isolation and structural elucidation of griffithine by 1d and 2d nmr techniques

MAGNETIC RESONANCE IN CHEMISTRY, VOL. 29, 1077-1083 (1991)

Isolation and Structural Elucidation of Griffithine by 1D and 2D NMR Techniques

Atta-ur-Rahman,* A m Pewin, M. Feroz, Shahnaz Perveen and M. I. Choudbary H.E.J. Research Institute of Chemistry, University of Karachi, Karachi-75270, Pakistan

Naim Hasan Department of Chemistry, Baluchistan University, Quetta, Pakistan

The isolation and structure elucidation of a novel dimeric alkaloid, griffithine, from the leafy shoots of Sophru griflthii Stock (Papilionaceae) is described. The alkaloid possesses a 12-membered ring system formed by the linking of C-10 and C-10' of the alkaloid with the N-1 and N-1' atoms, respectively. The structure was established on the basis of extensive 1D and 2D NMR studies. The relative stereochemistry at the chiral centres was determined by NOE mrence experiments. 10-0xosparteine was isolated for the first time from tbe leafy shoots of sophoru griflthii.

KEY WORDS Sophora griflthii Papilionaceae Grimthine Bisdipiperidine alkaloids 2D NMR HOHAHA HeteroCOSY HMBC

INTRODUCTION

Sophora griffithii is cultivated throughout the warm regions of east Asia, and is occasionally used as a diuretic, antipyretic and analgesic in indigenous medi- cine, and as an insecticide.'*2 A number of quinolizidine alkaloids and flavanoids have previously been isolated from this ~ l a n t . ~ - ~ This paper describes the isolation and structure determination of a novel dimeric alkaloid, griffithine (l), from the chloroform extract of the leafy shoots of S. griflthii.

CHO I

I CHO 1

* Author to whom correspondence should be addressed.

RESULTS AND DISCUSSION

Grifithine (1) was isolated (see Experimental Section for all details) as the major alkaloid from the chloroform extract of the leafy shoots of S. grz@thii. The electron impact mass spectrum of 1 showed the molecular ion at m/z 436.21 14 (calculated, 436.21 10) corresponding to the formula C2,H2,N404. The molecular ion was further confirmed by field desorption and fast atom bombard- ment mass spectrometry. The electron impact mass spectrum of 1 showed other fragment ions at m/z 232.1220 (C&,,&O,; calculated, 232.12 1 I), 21 8.1062 (C, ,H 14N202 ; calculated 2 18.1069, 190.1009 (CllHl,N20; calculated 190.1 106) and 72.0449 (C,H,NO; calculated 72.045 l), indicating the presence of a dipiperidine moiety. The UV spectrum (see Experimental) indicated the existence of pyridone m ~ i e t y . ~ - ~ The IR spectrum showed peaks for amide, C=O and a-pyridone, (2-0.

The 'H NMR spectrum (400 MHz) of I in CDCl, showed a downfield 1H double doublet at 6 4.40 for H-11B showing geminal coupling with the 1H double doublet at 6 2.91 for H-lla (Table 1, see also Fig. 1 for numbering). In the COSY-45 spectrum H-1 la and H-llB were coupled with each other and also with the 1H multiplet at 6 2.52 assigned to H-9a. When the multiplet for H-9a was irradiated the signals at 6 4.40 (H-11B) and 2.91 (H-lla) collapsed to doublets [J(lla, llg) = 12.8 Hz], indicating that the C-11 methylene is adjacent to C-9. Another 1H doublet at 6 4.06 assigned to H-lOB [J(lOB,lOa) = 14.2 Hz] showed geminal coupling with a double doublet for H-lOa at 6 3.83. The COSY-45 spectrum of 1 established the various 'H-'H connectivities shown in Table 1, and led to the fragment I.

The aromatic region of 1 showed three double doublets at 6 6.42, 6 7.24 and 6 6.04, which were assigned to

Received 1 1 September 1990 Accepted (revised) 16 February 1991

0749-1581/91/111077-07 $05.00 0 1991 by John Wiley & Sons, Ltd.

1078 A.-UR-RAHMAN ET AL.

Fragment I Fragment II

H-3, H-4 and H-5, respectively. When the signal at 6 7.24 (H-4) was irradiated the two double doublets at 6 6.42 (H-3) and 6 6.04 (H-5) collapsed into doublets [J(3,5) = 1.1 Hz], leading to fragment 11.

