.o

20 1 ~ 9 114.5 I Q . ~ inL9 11,i qn.9 COOMie - - a Determined in CDCI,.

20.3.

1n.8. Acetate: CO, 169.7; Me, 21.4.

Angelic ester: C-1, 166.11 C-2, 128.0; C-3, 1136.4; C-4, 16.2; C-2'.

Tiglic ester: C-11 166.4; (2-2, 129.6; C-3, 1135.11 C-4, 13,8; C-2'.

P, ** These assignments may be interchanged.

24 M. GRANDE ETAL.

to enhance JCH = 7 Hz) showed correlations between the carbonylic carbon of the tiglate and acetate groups with H-7 (6 4.98, t, J = 2 . 8 H z ) and.H-15 (6 5.31, t, J = 1.6 Hz), respectively, which confirms the structure and assignments given for this last compound. The remaining 2D correlations confirmed the assignments proposed but did not give any more infor- mation on the methylene carbon assignments.

Acknowledgements

We would like to thank Prof. K.-H. Kubeczka, University of Hamburg, and Dr. V. Formacek, Bruker Analytische Messtechnik Gmbh, Rheinstattet, for the facilities and the high-field (11.7T) 2D. NMR spectra of compound 9. One of US (M.J.M.1 greatly acknowl- edge the P.H.E.T. for a grant. This work has been completed with the financial support of the Junta de Castilla y Leon, Project Number 0704l90.

REFERENCES

Bendell, M. R. and Pegeb D. T. (1983). Proton-carbon~l3 two- dimensional chemical shift correlbtion spectroscopy using DEFT. J. Megn, Res. 53, 1144-1148.

Duc, D. K. M., Fetizonb M., Lazare, S., Grant, P. K.. Nicholls, MI J., Liaub HI T. L.. Francis, Mh J., Piossonb J., Bernasseau, J! MI, Roqpe, N1 F., Workulichb P. ML and Wenkert, E. (1981). 13C NMR spectroscopy of tetracarbocyclic diterpenes and relbted substances. Tetrahedron 37, 2371 ~2374.

Gonehllez. A. G., Fraga, 8. M,, Hernandez, M. GI and Hbnsonb J! R. (18811. The lBC NMR spectra of some ent-18-hydtoxykaur- 16-enes. Phytochemistry 20, 846-847.

Grande, Mt (1888). Secondary metabolites in umbellifers: diter- penoidb. In Le Ombrellifere: Ricerche ed Applicazioni, pp. 32-49. Societa ltaliana di Fitochimica.

Grandie, MI. Segura, MI and Mbnchefio, B. (1886). Niew kaurane diterpenoidb from the roots of Elseoselinum tenuifoliumr J. Net. Prod 49. 259-264.

Grande, M., Mancheiio, B. and Sanchez, M. J. (1889). Elescliepiol end elmcliepic acid, beyerane diterpenoidb from Eleeoselinum ssclepium. Phytochemistry 28, 1855- 1858.

Grande, MI, Mbcias, M. J., Manchefio, B., Segura, MI and Zarzo, A. (leela). New kaurane diterpenoidb from the aerial Darts of Distichoselinum tenuifolium. J. Nat. Prod. 54, 866-869.

Grandie, M.. MbncheAo, B. and 6anchez, MI J. ( l89lb). Elbsclepial end other tetracyclic diterpenoids from Elseoselinum asclepium. Ph ytochemistry 30, 1877- 1882.

Harisonb J. R., Siverns, M.. Piozzi, F. and Gavona, GL (1876). The "C NMR spectra of kaurenoid diterpenes. J. Chemt SOC., Perkin Trans. I 1114-1 ll V .

Hegnauer, R. (1873). Chemotaxonomie der Pflanzen, Vol. 6, pp. 564-629: Birkhauser Verlbg, Basel,

Hutchinson. MI, Lewer, P. and Mbcmillbn. J. (1864). CarbonplB NMR spectra of eighteen dlerivatives of ent-kaur-16-en-18- oic acid. J. Chem, SOC.. Perkin Trans. 12363-2366.

Maudbelly, A. A. and Ernst. R. R. (1877). Indirect detection of

magnetic resonance by internucliear two-dimensional spec- troscopy. Chem, Phys. Lett. 50, 368-372.

Mbdv, N! V. and Pellietier, S. W. (1978). "C NMR spectroscopy of atisene and beatchine-type C,,-diterpenoid alkabids from Aconitum and Garrya species. Tetrahedron 34, 242~2431.

Pascual Teresa, J! de, Pascuall M. die, Grande, C.. Jimenez, B.. Santos, J. and Grandie, M L (1878). Components qpimicos die umbellferas: Thapsia villbsa, Magydaris panacifolia, Ferulsgo brachyloba y Elseoselinum gumrniferum. XVlll Reunion Bienal die Ib R.S.E.F.Q. Simposio 30, 53-54.

Pinar, M., Rodtiguez, B. and Aliemanly, A. (1878). Gummiferolic acid1 a new ent-atis-16-ene diterpenoid from Margotia gum mifera. Phytochemistry In, 1637-1 1640.

Rodtiguez, B. and Pinar. M. 11979). Margotianin, a new diterpe- noid from Margotia gummifera. Phytochemistry 18, 8 9 1 ~ 893,

Rodriguez, B., Aliemenn/, A. and Pinar, ML (1878). Carbon-13 NMR spectra of some ent-beyerane derivatives. Tetrahedron Lett. 3069-3070.

Von CarstennrLichterfeldk, C.. Pascual, C., Pons, J!. Rabanali R. M., Rodtiguez, B. and Valberde, S. (1875). CarbonplB NMR spectra of ent-beyerane and ent-beyerene derivatives. Tetrahedkon Lett. 3569-3572.

Von Carstenn-Lichterfelde, C., Pascualb C., Rabanali R. M., Rodtiguez, B. and Valverde, S. (1877). "C NMR substituent effects in tetracyclic diterpenoids. Tetrahedron 33, 1889-

Wahlberg, 11, Enzellt C. R. and Rowe, J. W. (1875). ent-16-Kauren- 19.01 from coffee. Phytochemistry 114, 1677.

Wehrli, F. W. and Nishide, T. (1877). The use of carbonp1B NMR spectroscopy in natural Droducts chemistry. Fortschr. Chemt Org, Naturst. 36, 1B-229.

Yamasaki, K., Kohdb, Hi. Kobayashi, T.. Kasai, R. and Tanaka, 0. (1876). Structures of Stevia diterpene gllycosidies: Applications of "C NMR. Tetrahedron Lett. 1005-1008.

1882.