13cNuclear magnetic resonance spectroscopy

in the elucidation of structures of diterpenoid alkaloids'

BALAWANT
S. JOSHI,JOHNK. WUNDERLICH,
AND S. WILLIAM
PELLETIER~
Institutefor Natural Products Research and the School of Chemical Sciences, The University of Georgia, Athens, GA 30602, U.S.A.
Received May 28, 1986
This paper is dedicated to Dr. 0 . E. (Ted)Edwards

S. JOSHI,JOHNK. WUNDERLICH,
BALAWANT and S. WILLIAM
PELLETIER.
Can. J. Chem. 65,99 (1987).
I3C Nuclear magnetic resonance spectroscopy is an exceptionally useful tool for the structure determination of diterpenoid
alkaloids. A detailed study of the 'H and 13Cnmr spectra of aconitine and 3-deoxyaconitinehas permitted definite assignments
to all the carbon atoms of the molecule. Chemical shift revisions have been suggested for certain carbon atoms of the
C19-diterpenoidalkaloids. Chemical examination of Aconitum columbianum Nutt. ssp. columbianum, A. forrestii Stapf,
Delphinium tatsienense Franch., and D. vestitum Wall. resulted in the isolation of several new C19-diterpenoidalkaloids.
Can. J. Chem. Downloaded from www.nrcresearchpress.com by 64.107.14.30 on 11/10/14

The structure derivation of those alkaloids was based mainly on 13Cnmr spectroscopic evidence.

BALAWANT S. JOSHI,JOHN K. WUNDERLICH et S. WILLIAM PELLETIER. Can. J. Chem. 65,99 (1987).

La spectroscopic rmn du 13cest un outil d'une utilitt exceptionnelle pour determiner la structure des alcaloi'des diterp6niques
enCI3.Une Ctude dCtaillCe des spectres rmn du 'H et 13Cde l'aconitine et de la dCoxy-3 aconitine apermis d'attribuer d'une fason
definitive tous les dtplacements de tous les atomes de carbone de la mol~cule.Des revisions des dtplacements chimiques ont CtC
suggCrCes pour certains atomes de carbone d'alcaloi'des diterptniques en CI9.Un examen des produits chimiques contenus dans
les sources naturelles suivantes Aconitum columbianum Nutt. ssp. columbianum, A. forrestii Stapf, Delphinium tatsienense
Franch. et D . vestitum Wall. a permis d'isoler plusieurs nouveaux alcaloides diterptniques en CI9. Les structures de ces
alcaloides ont CtC dttermides en se basant principalernent sur des donntes de la rmn du 13c.
[Traduit par la revue]

To date, the structures of about 175 naturally occumng

For personal use only.

