Absolute configuration of menthene derivatives by vibrational circular dichroism

Article abstract of DOI:10.1021/acs.jnatprod.6b00491

The aerial parts of Ageratina glabrata afforded (-)-(3S,4R,5R,6S)-3,5,6-trihydroxy-1-menthene 3-O-β-d-glucopyranoside (1) and (-)-(3S,4S,6R)-3,6-dihydroxy-1-menthene 3-O-β-d-glucopyranoside (3). Acid hydrolysis of 1 yielded (+)-(1R,4S,5R,6R)-1,5,6-trihydr

Full text of DOI:10.1021/acs.jnatprod.6b00491

Article
pubs.acs.org/jnp
Absolute Configuration of Menthene Derivatives by Vibrational
Circular Dichroism
Julio C. Pardo-Novoa,† Hector M. Arreaga-Gonzalez,† Mario A. Gomez-Hurtado,†
́
́
́
Gabriela Rodríguez-García,† Carlos M. Cerda-García-Rojas,*,‡ Pedro Joseph-Nathan,‡
and Rosa E. del Río*,†
†Instituto de Investigaciones Químico Biolog
Michoacan 58030, Mexico
‡Departamento de Química, Centro de Investigacion
́
icas, Universidad Michoacana de San Nicolas
́
de Hidalgo, Ciudad Universitaria, Morelia,
́
́
y de Estudios Avanzados del Instituto Politecnico Nacional, Apartado 14-740,
́
Mexico City 07000, Mexico
S
* Supporting Information
ABSTRACT: The aerial parts of Ageratina glabrata afforded
(−)-(3S,4R,5R,6S)-3,5,6-trihydroxy-1-menthene 3-O-β-D-gluco-
pyranoside (1) and (−)-(3S,4S,6R)-3,6-dihydroxy-1-menthene
3-O-β-D-glucopyranoside (3). Acid hydrolysis of 1 yielded
(+)-(1R,4S,5R,6R)-1,5,6-trihydroxy-2-menthene (5) and
(+)-(1S,4S,5R,6R)-1,5,6-trihydroxy-2-menthene (6), while
hydrolysis of 3 yielded (+)-(3S,4S,6R)-3,6-dihydroxy-1-men-
thene (10), (+)-(1R,4S,6R)-1,6-dihydroxy-2-menthene (11), and
(+)-(1S,4S,6R)-1,6-dihydroxy-2-menthene (12). The structures
of the new compounds 1, 2, 5−9, and 11 were defined by 1D
and 2D NMR experiments, while the absolute configurations of
the series of compounds were determined by comparison of the
experimental vibrational circular dichroism (VCD) spectra of the
1,6-acetonide 5-acetate derived from 6 and of the 1,6-acetonide
derived from 12 with their DFT-calculated spectra. In addition, Flack and Hooft X-ray parameters of 10 permitted the same
conclusion. The results further led to the absolute configuration reassignment of 10 isolated from Brickellia rosmarinifolia, Mikania
saltensis, Ligularia muliensis, L. sagitta, and Lindera strychnifolia, as well as of 11 from Cacalia tangutica, as ent-11.
he Astereaceae family occupies an important niche in the
Mexican flora since many of its species are listed in Mexican
sagitta,9 and Lindera strychnifolia10 and 11 from Cacalia
tangutica.11
T
traditional medicine. This is the case for Ageratina glabrata
(Kunth) R.M.King & H.Rob. (Asteraceae), popularly known
as “chamizo blanco” (white greasewood), which is used as an
analgesic. Such biological activity was established using the
CH2Cl2 extract of the leaves.1 In previous chemical studies
of the nonpolar extracts from the aerial parts of A. glabrata,
the presence of thymol derivatives, as main constituents, was
evidenced,2−5 while to date chemical studies of the polar extracts
from this species have not been reported. In the present paper,
the structure elucidation of the new (−)-(3S,4R,5R,6S)-3,5,6-
trihydroxy-1-menthene 3-O-β-D-glucopyranoside (1) and the
isolation of the known (−)-(3S,4S,6R)-3,6-dihydroxy-1-menthene
3-O-β-D-glucopyranoside (3) are reported. Full characterization
of 1 and 3 was supported by derivatization and subsequent
spectroscopic studies of 2 and 4−13, while the absolute con-
figuration (AC) for the whole series was established by
comparison of the experimental and calculated vibrational circular
dichroism (VCD) spectra of 8 and 13, as well as by the Flack and
Hooft X-ray parameters of 10. Likewise, this study permitted
the reassignment of the AC of 10 isolated from Brickellia
rosmarinifolia,6 Mikania saltensis,7 Ligularia muliensis,8 Ligularia
RESULTS AND DISCUSSION
■
Successive column chromatography of the methanol extract
from the aerial parts of A. glabrata afforded (−)-(3S,4R,5R,6S)-
3,5,6-trihydroxy-1-menthene 3-O-β-D-glucopyranoside (1) and
(−)-(3S,4S,6R)-3,6-dihydroxy-1-menthene 3-O-β-D-glucopyra-
noside (3). The molecular formula of 1 was established as
C16H28O8 by HRESI/APCIMS, showing a sodium adduct
molecular ion at m/z 371.1678 (calcd as 371.1676 for
C16H28O8Na+). It showed a specific rotation value of −39 in
1
MeOH. Its H NMR data (400 MHz, methanol-d4, Table 1)
indicated H-2 as a multiplet at δH 5.69; the H-3, H-6, and
H-5 signals appearing at δH 4.19 (br d, J = 7.9 Hz), 3.81 (d, J =
3.8 Hz), and 3.55 (dd, J = 10.5, 3.8 Hz), respectively; an isopropyl
group at δH 2.17 (septd, J = 7.0, 3.3 Hz, H-8), 1.06 (d, J = 7.0 Hz,
CH3-9), and 1.05 (d, J = 7.0 Hz, CH3-10); and a vinylic methyl
group at δH 1.82 (br t, J = 1.4 Hz, CH3-7). In addition, the seven
Received: May 28, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00491
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
values J3,4 = 7.9 Hz, J4,5 = 10.5 Hz, and J5,6 = 3.8 Hz, as well as from
the NOESY correlations of H-3 with H-5 and H-6. The 1H and
13C NMR spectra were also measured in DMSO-d6 and in
pyridine-d5 in order to increase the pool of available data for
future comparative purposes (Table 2). In order to confirm the
presence of the hydroxy groups in 1, it was peracetylated to
afford compound 2. The HRESI/APCIMS spectrum showed an
[M + Na]+ ion at m/z 623.2322 (calcd as 623.2310 for
C28H40O14Na+), in agreement with the molecular formula
1
C28H40O14. The H NMR data (Table 1) showed six methyl
singlets within the δH 2.12−2.01 range, and the 13C NMR
spectrum displayed six carbonyl signals (δC 170.9−169.3) and six
methyl signals (δC 21.0−20.6), reminiscent of the incorporation
of six O-acetyl groups.
