Neoclerodane Diterpenoids from Scutellaria pontica - American

Neoclerodane Diterpenoids from Scutellaria pontica†. Benjamın Rodrıguez,*,‡ Marıa C. de la Torre,‡ Marıa-Luisa Jimeno,‡ Maurizio Bruno,§,...
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J. Nat. Prod. 1997, 60, 348-355

Neoclerodane Diterpenoids from Scutellaria pontica† Benjamı´n Rodrı´guez,*,‡ Marı´a C. de la Torre,‡ Marı´a-Luisa Jimeno,‡ Maurizio Bruno,§,⊥ Nadia Vassallo,⊥ MariaLuisa Bondı`,⊥ Franco Piozzi,§ and Orietta Servettaz| Instituto de Quı´mica Orga´ nica, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain, Dipartimento di Chimica Organica, Universita` di Palermo, Archirafi 20, 90123 Palermo, Italy, Istituto di Chimica e Tecnologia dei Prodotti Naturali-Consiglio Nazionale delle Ricerche (ICTPN-CNR), Archirafi 26, 90123 Palermo, Italy, and Dipartimento di Biologia, Universita` di Milano, Celoria 3, 20132 Milano, Italy Received November 8, 1996X

Seven novel neoclerodane diterpenoids, scupontins A-G, have been isolated from the Me2CO extract of the aerial parts of Scutellaria pontica (1-7), together with the known neoclerodanes scutalbin A and scutalpin M. Structures 1-7 were established by exhaustive NMR spectroscopic studies and chemical transformations. Scupontins A-D (1-4, respectively) and scupontins E (5) and F (6) possess unusual [(3′S,3′′S)-3′-[(3′′-acetoxybutyryl)oxy]butyryloxy and [(3′S,3′′S,3′′′S)3′-[[3′′-[(3′′′-hydroxybutyryl)oxy]butyryl]oxy]butyryl]oxy substituents, respectively, attached to the C-19 position of the neoclerodane framework. In the case of the 6R,7β-dibenzoate derivative 7 (scupontin G) its absolute configuration was established by the CD exciton chirality method. The neoclerodane diterpenes1 isolated from Scutellaria species (Labiatae) are of interest on account of their biological activity as insect antifeedants2-6 and as antifungal agents against plant pathogenic fungi.7 In continuation of our studies on Scutellaria plants6,8-10 we report here on the isolation and structure elucidation of seven new neoclerodane derivatives isolated from Scutellaria pontica C. Koch. Results and Discussion The Me2CO extract of the aerial parts of S. pontica was subjected to extensive chromatography (see Experimental Section) to yield the already known neoclerodane diterpenoids scutalbin A10 and scutalpin M,8 together with seven new substances, scupontins A-G, whose structures (1-7, respectively) were established as follows. Scupontin A (1) had the molecular formula C32H46O12, and its IR spectrum showed absorptions for hydroxyl (3470 cm-1), vinyl ether (3090, 1620 cm-1), and ester (1740, 1250 cm-1) groups. The 1H- and 13C-NMR spectra of 1 (Tables 1 and 2, respectively) revealed the presence of two acetoxyl groups (δH 2.02 and 1.93, both 3H, s; δC 170.6 s, 170.0 s, 21.21 q, and 21.17 q) and two 3-O-acylbutyric ester moieties [δH-3 5.32 qdd and 5.21 br sext, both 1H; δMe-4 1.30 d and 1.27 d, both 3H, J ) 6.2, 6.3 Hz, respectively; δC 169.7 s (C-1′), 169.4 s (C1′′), 40.8 t, double signal (C-2′ and C-2′′), 67.8 d (C-3′), 67.4 d (C-3′′), and 19.89 q and 19.87 q (C-4′ and C-4′′); see also Table 1 for the C-2 methylene protons]. In addition, 1 showed characteristic signals of a neoclerodane diterpene [δH 0.83 d, 3H, J ) 6.5 Hz (Me-17) and 0.95 s, 3H (Me-20); δC 16.4 q (C-17) and 14.1 q (C-20)] having a 4R,18-oxirane [δH 2.96 dd, 1H, Jgem ) 3.9 Hz, * To whom correspondence should be addressed. † Dedicated to the memory of the late Prof. F. Martı´n Panizo (19111996), CSIC, Madrid. ‡ Instituto de Quı´mica Orga ´ nica, CSIC, Madrid. § Dipartimento di Chimica Organica, Universita ` di Palermo. ⊥ Istituto di Chimica e Tecnologia dei Prodotti Naturali-Consiglio Nazionale delle Ricerche, Palermo. | Dipartimento di Biologia, Universita ` di Milano. X Abstract published in Advance ACS Abstracts, April 1, 1997.