The spin systems in 1 were further investigated by the application of HOHAHA experiment^^.'^ recorded with mixing times of 20, 60, 80 and 120 ms. The spectrum obtained with a mixing time of 20 ms closely resembled the COSY-45 spectrum, showing the direct connectivities, whereas with longer mixing intervals the magne- tization was seen to spread to more distant protons within the individual spin Thus H-1 1j3, (6 4.40) and H-l la (6 2.91) showed cross-peaks with each other and with H-8 (6 2.05), H-lOa (6 3.83), H-lOj3 (6 4.06), H-7a (6 3.12), H-13a (6 3.34) and H-138 (6 3.65) in the HOHAHA spectrum (Fig. 1). This established that protons attached to (2-11, (2-8, C-10, C-7 and C-13 are part of the same spin system. Other interactions

appeared between 6 4.06 (H-1OB) on the one hand and H-8a (6 2.05), H-7a (6 3.12), H-13a (6 3.34), H-13j3 (6 3.65), H- l la (6 2.91) and H-11j3 (6 4.40) on the other, in agreement with the structure assigned to fragment I.

The 'H NMR spectrum of 1 in CDCl, showed a doubling of the signals of the protons attached to C-11, C-13 and C-14 at room temperature. Variable temperature NMR studies were therefore carried out in dimethyl sulphoxide (DMSO) and C,D,N at 25, 30, 40, 55 and 90 "C in order to establish whether the doubling was due to an intrinsic asymmetry or to restricted rota- tion because of the presence of four amide functionalities. In DMSO the doubling of the signals disappeared at 90°C, thus confirming that the doubling was due to the double bond character introduced by the presence of the amide functionalities in the ring systems.

NOE difference measurements were carried out (Fig. 2) to determine the relative stereochemistry of 1 at the asymmetric centres. When H-9a (6 2.52) was irradiated, an 8.3% NOE was observed at 6 3.83 (H-lOa), a 6.3% NOE at 6 2.91 (H-lla) and an 8.7% NOE at 6 2.05 (H- 8), indicating a stereochemistry for the C-9 proton. Cor- responding NOES in the reverse directions were obtained on irradiation of the signals at H-lOa, H-l la and H-8 (Fig. 2). Irradiation of H-7 (6 3.12) resulted in a 6.6% NOE at 6 3.34 (H-l3a), an 8.7% NOE at 2.05 (H- 8) and a 5.9% NOE at 6 6.04 (H-5), indicating that the proton at C-7 was a oriented.

The broad-band decoupled 13C NMR spectrum of

Table 1. 'H NMR, 2D J-resolved and COSY45 data for griiffithine (1)'

Protons (ppm)

- 6.42 (H-3)

7.24 (H-4) 6.04 (H-5) 3.12 (H-7a.H-7a)

2.52 (H-9a/H-9'a) 2.05 (H-8)

3.83 (H-lOa/H-lOa)

4.06 (H -1 Op/H-l 0'8) 2.91 (H- l la )

4.40 (H-118) 3.34 (H-13a)

3.65 (H-138) 7.62 (H-14) 6.41 (H-3')

7.22 (H-4') 6.06 (H-5') 2.06 (H-8') 2.89 (H-1 l'a)

4.50 (H-11'8) 3.39 (H - 1 3'a)

3.52 (H-13'8) 7.56 (H-14')

Multiplicities, 'H-'H (J in Hz)

dd, 3, 4 (9.1 ) 3, 5 (1.1)

dd, 4, 5 (6.8) dd m m m

dd, 10a. 108 (14.2) 10a. 9a (3.8)

d dd, l l a , 118 (12.8)

l l a , 9a (1.9) dd, 118, 9a (2.0) dd, 13a. 138 (13.2)

13a. 7a (2.8) dd, 138, 7a (3.8)

dd, 3'. 4' (9.1) 3'. 5' (1.1)

dd, 4, 5 (6.8) dd m dd, l l 'a, 11'8 (12.8)

1 1 'a, 9'a (1.9) dd, 11 '8, 9'a (2.0) dd, 13'a. 1 3 8 (13.2)

13a, 7'a (2.8) dd, 13'8, 7'a (3.8)

S

S

COSY-interactions

7.25 (H-4)