The molecular formula of 3 was determined as C16H28O7 by
HRESI/APCIMS, showing an [M + Na]+ molecular ion at m/z
1
355.1735 (calcd as 355.1727 for C16H28O7Na+). Its H NMR
(400 MHz, methanol-d4) spectrum showed the characteristic
signals for a disubstituted menthene having one glucopyranosyl
unit. Compound 3 was previously reported from Ageratina
anisochroma12 and Telekia speciosa;13 however, the structure
1
elucidation was based only on the H NMR data in methanol-
d4.12 Herein, the 1H NMR data were also recorded in DMSO-d6
and pyridine-d5, while the 13C and 2D NMR spectra were also
measured in methanol-d4, DMSO-d6, and pyridine-d5 (Tables 1
and 2). These spectra were useful to confirm the connectivity
and the relative configuration of the molecule. Its 13C NMR
(100 MHz, methanol-d4) spectrum showed 16 signals, including
the C-1 and C-2 vinylic carbons at δC 138.5 and 127.1,
respectively, and two oxymethine carbon atoms at δC 76.4
(C-3) and 68.2 (C-6). The typical carbon signals of the
β-D-glucopyranosyl unit were observed in the δC 102.1−62.9
range. Analysis of the HSQC and HMBC spectra allowed the
unambiguous assignment of the 13C NMR data for 3 (Table 1
and Figures S29 and S30, Supporting Information). Peracetyla-
tion of 3 afforded 4,12 which confirmed the presence of the
five hydroxy groups. The HRESI/APCIMS spectrum showed
an [M + Na]+ ion at m/z 565.2267 (calcd as 565.2255 for
C26H38O12Na+) consistent with the molecular formula, C26H38O12.
1
The H NMR spectrum in CDCl3 was in agreement with
that reported,12 and the 13C NMR spectrum, which displayed
five carbonyl signals (δC 171.0−169.3) and five methyl signals
(δC 21.2−20.7), is fully assigned in Table 1.
Figure 1. Menthene glucopyranosides 1 and 3, isolated from Ageratina
glabrata, and their derivatives 2 and 4−13.
In order to determine the AC of 1 and 3 using the versatile
VCD methodology of the corresponding aglycones, these
compounds were subjected to acid hydrolysis.14 The specific
rotation and NMR spectra of the sugars arising from these
reactions established the D-glucopyranosyl moieties in 1 and 3,
as carried out for the glycosylated compounds of Ageratina
cylindrica.29 Acid hydrolysis of 1 gave the menthene triol
derivatives 5 and 6 (Scheme 1), while none of the expected 3,5,6-
triol derivatives could be detected. This transformation can take
place if, after hydrolysis of the glucose moiety (Scheme 1a),
the protonated hydroxy group at C-3 acts as a leaving group,
generating an allylic carbocation15 at C-3, which can migrate to
C-1 with the simultaneous shift of the double bond from the
C-1C-2 to the C-2C-3 position. Finally, a H2O molecule
may stabilize the carbocation at C-1 to yield 5 or 6, depending on
the face of the attack. Alternatively, a concerted mechanism could
also be possible, as depicted in Scheme 1b, in which the attack
at C-1 of a H2O molecule promotes the shift of the double
bond from the C-1C-2 to the C-2C-3 position with the
simultaneous departure of the protonated glucose moiety.
characteristic proton chemical shifts for a β-D-glucopyranosyl
moiety were observed (Table 1). Its 13C NMR spectrum showed
16 signals: the C-1 and C-2 vinylic carbon signals at δC 137.7 and
125.7, respectively; three oxymethines at δC 75.0 (C-3), 71.6
(C-6), and 70.8 (C-5); and the five remaining carbon signals of
the aglycone portion within the δC 46.5−19.3 range. The typical
chemical shifts for the carbons of the β-D-glucopyranosyl moiety
resonated in the δC 101.6−63.0 region (Table 1). In the COSY
spectrum of 1, H-3 (δH 4.19) displayed correlations with H-2
(δH 5.69) and H-4 (δH 1.84), while H-5 (δH 3.55) showed a
correlation with H-6 (δH 3.81) (Figure S2, Supporting
Information). The HSQC spectrum allowed the unambiguous
assignment of all the protonated carbons as listed in Table 1.
