MAGNETIC RESONANCE IN CHEMISTRY

Magn. Reson. Chem. 2000; 38: 201206

Note
Complete assignment of the
1
H and
13
C NMR spectra of four
triterpenes of the ursane, artane, lupane and friedelane
groups
Josinete S. Alves, Janiza C. M. de Castro, Maisa O. Freire, Emidio V. Leit ao da-Cunha,
Jos e Maria Barbosa-Filho and Marcelo Sobral de Silva

Laborat orio de Tecnologia Farmac eutica, Universidade Federal da Paraba, Cx. Postal 5009, 58051-970, Jo ao Pessoa, Paraba, Brazil
Received 14 September 1999; accepted 27 October 1999
ABSTRACT: The detailed
1
H and
13
C NMR assignments of the four known triterpenes 3-hydroxyurs-12-en-28-
oic acid, 3-oxo-24-methylenecycloartan, 3-oxo-11-hydroxy-20(29)lupen and 29-hydroxyfriedelin, isolated from three
Brazilian plants, were achieved by 1D and 2D techniques such as DEPT, HETCOR, HMQC, HMBC, COSY and
NOESY. Copyright 2000 John Wiley & Sons, Ltd.
KEYWORDS: NMR; 1D NMR; 2D NMR; Triterpenoids;
1
H and
13
C chemical shift assignments
INTRODUCTION
Two-dimensional NMR is becoming more and more a
routine tool in the elucidation of the chemical structures
of natural products. In the case of complex triterpenes,
experiments such as HETCOR, HMQC, HMBC, COSY
and NOESY allow the researcher to achieve unambiguous
assignments for all hydrogen and carbons.
Recent reports showed the results of the application
of 1D and 2D NMR techniques for the identication of
the structures and to determine the
1
H and
13
C NMR
assignments of some triterpenes of the series ursane,
1,2
artane,
38
lupane
9
and friedelane.
1
This paper describes the structure determination of four
triterpenes in these series, namely: 3-hydroxyurs-12-en-
28-oic acid (1) 3-oxo-24-methylenecycloartan (2) 3-oxo-
11-hydroxy-20(29)lupen (3) and 29-hydroxyfriedelin (4).
* Correspondence to: M. Sobral da Silva, Laborat orio de Tecnologia
Farmac eutica, Universidade Federal da Paraba, Cx. Postal 5009, 58051-
970, Jo ao Pessoa, Paraba, Brazil.
Contract/grant sponsor: CAPES.
Contract/grant sponsor: CNPq.
Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2000; 38: 201206
202 J. S. ALVES ET AL.
The complete assignment of all their hydrogen and car-
bons is also shown. The compounds used in this work
were isolated from Salzmania nitida (1), Krameria tomen-
tosa (2) and Maytenus obtusifolia (3 and 4).
EXPERIMENTAL
Materials
Sallzmania nitida D.C. (Rubiaceae) was collected in January 1998 near
the city of Santa Rita, Krameria tomentosa A. St Hill (Krameriaceae) in
August 1997 near the city of Campina Grande and Maytenus obtusifolia
(Celastraceae) in the city of Sap e, state of Paraba, Brazil. The plants
were identied by the botanist Maria de F atima Agra of the Universidade
Federal de Paraba. Voucher specimens (Agra 2986, Agra 1786 and Agra
3424, respectively) are deposited at the Herbarium Prof. Lauro Pires
Xavier of the UFPB.
The dried and ground roots of S. nitida (3400 g) were extracted in
a Soxhlet apparatus with 95% EtOH. The solvent was removed under
vacuum and yielded 180 g of extract. This extract was suspended in
water and subjected to partitioning with CHCl
3
and AcOEt. The AcOEt
fraction was subjected to column chromatography over silica gel with
hexaneCHCl
3
(1 : 1, fractions 1056) as eluent, yielding pimarane
diterpenes, and with CHCl
3
MeOH (9 : 1, fractions 103168), yielding
1 (185 mg).
Table 1. NMR data for 3-hydroxyurs-12-en-28-oic acid (1)
HMQC
1
H
1
H
C
C