Clg-diterpenoid alkaloids have been reported and a large

majority of these structures have been elucidated during the last
ten years (1). The structure determination of these complex
alkaloids has largely been possible because of the developments
in isolation methods (2) and advances in spectroscopic tech-
niques, e.g. proton nmr, carbon-13 nmr, high resolution mass
spectra, and X-ray analysis. However, by far the most useful
technique for the structure elucidation of these alkaloids has
been the application of 13C nmr spectroscopy. By a systematic
study of more than 100 naturally occumng Clg-diterpenoid
alkaloids and their derivatives, we have accumulated a data base
that has proved very useful in the derivation of structures of
newly isolated alkaloids.
Jones and Benn made the initial contribution to 13C nmr
spectroscopy of diterpenoid alkaloids (3). In 1976, Pelletier
and Djarmati studied the 13C nmr spectra of various closely
related aconitine-type alkaloids and suggested assignments of
all the carbon atoms making use of substituent effects (4).
Undoubtedly, these data on the 13C chemical shifts of various
functional groups in Clg-diterpenoid alkaloids have been useful
in structure determination of newly isolated alkaloids. Although
errors in assignment may be averaged out over a large number of
compounds having similar structural features, and the correct
structure may be derived in the case of new alkaloids, the
assigned chemical shifts may not be absolutely correct. We
therefore undertook a detailed study of the 13C nmr chemical
shift assignments for the well-known alkaloid aconitine (1A).
A variety of methods were used to obtain the 'H and 13cnmr
assignments, with the major emphasis on the use of the 2-D nrnr
technique (5). The assignments of the acetyl and benzoyl groups
have been well studied and there is no doubt about these values.
'presented at the R.H.F. Manske Symposium held during the
10th International Congress of Heterocyclic Chemistry in Waterloo,
Canada, August 15, 1985.
'~uthorto whom correspondence may be addressed.
1A R=OH Aconitine
First, homonuclear proton decoupling experiments were carried
out and then 'H-'H correlated (COSY) 2-D experiments,
without and with the addition of pyridine, to note the dispersion
of some signals. A summary of the results of the COSY
experiment is given in Table 1. A slice through the data-matrix
at 6 4.88 showed two off-diagonal elements: a doublet of
doublets at 6 2.90 and a doublet at 6 3.33. The correlation with
the doublet at 6 3.33 can best b e attributed to "w" coupling
between H-14 and H-16. A slice taken at 6 2.90 showed as an
off-diagonal element, a doublet at 6 4.88 (H-14) and a doublet
of doublets of doublets at 6 2.33. Assuming that H-9 appears at
6 2.9, the signal at 6 2.33 should be assigned to H-10. A slice
taken at 6 4.48 showed, as off-diagonal elements, a doublet at
6 4.39 and a doublet at 6 3.33. Based on earlier evidence, since
H-16 appears at 6 3.33, the signal at 6 4.48 must b e assigned to
H-15 and the doublet at 6 4.39 should be due to C(15)-OH.
By this process most of the proton assignments were made as
shown in Table 1. These 'H nrnr assignments are essentially
in agreement with the reported values of Katz and Rudin for
aconitine (6).
A C/H correlation experiment was performed on aconitine
without the addition of pyridine. The quaternary carbon
resonances will not appear since the experiment relies on a
polarization transfer step to observe the C/H correlation. A slice
taken at the downfield carbon resonance at 90.1 ppm showed a
correlation with the H-16 resonance at 6 3.33, indicating that the
signal at 90.1 should be assigned to C(16). Similarly, the signal
 100 CAN. J. CHEM.VOL. 65, 1987

TABLE1 . COSY experiment on aconitine l A a 2. C/H Correlation of aconitine l A a

TABLE

Proton Off-diagonal Proton Proton correlation

Slice (6) assignment elements assignment Carbon (ppm) Assignment (PP~)
4.88 d 3.33 d -
2.90 dd 3.33 d
4.48 dd 4.39 d 4.04 brd
3.33 d 3.16 dd
4.39 d 4.48 dd 4.88 d
4.04 brd 2.84 brs 4.48 d
2.10 brd 3.63 d, 3.49 d
3.63 d 3.49 d -
3.33 d 4.88 d 3.77 dd
4.48 dd 3.76 s
2.90 dd 4.88 d 3.11 brs
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2.33 ddd 3.30 s

2.73 dq 2.39 dq 3.17 s
1.10 t 3.27 s
2.34 dddb 3.77 dd -
3.16 dd 2.73 dq, 2.39 dq
2.02 ddd 2.80 m, 2.37 m
2.10 brd 4.04 brd 2.10 brd
3.11 brs 2.84 brs
2.90 dd
a N ~ For ~ : 1 , 2, and 3 the 2D-nmr spectra were taken on a JEOL
~ Tables -
FX-270 MHz instrument using JEOLFAFT-842D software. Aconitine (90 mg) 2.33 ddd
and 3-deoxyaconitine (79.8 mg) were dissolved in 0.5 mL or 1 mL of CDCI, 2.74 m, 2.12 m
for 5-mm or 10-mm (od) nrnr tubes, respectively. All chemical shifts are 2.34 ddd, 2.02
reported in pprn (6) relative to internal tetrarnethylsilane;s: singlet, d: doublet, -
For personal use only.

t: triplet, q: quartet, br: broad, m: multiplet.

bCOSY experiment after addition of pyridine.
"See NOTE,Table 1.

