MAGNETIC RESONANCE IN CHEMISTRYMagn. Reson. Chem.2000;38: 802–804

Note

Complete assignments of 1H and 13C NMR spectra of fourhispanane diterpenoids

Benjamın Rodrıguez1∗ and Giuseppe Savona2

1 Instituto de Quımica Organica, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain2 Dipartimento di Chimica Organica ‘E. Paterno,’ Universita di Palermo, Parco D’Orleans 2, I-90128 Palermo, Italy

Received 10 January 2000; revised 10 April 2000; accepted 12 April 2000

ABSTRACT: An NMR study of four hispanane diterpenoids is described. In addition to conventional 1D NMRmethods, 2D shift-correlated experiments [1H,1H-COSY, 1H,1H-TOCSY, 1H,13C-HSQC-1J(C,H). 1H,13C-HMBC-nJ(C,H) (n D 2 and 3)] and 2D1H,1H-NOESY were used for the complete and unambiguous1H and 13C chemicalshift assignments of these diterpenoids. Copyright 2000 John Wiley & Sons, Ltd.

KEYWORDS: NMR; 1H NMR; 13C NMR; 1D NMR; 2D NMR; diterpenes; hispanane derivatives

INTRODUCTION

The structures of methyl hispanonate (1) and methylhispaninate (2), isolated fromBallota hispanica Neck.ex Nim. (Labiatae), were established 20 years ago bychemical transformations and IR, UV and1H and 13CNMR spectroscopic studies.1 Shortly afterwards, a single-crystal x-ray diffraction analysis confirmed structure1 formethyl hispanonate.2

The structures of1 and 2 are particularly interestingbecause they belong to the very small group of nat-ural diterpenoids that exhibit a hispanane hydrocarbonskeleton,1,3 whose biogenetic pathway has been the subjectof some speculation.3,4 Complete1H NMR assignmentsof 1, 2 and4 have not been performed previously,1 sincemost of the1H signals of the A- and B-rings appear ina narrow chemical shift range. Moreover, the previousassignments of the13C NMR spectra of these hispananederivatives1 were performed only on the basis of gen-eral chemical shift arguments and by comparison with thereported data for other pimarane, isopimarane and abi-etane diterpenoids, thus suggesting that some of thoseassignments need to be re-examined. These facts stimu-lated us to perform the complete assignment of the1Hand 13C NMR spectral data of1 and 2, together withthose of the 11,12-didehydro derivative (3) of the formerand the 9 ,16 -peroxide (4) of the latter,1 in the hopethey could be useful for future assignments of relatedcompounds.

* Correspondence to: B. Rodrıguez, Instituto de Quımica Organica,CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain; e-mail: iqor107@iqog.csic.esContract/grant sponsor: Comision Interministerial de Ciencia y Tec-nologıa (CICYT); Contract/grant number: AGF98-0805.Contract/grant sponsor: Ministero dell’Universita e della Ricerca Sci-entifica e Tecnologica (MURST).

RESULTS AND DISCUSSION

For the complete assignment of the1H and13C NMR spec-tra of 1–4 (Tables 1 and 2, respectively), a combinationof two-dimensional COSY, TOCSY, HSQC, HMBC andNOESY experiments was carried out. Our strategy in the1H and 13C signals assignment of these compounds wasas follows.

In all compounds (1–4), the carbonyl carbon of themethyl ester group (υ 177.7–177.6) showed HMBC con-nectivities with a methine proton (double doublet atυ1.31–1.83, H-5), a methylene proton (H-3, see Table 1)and two methyl groups, one of them belonging to theester group (singlet atυ 3.60–3.66) and the other one(singlet atυ 1.19–1.26) attached to a quaternary carbonatom (υ 43.8–45.3, HMBC two bond connectivity, C-4).These results established that the carbomethoxyl and theC-methyl groups were geminal and attached to the C-4position. The relative configuration of this carbomethoxyl-methyl grouping must be (equatorial, C-18) for the

Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem.2000;38: 802–804

2D NMR OF SOME HISPANANE DITERPENES 803

Table 1. 1H NMR chemical shifts (υ, ppm) and coupling constants (JH,H Hz) for 1–4a

