Received: 13 November 2018 Revised: 16 January 2019 Accepted: 17 January 2019
DOI: 10.1002/mrc.4839
LETTER ‐ SPECTRAL ASSIGNMENT
1
H and 13C NMR reassignment of some chemical shifts of
lantanilic acid and camaric acid
1 | INTRODUCTION
Lantana camara L. is a plant native to the Americas,
which has been spread around the world as an ornamental
flower thanks to its beauty and easy handling.[1] L. camara
is also used as a medicinal plant to treat different patholog-
ical conditions, including respiratory, gastrointestinal,
dermal, hepatic, neurological, inflammatory, infectious,
and rheumatic diseases.[1,2] Due to its high medicinal
value, this plant has been studied to obtain its active com-
pounds. Many kinds of molecules have been isolated from
this species, from which 74 are triterpenes. These second-
ary metabolites can be grouped according to their base
skeletons: lupane, ursane, euphane, and oleanane, the last
one being the most extensive.[3] Lantanilic and camaric
acids are among the oleanane triterpenes synthesized
by L. camara. Both molecules are isomers and differ only
in the position of a single methyl substituent.
The structure and stereochemistry of lantanilic acid was
reported in 1976 by Barua and coworkers.[4] This work
reports the 1H chemical shifts only for some functional
groups. Later, in 1995, Siddiqui and collaborators achieved
the complete assignment of the 1H and 13C signals of
lantanilic acid and a new triterpenoid also isolated from
L. camara, camaric acid.[5] The subsequent publications
about the nuclear magnetic resonance (NMR) structural
elucidation of lantanilic and camaric acids refer to the work
of Siddiqui et al.[5] However, recently, our group purified
two triterpenes from L. camara and performed their
structural characterization; the 1H and 13C NMR chemical
shifts of both molecules were consistent with those reported
by Siddiqui and coworkers in 1995, but we realized that
some were interchanged (2↔15, 4↔8, 24↔26, and
27↔30). In the present paper, we provide the unambiguous
assignment of 1H and 13C NMR spectroscopic data of
lantanilic and camaric acids.
2 | R E S U L T S AN D D I S C U S S I O N
A mixture of triterpenes was isolated from the aerial parts
of L. camara by open column chromatography. The
320 © 2019 John Wiley & Sons, Ltd.
components of this mixture were subjected to high‐
performance liquid chromatography (HPLC) analysis and
separation, which yielded two white amorphous solids
with HR‐DART‐MS m/z 569.38421 (C35H52O6 [M + H]+).
NMR experiments (1H, 13C, COSY, HSQC, and HMBC)
were acquired for both structures (Figures S1–S10). The
analysis of all spectra led the elucidation of two isomeric
compounds, corresponding to 3β,25‐epoxy‐3α‐hydroxy‐
22β(3,3dimethylacryloyloxy)‐12‐oleanen‐28‐oic acid
(lantanilic acid, 1) and 3β,25‐epoxy‐3α‐hydroxy‐22β(2‐
methyl‐2Z‐butenoyloxy)‐12‐oleanen‐28‐oic acid (camaric
acid, 2; Figure 1). Nevertheless, we identified some
different assignments with respect to previous reports.
Lantanilic acid was isolated and characterized by
NMR for the first time by Barua et al.[4] This triterpene
was obtained from the petrol extract from L. camara
leaves. In that publication, NMR analyses were
performed in CDCl3 using a 60‐MHz spectrometer. The
authors reported two doublets δH 4.22 and δH 3.89
(J = 9 Hz, 1H), which were assigned to H‐25a and
H25b, respectively. Also, a triplet δH 5.01 corresponding
to H‐22 was reported, whereas singlets δH 0.77, δH 0.88,
δH 0.95, δH 1.01, and δH 1.14 were only attributed to six
tertiary methyl groups. Besides, the multiplet δH 5.56
was assigned to the vinyl proton H‐2′ and signals δH
1.83 (d, J = 0.5 Hz, 3H) and δH 2.12 (d, J = 0.5 Hz, 3H)
were assigned to the β‐methyl groups. In that publication,
no additional information regarding the 1H and 13C NMR
chemical shifts is given. The next report about the total
assignment for the NMR spectra of lantanilic acid was
published by Siddiqui and colleagues.[5] In that paper,
authors report the 13C and 1H NMR data of five
pentacyclic triterpenoids from L. camara aerial parts,
including lantanilic and camaric acids. 1H and 13C NMR
spectra (COSY‐45, NOESY, J‐resolved, and hetero‐COSY)
were acquired in CDCl3 using a 300‐MHz spectrometer.