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J18B,3R ) 2.3 Hz (HB-18) and 2.25 d, 1H, Jgem ) 3.9 Hz (HA-18); δC 62.3 s (C-4) and 48.4 t (C-18)], an esterified 6R-hydroxyl group (δH-6β 4.63 dd, J6β,7R ) 11.6 Hz, J6β,7β ) 4.6 Hz; δC-6 71.9 d), a C-19 acyloxy grouping (δ 4.80 d and 4.39 d, Jgem ) 12.4 Hz; δC-19 62.3 t), and a tetrahydrofurofuran moiety involving the C-11-C-16 carbons. The presence of a C-14,C-15 olefinic double bond in this structural part was revealed by the 1HNMR signals at δ 4.79 (1H, t, J14,13 ) J14,15 ) 2.4 Hz, H-14) and 6.45 (1H, t, J15,13 ) J15,14 ) 2.4 Hz, H-15), and the carbon atom resonances at δ 101.8 d (C-14) and 146.9 d (C-15). (See Tables 1 and 2 for the remaining proton and carbon resonances of the C-11-C-16 fragment). All the above-mentioned functionalities, except for those of the 3-O-acylbutanoates, have been found in several neoclerodane derivatives previously isolated from Scutellaria species.2,3,6-10 In addition, scupontin A (1) possessed a secondary hydroxyl group attached to the neoclerodane nucleus (δC 68.7 d, geminal proton as a broad multiplet at δ 3.70, W1/2 ) 22 Hz). This hydroxyl group must be placed at the C-2 position because the TOCSY spectrum of 1 showed a structural fragment in which the C-1 and C-3 methylene protons, the C-10 methine proton, and the geminal proton of this hydroxyl group are involved. Double resonance experiments indicated that the C-2 hydroxyl group is equatorial, because irradiation at its geminal proton (δ 3.70 m) transformed the signal of the H-3R axial proton (δ 2.10 td, J3R,3β ) J3R,2β ) 11.8 Hz, J3R,18B ) 2.3 Hz) into a double doublet (J3R,3β ) 11.8 Hz, J3R,18B ) 2.3 Hz), thus establishing a trans-diaxial relationship between these two protons.11 In agreement with the above conclusions, treatment of 1 with Ac2O-pyridine yielded a derivative (8, C34H48O13) for which the IR spectrum was devoid of any hydroxyl absorption and whose 1H-NMR spectrum was identical to that of 1, except for the presence of an additional acetoxyl group (δ 2.00, 3H, s) and the downfield resonance of the H-2β proton (δ 4.73 m). Comparison of the 13C-NMR spectra of 1 and 8 further supported11 the presence of a 2R-hydroxyl group in scupontin A [Table 2, acetylation shifts: ∆δ + 2.0 ppm

© 1997 American Chemical Society and American Society of Pharmacognogy

Nuclerodane Diterpenoids from Scutellaria Table 1.