The HMBC spectrum established the positional assignment
since H-6 correlated with C-1, C-2, C-5, and C-7, while H-3
correlated with C-2, C-4, and the anomeric carbon (C-1′). This
analysis indicated the presence of a trihydroxylated menthene
skeleton bearing a glucopyranosyloxy unit at C-3. The relative
configuration of 1 was deduced from the vicinal coupling constant
B
DOI: 10.1021/acs.jnatprod.6b00491
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Table 1. NMR Data for Menthene Glucopyranosides 1 and 3 in Methanol-d4 and 2 and 4 in CDCl3 (δ in ppm)
a
b
a
b
1
2
3
4
position
δC
δH, mult (J in Hz)
HMBC (H→C)
δC
δH, mult (J in Hz)
HMBC (H→C)
δC
δC
1
137.7
125.7
75.0
46.5
70.8
71.6
20.8
27.0
21.4
19.3
101.6
75.1
78.1
71.9
77.8
63.0
132.6
127.1
74.8
42.8
69.4
68.7
20.2
25.2
20.78
17.7
98.6
71.5
73.0
68.6
71.8
62.4
138.5
127.1
76.4
40.8
30.5
68.2
20.7
26.3
21.4
17.1
102.1
75.0
78.2
71.8
77.7
62.9
134.9
127.8
75.9
40.0
26.4
69.4
20.3
25.0
20.64
16.4
98.8
71.5
73.0
68.7
71.7
62.4
2
5.69, m
4, 6, 10
1, 2, 4, 7, 1′
3, 8
5.64, m
4, 6, 10
3
4.19, br d (7.9)
1.84, m
4.10, br d (9.4)
2.03, m
1, 2, 4, 7, 1′
3, 6, 7
4
5
3.55, dd (10.5, 3.8)
3.81, d (3.8)
3, 4, 6
4.89, dd (11.4, 4.1)
5.39, br d (4.1)
1.71, br s
3, 4, 6, Ac-5
1, 2, 4, 5, 10, Ac-6
1, 2, 6
6
1, 2, 4, 10
1, 2, 6
7
1.82, br t (1.4)
2.17, septd (7.0, 3.3)
1.06, d (7.0)
8
3, 4, 5, 8, 9
4, 7, 9
2.10, m
4, 8, 9
9
0.98, d (6.8)
0.89, d (6.8)
4.64, d (8.2)
4.96, dd (9.4, 8.2)
5.22, t (9.4)
5.05, dd (10.0, 9.4)
3.68, m
4, 7, 9
10
1′
2′
3′
4′
5′
6′a
6′b
Ac-5
1.05, d (7.0)
4, 7, 8
4, 7, 8
4.43, d (8.0)
3, 5′
1′, 3′
2′, 4′
5′, 6′
3, 3′, 5′
3.15, dd (9.0, 8.0)
3.37, dd (9.0, 8.0)
3.31, m
1′, 3′, Ac-2′
2′, 4′, Ac-3′
3′, 5′, 6′, Ac-4′
1′, 3′, 4′,
4′, 5′, Ac-6′
4′, 5′, Ac-6′
3.26, m
3.86, dd (11.7, 2.2)
3.68, dd (11.7, 5.3)
4′, 5′
4′, 5′
4.16, m
4.15, m
170.2
20.60
170.9
20.97
169.3
20.60
170.3
20.94
169.4
20.72
170.6
20.67
170.4
20.60
2.01, s
2.12, s
2.00, s
2.06, s
2.04, s
Ac-5
Ac-6
Ac-2′
Ac-3′
Ac-4′
Ac-6
Ac-2′
Ac-3′
Ac-4′
Ac-6′
169.3
20.68
170.6
20.71
169.4
20.62
171.0
21.24
2.03, s
Ac-6′
a
b
Measured at 400 MHz for H and 100 MHz for 13C using TMS as the internal reference. Measured at 300 MHz for H and 75.4 MHz for 13C.
1
1
a
Table 2. NMR Data for Menthene Glucopyranosides 1 and 3 in DMSO-d6 and in Pyridine-d5 (δ in ppm)
b
c
b
c
1
1
3
3
position
δC
δH, mult (J in Hz)
5.52, br s
δC
δH, mult (J in Hz)
6.03, br s
δC
δH, mult (J in Hz)
δC
δH, mult (J in Hz)
1
135.1
125.1
73.4
137.1
126.4
75.1
137.1
125.5
74.4
138.9
126.6
75.9
2
5.49, br s
6.01, br s
3
3.98, br d (9.4)
4.54, br d (9.1)
3.93, br d (9.3)
1.64, m
4.44, br d (9.0)
2.32, m
4
44.1
1.64, ddd (11.3, 9.4, 1.8)
3.27, dd (11.3, 3.8)
46.4
2.44, ddd (10.7, 9.1, 1.9)
3.86, br d (10.7)
39.0
40.7
5β
5α
6
68.9
70.7
29.6
1.26, td (13.3, 4.1)
1.57, dt (13.3, 2.6)
3.76, br s
31.1
1.55, td (13.3, 4.1)
2.05, dt (13.3, 2.8)
4.25, br s
69.9
20.6
24.7
20.9
18.3
100.3
73.5
77.1
70.5
76.7
61.5
3.60, d (3.8)
1.68, br s
71.6
21.6
26.5
22.3
19.3
102.5
75.7
79.2
72.7
78.9
63.8
4.22, br s
65.7
20.5
24.7
20.9
16.5
100.9
73.5
77.1
70.4
76.7
61.4
67.5
21.4
26.1
21.6
17.4
103.0
75.6
79.2
72.5
78.7
63.6
7
1.91, br s
1.67, br s
1.92, br s
8
2.15, m
2.80, septd (7.0, 1.9)
1.32, d (7.0)
1.32, d (7.0)
5.09, d (8.5)
4.04, t (8.5)
4.32, t (8.5)
4.23, t (8.5)
4.00, m
2.08, septd (6.9, 3.1)
0.84, d (6.9)
0.73, d (6.9)
4.22, d (8.3)
2.90, t (8.3)
3.13, t (8.3)
3.03, m
2.54, septd (6.9, 3.0)
0.94, d (6.9)
0.93, d (6.9)
5.07, d (8.0)
4.05, t (8.0)
4.33, t (8.0)
4.30, t (8.0)
3.98, m
9
0.96, d (7.2)
0.93, d (7.2)
4.24, d (8.5)
2.89, t (8.5)
3.13, t (8.5)
3.03, dd (9.6, 8.5)
3.07, m
10
1′
2′
3′
4′
5′
6′a
6′b
3.06, m
3.63, dd (11.5, 1.6)
3.38, dd (11.5, 5.8)
4.59, br d (11.7)
4.40, dd (11.7, 4.4)
3.64, br d (11.2)
4.57, br d (11.0)
d
3.39, br d (11.2)
4.41, br d (11.0)
a
b
c
d
Measured at 300 MHz for 1H and 75.4 MHz for 13C using TMS as the internal reference. In DMSO-d6. In pyridine-d5. Partially overlapped with
the HOD signal.