H
HMBC COSY NOESY
4 39.5 3H-23, 3H-24
8 40.4 H-9, 3H-26, 2H-6,
2H-11, 3H-27
10 37.7 H-5, H-9,
3H-25, 2H-6
13 139.7 H-18, H-15, 3H-27
14 43.0 3H-27, H-9, H-12,
2H-15, H-16, H-18,
3H-26
17 48.5 2H-16, H-18, 2H-22,
2H-21
28 180.0 2H-16, H-18
CH
3 78.6 3.48(dd, J D 6.5, 9.5 Hz) 3H-23, 3H-24 2H-2 H-5, 3H-23
5 56.3 0.88 3H-23, 3H-24, 2H-6
3H-25
9 48.5 1.65 (t, J D 7.8 Hz) 2H-11, H-12, H-25, 2H-11
3H-26
12 126.1 5.52 (br s) H-18 2H-11 H-18, H-11
18 54.0 2.67 (d, J D 11.6 Hz) H-19, H-12, H-22, H-19
3H-29
19 39.9 1.50 H-18, 3H-29, 3H-30 H-18, H-20
20 39.9 1.03 3H-30, H-18, 3H-29 H-19
CH
2
1 39.8 0.98, 1.58 (m) 2H-2, H-9, 3H-25 2H-2
2 28.6 1.80, 1.90 (m) H-3 2H-1, H-3
6 19.2 1.39 (H-6) H-5 H-5, 2H-7
1.60 (H-6)
7 34.0 1.39, 1.49 (m) 3H-26 2H-6
11 24.1 1.95 (H-11, H-11) H-9, H-12 H-9, H-12
15 29.1 1.23 (H-15) H-16, 3H-27 2H-16 3H-26
2.35 (dt, J D 4.3, 11.5
Hz, H-15)
16 25.4 2.02 (H-16) H-15, H-18 2H-15 3H-27
2.13 (dt, J D 4.8, 11.5 Hz, H-16)
21 31.5 1.48, 1.99 (m) 3H-30
22 37.9 1.48 (H-22)
1.99 (H-22)
CH
3
23 29.3 1.28 (s) H-3, 3H-24
24 17.0 1.05 (s) H-3, 3H-23
25 16.1 0.92 (s) H-9
26 17.9 1.08 (s) H-9
27 24.4 1.27 (s) 2H-15
29 18.0 1.03 (d, J D 7.6 Hz) H-19, H-18
30 21.9 0.98 (d, J D 7.0 Hz)
Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2000; 38: 201206
ASSIGNMENT OF NMR SPECTRA OF FOUR TRITERPENES 203
The dried and ground root bark of K. tomentosa (2300 g) was
extracted in a Soxhlet apparatus rst with hexane and then with CHCl
3
.
The hexane extract (20 g) was subjected to column chromatography over
silica gel with hexaneAcOEt (9 : 1, fractions 915) as eluent, yielding
2 (125 mg), and hexaneAcOEt (8 : 2, fractions 70145), yielding the
nor-neolignan rathaniafenol I
10
and the neolignan conocarpane.
11
The dried and ground stem of M. obtusifolia (4500 g) was extracted
in a Soxhlet apparatus rst with CHCl
3
, followed by AcOEt and MeOH.
The CHCl
3
extract (35 g) was subjected to column chromatography over
silica gel with CHCl
3
MeOH (97 : 3, fractions 4798) as eluent and
CHCl
3
MeOH (95 : 5, fractions 121168) to yield 3 (85 mg) and 4
(50 mg), respectively.
NMR spectra
1
H and
13
C NMR experiments were performed on a
Bruker ARX 500 spectrometer operating at 500.1 MHz for
hydrogen and 125.75 MHz for
13
C, using C
5
D
5
N or CDCl
3
as solvent. Solutions were prepared with 0.5 ml of C
5
D
5
N
(1) or CDCl
3
(24) and 1520 mg of the triterpenes.
The solvent was also used as internal reference. For
all experiments the temperature was stabilized at 298 K.
For the NOESY experiments, samples were degassed
by bubbling nitrogen through the solution and tting a
PTFE serum cap. The 2D experiments were acquired and
processed with the software provided by Bruker on a
Silicon Graphics computer.
Typical acquisition and processing conditions for COSY
and NOESY experiments were relaxation delays of 12 s,
5121024 t
1
increments, 10242048 t
2
points and a
sweep width of 6 ppm. Sine-bell square and shifted
(/4, /6 and /8) apodization functions were used for
Table 2. NMR data for 3-oxo-24-methylenecycloarane (2)
HMQC
1
H
1
H
C
C