1H-NMR

proton(s) H-1R H-1β H-2R H-2β H-3R H-3β H-6β H-7R H-7β H-8β H-10β H-11R HA-12 HB-12 H-13β HA-14 HB-14 HA-15 HB-15 H-16β Me-17 HA-18e HB-18f HA-19 HB-19 Me-20 OH-2R OAc-2R OAc-6R OAc-3′′ or 3′′′ HA-2′ HB-2′ H-3′ Me-4′ HA-2′′ HB-2′′ H-3′′ Me-4′′ HA-2′′′ HB-2′′′ H-3′′′ Me-4′′′

Journal of Natural Products, 1997, Vol. 60, No. 4 349

Spectral Data of Compounds 1-3, 5, 8, 10, and 12 1

2

1.58 (m)a 2.44 (m)a

3

3.70 (m)b 2.10 (td) 1.40 (ddd) 4.63 (br dd) 1.60 (m)a 1.46 (m)a 1.46 (m)a 1.58 (dd) 4.01 (dd) 1.63 (br dd) 1.71 (ddd) 3.55(qt) 4.79 (t)

a a a a a a 4.67 (br dd) a a a a 4.01 (dd) a a 3.55 (m)c 4.80 (t)

6.45 (t)

6.45 (dd)

6.03 (d) 0.83 (3H, d) 2.25 (d) 2.96 (dd) 4.39 (br d) 4.80 (d) 0.95 (3H, s) 2.23 (br)

6.00 (d) 0.82 (3H, d) 2.19 (d) 2.96 (dd) 4.41 (br d) 4.84 (d) 0.94 (3H, s)

1.93 (3H, s) 2.02 (3H, s) 2.54 (dd) 2.66 (dd) 5.32 (qdd) 1.30 (3H, d) 2.45 (dd) 2.60 (dd) 5.21 (br sext) 1.27 (3H, d)

1.94 (3H, s) 2.01 (3H, s) 2.54 (dd) 2.68 (dd) 5.32 (br sext) 1.29 (3H, d) 2.45 (dd) 2.58 (dd) 5.23 (br sext) 1.27 (3H, d)

5

8

10

12

∼1.65a ∼2.45a

∼1.60a ∼2.40a

∼1.60a ∼2.50a

∼1.60a ∼2.45a

∼1.60a ∼2.50a

3.70 (m)b ∼2.10a ∼1.40a 4.62 (br dd) ∼1.60a ∼1.45a ∼1.45a ∼1.60a 4.09 (dd) ∼1.60a ∼1.65a 2.87 (m)d a a 3.85 (m) 3.85 (m) 5.65 (d) 0.86 (3H, d) 2.25 (d) 2.96 (dd) 4.39 (br d) 4.81 (d) 0.94 (3H, s) a

3.70 (m)b 2.12 (td) ∼1.40a 4.62 (br dd) ∼1.60a ∼1.45a ∼1.45a ∼1.60a 4.02 (dd) ∼1.60a ∼1.70a 3.55 (m)c 4.79 (t)

4.73 (m)b 2.16 (td) ∼1.50a 4.62 (br dd) ∼1.60a ∼1.50a ∼1.50a ∼1.60a 4.01 (dd) ∼1.60a ∼1.70a 3.60 (m)c 4.79 (t)

4.68 (m)b 2.21 (td) ∼1.35a 4.63 (br dd) ∼1.65a ∼1.50a ∼1.40a ∼1.60a 4.15 (dd) ∼1.60a ∼1.90a 3.25 (m)d 2.40 (dd) 2.89 (dd)

4.67 (dddd) 2.21 (td) 1.36 (ddd) 4.63 (br dd) 1.64 (br q) 1.48 (br dt) 1.40 (ddq) 1.60 (dd) 4.15 (dd) ∼1.60a ∼1.90a 3.25 (m)d 2.39 (dd) 2.88 (dd)

6.45 (dd)

6.43 (dd)

6.04 (d) 0.83 (3H, d) 2.26 (d) 2.96 (dd) 4.36 (br d) 4.83 (d) 0.96 (3H, s) a

5.97 (d) 0.81 (3H, d) 2.28 (d) 2.96 (dd) 4.39 (br d) 4.84 (d) 0.95 (3H, s)

6.01 (d) 0.86 (3H, d) 2.29 (d) 2.98 (dd) 4.40 (br d) 4.83 (d) 0.95 (3H, s)

6.01 (d) 0.86 (3H, d) 2.28 (d) 2.98 (dd) 4.42 (br d) 4.83 (d) 0.96 (3H, s)