The chemical shifts and their assignments reported by
Siddiqui are summarized in Table 1. Years after this work,
more studies have been conducted on 1 and 2, and they
refer to Siddiqui's article for spectral assignments.[6–16]
However, our analysis indicated that eight signals were
wrongly assigned.
wileyonlinelibrary.com/journal/mrc Magn Reson Chem. 2019;57:320–325.
LETTER ‐ SPECTRAL ASSIGNMENT
FIGURE 1 Structures of lantanilic and camaric acids, 1 and 2,
respectively
The following two‐ and three‐bond 1H–13C HMBC
correlations confirmed the new assignment (Figure 2).
The correlations from δH 1.01 (H‐30) to δC 33.88 (C‐29),
δC 30.51 (C‐20), δC 45.91 (C‐19), and δC 37.93 (C‐21), as
well as the correlations from δH 1.74 (H‐19a), δH 1.75
(H‐21a), and δH 1.49 (H‐21b) and δH 0.88 (H‐29) to δC
26.38 (C‐30), confirmed the new C‐30 assignment. On
the other hand, the correlation from δH 1.16 (H‐27) to
δC 38.88 (C‐8), δC 42.12 (C‐14), and δC 27.93 (C‐15)
supported the reassignment of H‐27.
The 1H–13C HMBC correlations from δH 0.96 (H‐24)
to δC 27.37 (C‐23), δC 98.94 (C‐3), 40.41 (C‐4), and δC
50.46 (C‐5) plus the correlations from δH 1.21 (H‐5)
and δH 1.03 (H‐23) to δC 18.40 (C‐24) confirmed the
C‐24 reassignment. Meanwhile, the correlations from
δH 0.77 (H‐26) to δC 38.88 (C‐8), δC 42.12 (C‐14), δC
42.22 (C‐9), and δC 31.21 (C‐7) and from δH 1.69 (H‐9)
and δH 1.36 (H‐7) to δC 17.46 (C‐26) confirmed the
C‐26 reassignment.
For the methylene groups: the 1H–13C HMBC
correlations from δH 2.16 (H‐2a) to δC 34.85 (C‐1) and
δC 98.94 (C‐3), from δH 1.71 (H‐2b) to δC 35.24 (C‐10)
and δC 34.85 (C‐1), and from δH 2.15 (H‐1a) and δH
1.20 (H‐1b) to δC 29.37 (C‐2) confirmed the new C‐2
assignment. With this new assignment for 2, the
assignment for 15 was established as follows: the
correlations from δH 1.52 (H‐15a) and δH 1.23 (H‐15b)
to δC 25.46 (C‐27) and δC 42.12 (C‐14), from H‐15a to
δC 24.25 (C‐16), and from H‐15b to δC 50.94 (C‐17)
and likewise from δH 1.87 (H‐16) and δH 1.16 (H‐27)
to δC 27.93 (C‐15) confirmed the C‐15 reassignment.
Additionally, 1H–1H COSY coupling between δH 2.16
FIGURE 2 Key 1H‐13C HMBC and 1H‐1H COSY correlations of
1 and 2
321
and 1.71 (H‐2a and 2b) with δH 2.15 and 1.20 (H‐1a
and H‐1b) and, furthermore, δH 1.52 and 1.23 (H‐15a
and H‐15b) with δH 1.87 (H‐16) reinforced the
reassignment of H‐2 and H‐15.
For the quaternary carbon reassignments, the 1H–13C
HMBC correlations from δH 1.21 (H‐5), δH 1.03 (H‐23),
and δH 0.96 (H‐24) to δC 40.41 (C‐4) and from δH 1.36
(H‐7), δH 1.69 (H‐9), δH 2.02 (H‐11a), δH 1.79 (H‐11b),
δH 0.77 (H‐26), and δH 1.16 (H‐27) to δC 38.88 (C‐8)
support C‐4 and C‐8 reassignment, respectively.
A similar analysis was performed for compound 2.
The complete 13C and 1H assignment for both structures
is given in Table 1.