6.42 (H-3). 6.04 (H-5) 7.25 (H-4) 2.05 (H-8). 3.34 (H-l3a). 3.65 (H-138) 2.52 (H-9a). 3.12 (H-7a) 2.05 (H-8). 2.91 (H- l la) , 3.83 (H-lOa),

4.06 (H-lOB), 4.40 (H-118) 4.06 (H-108). 2.52 (H-90)

3.83 (H-lOa), 2.52 (H-9a) 4.40 (H-l lB), 2.52 (H-9a)

2.91 (H- l la) , 2.52 (H-9a) 3.12 (H-7a). 3.65 (H-138)

3.34 (H-l3a). 3.12 (H-7a) - 7.22 (H-4')

6.41 (H-3'). 6.06 (H-5') 7.22 (H-4') 3.12 (H-7'a). 2.52 (H-9a) 2.52 (H-9'a), 4.50 (H-11'8)

2.52 (H-9'a). 2.89 (H-l l 'a) 3.12 (H-7'a), 3.52 (H-13'8)

3.12 (H-7'a). 3.39 (H-13'a) -

a Spectra were recorded in CDCI, using TMS as reference.

ISOLATION AND STRUCTURAL ELUCIDATION OF GRIFFITHINE BY ID AND 2D NMR TECHNIQUES 1079

8 .O 7.0 6.0 5 .0 4.0 3.0

I 1 - -r?m

7. 0 1.0

- m

i . o

2 . 0

3 . 0

A . 0

5 . 0

6 . 0

I

7. 0

8 . 0

PPM

Figure 1. HOHAHA spectrum of 1 recorded at 120 ms.

grifithine (1) (CDCl,, 100 MHz) (Table 2) showed 24 carbon signals."*'* The DEPT experiment established that there were twelve methylene and eight methine carbon signals, and therefore the remaining carbons must be quaternary in nature. The six downfield methylene carbons resonated at 6 48.66, 48.89, 47.19, 46.84, 52.17 and 53.48, which were assigned with the help of COSY-45 and heteroCOSY experiments to C-10, C-10, C-11, C-ll', C-13 and C-13', respectively. The two downfield methine signals at 6 161.08 and 161.99 were assigned to C-14 and (2-14, respectively.

The 2D heteroCOSY spectrum of 1 showed that the carbon resonating at 6 47.19 (C-11) had a one-bond coupling with the protons at 6 4.40 (H-11B) and 6 2.91 (H-1 la). Similarly, the carbon at 6 34.70 (C-7) showed a cross-peak with the proton at 6 3.12 (H-74. Another cross-peak for C-9 (6 26.82) was due to one-bond coupling with the proton at 6 2.52 (H-9a). The aromatic carbons at 6 117.66 (C-3), 138.58 (C-4) and 105.61 (C-5) showed cross-peaks with protons at 6 6.42 (H-3), 6 7.24 (H-4) and 6 6.04 (H-5), respectively. With the help of data obtained from COSY-45, NMR and 2D het- eroCOSY, we formulated the fragments I11 and IV.

I

I I I H5

Fragment Ill Fragment IV

The HBMC spectrum of 1 was very informative and the interactions in the spectrum helped to join the individual fragments. It showed that the quaternary carbon absorbing at 6 148.65 was coupled to the protons at 6 6.04 (H-5), 6 7.24 (H-4), 6 3.34 (H-l3a), 6 3.12 (H-7a) and 6 2.05 (H-8); it was therefore assigned to C-6, adjacent to C-5 and C-7, leading to fragment V.

Other important long range carbon-proton interactions appeared between the carbon absorptions at 6 163.30 and the protons absorbing at 6 6.42 (H-3) and 7.24 (H-4), indicating that fragment IV was joined at the other end with the quaternary carbon at 6 163.30 assigned to C-2 (Fragment VI).

The downfield methine at 6 161.08 (C-14) showed long-range interactions with H-11j3 (6 4.40), H-13a (6

1080 A.-UR-RAHMAN ET AL.

Figure 2. NOE measurements of griffithine (1).

3.34), H-138 (63.65) and H-l la (6 2.91) in its HMBC spectrum, thus confirming that the formyl group was situated at N-12; this established the structure of half of 1 (fragment VII).