2.00 (3H, s) 1.93 (3H, s) 2.01 (3H, s) 2.53 (dd) 2.68 (dd) 5.30 (br sext) 1.28 (3H, d) 2.44 (dd) 2.57 (dd) 5.22 (br sext) 1.26 (3H, d)

2.01 (3H, s) 1.95 (3H, s) 2.03 (3H, s) 2.54 (dd) 2.70 (dd) 5.30 (br sext) 1.30 (3H, d) 2.45 (dd) 2.59 (dd) 5.23 (br sext) 1.27 (3H, d)

2.00 (3H, s) 1.95 (3H, s) 2.02 (3H, s) 2.55 (dd) 2.69 (dd) 5.30 (br sext) 1.30 (3H, d) 2.46 (dd) 2.58 (dd) 5.26 (br sext) 1.26 (3H, d)g 2.45 (dd) 2.59 (dd) 5.24 (br sext) 1.27 (3H, d)g

1.93 (3H, s) 2.02 (3H, s) 2.54 (dd) 2.66 (dd) 5.30 (qdd) 1.30 (3H, d) 2.45 (dd) 2.60 (dd) 5.21 (br sext) 1.27 (3H, d)

1.93 (3H, s) 2.54 (dd) 2.66 (dd) 5.33 (br sext) 1.31 (3H, d) 2.51 (dd) 2.60 (dd) 5.28 (br sext) 1.29 (3H, d) 2.55 (dd) 2.65 (dd) 4.20 (br sext) 1.22 (3H, d)

JH,H (Hz)

1h

2h,i

3h,j

5h

8h

10h

12h

1R,2β 1R,10β 1β,2β 1β,3β 1β,10β 2β,3R 2β,3β 3R,3β 6β,7R 6β,7β 7R,7β 7R,8β 7β,8β 8β,17 11R,12A 11R,12B 12A,12B 12A,13β 12B,13β 13β,14A 13β,14B 13β,15 13β,16β 14A,14B 14A,15A 18A,18B 18B,3R 19A,19B 19A,6β 2′A,2′B 2′A,3′ 2′B,3′ 3′,4′ 2′′A,2′′B 2′′A,3′′ 2′′B,3′′ 3′′,4′′ 2′′′A,2′′′B 2′′′A,3′′′ 2′′′B,3′′′ 3′′′,4′′′

k 10.1 k 1.8 2.0 11.8 4.3 11.8 11.6 4.6 a a a 6.5 4.5 11.7 12.0 < 0.3 8.1 2.4

a a a a a a a a 11.5 4.8 a a a 6.4 4.8 11.5 a k k 2.6

k a k a a 11.7 k 11.7 11.5 4.5 a a a 6.5 4.5 11.7 a k k 2.7

a a a a a 12.2 k 12.2 11.5 4.6 a a a 6.5 4.6 11.5 a k k 2.7

k a k a a 12.0 k 12.2 11.5 4.7 a a a 6.5 5.6 11.2 a k k 4.0 10.4

12.7 12.7 5.2 1.6 2.4 12.2 4.9 12.2 11.5 4.7 13.7 13.0 4.5 6.7 5.5 11.2 a k k 4.0 10.5

2.4 6.2

2.4 6.2

2.0 6.2

2.1 6.3

5.5 18.7

5.5 18.7

2.4 3.9 2.3 12.4 < 0.2 15.6 5.4 7.7 6.2 15.7 6.1 7.2 6.3

2.6 3.9 2.3 12.1 < 0.5 15.7 5.8 7.5 6.0 15.4 5.8 7.5 6.2

k a k a a k k a 11.2 4.6 a a a 6.4 5.4 11.4 a k k k k 0 5.1 a a 3.9 2.2 12.4 < 0.5 15.6 5.4 7.7 6.3 15.7 6.1 7.1 6.3