The molecular formula of 5 was established as C10H18O3 by
HRESI/APCIMS, displaying a molecular ion at m/z 186.1256
(calcd as 186.1251 for C10H18O3 ). Its H NMR (400 MHz,
CDCl3) spectrum displayed two vinylic proton signals at δH 5.62
(ddd, J = 10.1, 2.2, 1.4 Hz, H-2) and 5.68 (dd, J = 10.1, 2.1 Hz, H-3),
the signals for the oxymethine hydrogens appeared at δH 3.97
+
1
C
DOI: 10.1021/acs.jnatprod.6b00491
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Scheme 1. Putative Reaction Mechanisms for the Hydrolysis
of 1 To Yield Trihydroxymenthene Derivatives 5 and 6:
(a) Stepwise Mechanism Involving Allylic Carbocations and
(b) Concerted Mechanism
(dd, J = 8.4, 2.7 Hz, H-5) and 3.73 (dd, J = 2.6, 1.3 Hz, H-6), the
H-4 methine showed a mutliplet at δH 2.16, the isopropyl group
showed characteristic signals at δH 2.11 (m, H-8), 1.04 (d, J =
6.8 Hz, H-9), and 0.85 (d, J = 6.8 Hz, H-10), and the 7-methyl
group was observed as a sharp singlet at δH 1.40 (Table 3). Its 13C
NMR (100 MHz, CDCl3) spectrum showed 10 signals (Table 4),
of which the two vinyl carbon signals appeared at δC 131.0 (C-2)
and 129.4 (C-3), and the signals for three oxymethine carbon
atoms resonated at δC 77.1 (C-6), 68.4 (C-5), and 71.3 (C-1).
The COSY spectrum confirmed the vicinity of H-5 (δH 3.97) with
H-6 (δH 3.73) and H-4 (δH 2.16). The unambiguous assign-
ment of the 13C NMR spectrum was done via the HETCOR
and HMBC spectra, which also established the positions of
the functional groups in the menthene skeleton. The NOESY
data confirmed the relative configuration through correlations
between H-5 and H-6, as well as between H-4 and CH3-7,
indicating the cis relationship between the hydroxy groups at C-5
and C-6 and between H-4 and CH3-7 (Figure S51, Supporting
Information).
In turn, 6 displayed an [M + Na]+ ion at m/z 209.1156 (calcd
as 209.1148 for C10H18O3Na+), in agreement with the molecular
1
formula, C10H18O3. Although the H and 13C NMR spectra
resembled those of 5 (Tables 3 and 4), there were evident
changes in the chemical shifts of the H-4 (δH 2.19), H-7 (δH
1.29), and H-8 (δH 1.83) signals. The COSY spectrum confirmed
the vicinity of H-5 (δH 3.92) to H-4 (δH 2.19) and H-6 (δH 3.66),
while the 13C NMR (100 MHz, CDCl3) spectrum exhibited
10 signals, of which the chemical shifts at δC 132.4 (C-2),
127.1 (C-3), 75.0 (C-6), 71.0 (C-5), and 71.1 (C-1) showed clear
differences when compared with those observed for 5. The
HETCOR and HMBC spectroscopic data were sufficient to
allow the full assignment of all 13C NMR signals. The HMBC
spectrum displayed correlations of H-6 (δH 3.66) with C-1 (δC
71.1), C-2 (δC 132.4), and C-4 (δC 47.4), while H-5 (δH 3.92)
showed correlations with C-3 (δC 127.1) and C-4 (δC 47.4). The
relative configuration was established using the NOESY
correlations of H-6 with H-5 and H-7 indicative of the all-cis
D
DOI: 10.1021/acs.jnatprod.6b00491
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Table 4. 13C NMR Data for Menthene Derivatives 5−13 in CDCl3 (δ in ppm)
a
a
a
b
b
b
a
b
position
5
6
7
8
9
11
12
13
1
71.3
131.0
129.4
44.6
68.4
77.1
25.5
27.0
20.8
17.7
71.1
132.4
127.1
47.4
71.0
75.0
26.6
28.6
21.2
19.0
80.0
131.3
125.0
43.7
69.3
82.4
24.9
25.9
20.8
17.0
108.6
27.7
27.5
109.0
131.6
124.7
40.3
70.6
80.0
24.4
26.1
20.5
17.0
80.2
27.7
27.5
170.8
21.3
71.9
134.9
127.7
45.1
74.4
82.3
23.5
30.6
20.1
19.0
108.3
26.8
25.0
70.9
131.9
132.1
38.4
28.7
73.4
23.5
31.8
19.9
19.8
70.2
131.9
131.1
37.5
29.3
72.7
27.0
31.5
19.6
19.4
77.5
130.9
130.2
35.6
26.6
78.5
24.8
31.2
19.3
19.0
107.3
27.6
27.5
2
3
4
5
6
7
8
9
10
11
12
13
Ac-5
a
b
Measured at 100 MHz. Measured at 75.4 MHz using TMS as the internal reference.
1
orientations of the hydroxy groups at C-1, C-5, and C-6
(Figure S57, Supporting Information).
of +10 (c 0.54, MeOH). The H and 13C NMR data were
identical to those obtained for the compound isolated from
Ligularia sagitta,9 although no specific rotation values were
reported. Comparison of the AC and the specific rotation data of
10 and ent-10 with reported data showed some inconsistencies.