3 | EXPERIMENTAL
3.1 | General
A Waters™ HPLC‐PDA (pump 600, controller 600S, and
PDA detector 2998) was used for the chromatographic
separation of 1 and 2. Reverse phase chromatographic
column employed was a Waters™ RP‐18 Xterra Shield,
125 Å, 3.5 μm, 4.6 × 150 mm. For DART‐MS, a JEOL
AccuTOF JMS‐T100LC DART was used; in m/z positive.
Measurements of NMR spectra are described below.
3.2 | Collection and identification of the
vegetal material
L. camara (L.) aerial parts were collected in the gardens
of the Faculty of Chemistry, Universidad Autónoma de
Querétaro, Cerro de las Campanas s/n, Las Campanas,
Querétaro, México (20°35′25.4″N 100°24′38.3″W), during
the months of March and April, 2014. L. camara (L.)
was identified by Dr. Heike Vibrans, and a herbarium
specimen (voucher: 1414005) was deposited at National
Herbarium of Mexico (MEXU).
3.3 | Extraction and isolation
The plant material (6.5 kg) was dried at room temperature
for 2 weeks, and then it was ground in an electric mill
(IKA MF 10, mesh pore diameter 0.5 mm). The powder
obtained was extracted by two weekly subsequent
macerations in dichloromethane. The dichloromethane
was evaporated in a rotatory evaporator, and the crude
extracts were pooled (122 g) and fractionated by open
column chromatography (stationary phase: silica gel 60 Å,
70–230 mesh, 63–200 μm). The column was eluted with
hexane:ethyl acetate (100:0, 98:2, 95:5, 9:1, 8:2, 7:3, 6:4, 1:1,
0:100) and finally, methanol (100%). This procedure yielded
TABLE 1 Reassignment of chemical shifts (δ in ppm) of 1 and 2 at 298 K in chloroform‐d
322

Lantanilic acid (1) Camaric acid (2)

This work Siddiqui et al. This work Siddiqui et al.
Position δH, mult., (J in Hz) δC δH, mult., (J in Hz) δC δH, mult., (J in Hz) δC δH, mult., (J in Hz) δC
a a
1a 2.15 , m 34.85 2.12, m 34.7 2.15 , m 34.86 2.10, m 34.7
1b 1.20a, m 1.20, m 1.20a, m 1.20, m
2a 2.16a,b, m 29.37b 1.40, m 27.8 2.15a,b, m 29.52b 1.40, m 27.8
2b 1.71a,b, m 1.19, m 1.71a,b, m 1.20, m
3 — 98.94 — 98.9 — 98.78 — 98.9
b b
4 — 40.41 — 38.4 — 40.41 — 38.4
a a
5 1.21 , m 50.46 1.20, m 50.5 1.21 , m 50.49 1.20, m 50.4
6 1.52a, m 19.86 1.50, m 19.8 1.52a, m 19.85 1.50, m 19.7
7 1.36, m 31.21 1.36, m 31.1 1.37, m 31.20 1.37, m 31.0
b b
8 — 38.88 — 40.3 — 38.47 — 40.3
a,b b a,b b
9 1.69 , m 42.22 1.70, m 42.1 1.70 , m 42.21 1.70, m 42.0
10 — 35.24 — 35.2 — 35.27 — 35.2
11a 2.02, m 23.90 2.00, m 23.8 2.01, s 23.90 2.00, m 23.8
11b 1.79, m 1.80, m 1.81, m 1.80, m
12 5.39, t (3.6) 122.82 5.34, t (3.50) 122.6 5.41, br t (3.4) 122.95 5.36, t (3.50) 122.8
13 — 143.06 — 143.1 — 143.03 — 143.1
14 — 42.12b — 42.1 — 42.12b — 42.0
a,b b a,b b
15a 1.52 , m 27.93 2.12, m 29.5 1.52 , m 27.92 2.15, m 29.5
15b 1.23b, m 1.70, m 1.25b, m 1.70, m
16 1.87, td (13.4, 3.5) 24.25 1.86, m 24.2 1.89, td (13.5, 3.4) 24.32 1.85, m 24.3
17 — 50.94 — 50.9 — 50.91 — 50.8
18 3.04, dd (13.8, 4.0) 39.40 3.00, dd (13.30, 4.00) 39.3 3.06, dd (14.0, 4.1) 39.42 3.03, dd (13.80, 3.70) 39.2
a a
19a 1.74 , m 45.91 1.70, m 45.9 1.74 , m 45.89 1.73, m 45.9
19b 1.29, m 1.25, m 1.29, m 1.27, m
20 — 30.51 — 30.1 — 30.21 — 27.9
a a
21a 1.75 , m 37.93 1.75, m 37.8 1.76 , m 38.00 1.75, m 37.9
21b 1.49, m 1.47, m 1.52a, m 1.50, m
22α 5.03, t (3.2) 75.35 4.98, t (3.25) 75.4 5.09, t (3.2) 76.05 5.00, t (3.00) 76.1
23 1.03, s 27.37 0.99, s 27.3 1.03, s 27.39 1.01, s 27.3
(Continues)
LETTER ‐ SPECTRAL ASSIGNMENT
 LETTER ‐ SPECTRAL ASSIGNMENT

TABLE 1 (Continued)

Lantanilic acid (1) Camaric acid (2)

This work Siddiqui et al. This work Siddiqui et al.