2.7 3.8 2.1 12.2 < 0.5 15.6 5.4 7.7 6.2 15.7 6.1 7.3 6.3 15.7 5.0 7.9 6.5

2.7 3.9 2.2 12.3 < 0.5 16.0 5.7 7.4 6.3 15.4 5.7 7.5 6.4

3.9 2.1 12.3 < 0.5 15.9 5.7 7.5 6.3 15.3 5.8 7.5 6.4

4.0 2.2 12.5 < 0.5 16.0 5.7 7.5 6.2 15.4 5.7 7.5 6.2 15.5 5.7 7.5 6.2

a This is an overlapped signal. b This signal shows a W c d e 1/2 ) 22 Hz. This signal shows a W1/2 ) 15 Hz. This signal shows a W1/2 ) 24 Hz. This is the exo hydrogen with respect to ring B. f This is the endo hydrogen with respect to ring B. g These assignments may be interchanged. h In all compounds the J1R,1β value was not measured due to overlapping. i In this compound the J values that concern the H-2R and H-2β protons were not measured due to overlapping of these signals. j In this compound J values concerning the 2H-14 and 2H-15 protons were not determined. k Value was not determined.

350

Journal of Natural Products, 1997, Vol. 60, No. 4

Table 2.

13C-NMR

carbon

Rodrı´guez et al.

Spectral Data of Compounds 1-3, 5, 8, 10, and 12 1

2

3

5a

8

10

12

32.0 (t) 69.0 (d) 41.5 (t)

28.1 (t) 70.7 (d) 38.3 (t) 62.1 (s) 44.7 (s) 71.9 (d) 33.4 (t) 36.4 (d) 40.1 (s) 44.4 (d) 84.7 (d) 31.2 (t) 46.0 (d) 101.9 (d) 146.9 (d) 107.7 (d) 16.1 (q) 48.3 (t) 61.9 (t) 14.1 (q) 169.9 (s) 21.1 (q) 170.0 (s) 21.2 (q) 170.4 (s) 21.1 (q) 169.7 (s) 40.9 (t)b 67.6 (d) 19.9 (q) 169.2 (s) 41.1 (t)b 67.2 (d) 19.9 (q)

27.9 (t) 70.5 (d) 38.0 (t) 61.9 (s) 44.4 (s) 71.5 (d) 33.1 (t) 36.1 (d) 40.2 (s) 43.7 (d) 84.5 (d) 31.9 (t) 38.0 (d) 35.1 (t) 175.0 (s) 106.7 (d) 16.0 (q) 48.2 (t) 61.6 (t) 13.7 (q) 169.8 (s) 21.0 (q) 170.0 (s) 21.0 (q) 170.5 (s) 21.1 (q) 169.6 (s) 40.9 (t) 67.4 (d) 19.7 (q) 169.2 (s) 40.7 (t) 67.1 (d) 19.7 (q)

28.0 (t) 70.7 (d) 38.1 (t) 62.1 (s) 44.6 (s) 71.6 (d) 33.2 (t) 36.2 (d) 40.3 (s) 43.8 (d) 84.6 (d) 32.0 (t) 38.1 (d) 35.3 (t) 175.0 (s) 106.8 (d) 16.2 (q) 48.3 (t) 61.7 (t) 13.9 (q) 170.0 (s) 21.1 (q) 170.1 (s) 21.1 (q) 170.6 (s) 21.3 (q) 169.7 (s) 40.9 (t) 67.6 (d) 19.7 (q) 169.2 (s) 40.9 (t) 67.5 (d) 19.8 (q) 169.1 (s) 40.8 (t) 67.2 (d) 19.9 (q)

C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-17 C-18 C-19 C-20 2R-0Ac

32.1 (t) 68.7 (d) 41.0 (t) 62.3 (s) 44.2 (s) 71.9 (d) 33.4 (t) 36.1 (d) 39.9 (s) 44.5 (d) 84.2 (d) 31.3 (t) 46.0 (d) 101.8 (d) 146.9 (d) 107.6 (d) 16.4 (q) 48.4 (t) 62.3 (t) 14.1 (q)