The first report of the AC determination of 10 (lit.16 [α]D +28)
referred to its preparation by reduction of 3,6-endoperoxide-1-
menthene isolated from Chenopodium multifidum.16 The authors
assumed the (+)-(3S,4S,6R)-3,6-dihydroxy-1-menthene (10)
structure by comparison of the specific rotation with that of
(−)-ent-10, which was prepared from (−)-(R)-α-phellandrene.17
The same reaction sequence was also used to define the
configuration of several menthene derivatives.18−20 Nonetheless,
in a chemical study of Mikania saltensis,7 ent-10 was reported
with a dextrorotatory specific rotation of +114 (at unspecified
wavelength), contrasting with the results from the study of 10
from Chenopodium multifidum.16 In more recent papers on the
medicinal plants Brickellia rosmarinifolia,6 Ligularia muliensis,8
Ligularia sagitta,9 and Lindera strychnifolia10 and on the mixture
of Amomum tsao-ko, Artemisia scoparia, Chrysanthemum indicum,
Eupatorium fortunei, and Houttuynia cordata contained in a
Chinese multiherb remedy,21 the AC of the 3,6-dihydroxy-1-
menthene 10 was defined on the basis of the data given in the
chemical study of Mikania saltensis.7
To gain chemical support for the positional assignment of the
hydroxy groups at C-1 in 5 and 6, the corresponding acetonide
derivatives were prepared. Thus, triol 6 afforded acetonide 7,
established as C13H22O3 by HRESI/APCIMS, displaying an
[M + NH4]+ ion at m/z 244.1908 (calcd as 244.1907 for
C13H22O3 + NH4 ). Its 1H NMR (400 MHz, CDCl3) spectrum
+
displayed a significant low-field shift of H-6 (δH 4.12) and
showed two methyl signals at δH 1.41 and 1.38 arising from the
acetonide moiety. The signal for H-5 was observed at δH 3.58 as a
broad doublet (J = 9.9 Hz) (Table 3), while the COSY spectrum
showed correlations of H-5 (δH 3.58) with H-6 (δH 4.12) and
H-4 (δH 2.28). The 13C NMR (100 MHz, CDCl3) spectrum
displayed the three signals at δC 108.6 (C), 27.7 (CH3), and 27.5
(CH3) of the acetonide moiety. Significant differences in the C-1
and C-6 chemical shifts of 7 with respect to 6 were observed,
confirming that the acetonide group was located at C-1 and C-6
and not at C-5 and C-6 (Table 4). The HETCOR and HMBC
spectra facilitated the unequivocal assignment of the carbon
signals of the menthene skeleton of 7 (Figures S65 and S66,
Supporting Information), while the presence of the C-5 hydroxy
group was chemically confirmed by formation of the
corresponding acetylated acetonide menthene derivative 8
(Tables 3 and 4). In turn, menthene triol 5 gave acetonide 9.
Compound 11 was obtained as colorless oil, whose molecular
formula was confirmed as C10H18O2 by HRESI/APCIMS
displaying an [M + NH4]+ ion at m/z 188.1637 (calcd as
1
Its H NMR spectrum (300 MHz, CDCl3) showed significant
differences in the chemical shifts in comparison with 5. Both H-2
and H-3 appeared as double doublets at δH 5.79 (1H, dd, J = 10.0,
3.6 Hz), and 5.57 (1H, dd, J = 10.0, 2.6 Hz). The oxymethine
hydrogen atoms resonated at δH 4.13 (1H, dd, J = 7.6, 5.5 Hz,
H-5) and 4.03 (1H, d, J = 7.6 Hz, H-6), which indicated the
presence of the acetonide group at OH-5 and OH-6. The two
signals for the acetonide methyl groups were observed at δH 1.48
(d, J = 0.6 Hz) and 1.38 (d, J = 0.6 Hz) (Table 3). The 13C NMR
spectrum (75.4 MHz, CDCl3) showed 13 signals, including the
carbon signals for the acetonide group at δC 108.3 (C), 26.8
(CH3), and 25.0 (CH3). Significant differences for C-5 (δC 74.4)
and C-6 (δC 82.3) were observed in comparison with those of 5,
suggesting the formation of the C-5/C-6 acetonide (Table 4).
In contrast to compound 1, acid hydrolysis of menthene
glucoside 3 gave the expected menthenediol 10, together
with epimeric compounds 11 and 12. Diol 10 was obtained as
colorless needles (mp 166−168 °C) and had a specific rotation
+
188.1645 for C10H18O2 + NH4 ). This compound had a specific
rotation of +58 (c 0.9, CHCl3). Its structure was supported by the
1H, 13C, and 2D NMR data. In particular, the NOESY spectrum
showed a cross-peak between H-4 and CH3-7, which accounted
for their cis relationship. The H and 13C NMR spectroscopic
1
data of 11 (Tables 3 and 4) were similar to those of ent-11 (lit.22
[α]D −46, c 1.0, CHCl3), which was obtained by microbial
transformation of (−)-(R)-α-phellandrene.22 However, in the
chemical study of Cacalia tangutica,11 11 is reported to possess
a levorotatory specific rotation of −24 (c 1.38, CHCl3), which is
in disagreement with the current results.
The menthenediol 12 was obtained as colorless oil with a
specific rotation of +43 (c 0.65, CHCl3). The NMR spectros-
copic data (Tables 3 and 4) were similar to those of an isolate
from the essential oil of Chenopodium multifidum (lit.23 [α]D +40,
c 0.92, CHCl3). The relative configuration of 12 was confirmed
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Journal of Natural Products
Article
by NOESY (Figure S93, Supporting Information) data, since
a cross-peak was observed between H-6 and CH3-7. Also, the
corresponding acetonide 13 showed [α]D +13 (c 0.57, CHCl3).
This derivative was also prepared in the C. multifidum study;
however, surprisingly the specific rotation was very different
(lit.23 [α]D −143, c 0.25, CHCl3) from that of our compound.
Because of all these discrepancies, the AC of natural and
prepared menthene derivatives 1−13 needed more scrutiny,
and therefore the determination of the AC by VCD of selected
derivatives was undertaken. This spectroscopic technique
requires a comparison of both the experimental and calculated
VCD and IR spectra and has been successfully employed in the
AC determination of many monoterpenes.24,25 Molecular
models of (1S,4S,5R,6R)-8 and (1S,4S,6R)-13 were built, and
their conformer distribution was explored with the Monte Carlo
protocol using the Merck Molecular Force Field.26 An energy
window of 10 kcal/mol produced nine conformers for 8 and six
for 13. Those conformations within a 5 kcal/mol energy range,
three for 8 and five for 13, were subjected to single-point DFT
energy calculation at the B3LYP/6-31G(d) level of theory,
followed by geometry optimization at the B3LYP/DGDZVP
level27 for those structures within an energy range of 3 kcal/mol.