H
HMBC COSY
3 217.5 H-2, 3H-28
4 50.2 3H-29, 3H-28
9 21.0 2H-1
10 25.9 2H-6
13 45.3 H-8, 3H-18
14 48.7 3H-18
24 157.5 3H-26, 3H-27
CH
5 48.4 1.66 2H-19 2H-6
8 47.8 1.56 3H-30
17 52.2 1.59 3H-21 H-20, 2H-16
20 36.0 1.38 3H-21 H-17, 2H-22
25 33.7 2.20 3H-26, 3H-27 3H-26, 3H-27
CH
2
1 33.4 1.85 (H-1) 2H-2 2H-2
1.52 (H-1)
2 37.4 2.28 (H-2) 2H-1 2H-1
2.70 (ddd, J D 2.2, 6.6 e
14.1 Hz, H-2)
6 21.5 0.91 (H-6) H-5 H-5, 2H-7
1.51 (H-6)
7 28.1 1.10 (H-7) 2H-6 2H-6
1.35 (H-7)
11 26.7 1.15 (H-11) H-8, 2H-9 2H-12
2.00 (H-11)
12 32.7 1.62 3H-18, 3H-30 2H-11
15 34.9 1.30 2H-16
16 25.8 1.29 (H-16) 2H-15, H-17
1.90 (H-16)
19 29.5 0.60 (d, J D 4.2 Hz)
0.80 (d, J D 4.2 Hz)
22 35.5 1.10, 1.55 3H-21 2H-23
23 31.2 1.85, 2.10 2H-22
31 105.9 4.64 (sl) H-25
4.69 (sl)
CH
3
18 18.0 1.06 (s) 2H-12, H-17
21 18.3 0.91 (d, J D 6.3 Hz) H-17 H-20
26 22.0 1.02 (d, J D 5.3 Hz) H-25, 3H-27 H-25
27 21.8 1.05 (d, J D 5.3 Hz) H-25, 3H-26 H-25
28 20.7 1.12 (s) H-8, 2H-15
29 22.1 0.92 (s) H-5, 3H-28
30 19.3 1.07 (s) H-5, 3H-29
Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2000; 38: 201206
204 J. S. ALVES ET AL.
processing. The mixing time in the NOESY experiments,
generally set to 1.21.5 s, was also varied between 0.8
and 2 s without a substantial change in the results. For
1
H
13
C (
13
C detected) and
13
C
1
H (
1
H detected) cor-
relations the same relaxation delay was used, 5121024
t
1
increments, 10242048 t
2
points and a sweep width of
6 ppm for
1
H and 180 ppm for
13
C. Lorentzian and Gaus-
sian deconvolutions were generally used in the processing.
The number of scans was set for an overall acquisition
time of about 814 h.
RESULTS AND DISCUSSION
The structure elucidation of the four triterpenes was
based on the assignments of the NMR spectra, which
was conrmed by the 2D experiments (COSY, NOESY
HMQC and HMBC). The
1
H and
13
C chemical shifts and
the correlations of the hydrogen and carbon atoms are
given in Tables 14.
For compounds 14 a combination of BB and DEPT
13
C NMR was used to differentiate the signals belonging
to quaternary carbons, methine, methylene and methyl
groups. For 1, 3 and 4 thirty carbons were assigned,
whereas for 2, 31 carbon atoms were assigned. The
NMR data obtained were analogous to those published
previously.
19
For this reason, instead of presenting a
detailed discussion of the results, only some peculiarities
are presented.
In triterpene 1, the deshielding effect on C-5 and C-21
and the shielding effect on C-4 are related to the absence
of hydroxyl and hydroxymethylene in comparison with
the model compound.
1
On the other hand, the systematic
shielding effect of the hydrogen atoms H-3 (
H
3.48), H-16
(
H
2.02 and 2.13) can be attributed to the aromatic solvent
used for recording the spectrum (Table 1).
Table 3. NMR data for 3-oxo-11-hydroxy-20(29) lupen (3)
HMQC
1
H
1
H
C
C

H
HMBC COSY NOESY
3 218.9 H-1, 2H-2, 3H-23
4 47.6 2H-6, 3H-23, 3H-24
8 43.0 2H-6, 3H-26, 3H-27
10 38.2 H-1
14 42.6 2H-16, 3H-27
17 42.4 2H-16
20 150.0 3H-30
CH
5 54.7 1.48 H-1, 3H-23 or 3H-25, 2H-6
9 54.9 3H-26
11 70.2 3.88 (m) H-9 2H-12 H-13, 3H-25, 3H-26
13 37.2 1.81 3H-27 H-18
18 47.7 1.36 H-16, 3H-28 H-13, H-19
19 47.7 2.30 (m) 2H-29, 3H-30 H-18
CH
2
1 42.1 1.62 (H-1) 2H-2, 3H-25 2H-2 H-7, 3H-27
2.60 (m, H-1)
2 34.1 2.38 (H-2, H-2) 2H-1 3H-24
6 19.5 1.43 (H-6, H-6) H-5, 2H-7 H-5, 2H-7
7 35.5 1.50 (H-7), 3H-26 2H-6 H-1, 3H-27
1.38 (H-7)
12 27.3 1.22 (H-12) H-18 H-11
1.95 (m, H-12)
15 37.4 1.03 (H-15) 3H-27 2H-16
1.60 (H-15)
16 34.1 1.41 (H-16, H-16) 3H-28 2H-15 3H-27
21 29.5 1.35 (H-21) 2H-22 2H-22
1.92 (m, H-21)
22 39.7 1.20 (H-22) 2H-16, 3H-28 2H-21
1.40 (H-22)
29 109.8 4.56 (sl) 3H-30
4.68 (sl)
CH
3
23 27.2 0.98 (s) H-1
24 20.6 0.79 (s) H-5 H-2
25 16.7 1.02 (s) H-5 or H-9 H-11
26 16.8 1.04 (s) H-9 H-11
27 14.2 0.96 (s) H-7, H-16
28 18.1 1.05 (s) 2H-22
30 19.3 1.65 (s) 2H-29
Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2000; 38: 201206
ASSIGNMENT OF NMR SPECTRA OF FOUR TRITERPENES 205
Table 4. NMR data for 29-hydroxyfriedelin (4)
HMQC
1
H
1
H
C
C