Proton 1 2 3 4 JH,H 1 2 3 4

1˛ 1.21 ddd 1.14 ddd 1.20 ddd 1.97 ddd 1˛,1ˇ 13.2 13.0 13.6 13.01ˇ 1.99 dddd 1.91 ddd 2.22 mb 1.40 dddd 1,2˛ 4.2 3.8 3.6 3.72˛ 1.57 ddddd 1.55 ddddd 1.62 ddddd 1.50 ddddd 1˛,2ˇ 13.2 13.0 13.2 13.62ˇ 1.89 ddddd 1.87 ddddd 1.99 ddddd 1.81 ddddd 1ˇ,2˛ 2.8 2.9 2.8 3.73˛ 1.01 td 1.01 td 1.01 td 1.08 td 1ˇ,2ˇ 3.8 3.8 3.8 3.13ˇ 2.22 dddd 2.22 mb 2.24 mb 2.07 dddd 2,2ˇ 13.6 13.6 14.1 13.85˛ 1.32 dd 1.31 dd 1.38 dd 1.83 dd 2˛,3˛ 4.4 4.3 4.3 3.76˛ 2.08 dddd 2.05 dddd 2.25 mb 2.27 mb 2˛,3ˇ 2.8 2.8 2.8 3.76ˇ 1.64 dddd 1.73 dddd 1.76 dddd 1.95 dddd 2ˇ,3˛ 13.5 13.5 13.6 13.47˛ 2.79 br dc 2.22 mb 2.67 ddd 2.43 ddd 2,3ˇ 3.8 3.8 3.8 3.17ˇ 2.08 mc 2.22 mb 3.13 dd 2.53 dddd 3,3ˇ 13.5 13.5 13.6 13.411 2.50 mb 2.29 mb,c 7.08 d 2.24 mb 5˛,6˛ 0.8 1.8 1.5 8.011 2.50 mb 2.29 mb,c — 2.13 ddd 5 ,6ˇ 13.0 12.4 12.4 11.412 2.63 mb 2.50 mc 7.20 dd 2.28 dddd 6,6ˇ 13.8 13.5 13.9 13.712 2.63 mb 2.66 mc — 2.77 dddd 6,7˛ 6.4 2.0 6.9 1.614 6.31 d 5.78 dddd 6.72 dd 5.63 ddd 6˛,7ˇ 1.3 4.2 0.0 8.715 7.45 d — 7.75 d — 6,7˛ 1.3 7.3 12.4 9.717 — 5.90 dd — 5.95 dd 6ˇ,7ˇ 5.4 1.2 5.0 10.8Me-18 1.20 s 1.21 s 1.26 s 1.19 s 7˛,7ˇ 16.3 —e 19.8 15.1Me-20 0.86 s 0.81 s 1.12 s 1.15 s 11˛,11 —e —e — 15.819-COOMe 3.60 s 3.62 s 3.65 s 3.66 s 11˛,12 —e —e 12.0 5.3

11 ,12 —e —e — 3.411 ,12 —e —e — 13.811 ,12 —e —e — 6.112 ,12 —e —e — 17.914, 15 1.8 — 1.9 —1ˇ,3ˇ 1.3 0 —e 1.87˛,17 — 0 — 07ˇ,17 — 0 — 2.412 ,14 0 1.6 1.0 2.312 ,14 0 1.6 — 0.814, 17 — 1.6 — 0

a All these assignments were in agreement with HSQC, HMBC and NOESY spectra.b Overlapped signals;υ values were measured from the HSQC spectra.c These protons appeared as broadened signals with poor resolution.d These assignments are changed with respect to those reported previously.1

e These coupling constants were not measured owing to overlapping and/or broadening of the signals.