This procedure yielded three conformers for both 8 and 13,
corresponding to the three rotamers of the isopropyl group,
as depicted in Figures 2 and 3, where it can be visualized that
Figure 3. Most relevant conformers of (+)-(1S,4S,6R)-1,6-dihydroxy-2-
menthene 1,6-acetonide (13), accounting for 99.9% of the conforma-
tional population.
Table 5. Thermochemical Analysis of (+)-(1S,4S,5R,6R)-5-
Acetyloxy-1,6-dihydroxy-2-menthene 1,6-Acetonide (8) and
(+)-(1S,4S,6R)-1,6-Dihydroxy-2-menthene 1,6-Acetonide (13)
a
b
c
d
e
f
conformer ΔEMMFF
%
ΔEDFT
ΔEOPT
ΔGOPT
%
OPT
MMFF
80.6
17.8
1.5
8a
0.00
0.89
2.35
0.00
0.07
0.85
0.00
1.26
2.70
0.00
0.05
0.47
88.6
10.4
0.9
0.00
1.76
3.19
0.00
0.25
0.62
94.7
4.7
8b
8c
0.4
13a
13b
13c
47.1
41.2
11.2
42.3
38.4
19.0
50.1
32.4
17.4
a
Molecular mechanics energies relative to 8a, EMMFF = 42.183 kcal/mol,
b
and to 13a, EMMFF = 37.924 kcal/mol. Population in % calculated from
c
the MMFF energies according to ΔEMMFF ≈ − RT ln K. Single-point
B3LYP/6-31G(d) energies relative to 8a, E6‑31G(d) = −556 589.884
d
kcal/mol, and to 13a, E6‑31G(d) = −413 573.847 kcal/mol. Popula-
tion in % calculated from B3LYP/6-31G(d) energies according to
e
ΔE6‑31G(d) ≈ − RT ln K. Gibbs free energies relative to 8a, GDGDZVP
=
−556 438.652 kcal/mol, and to 13a, GDGDZVP = −413 441.275 kcal/mol.
f
Population in % calculated from Gibbs free energies according to
ΔG = −RT ln K.
Figure 2. Most relevant conformers of (+)-(1S,4S,5R,6R)-5-acetyloxy-
1,6-dihydroxy-2-menthene 1,6-acetonide (8), accounting for 99.8% of
the conformational population.
of 8 and 13, respectively, while Figures 4 and 5 demonstrate the
good agreement of the experimental and calculated VCD and IR
spectra for 8 and 13, respectively.
Visual comparison of experimental and calculated VCD
spectra is in general sufficient to decide if the calculated
enantiomer is the correct one, although statistical methods are
helpful to quantitatively evaluate the agreement. The quantitative
approximation of the spectral similarity was accomplished by
using the CompareVOA algorithm28 (BioTools Inc., Jupiter, FL,
USA), which compares the integrated overlap of the experimental
and calculated data. The optimal anharmonicity factors (anH)
the cyclohexene ring exists in a single conformation. Table 5
summarizes the thermochemical data for the 8a−c and 13a−c
conformers, respectively. The IR/VCD frequencies were cal-
culated for the three rotamers, and the conformational popula-
tions were obtained by means of the ΔG = −RT ln K equation to
generate the Boltzmann-averaged IR and VCD spectra. Figures 2
and 3 show the minimum energy structures of the conformers
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Article
Figure 4. Comparison of the experimental and B3LYP/DGDZVP-
calculated VCD and IR spectra of (+)-(1S,4S,5R,6R)-5-acetyloxy-1,6-
dihydroxy-2-menthene 1,6-acetonide (8).
Figure 5. Comparison of the experimental and B3LYP/DGDZVP-
calculated VCD and IR spectra of (+)-(1S,4S,6R)-1,6-dihydroxy-2-
menthene 1,6-acetonide (13).
and the VCD spectroscopic similarities for the correct (SE) and
the incorrect enantiomers (S−E) of each compound are listed
in Table 6, together with the enantiomer similarity index (ESI)
values, indicating that the structures of (1S,4S,5R,6R)-8 and
(1S,4S,6R)-13 have the correct AC [100% confidence (C)]
according to the employed algorithm.28
Table 6. Confidence Level Data for the IR and VCD Spectra of
8 and Two Epimers of 13, Calculated at the B3LYP/DGDZVP
Level of Theory
a
b
c
d
e
f
compound
anH
SIR
SE
S−E
ESI
C (%)
During the exploration of VCD spectroscopy of natural
products we have found that a specific spectrum is not straight-
forwardly comparable to others, especially if there are changes in
one stereogenic center or when an additional stereogenic center
is added. These differences are valuable because the method,
besides distinguishing between enantiomers, is also capable of
differentiating among stereoisomers, as has been done for mono-
terpenes,29 sesquiterpenoids,30,31 diterpenoids,32 and a triterpe-
noid,33 or between closely related compounds as in the present
study. The normal modes of vibration of the calculated VCD
bands for 8 and 13 were contrasted with those found in their
respective experimental spectra. Also, the main differences
between the experimental spectra of 8 and 13 were in good
agreement with the expected vibrational changes when going
from one compound to the other. The negative band labeled as
10 in compound 8 (Figure 4) is due to the O−CO stretching of
the acetyl group, which is absent in 13 (Figure 5). In compound
13, negative bands 4 and 10, 11 (Figure 5) are mainly due to
(1S,4S,5R,6R)-8
(1S,4S,6R)-13
(1R,4S,6R)-13
0.98
0.98
0.98
96.5
92.6
72.6
81.7
85.4
20.2
11.9
8.0
69.8
77.4
100
100
g
g
47.1
c
−26.9
44
a
b
Anharmonicity factor. IR spectra similarty. VCD spectra similarity
d
for the correct enantiomer. VCD spectra similarity for the incorrect
e
enantiomer. Enantiomer similarity index calculated as SE − S−E
.
f
g
Confidence level for the configurational assignments. These values
indicate that the (1R,4S,6R)-stereoisomer is not the correct one for 13
and that its experimental and calculated VCD spectra do not match.
bending of the C-5 methylene (scissoring and wagging,
respectively), while the positive band 16 arises from the twisting
vibration of this same methylene group. In addition, the negative
band labeled as 17 in 13 originates from vibrations associated
with the C-4−C-5 and C-5−C-6 stretching motions, which are
modified in 8. The positive bands labeled as 11 in 8 and 13 in 13
are due to the O−C−O stretching of the acetonide groups
present in both compounds. To facilitate this comparison,
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Figure 6. X-ray crystal structure of (+)-(3S,4S,6R)-3,6-dihydroxy-1-menthene (10).