22.2 (t) 25.0 (t) 32.7 (t) 64.9 (s) 45.5 (s) 71.9 (d) 33.4 (t) 36.2 (d) 40.0 (s) 48.4 (d) 84.5 (d) 31.2 (t) 46.0 (d) 101.9 (d) 146.9 (d) 107.7 (d) 16.4 (q) 48.6 (t) 61.9 (t) 14.1 (q)

31.9 (t) 68.6 (d) 41.8 (t) 62.3 (s) 44.2 (s) 71.9 (d) 33.4 (t) 35.6 (d) 40.3 (s) 44.1 (d) 84.7 (d) 32.4 (t) 42.0 (d) 32.5 (t) 68.2 (t) 107.6 (d) 16.4 (q) 48.3 (t) 62.1 (t) 13.9 (q)

6R-OAc

170.0 (s) 21.17 (q) 170.6 (s) 21.21 (q) 169.7 (s) 40.8 (t) 67.8 (d) 19.87 (q)c 169.4 (s) 40.8 (t) 67.4 (d) 19.89 (q)c

170.1 (s) 21.1 (q) 170.1 (s) 21.3 (q) 169.9 (s) 41.0 (t) 67.6 (d) 19.8 (q) 169.3 (s) 40.8 (t) 67.2 (d) 19.9 (q)

169.9 (s) 21.0 (q) 170.3 (s) 21.0 (q) 169.6 (s) 40.8 (t) 67.7 (d) 19.8 (q) 169.3 (s) 40.8 (t) 67.2 (d) 19.8 (q)

3′′- or 3′′′-OAc C-1′ C-2′ C-3′ C-4′ C-1′′ C-2′′ C-3′′ C-4′′ C-1′′′ C-2′′′ C-3′′′ C-4′′′

72.0 (d) 33.0 (t) 36.0 (d) 44.5 (d) 84.0 (d) 31.0 (t) 46.0 (d) 102.0 (d) 147.0 (d) 108.0 (d) 16.0 (q) 48.0 (t) 62.0 (t) 13.5 (q)

20.0 (q)

41.0 (t) 68.0 (d) 19.5 (q) 41.0 (t) 68.0 (d) 19.5 (q) 41.0 (t) 65.0 (d) 22.0 (q)

a Only protonated carbons were measured from the HMQC spectrum; δ values for 5 are accurate to ( 0.5 ppm. C may be reversed.

for the C-2 R-carbon, and -4.0 and -2.7 ppm for the β-carbons (C-1 and C-3, respectively)]. The location of the two acetates and the two 3-Oacylbutyrates of scupontin A as well as their arrangement were unambiguously established from the HMBC spectrum. The H-6β proton of 1 (δ 4.63 br dd) showed a correlation through three bonds with a carbonyl carbon belonging to an acetoxyl group (δ 170.0 s), thus establishing that one of the acetates is attached to the C-6R position. This assignment, together with all the above deductions, implied that 1 possesses a [3′-[(3′′acetoxybutyryl)oxy]butyryl]oxy substituent at the C-19 position. This conclusion was corroborated by the TOCSY and HMBC spectra of 1, which allowed the assignment of both 3-O-acylbutyrate fragments (see Tables 1 and 2) and showed connectivities, among others, between the C-19 methylene protons (δ 4.39 and 4.80) and the carboxyl carbon of the first 3′-O-acylbutyrate (δ 169.7 s), between the H-3′ proton (δ 5.32) of this substituent and the carboxyl carbon of the second 3′′-O-acylbutyrate (δ 169.4 s), and finally, between the H-3′′ proton (δ 5.21) of this last group and the carbonyl carbon corresponding to the other acetoxyl group (δ 170.6 s) of scupontin A. Moreover, the observed connectivities between the H-3′ and H-3′′ protons and the C-1′ and C-1′′ carboxyl carbons, respectively, further supported the arrangement of the ester groups. All the 1H- and 13C-NMR data and the TOCSY, HMQC, and HMBC spectra were in complete agreement with structure 1 for scupontin A and allowed the unequivocal