0

I I

Fragment V Fragment VI

0

I Q//c\H

Fragment VII

The various proton-carbon long-range connectivities are given in Table 2. Grifithine (1) is therefore a dimer of k~raramine,’.~ the dipiperidine lupine alkaloid from Sophoru$avescens. A comparison with the spectral data of k~raramine’ ,~ showed a close resemblance with the data for 1 except for the absence of the C-10 hydroxy- methylene group absorbing at 6 65.4 in the 13C NMR spectrum of kuraramine, thus suggesting that C-10 and C-10 of kuraramine were linked with N-1 and N-1’ of the pyridone moieties. On this basis, griffthine was assigned the structure 1.

10-Oxosparteine (2), a colourless amorphous material, was isolated from the leafy shoots of Sophoru griffithii for the first time. It had previously been isolated from another ~ource . ’~ The IR, mass and NMR spectra were identical with those of the known com- pound. The electron impact mass spectrum exhibited the molecular ion peak at m/z 248.1889 (calculated 248.1888), corresponding to the formula C,,H,,N20, and the major ions at m/z 220.1940 (C,,H,,N,; calculated, 220.1939), 110.0964 (C,H,,N; calculated, 110.0769), 98.0958 (C6H,,N; calculated, 97.0891) and

The ‘H NMR spectrum of 2 (CDCl,, 400 MHz) showed a downfield 1H quintet of doublets at 6 4.72

96.0799 (C6H ,,N ; calculated, 96.08 13).

ISOLATION AND STRUCTURAL ELUCIDATION OF GRIFFITHINE BY 1D AND 2D NMR TECHNIQUES 1081

H

2

which showed geminal coupling with the 1H double doublet at 6 2.45, the signals being assigned to H-2a and H-28, respectively. The C-68 proton resonated as a broad doublet at 6 3.21 and showed cross-peaks in its COSY-45 spectrum with H-51% and H-58 which resonated as a 2H multiplet centred at 6 1.52. H-17a resonated as a broad triplet at 6 2.75 whereas H-178 resonated at 6 2.21 as a triplet of doublets. H-17a and

H-178 showed cross-peaks with each other and with H-78, resonating as a multiplet (6 1.68) in its COSY-45 spectrum.

The spin systems present in 2 were further investigated by a HOHAHA experiment. The HOHAHA spectrum of 2 recorded with a mixing time of 120 ms showed that H-98 (6 2.25) was coupled with H-l7a (6 2.75), H-178 (6 2.21), H-78 (6 1.68), H-12a (6 1.45) and H-128 (6 1.25). Similarly, H-68 (6 3.21) showed cross- peaks with H-5a (6 1.52), H-4a (6 1.85), H-48 (6 1.55), H-3a (6 1.59) and H-38 (61.46) in its HOHAHA spectrum, thus confirming the assignments of the protons present in ring A of 2. Other important cross-peaks appeared between the proton at 6 2.75 (H-1%) with H-12a (6 1.45), H-13a (6 1.80), H-138 (6 1.39) and H-lla (6 1.85), thus confirming the assignments of the C-15, C-14, C-13, C-12 and C-11 protons.

The "C NMR spectrum (broad-band decoupled and DEPT) of 2 showed the presence of four methine, ten methylene and one quaternary carbon signals. The

Table 2. "C NMR (BB, DEP") and HMBC data of griffitbine (lYvb

Carbon Carbon chemical Multiplicities No. shift (ppm) (DEPT) 'H-"C long-range connectivities (HMBC)

- 2 163.30 S 6 7.25 (H-4), 6 6.42 (H-3) 3 11 7.66 d 6 6.04 (H-5) 4 138.58 d 6 6.42 (H-3) 5 105.61 d 6 6.42 (H-3). 6 6.04 (H-5) 6 148.65 S 6 2.05 (H-5). 6 2.91 (H- l la) , 6 3.34 (H-1301).

7 34.70 d 6 2.05 (H-8). 6 2.91 (H- l la) , 3.65 (H-138).

8 26.45 t 64.40 (H- l lp ) , 3.34 (H-l3a), 6 3.65 (H-138),

6 6.04 (H-5), 6 7.25 (H-4)

64.40 (H- l lb ) , 66.04 (H-5)

6 3.83 (H-lOa), 6 4.06 (H-108). 6 3.12 (H-7a) 9 26.82 d 6 3.65 (H-138). 6 4.06 (H-108)

10 48.66 t 6 2.05 (H-8). 6 2.89 (H- l la) , 11 47.1 9 t 6 4.06 (H-108). 6 3.83 (H-lOa), 6 2.05 (H-8).