83.4 pprn was assigned to C(6) as it was correlated with H-6, TABLE3. DIFNOE experiment on aconitine lAa
appearing at 6 4.04. Using the same approach, assignments
were made for C(l), C(3), C(14), C(15), C(17), and C(18) Assignment
(Table 2). The signals at 48.9 and 47.0 pprn showed correlation Nuclear Overhauser
with the methylene proton resonances of an N-ethyl group and OMe 'H signal enhancement ( 6 ) 'H I3c
C(19), respectively. The methine carbons appearing at 46.9,
44.8, and 44.2 pprn were correlated with H-5, H-7, and H-9 H-15 61.1
OH-15 -
resonating at 2.1, 2.84, and 2.9, respectively. The doublet at OH- 13 -
40.9 pprn was assigned to C(10) as it showed an overlap with the H-16 -
H-10 resonance at 6 2.33. The remaining chemical shifts for - 59.1
C(2) and C(12) were assigned to the signals appearing at 33.9 H- 1 55.9
and 35.9 ppm, respectively. H-6 58.0
The DIFNOE (difference nuclear Overhauser enhancement) C(8)-OAC
technique was used for assigning the methoxyl protons.
Irradiation at 6 3.76 gave four enhanced signals, due to H-15, "See NOTE, Table 1 .
15-OH, 13-OH, and H-16, suggesting that the 6 3.76 signal
belongs to the 16'-methoxyl group. The C/H correlation shows have been assigned incorrectly. One such error has been noticed
that the signal at 61.1 pprn can be assigned to the 16'-methoxyl. in the chemical shift assignments for C(10) and C(13). For most
The remaining three methoxyls were similarly assigned (Table 3). of the alkaloids, the higher field resonance appearing in the
These investigations provided accurate 13cchemical shift range 37-43 pprn has been ascribed to C(10) and the lower
assignments for all the carbon atoms of aconitine. Table 4 field signal around 43-46 pprn to C(13), when neither of these
shows a comparison of the previously assigned values (4) and carbons bears an oxygen function. It is well established that
the revised chemical shift assignments. By a similar set of within a group of closely related compounds, a given substituent
experiments, the 'H and 13C chemical shifts of 3-deoxyaconi- produces remarkably similar effects on the shielding of the
tine ( 1 B ) were derived (Table 4). carbons at or near the site of substitution. Thus, when a
On the basis of published data of the 13C nmr spectra of more hydrogen is replaced by a hydroxyl group, a downfield shift of
than 160 Clg-diterpenoid alkaloids and their derivatives, we about 7-8 pprn is observed on the p carbon (p-effect) and an
have prepared a table indicating the general chemical shift upfield shift of 2-4 pprn on the y carbon (y-effect).
ranges of all the carbon atoms (1). These data are only These generalized P- and y-effects are not observed in the
approximations but are useful in studying the structures of published chemical shifts of C(10) and C(13) in closely related
newly isolated alkaloids. C19-diterpenoid alkaloids; e.g. placement of a hydroxyl at C(9)
Even when correct structures may be derived on the basis of in monticoline (2) leads to a related alkaloid ranaconine (3). The
these chemical shift values, the individual carbon atoms could chemical shifts for C(10) and C(13) in monticoline (2) have
 JOSH1 ET AL.

TABLE4 . 13cChemical shift assignments of aconitine ( 1 A ) and Me -----r o ~ e

3-deoxyaconitine ( l B ) _--. --OH
, ,':
t : ' ", ------
Aconitine Deoxyaconitine :I ' H
Carbon New Original ( 4 ) New Original (4) :,,M e
0--' M~ OH

5 Lappaconine 6 Gadesine
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7 Nevadenine

C(13) are reversed, then the expected p- and y-effects are

observed. We have therefore suggested that the earlier assigned
chemical shifts for C(10) and C(13) of many of the C19-diter-
penoid alkaloids should be reversed, assigning the downfield
signal occurring around 43-46 pprn to C(10) and the upfield
signal around 37-43 pprn to C(13) (11).
Another assignment that needs to be corrected in the literature
is that of C(l) and C(19) in gadesine (6) (1, 12), nevadenine (7)
(13), and other alkaloids containing a 1,19-oxygen bridge. The
low field signals at 89.0 and 87.6 pprn respectively in 6 and 7
should be assigned to C(19) and the upfield signals at 68.3
For personal use only.