1
at 300 or 400 MHz for H and at 75.4 or 100 MHz for 13C on a
Varian Mercury 300 or 400 spectrometer, respectively, from CDCl3,
methanol-d4, DMSO-d6, or pyridine-d5 solutions using tetramethylsilane
(TMS) as internal reference. Chemical shift values are reported in ppm,
and coupling constants (J) are in Hz. HRESIMS data were acquired on
an Agilent LCTOF instrument at the UCR Mass Spectrometry Facility,
University of California, Riverside. Silica gel 230−400 mesh (Merck)
was used for column chromatography.
overlays of the calculated and experimental VCD spectra of
compounds 8 and 13, as well as superimposition of both
experimental spectra, are included in the Supporting Information
(Figures S103−S105, respectively). The VCD methodology is
sensitive enough to differentiate between epimers because the
change in one stereogenic center is immediately reflected by
the CompareVOA values.29−33 This was tested again for 13 by
calculating the VCD spectrum of its C-1 epimer since this
stereogenic center was created from C-1 in 10. The results were
conclusive, as can be observed in Table 6 when comparing the
correct (1S,4S,6R)-13 vs the incorrect (1R,4S,6R)-13 dister-
eoisomer.
In addition, a single crystal of 10 was obtained by recrystalliza-
tion in acetone and was measured on an X-ray diffractometer
equipped with Cu Kα monochromated radiation and collecting
one-half of the data sphere. The structure was solved by direct
methods and refined to a discrepancy index of 3.3%. The data
set was also used to calculate the Flack parameter,34 which for the
enantiomer shown in Figure 6 was x = 0.0(3), and the Hooft
parameter,35 which was y = 0.09(10). For the inverted structure,
these parameters were x = 1.0(3) and y = 0.91(10), respectively,
confirming that the correct enantiomer is the one depicted in
Figure 6. In a previous study, the X-ray diffraction analysis of 10
was undertaken, but its AC was not determined.36
Plant Material. Ageratina glabrata (Kunth) R.M.King & H.Rob.
specimens were collected during the flowering stage, on February 28,
́
2012, near km 4.5 of the Patzcuaro−Santa Clara del Cobre federal road
no. 200, N 19°29.516′ W 101°35.273′ at 2285 m above sea level.
A voucher specimen (No. 226133) was deposited at the Herbarium
of Instituto de Ecología, A. C., Centro Regional del Bajío, Pat
Michoacan, Mexico, where Prof. Jerzy Rzedowski identified the plant
material.
́
zcuaro,
́
Extraction and Isolation. Leaves (500 g), flowers (460 g), and
stems (470 g) from A. glabrata were individually macerated with hexanes
(3 × 2 L), CH2Cl2 (3 × 2 L), and MeOH (3 × 2 L) at room temperature
for 3 days each. Filtration and evaporation of the MeOH extracts yielded
80 g (16%) from leaves, 83 g (18%) from flowers, and 38 g (8%)
from stems. Aliquots of extracts from leaves (40 g), flowers (20 g),
and stems (35 g) were column-chromatographed using EtOAc−
MeOH mixtures in ascending polarity, affording 3 (100 mg, 0.25%)
at 4:1 EtOAc−MeOH and 1 (40 mg, 0.1%) at 7:3 EtOAc−MeOH.
An additional purification of 1 employing alumina as the support and
acetone as the eluent was required.
In summary, the AC of the new compounds 1, 2, 5−9, and 11
was defined by the VCD methodology and X-ray diffraction
analysis. The data provided herein for the AC of (+)-(3S,4S,6R)-
3,6-dihydroxy-1-menthene (10) indicate that this substance,
previously isolated from Brickellia rosmarinifolia,6 Ligularia
muliensis,8 Ligularia sagitta,9 Lindera strychnifolia,10 and Mikania
saltensis,7 as well as from the Chinese multiherb remedy,21
needs AC reassignment according to its specific rotation data to
agree with either (+)-(3S,4S,6R)-10 or (−)-(3R,4R,6S)-10. Also,
the AC of 11, isolated from Cacalia tangutica,11 is reassigned as
(−)-(1S,4R,6S), while 11 from the present study corresponds to
the (+)-(1R,4S,6R) enantiomer. The AC of 13 was established
as (+)-(1S,4S,6R) from its dextrorotatory specific rotation.
The present results are useful to assign the AC for menthene
derivatives since the employed methods have been proved to be
accurate.
Acetylation Reactions. Aliquots of 1 (20 mg) or 3 (50 mg) were
dissolved in pyridine (1 mL), and Ac2O (1 mL) was added. Each reaction
mixture was heated at 45 °C for 4 h. In turn, a solution of 7 (20 mg)
in pyridine (1 mL) was mixed with Ac2O (1 mL) and left at room
temperature for 3 h. Each reaction mixture was poured over ice−H2O
and extracted with CH2Cl2, and the organic layer was washed with
aqueous 10% HCl, H2O, aqueous NaHCO3, and H2O, dried, filtered, and
evaporated. The crude reaction products from 2 (17 mg) or 4 (45 mg)
were column-chromatographed using silica gel. Elution with hexanes−
EtOAc (4:1) afforded 2 (15 mg, 44%), while elution with hexanes−
EtOAc (9:1) afforded 4 (40 mg, 44%). The residue from the acetylation
of 7 was filtered and evaporated to yield 8 (11 mg, 46%).
(−)-(3S,4R,5R,6S)-3,5,6-Trihydroxy-1-menthene 3-O-β-D-gluco-
pyranoside (1): colorless oil; [α]589 −39, [α]578 −41, [α]546 −49 (c 0.9,
MeOH); IR νmax 3372, 2922, 1652 cm−1; 1H NMR (400 MHz,
methanol-d4) and 13C NMR (100 MHz, methanol-d4) data, see Table 1;
NMR data in pyridine-d5 and DMSO-d6, see Table 2; HRESI/APCIMS
m/z 371.1678 [M + Na]+ (calcd for C16H28O8 + Na+, 371.1676).