13 52.1 7 t 64.40 (H-11/3), 6 3.83 (H-lOa), 64.04 (H-lob),

14 161.08 d 6 3.65 (H-138). 6 3.34 (H-l3a), 64.40 (H-lib),

6 3.12 (H-7a), 6 3.65 (H-138). 6 7.62 (H-14)

6 2.05 (H-8)

62.91 (H- l la ) 2' 163.35 S 6 6.06 (H-5') 3' 117.99 d 6 6.06 (H-5') 4' 138.82 d 6 6.06 (H-5'). 6 6.41 (H-3') 5' 105.40 d 66.41 (H-3') 6' 147.95 S 6 2.06 (H-8'), 6 2.89 (H-l l 'a), 6 3.39 (H-l3'a),

7' 8' 26.50 t 6 4.50 (H-11'/3), 6 2.89 (H-l l 'a), 6 4.06 (H-10'8).

9' 27.22 d 6 3.12 (H-7'a). 6 4.06 (H-lOB), 6 3.52 (H-138).

6 6.06 (H-5'). 6 7.22 (H-4), 3.12 (H-7'a) 6 6.06 (H-5). 6 2.06 (H-8'). 6 2.89 (H-l l 'a) d

6 3.52 (H-138)

6 3.83 (H-lO'a)

6 3.52 (H-13'8). 6 3.39 (H-13'a) 10 48.89 t 6 2.06 (H-8). 6 4.50 (H-11'8). 6 2.89 (H-ll'a),

11' 46.84 t 64.06 (H-10'8). 3.83 (H-lO'a), 63.12 (H-7'a).

13' 53.48 t 64.50 (H- l l 'p) , 64.06 (H-10'8). 63.83 (H-lO'a),

14' 161.99 d 6 4.50 (H-ll'fl), 6 2.89 (H-ll'a), 6 3.39 (H-l3'a),

6 2.06 (H-8)

6 2.06 (H-8')

6 3.52 (H-13'8)

a 'H-'"C one-bond connectivities were determined by employing the HeteroCOSY experiment.

Recorded in CDCI, using TMS as internal reference.

1082 A,-UR-RAHMAN ET AL.

downfield methylene carbons at 6 63.66, 56.79 and 42.41 were assigned to C-15, C-17 and C-20, respectively. The two methine carbons at 6 61.27 and 64.90 were due to C-6 and C-11, respectively. The carbon at 6 169.50 was due to C-10.

In order to confirm the one-bond proton-carbon connectivities, an HMQC experiment was carried out which showed that the carbon which resonated at 6 43.93 (C-9) was coupled with the proton at 6 2.25 (H-98). Similarly, the carbon at 6 56.79 (C-15) showed cross- peaks with the protons at 6 1.82 (H-l5a) and 6 2.65 (H- 154, whereas the carbon at 6 42.41 (C-2) was coupled with protons at 6 4.72 (H-2a) and 6 2.45 (H-2B). C-6 (6 61.27) was connected with the proton at 6 3.21 (H-68) through one-bond coupling, whereas C-11 (6 64.90) was coupled with the proton at 6 1.85 (H-ll&. The various carbon-proton connectivities are given in Fig. 3.

EXPERIMENTAL

The UV spectra were recorded on a Shimadzu UV-240 spectrometer and the IR spectra on a JASCO IRA-I spectrometer. The mass spectra were recorded on Varian MAT 112 and 312 double-focusing mass spec- trometers connected to DEC PDP 11/34 computer systems. The NMR spectra were recorded on a Bruker AM 400 NMR spectrometer. Optical rotations were recorded on a Polartronic Universal Australian stan- dard K-157 spectropolarimeter.

The leafy shoots of Sophora grqjthii were collected from Quetta (Pakistan) and identified by Saud Omar, Department of Botany, University of Karachi, where a sample specimen was deposited (Herbarium Voucher No. 34247, KUH).