b,c

These assignments

assignment of all the protons and carbons of this new diterpenoid (see Tables 1 and 2). Reduction of 1 with LiAlH4 gave a complex mixture of products from which enantiomerically pure (S)-(+)1,3-butanediol was isolated and identified12 by its specific rotation value ([R]26D +29.2°)13 and gas chromatographic analysis on a chiral column (see Experimental Section), thus establishing a 3′S,3′′S absolute configuration for the [3′-[(3′′-acetoxybutyryl)oxy]butyryl]oxy moiety of scupontin A (1). The absolute configuration of the clerodane part of 1 was not ascertained. However, on biogenetic grounds, it is reasonable to assume that it belongs to the neoclerodane series,1 like scupontin G (7, see below), cooccurring in the same plant, and other diterpenoids previously found in Scutellaria species.6,8,9,14 The molecular rotation values of 1 ([M]D -196°) and its 2R-Oacetyl derivative (8, [M]D -265°) suggested a steroidlike absolute configuration15 such as in 1 for scupontin A. Scupontin B (2, C32H46O11) is the 2-deoxy derivative of scupontin A (1, C32H46O12). The IR spectrum of 2 was devoid of hydroxyl absorptions, and the only differences observed between the 1H- and 13C-NMR spectra of 1 and 2 were in agreement with the absence in the latter of the 2R-hydroxyl group of the former [2: no signal of the H-2β proton downfield; δC at 22.2 t (C-1), 25.0 t (C-2), 32.7 t (C-3), 64.9 s (C-4), and 48.4 d (C-10); 1: δH-2β 3.70 m; δC at 32.1 t (C-1), 68.7 d (C-2), 41.0 t (C-3), 62.3 s (C-4), and 44.5 d (C-10)]. Moreover, the TOCSY and

Nuclerodane Diterpenoids from Scutellaria

Journal of Natural Products, 1997, Vol. 60, No. 4 351

Chart 1

HMBC spectra of scupontin B were only compatible with a structure such as 2 for this diterpenoid. The 14,15-dihydro derivative of 1 was also present in the Me2CO extract of S. pontica. This diterpenoid (3, scupontin C, C32H48O12) showed 1H- and 13C-NMR spectra almost identical to those of 1 (see Tables 1 and 2). The spectroscopic differences between these compounds are due to the presence in 3 of a hexahydrofurofuran moiety3,6 [C-15 methylene protons at δ 3.85 m, 2H; δC 42.0 d (C-13), 32.5 t (C-14), and 68.2 t (C-15)] instead of the tetrahydrofurofuran part of 1 (see above and Tables 1 and 2). As in the case of 1 and 2, the TOCSY and HMBC spectra of scupontin C confirmed the location and arrangement of its substituents as depicted in formula 3. Scupontin D (4) was homogeneous on TLC, and its 1H-NMR spectrum (see Experimental Section) showed essentially the same signals as those present in the 1HNMR spectrum of 1. The observed differences between the 1H-NMR spectra of 4 and 1 were in agreement with the former being a 1:1 mixture of the C-15 epimers of the 14,15-dihydro-15-hydroxy derivative of the latter. Thus, in 4 the H-11, H-15, and H-16 protons appeared as pairs of signals10 [δ 4.57 dd and 3.96 dd, 0.5 H each, J ) 11.5, 4.1 Hz (H-11R in the 15S and 15R epimer, respectively); 5.61 and 5.50, 0.5 H each, both br d, J ) 4.3, 4.6 Hz, respectively (H-15); 5.81 and 5.76, 0.5 H each, both d, J ) 5.4 Hz (H-16)] instead of the single signals corresponding to the H-11, H-15, and H-16 protons of 1 (see above and Table 1); the rest of the spectrum was identical in both compounds. Treatment of 4 with Ac2O-pyridine gave 9 as a 7:3 mixture of 15-exo and 15-endo epimers, respectively.16 Oxidation of 9 with an excess of Jones reagent yielded the 15,16-γ-lactone derivative 10 (C34H48O14, νmax 1790 cm-1; δH-16 6.01, 1H, d, J16,13 ) 5.5 Hz; δC-15 175.0 s) by an initial hydrolysis of the acetate group at the C-15