C-methyl group and (axial, C-19) for the carbomethoxylsubstituent, as was rigorously established from the chem-ical shift values of the C-18 (υ 28.2–28.4) and C-19 (υ177.6–177.7) carbons.5,6

Further extension of the C-3, C-4, C-5, C-18 and C-19structural fragment was sorted out since the H-5˛ protonshowed contour peaks with the C-1, C-3, C-4, C-6, C-7,C-10 and C-20 carbons, the H-3˛ proton (υ 1.01–1.08)was connected, among others, with the C-2 carbon (υ18.9–19.9) and the C-8 carbon showed connectivities withthe C-6 and C-7 methylene protons. In addition, the C-20methyl protons (singlet atυ 0.81–1.15, see Table 1) werecorrelated with the C-1, C-5, C-9 and C-10 carbons, thusconfirming their assignments. Moreover, the COSY andTOCSY experiments were in agreement with the aboveassignments for the protons belonging to the A- andB-rings.

The remaining1H and 13C NMR assignments corre-sponding to C- and D-rings of1–4 (Tables 1 and 2) wereachieved taking into account the previous identificationof the C-7–C-10 carbons (see above) and the obvious

assignment of some of the protons and carbons of the˛,ˇ-disubstituted furan in1 and3 and the ,ˇ-unsaturated -lactone in2 and 4. The C-11 methylene protons of1,2 and 4, and also the C-11 olefinic proton of3, wereidentified from their connectivities with the C-8 and C-10carbons observed in the HMBC spectra. The C-14 furanic(1 and3) or olefinic (2 and4) proton showed, among oth-ers, connectivity with the C-12 carbon and, in the case of2, 3 and4, allylic couplings were observed (see Table 1)between the C-12 and C-14 protons. The C-13 olefiniccarbon was identified in all the diterpenoids (1–4) by itsconnectivity with the C-11 protons and, in the case of2and4, by an additional connectivity with the C-17 olefinicproton, which in turn showed a cross peak with the C-7carbon, whereas the C-17 carbonyl carbon of1 and3 dis-played connectivities with the C-7 methylene protons. TheC-15 furanic (1 and 3) or lactonic carbon (2 and 4) wasassigned by its characteristic chemical shift [υ 145.8 d and147.2 d for1 and3, respectively, HSQC correlation withthe˛-furan protons atυ 7.45 d and 7.75 d, respectively;υ169.4 s (2) and 170.3 s (4)] and by its connectivity with

Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem.2000;38: 802–804

804 B. RODRIGUEZ AND G. SAVONA

Table 2. 13C NMR chemical shifts (υ, ppm) for 1–4a

Carbon 1 2 3 4

1 36.3 tb,c 36.7 tc,d 39.2 t 32.0 t2 19.4 t 19.4 t 19.9 t 18.9 t3 37.4 tc 37.4 tc 36.9 t 37.7 t4 43.8 s 43.8 s 44.1 s 45.3 sc

5 53.0 d 52.9 d 52.6 d 43.2 d6 20.1 t 20.7 t 20.0 t 21.1 t7 28.1 td 34.1 t 31.1 t 26.8 t8 134.7 s 126.2 s 145.8 s 150.6 s9 154.0 se 147.9 s 154.2 s 88.7 s10 41.3 s 40.8 s 42.6 s 42.7 sc

11 27.6 tf 25.0 t 129.2 d 24.9 t12 25.0 tf 27.0 t 124.4 d 22.3 t13 135.7 s 151.2 s 129.5 s 168.3 s14 112.7 d 113.6 de 111.1 d 111.9 de

15 145.8 d 169.4 s 147.2 d 170.3 s16 149.6 se 158.1 s 154.4 s 104.7 s17 183.1 s 116.5 de 175.2 s 119.1 de

18 28.2 q 28.3 q 28.3 q 28.4 q19 177.7 s 177.7 s 177.6 s 177.7 s20 16.7 q 16.9 qd 20.2 q 20.4 qCOOCH3 51.2 q 51.2 q 51.3 q 51.4 q

a All these assignments were in agreement with HSQC andHMBC spectra.b Multiplicities were established from the HSQC spectra.c,e,f Within each column, assignments with the same superscriptare reversed with respect to those reported previously.1 For 3 noprevious13C NMR data are available.d These carbons appeared as slightly broadened signals.