and 69.4 pprn should be assigned to C(l) for these compounds

. signal around 90 pprn (14).
a.bValues may be interchanged.
been assigned the values 37.1 and 42.0 ppm, respectively (7). In
ranaconine (3), C(10) and C(13) have been assigned 37.5 and
5 1.1 pprn (8), which point to a downfield shift of 0.4 pprn on
C(10) (p-effect) and a downfield shift of 9.1 pprn on C(13)
(y-effect). Similarly, the published values for sachaconitine (4)
(9) and lappaconine (5) (10) indicate an upfield shift of 1.1 pprn
on C(10) and a downfield shift of 4.9 pprn on C(13) when C(9)
is substituted with a hydroxyl group contrary to the expected
p- and y-effects. If the presently assigned values of C(10) and
instead of the reversed assignments reported. The C(19) signal
generally appears in the range 52.0-52.5 pprn when it does not
bear an oxygen function. When it does, a downfield shift of
around 40 pprn would be expected and C(19) should show a
Recent examples of our application of 13Cnmr spectroscopy
in the field of diterpenoid alkaloid structure elucidation include
the following:
Alkaloids of Delphinium tatsienense Franch.
From the roots of Delphinium tatsienense Franch., a Chinese
medicinal plant, more than ten alkaloids were isolated and their
structures determined mainly on the basis of I3C nmr spectro-
scopy. Tatsiensine, C27H39N07ran amorphous alkaloid, was
formulated as 8 on the basis of 'H nmr and 13C nrnr evidence
(15). The structure of tatsinine (9), C22H35N06,mp 132-