H
HMBC COSY
3 212.1 2H-2, H-4, 3H-23
5 42.1 3H-24
9 37.3 3H-25
13 39.8 3H-27, 3H-26
14 38.1 3H-26, 3H-27
17 29.7 3H-28
20 33.0 3H-29, 3H-30
CH
4 58.1 2.20 (q, J D 6.6 Hz) 3H-23, 3H-24 3H-23
8 53.3 1.34 (t, J D 6.4 Hz) 3H-26 2H-7
10 59.3 1.47 H-4, 3H-28
18 41.7 1.55 3H-27, 3H-28
CH
2
1 22.6 1.65 (H-1) H-10 H-2
1.90 (H-1)
2 41.2 2.38 (H-2) H-4 H-1
2.48 (H-2)
6 41.4 1.69 (H-6, H-6) 3H-4
7 18.1 1.38 (H-7, H-7) H-8
11 35.5 1.40 (H-11, H-11) 3H-25
12 29.7 1.10 (H-12) 3H-27
1.42 (H-12)
15 32.6 1.25 (H-15) 3H-26
1.50 (H-15)
16 35.8 1.21 (H-16) 3H-28
1.50 (H-16)
19 30.5 1.28 3H-27
21 27.7 1.30 3H-30, 2H-29
22 39.4 0.95 (H-22) 3H-28
1.30 (H-22)
29 74.6 3.23 (sl) H-21, 3H-30
CH
3
23 6.7 0.80 (d, J D 6.6 Hz) H-4 H-4
24 14.5 0.65 (s) H-4
25 17.8 0.81 (s) H-8, 2H-11
26 18.3 0.98 (s) H-15
27 20.6 0.96 (s)
28 32.0 1.15 (s) 2H-16
30 25.7 0.97 (s) 2H-29
The starting point for the unambiguous assignment
of all
1
H and
13
C spectra of the cycloartan 2 was the
positioning of the carbonyl at C-3 and of the double bond
between C-24 and C-31 which were obtained through
HMBC, the former by the correlation between C-3 and the
hydrogen H-2 (
H
2.70) and with the methyl hydrogens
3H-28, and the latter by the correlations of C-24 and
C-25 with the methyl hydrogens 3H-26 and 3H-27 and
also of C-31 with H-25. This analysis was conrmed by
comparison of the chemical shifts of similar triterpenes
reported in the literature and by HMQC and COSY
experiments (Table 2).
The same procedure was used to assign all the
1
H
and
13
C chemical shifts of 3, and in addition to these
assignments, the NOESY experiment also conrmed the
equatorial conguration of the hydroxyl group in C-11,
through the correlation between H-11 and H-13, 3H-25
and 3H-26 (Table 3). In 4, HMBC gave important infor-
mation for the assignments of the chemical shifts of all
the hydrogens and carbons, conrming the localization the
hydroxymethylene group in C-20 through the correlation
between the hydrogens 2H-21 and 3H-30 with this carbon
(Table 4).
In conclusion, we were able to obtain complete NMR
spectral assignments for the four triterpenes 14, allowing
the unambiguous assignment of the structures of the
compounds.
Acknowledgments
The authors gratefully acknowledge CAPES and CNPq for nancial
support and thank Dr Edilberto Rocha Silveira of CENAUREM (Centro
Nordestino de Aplica c ao e Uso de RMN) for providing the NMR
spectra.
Copyright 2000 John Wiley & Sons, Ltd. Magn. Reson. Chem. 2000; 38: 201206
206 J. S. ALVES ET AL.
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