H-14 (1–4). Finally, the assignment of the C-16 carbonwas supported by its cross peaks with H-12 (1, 3 and4),H-14 (1–4) and H-15 (1 and3).

In all the compounds studied, the A-ring possesses achair conformation (4C1), as was deduced from the vicinalcoupling constant values of the C-1, C-2 and C-3 methyleneprotons (see Table 1), but the B-ring shows a differentconformation in each substance. In1 the B-ring has ahalf-chair conformation (10C7), whereas in2, 3 and 4 ithas a twisted-boat conformation with the flaps at C-6 andC-9 (6.9B), at C-5 and C-8 (B5,8), and at C-7 and C-10(7,10B), respectively. These conclusions are in agreementwith the 3J values observed for the C-5 methine and C-6and C-7 methylene protons (Table 1), and also with theallylic coupling between the H-7and H-17 protons in4(4J D 2.4 Hz) and with the NOESY spectra (e.g. strongNOE cross peaks between Me-20 and H-6ˇ in 1–3 andbetween Me-20 and H-7in 4; weak NOE between Me-20and H-6 in 4 and between Me-20 and H-7ˇ in 3; no NOEbetween Me-20 and H-7in 2). The relative configurationof the C-11 and C-12 methylene protons of4 (see Table 1)was determined on the basis of the observed allylic couplingbetween the H-14 and H-12˛ and H-12 protons (4J D 2.3and 0.8 Hz, respectively) and the large3J value (13.8 Hz)between the H-12 and H-11 protons. It is of interest toindicate that the conformations deduced in this study forA- and B-rings of methyl hispanonate (1) are identical withthose determined by an x-ray diffraction analysis.2

In comparison with the study on1–4 made 20 yearsago,1 this work provides the complete assignment of the

1H NMR spectra and the amendment of the previousassignments1 for the H-14 and H-17 protons in2 and 4,which must be reversed (Table 1). Moreover, the presentunequivocal analysis of the13C NMR spectra of1, 2 and4corrects some previous assignments,1 as shown in Table 2.

EXPERIMENTAL

Samples of hispanane derivatives

The samples of1, 2 and3 used for this study were smallamounts of crystals that remained from the original work,1

whereas4 was obtained for this work from2 as describedpreviously.1

NMR spectra

1H and 13C NMR spectra were recorded on a VarianINOVA-400 spectrometer operating at 400 and 100 MHz,respectively, using CDCl3 as solvent at 22°C. Chemicalshifts are given on theυ scale and were referenced toresidual CHCl3 at 7.25 ppm for proton and to the solvent at77.00 ppm for carbon. One-dimensional1H and13C NMRspectra were acquired with standard conditions. The pulseprograms of the COSY, TOCSY, NOESY, HSQC andHMBC experiments were taken from the Varian softwarelibrary. The COSY and NOESY 2D NMR spectra wereacquired in the phase-sensitive mode. Data were collectedin a 1024ð 256 matrix with a spectral width of 2485 Hzand a 2 s recycle delay and processed in a 1024ð 1024matrix. The NOESY spectra were generated with a mixingtime of 0.5 s. The TOCSY experiments were acquired withmixing times of 20–80 ms and processed in the phase-sensitive mode using parameters very similar to thosegiven above for the COSY and NOESY experiments. Thedata for the HSQC spectra were collected in a 1024ð 256matrix with a spectral width of 2485 Hz in the protondomain and 10 000 Hz in the carbon domain and processedin a 1024ð 512 matrix. The null time-following theBIRD pulse was 400 ms. The HMBC experiments wereoptimized for long-range coupling constants of 8 Hz andthe data were processed using parameters very similar tothose used in the HSQC experiments.

Acknowledgements

This work was supported by funds from the Spanish Comision Inter-ministerial de Ciencia y Tecnologıa (CICYT) (grant No. AGF98-0805)and from the Italian Ministero dell’Universita e della Ricerca Scientificae Tecnologica (MURST).

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Magn. Reson. Chem.1993;31: 841.6. Miguel del Corral JM, Gordaliza M, Salinero MA, San Feliciano A.

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Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem.2000;38: 802–804