8 R=Ac Tatsiensine 9 Tatsinine

133"C, was proposed solely on the basis of 13Cnmr spectral data

(16). This structure has recently been confirmed by an X-ray

6:;-rMe
crystal structure determination (17). The isolation and structure
2 Monticoline
3 Ranaconine determination of another amorphous alkaloid, C25H41N07(lo),
designated as deltatsine, was based on 'H nrnr and 13cnmr
spectroscopic data and a chemical correlation with delcosine
(11) (18). The other C19-diterpenoidalkaloids from D. tatsien-
Et- --,r4 ,, ___--- ense, whose structures were determined by 13C nmr spectra,
Y
'. H were browniine, delcosine, lycoctonine, and the C20-diterpenoid
alkaloids ajaconine, hetisine, and hetisinone (15). A C2,-diter-
4 Sachaconitine penoid alkaloid, C21H33N03,mp 181-182"C, isolated from
 CAN. J. CHEM.
OMe
10 R=Me D e l t a t s i n e 12 D i c t y z i n e
11 R = H D e l c o s i n e
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this plant, was shown to be identical with dictyzine (12), first
obtained from D. dictyocarpum DC. The structure 13proposed
for this alkaloid based on chemical studies (19) was revised to
12 from an X-ray crystal structure determination (20). We
derived the structure of this alkaloid from its 13Cnmr spectrum,
which showed the following signals (CDC13 ;TMS): C(l) 40.2t,
C(2) 20.8t, C(3) 27.7"t, C(4) 34.4s, C(5) 44.2d, C(6) 26.6"t,
C(7) 36.2d, C(8) 42.0s, C(9) 52.8d, C(10) 45.6s, C(11) 21.9t,
C(12) 42.8d, C(13) 23.lt, C(14) 26.6"t, C(15) 86.7d, C(16)
79.8s, C(17) 59.8t, C(18) 23.6q, C(19) 67.8t, C(20) 73.5d ppm
For personal use only.
(" these values may be interchanged).
I basis of 13C nmr spectral data.
From the roots of another Chinese medicinal plant, Aconitum
forrestii Stapf, we had isolated two new C19-diterpenoid
I
I alkaloids forestine (14) and foresticine (15) together with three
1
known alkaloids chasmanine, talatizamine, and yunaconitine
I
(21). These alkaloids were isolated from the crude alkaloid
I fraction obtained at pH 8 and the structures were derived mainly
on 13Cnmr spectroscopic evidence. From the alkaloidal fraction
1 taken at pH 5-6, we have isolated four new alkaloids acoforine
1 (16), acoforesticine (17), acoforestine (18), and acoforestinine
1 /
(19), besides crassicauline A (20) and yunaconitine (21).
Et-
14 R = H F o r e s t i n e 15 F o r e s t i c i n e
20 R = A c C r a s s i c a u l i n e A
, Except for acoforestine, mp 203-204"C, the three alkaloids 16,
1 17, and 19 are amorphous. The structures of all these alkaloids
I were derived from their 13C nmr spectral data and correlation
studies. Acoforine (16) was shown to be identical with
14-acetylcolumbidine, prepared from columbidine (vide infra).
When acoforesticine (17) was acetylated with acetic anhydride
1
and p-toluenesulfonic acid it gave the known alkaloid foresaco-
l
nitine (vilmorrianine C) (22). Heating crassicauline A (20) with
ethanol in a sealed tube at 130°C afforded acoforestine (18). As
the structure of 20 was based only on spectral and chemical
evidence, an X-ray crystal structure determination of acofores-
tine was carried out, confirming the structure proposals 18 and
20 for these alkaloids (22).
17 R = H A c o f o r e s t i c i n e 19 R=Et Acoforestinine
22 R = A c Foresaconitine 21 R = A c Yunaconitine
C19-Diterpenoid alkaloids containing a methoxyl or an
ethoxyl group at C(8) have been reported in the literature,
e.g. alkaloid A, ambiguine, puberaconitidine, septentrionine,
columbidine. The question as to whether these alkaloids
containing an alkoxyl group at C(8) are artefacts or are present
in the plants cannot be answered with certainty until the
extraction procedures are repeated, avoiding the use of metha-
nol and ethanol during the isolation.
The conversion of the 8-acetoxy group in C19-diterpenoid
alkaloids to afford the 8-alkoxy compounds can be considered
as a Grob type fragmentation, when the free electron pair on the
nitrogen atom is oriented periplanar with respect to the CP,Cy
bond that undergoes cleavage (23).
Aconitum columbianum Nutt. ssp columbianum, growing in
the Western parts of the USA, afforded the known alkaloids
cammaconine, deltaline, dictyocarpine, talatizamine (23), 8 - 0 -
methyl talatizamine, and a new alkaloid columbidine (24) (1 1).
The structures of all the alkaloids were derived mainly on the
Delphinium vestitum Wall., growing in the Western Hima-
layas, was found to contain two closely related alkaloids
delvestine (25) and delvestidine (26), the structures of which
OMe
16 d = ~ c R'=E~
; Acoforine
23 ~ = R ' = H Talatizarnine
24 d = H ; R'=E~ C o l u m b i d i n e 25 R = H Delvestine
26 R = M e Delvestidine
were based on 13C nmr spectroscopic data and correlation with
gigactonine and lycoctonine, respectively (24).
In summary, our work indicates that a data bank of 13cnrnr
spectra of C19-diterpenoid alkaloids is valuable in deriving
structures of newly isolated compounds of this class. Also,
in the case of amorphous alkaloids, where an X-ray crystal
structure determination is not possible, I3C nmr spectroscopy is
a very useful tool for derivation of structure.
1. S. W. PELLETIER, N. V. MODY,B. S . JOSHI,and L. C.
SCHRAMM. In Alkaloids: chemical and biological perspectives.
Vol. 2 . Edited by S.W. Pelletier. John Wiley, New York. 1984.
Chapt. 5 . p. 205.
2. S. W. PELLETIER, B. S. JOSHI, and H. K. DESAI. In Advances in
medicinal plant research. Wissenschaftliche Verlagsgesellsehaft
mbH, Stuttgart. 1985. pp. 153-195; H. K. DESAI, B. S. JOSHI,
A. M. PANU,and S. W. PELLETIER. J. Chromatogr. 322, 223
(1985).
3. A. J. JONES and M. H. BENN.Can. J. Chem. 51, 486 (1973).
4 . S. W. P E L L E T I E RDJARMATI.
~ ~ ~ Z . J. Am. Chem. Soc. 98,2626
(1976).
 JOSH1 ET AL. 103