Extraction and purification

Isolation of griffithine (1). Air-dried leafy shoots (5.0 kg) of Sophora grifithii were crushed, extracted with ethanol (20 1) and the extract was concentrated to a crude gum (150 g). The gum was acidified with 10% acetic acid and extracted with CHCI, (20 I). The acidic aqueous layer was then basified to pH 9 with ammonia solution and again extracted with CHCl, (15 1) to afford the crude alkaloids (80 g). The crude alkaloidal portion was then subjected to column chromatography on alumina (1.5 kg). Initially the elution was carried out with light petroleum-CHC1, (1 : 1) and then with increasing pol- arities of light petroleum-CHC1, , CHCl, and CHCI,-CH,COCH, mixtures. The fraction obtained on elution with CHCI, (6.7 g) was rechromatographed on column packed with TLC-grade alumina. Elution was carried out with light petroleum-CHCI, (2 : l), which afforded a fraction containing 1 as the major alkaloid. Grifithine (1) was purified on a Chromatotron, (circular plate chromatography), elution being with CHCl,, to yield alkaloid 1, 36.3 mg, as a cream- coloured solid mass, [aID = +217" (c, 0.03, MeOH); UV, I,,,. (MeOH, log E ) 230 (3.56), 308 (3.09) nm; IR, vmaX (CHCI,) (cm-') 1645 (C-0), 1650, 1550 (a-pyridone, C=O); 'H NMR (CDCI,, 400 MHz) 6 values in Table 1; "C NMR, results in Table 2.

Isolation of 10.oxosparteine (2). Air-dried leafy shoots of S. griflthii were extracted with ethanol (20 1) and the extract was concentrated to a crude gum (1.25 kg). The gum was acidified with 10% acetic acid and extracted with CHCl, (10 1). The aqueous layer was then basified with ammonia solution to pH 9. The crude alkaloids present in this basic fraction were extracted with CHC1, (20 I), which yielded the crude alkaloids (88.26 g). This

Figure 3. HMQC spectrum of 10-oxospartiene (2)

ISOLATION AND STRUCTURAL ELUCIDATION OF GRIFFITHINE BY 1D AND 2D NMR TECHNIQUES 1083

crude alkaloidal fraction was subjected to chromatography on alumina (1.5 kg). Initially the elution was carried out with light petroleum-CHC1, (1 : 1) and then with CHCI,, CH,COCH,, CH,COCH, and MeOH. The fractions obtained with light petroleum-CHCI, (30: 70 to 10: 90) were combined (10.5 g) and loaded on a column packed with TLC-grade silica gel, elution being carried out with CHC1,-light petroleum (3 : 1). A fraction containing 10-oxospartine (2) was purified on precoated alumina cards, elution being with CHC1,-light petroleum (3 : 1). IR, v,,, (CHCI,) (cm- I )

1650 (C-0); ELMS, m/z (rel. int.,%): 248 (40), 220 (30), 150 (20), 136 (401, 137 (20), 110 (60), 98 (90), 97 (100); 'H NMR (CDCI,, 400 MHz), 6 1.25 (lH, m, H-128), 1.35 (lH, m, H-48), 1.36 (lH, m, H-58), 1.39 (lH, m, H-138),

1.45 (lH, m, H-124, 1.46 (lH, m, H-34, 1.52 [lH, dd, J(5d, 54, J(5a, 68) = 11.0 Hz, 4 5 4 48) = 6.0 Hz, H-5a], 1.55 (lH, m, H-48), 1.55 (lH, m, H-84, 1.59 (lH, m, H-~Lx), 1.60-162 (2H, m, H-14a/H-148), 1.62 (lH, m, H-88), 1.80 (lH, m, H-l3a), 1.82 [lH, dd, J(l58, 15a) = 11.5 Hz, J(l58, 148) = Hz, H-15fl, 1.85 (2H, m, H-4a/H-lla), 2.07 [lH, ddd, J(8a, 88) = 12.0 Hz, 4 8 4 98) = 6.8 Hz, J(8q 78) = 6.0 Hz, H-8a], 2.21 [lH, dt, J(178, 17a) = 11.5 Hz, J(178, 78) = 2.5 Hz, H-17fl, 2.25 (lH, dd,), 2.45 [lH, dd 428, 2 4 = 14.6 Hz, J(28, 38) = 2.8 Hz, H-2fl, 2.65 (lH, H-l5a), 2.75 [lH, brt, J(17a, 178) = 11.5 Hz, H-17~~1, 3.21 [lH, brd, J(H-68, 5a) = 11.0 Hz, H-681, 4.72 [lH, d, 4 2 4 28) = 14.6 Hz, H-2aJ.

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isolation and structural elucidation of griffithine by 1d and 2d nmr techniques

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