Position δH, mult., (J in Hz) δC δH, mult., (J in Hz) δC δH, mult., (J in Hz) δC δH, mult., (J in Hz) δC
b b b b
24 0.96 , s 18.40 0.73, s 17.4 0.97 , s 18.39 0.75, s 17.3
25a 4.25, dd (9.1, 3.2) 68.33 4.20, dd (8.75, 2.29) 67.9 4.25, dd (8.6, 2.7) 67.89 4.21, dd (8.38, 2.28) 67.7
25b 3.91, dd (9.1, 1.5) 3.88, d (8.75) 3.90, dd (8.6, 1.5) 3.88, dd (8.38, 1.10)
26 0.77b, s 17.46b 0.93, s 18.3 0.77b, s 17.38b 0.99, s 18.3
b b b b
27 1.16 , s 25.46 0.97, s 26.3 1.16 , s 25.47 0.99, s 26.2
28 — 177.27 — 178.0 — 177.08 — 178.9
29 0.88, s 33.88 0.85, s 33.7 0.90, s 33.84 0.88, s 33.7
b b b b
30 1.01 , s 26.38 1.10, s 25.4 1.01 , s 26.26 1.14, s 25.4
1′ — 165.50 — 165.4 — 166.60 — 166.5
2′ 5.57, hept (1.2) 116.08 5.54, br s 116.1 — 127.85 — 127.9
3′ — 157.50 — 156.8 6.01, qq (7.3, 1.5) 138.92 5.97, qq (7.24, 1.52) 138.2
4′ 1.85, d (1.1) 27.58 1.82, s 27.3 1.97, dq (7.3, 1.5) 15.80 1.94, dq (7.24, 1.52) 15.6
a
5′ 2.14 , d (1.1) 20.38 2.10, s 20.2 1.81, quint (1.5) 20.65 1.78, quint (1.52) 20.5
a
Overlapped signals.
b
Reassigned signals.
323
324
325 fractions, which were pooled according to their
chromatographic similarity in 18 final fractions (FI‐FXVIII).
Lantanilic acid and camaric acid were present in FXV
(elution system: hexane/ethyl acetate, 6:4 and 1:1). FXV
had a powdery green‐whitish appearance due to the
presence of chlorophylls and a white powder. Chlorophylls
were removed by washes with ethyl acetate, and the
white powder was isolated, which consisted of lantanilic
and camaric acids. Both triterpenes were separated by
repeated injections in HPLC‐PDA (isocratic mobile phase:
acetonitrile 60%, 40% water with 0.1% of TFA; flux
chromatographic program: 0–3 min, 1 ml/min; 3–10 min,
1.8 ml/min, 10–13 min: 1 ml/min). The retention time of
lantanilic acid (18.5 mg) was 11.3 min, whereas for camaric
acid (8.5 mg) was 12.3 min.
3.4 | NMR spectroscopy
NMR spectra were acquired on an Avance III HD 700
spectrometer operating at a 1H frequency of
699.95 MHz (Bruker, Billerica, MA, USA) equipped with
a 5‐mm z‐axis gradient TCI cryoprobe. NMR experiments
were recorded using standard Bruker pulse sequences at
the concentrations of 26.42 (1) and 12.14 (2) mg/ml in
5‐mm NMR tubes at 298 K. The chemical shifts (δ) are
reported in ppm relative to the solvent resonance as the
internal standard (CDC13: δH = 7.26, δC = 77.16).