hemiacetalic position, as a consequence of the acidity of the reagent,6,9,10 followed by oxidation of the resulting 15,16-hemiacetal group to the corresponding γ-lactone.10 Treatment of the 2R-O-acetyl derivative (8) of scupontin A with Jones’ reagent also yielded 10 by oxidation of a 15,16-hemiacetal, which must have been formed from the 14,15-vinyl ether of the tetrahydrofurofuran moiety of 8, because it is known10,17 that the 14,15-double bond of these diterpenoids is very sensitive even to weak acidic conditions. This correlation established the structure depicted in 4 for scupontin D. Scupontin E (5, C34H50O13) showed hydroxyl absorption (3460 cm-1) in its IR spectrum. Its 1H- and 13CNMR spectra (Tables 1 and 2, respectively) were almost identical to those of 1 and the observed differences were consistent with the presence in the former of a third 3-hydroxybutyric ester group [δH 2.55, 1H, dd, J ) 15.7, 5.0 Hz (HA-2), 2.65, 1H, dd, J ) 15.7, 7.9 Hz (HB-2), 4.20, 1H, br sext (H-3), and 1.22, 3H, d, J ) 6.5 Hz (Me-4); δC 41.0 t (C-2), 65.0 d (C-3), and 22.0 q (C-4); see also Tables 1 and 2] instead of one of the acetates of 1 (see above). The attachment of the acetate group of 5 (δ 1.93, 3H, s) at the C-6R-position was in agreement with its unusual upfield resonance due to the shielding effect of the spatially close 4R,18-oxirane, such as in 1-4 (see Table 1). Consequently, scupontin E possessed structure 5. Like scupontin D (4), scupontin F (6) was a 1:1 mixture of the C-15 epimers of a 15,16-hemicetal. Its 1H-NMR spectrum (see Experimental Section) was very similar to that of 5, both showing the same differences as those found between the 1H-NMR spectra of 4 and 1 (see above). Treatment of 6 with Ac2O-pyridine yielded 11 as a 7:3 mixture of the 15-exo and 15-endo forms, respectively.16 Reaction of 11 with Jones’ reagent gave the 15,16-γ-lactone 12 (C38H54O16, νmax 1790 cm-1; δH-16 6.01, 1H, d, J16,13 ) 5.5 Hz; δC-15 175.0 s), which was

352

Journal of Natural Products, 1997, Vol. 60, No. 4

Table 3.

1H-

and

13C-NMR

Rodrı´guez et al.

Spectral Data of Compound 7

proton(s)

7

JH,H (Hz)

7

carbon

7

carbon(s)

7

H-1R H-2R H-2β H-3R H-3β H-6β H-7R H-10β H-11R H-11β H-14 HA-16 HB-16 Me-17 HA-18 HB-18 Me-19 Me-20 OH-8βc OH-12Rc H-2′,6′ H-3′,5′ H-4′ H-2′′,6′′ H-3′′,5′′ H-4′′

4.39 (td) 2.14 (m)a 1.48 (dddd) 2.37 (br td) 2.20 (m)a 6.02 (d) 5.84 (d) 1.98 (d) 1.65 (d) 2.17 (dd)b 6.10 (t) 4.84 (dd) 4.91 (dd) 1.11 (3H, s) 4.64 (br s) 4.79 (br s) 1.45 (3H, s) 1.56 (3H, s) 1.60 (s) 2.72 (d) 7.89 (2H, dd) 7.34 (2H, t) 7.48 (tt) 7.74 (2H, dd) 7.24 (2H, t) 7.39 (tt)

1R,2R 1R,2β 1R,10β 2R,2β 2R,3R 2R,3β 2β,3R 2β,3β 3R,3β 3R,18 6β,7R 11R,11β 11β,OH(12)c 14,16A 14,16B 16A,16B 18A (W1/2) 18B (W1/2) 2′,3′ 2′,4′ 2′,5′ 3′,4′ 2′′,3′′ 2′′,4′′ 2′′,5′′ 3′′,4′′

5.4 10.7 10.7 13.4 4.4 a 13.9 4.6 13.9