5. J. WUNDERLICH. Ph.D. thesis, University of Georgia, Athens, 16. J. A. GLINSKI,B. S. JOSHI,S. Y. CHEN,and S. W . PELLETIER.
Georgia. 1985. Tetrahedron Lett. 25, 121 1 (1984).
6. A. KATZand H. RUDIN.Helv. Chim. Acta, 67, 2017 (1984). 17. J. A. MADDRY.Ph.D. Thesis, University of Georgia, Athens,
7. E. F. AMETOVA, M. S. YUNUSOV, V. E. BANNIKOVA, N. D. Georgia. 1985.
ABDULLAEV, and V. A. TEL'NOV.Khim. Prir. Soedin. 466 18. B. S. JOSHI,J. A. GLINSKI,H. P. CHOKSHI, S. Y. CHEN,S. K.
(1981). SRIVASTAVA, and S. W. PELLETIER. Heterocycles, 22, 2037
8. S. W. PELLETIER, N. V. MODY,A. P. VENKOV,and N. M. (1984).
MOLLOV. Tetrahedron Lett. 5045 (1978). 19. B. J. SALIMOV, M. S. YUNUSOV, Y. V. RASHKES, and S. Y.
9. S. W. PELLETIER, N. V. MODY,and N. KATSUI.Tetrahedron Y u ~ u s o vKhirn.
. Prir. Soedin. 812 (1979).
Lett. 4027 (1977); C. R. YANG,D. Z. WANG,D. G. WU, X. J. 20. B. TASHKHADZHAEV. Khim. Prir. Soedin. 812 (1979); B. J.
HAO,and H. ZHOU.Acta Chim. Sin. 39, 445 (198 1). SALIMOV, B. TASHKHODZHAEV, and M. S. Y u ~ u s o v .Khim.
10. S. W. PELLETIER, N. V. MODY,and R. S. SAWHNEY. Can. J. Prir. Soedin. 86 (1981).
Chern. 57, 1652 (1979). 21. S. W. PELLETIER, S. Y. CHEN,B. S. JOSHI,and H. K. DESAI.
11. S. W. PELLETIER, S. K. SRIVASTAVA, B. S. JOSHI,and J. D. J. Nat. Prod. 47, 474 (1984).
OLSEN.Heterocycles, 23, 331 (1985). 22. S. W. PELLETIER, B. S. JOSHI,J. A. GLINSKI, H. P. CHOKSHI,
12. A. G. GONSALEZ, G. DE LA FUENTE,R. DIAZ,J. FAYOS,and S.-Y. CHEN,K. BHANDARY, and K. GO. Heterocycles. In press.
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M. MARTINEZ-RIPOLL. Tetrahedron Lett. 79 (1979). 23. C. A. GROB, H. R. KIEFER,H. LUTZ, and H . WILKENS.
13. A. G. GONSALEZ, G. DE LA FUENTE,T. ORRIBO,and R. D. Tetrahedron Lett. 290 1 ( 1964).
ACOSTA.Heterocycles, 23, 2979 (1985). 24. H. K. DESAI,B. S. JOSHI,and S. W. PELLETIER. Heterocycles,
14. E. BREITMAIER and W. VOELTER.' 3 ~ spectroscopy.- ~ ~ ~ 23, 2483 (1985).
Verlag Chernie, Weinheim, New York. 1978. p. 153.
15. S. W. PELLETIER, J. A. GLINSKI,B. S. JOSHI,and S. Y. CHEN.
Heterocycles, 20, 1347 (1983).
For personal use only.