Coupling constants (J) are given in Hertz. The pulse
conditions were as follows: for 1H NMR spectrum
(pulse sequence = zg30), spectral width (sw) = 14,098 Hz,
relaxation delay (d1) = 2 s, π/2 pulse for 1H (p1) = 7.8 μs,
acquisition time (aq) = 2.3 s, number of data
points (TD) = 64 K, spectral resolution
(HZpPT) = 0.22 Hz/point, number of scans (ns) = 16,
and dummy scans (DS) = 2; for 13C NMR spectrum
(pulse sequence = zgpg30), sw = 43,103 Hz, d1 = 5 s,
π/2 pulse for 13C (p3) = 11.2 μs, aq = 1 s, TD = 84 K,
HZpPT = 0.66 Hz/point; for gradient selected
multiple quantum filter 1H‐1H COSY (pulse
sequence = cosygpmfppqf), d1 = 1.3 s, sw = 6,849 Hz
(F1) and 6,849 Hz (F2), aq = 0.3 s, ns per increment = 8,
TD = 4 K × 128 (t2 × t1), HZpPT = 3.34 Hz/point; for
gradient selected HSQC spectra with multiplicity
editing (pulse sequence = hsqcedetgpsisp2.3), d1 = 1.9 s,
sw = 6,849 Hz (F1) and 26,455 Hz (F2), aq = 0.15 s,
ns per increment = 8, TD = 2 K × 128 (t2 × t1),
1
JCH optimized for 145.0 Hz, HZpPT = 6.69 Hz/point
for 1H and 25.83 Hz/point for 13C; for gradient
selected HMBC spectra with multiplicity editing (pulse
sequence = hmbcetgpl3nd), d1 = 1.4 s, sw = 6,849 Hz
(F1) and 42,373 Hz (F2), aq = 0.3 s, ns per increment = 16,
TD = 4 K × 160 (t2 × t1),), long‐range nJCH optimized
LETTER ‐ SPECTRAL ASSIGNMENT
1
for 8.0 Hz, HZpPT = 3.34 Hz/point for H and
20.69 Hz/point for 13C.
4 | C ON C L U S I ON S
Two related triterpenes, lantanilic acid (1) and camaric acid
(2), were isolated from the aerial parts of L. camara.
Comparison of our NMR assignment analysis with the
existing reports of these two compounds showed that some
data were interchanged. Through the 1D and 2D NMR
spectroscopic analysis, the unambiguous reassignment
for the signals of both triterpenes was achieved. Because
there are various pentacyclic triterpenes related to these
secondary metabolites, the present work constitutes an
important key to the revision of the NMR assignment of
this kind of natural products.
ACKNOWLEDGMENTS
R.D.‐A. would like to thank Consejo Nacional de Ciencia
y Tecnología (CONACYT) for her PhD scholarship (Grant
298060). This work was supported by grant FOFI‐UAQ
(Grant FCQ‐2018‐11) assigned to A.R. This study made
use of UNAM's NMR lab: LURMN at IQ‐UNAM, which
is funded by CONACYT Mexico (Grant 0224747) and
UNAM.
ORCID
Ronna Delgado‐Altamirano https://orcid.org/0000-0003-
3265-4554
Alejandra Rojas https://orcid.org/0000-0001-6131-9013
Nuria Esturau‐Escofet https://orcid.org/0000-0002-0915-
5346
Ronna Delgado‐Altamirano1,2
Alejandra Rojas1
Nuria Esturau‐Escofet3
1
Laboratorio de Investigación Química y Farmacológica de
Productos Naturales, Facultad de Química, Universidad
Autónoma de Querétaro, Querétaro, México
2
Posgrado en Ciencias Químico Biologicas, Facultad de
Química, Universidad Autónoma de Querétaro, Querétaro,
México
3
Instituto de Química, Universidad Nacional Autónoma de
México, Ciudad de México, México
Correspondence
Nuria Esturau‐Escofet, Instituto de Química, Universidad
Nacional Autónoma de México. Circuito Exterior s/n,
Ciudad Universitaria, Delegación Coyoacán, Ciudad de
México, 04510 México.
Email: nesturau@iquimica.unam.mx
LETTER ‐ SPECTRAL ASSIGNMENT 325

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