(−)-(3S,4S,5R,6S)-5,6-Diacetyloxy-3-hydroxy-1-menthene 3-O-
(2′,3′,4′,6′-tetra-O-acetyl)-β-D-glucopyranoside (2): colorless oil;
[α]589 −9, [α]578 −9, [α]546 −10, [α]436 −16, [α]365 −20 (c 0.6, CHCl3);
IR νmax 1754 (OAc) cm−1; 1H NMR (300 MHz, CDCl3) and 13C NMR
(75.4 MHz, CDCl3) data, see Table 1; HRESI/APCIMS m/z 623.2322
[M + Na]+ (calcd for C28H40O14 + Na+, 623.2310).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined on a Fisher-Johns apparatus and are uncorrected.
Optical rotations were recorded in CHCl3 or MeOH solutions on a
PerkinElmer 341 polarimeter. 1D and 2D NMR spectra were measured
H
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(−)-(3S,4S,6R)-3,6-Dihydroxy-1-menthene 3-O-β-D-glucopyrano-
side (3): colorless oil; [α]589 −58, [α]578 −61, [α]546 −69 (c 1.6,
MeOH); IR νmax 3383, 2953, 1646 cm−1; 1H NMR (400 MHz,
methanol-d4) and 13C NMR (100 MHz, methanol-d4) data, see Table 1;
NMR data in pyridine-d5 and DMSO-d6, see Table 2; HRESI/APCIMS
m/z 355.1735 [M + Na]+ (calcd for C16H28O7 + Na+, 355.1727).
(−)-(3S,4S,6R)-6-Acetyloxy-3-hydroxy-1-menthene 3-O-(2′,3′,4′,6′-
tetra-O-acetyl)-β-D-glucopyranoside (4): amorphous powder; mp
108−110 °C; [α]589 −35, [α]578 −48, [α]546 −56, [α]436 −90, [α]365
−133 (c 0.6, CHCl3); IR νmax 1752 (OAc) cm−1; 1H NMR (300 MHz,
CDCl3) and 13C NMR (75.4 MHz, CDCl3) data, see Table 1;
HRESI/APCIMS m/z 565.2267 [M + Na]+ (calcd for C26H38O12 + Na+,
565.2255).
(+)-(3S,4S,6R)-3,6-Dihydroxy-1-menthene (10): colorless needles;
1
mp 166−168 °C (MeOH); [α]D = +10 (c 0.5, MeOH). H and 13C
NMR data were identical to those reported.8
(+)-(1R,4S,6R)-1,6-Dihydroxy-2-menthene (11): colorless oil;
[α]589 +58, [α]578 +61, [α]546 +69, [α]436 +122 (c 0.9, CHCl3); IR
1
νmax 3600, 2961, 2874 cm−1; H NMR (300 MHz, CDCl3) and 13C
NMR (75.4 MHz, CDCl3) data, see Tables 3 and 4; HRESI/APCIMS
+
m/z 188.1637 [M + NH4]+ (calcd for C10H18O2 + NH4 , 188.1645).
(+)-(1S,4S,6R)-1,6-Dihydroxy-2-menthene (12): [α]D +40 (c 0.92,
CHCl3) and 1H NMR (60 MHz, CCl4) data as per ref 23; colorless oil;
[α]589 +43, [α]578 +45, [α]546 +52 (c 0.7, CHCl3); 1H NMR (300 MHz,
CDCl3) and 13C NMR (75.4 MHz, CDCl3) data, see Tables 3 and 4.
(+)-(1S,4S,6R)-1,6-Dihydroxy-2-menthene 1,6-acetonide (13): col-
orless oil; [α]589 +13, [α]578 +14, [α]546 +16, [α]436 +31 (c 0.6, CHCl3);
1H NMR (300 MHz, CDCl3) and 13C NMR (75.4 MHz, CDCl3) data,
see Tables 3 and 4; HREIMS m/z 195.1387 [M − CH3]+ (calcd for
Acid Hydrolysis of 1. A solution of 1 (45 mg) in aqueous 2% HCl
(3 mL) was stirred at room temperature during 30 min. The reaction
mixture was extracted with EtOAc, and the organic layer was washed
with H2O, aqueous NaHCO3, and H2O, dried, filtered, and evaporated.
The residue was column-chromatographed using silica gel and hexanes−
EtOAc mixtures as the eluent. Fractions 5−8 (hexanes−EtOAc, 1:4)
afforded 6 (10 mg, 41%), and fractions 10−13 (hexanes−EtOAc, 1:4)
gave 5 (9 mg, 37%). To recover the sugar fraction, the aqueous layer
was neutralized with 10% KOH, extracted with n-BuOH, and washed
with H2O. The solvent was removed under reduced pressure to yield a
mixture of α- and β-D-glucopyranose in a 36:64 ratio (1H NMR), which
was purified by column chromatography using silica gel and eluting with
AcOEt−MeOH, 1:1. The residue was dissolved in H2O and filtered
throughout a 0.2 μm nylon membrane. The sugar identity was verified
+
C12H19O2 , 195.1380).
X-ray Diffraction Analysis of 10. The data were collected on a
Bruker-Nonius CAD4 diffractometer using Cu Kα monochromated
radiation (λ = 1.541 84 Å) at 293(2) K in the ω/2θ scan mode. Crystal
data were C10H18O2, M = 170.24, monoclinic, space group C2,
a = 17.843(4) Å, b = 7.112(1) Å, c = 8.102(2) Å, β = 102.17(3) deg,
V = 1005.1(3) Å3, Z = 4, ρ = 1.125 mg/mm3, μ = 0.604 mm−1, total
reflections =1650, unique reflections 1353 (Rint 0.01%), observed
reflections 1304. The structure was solved by direct methods using the
SHELXS-97 program included in the WinGX v1.70.01 crystallographic
software package. For the structural refinement, the non-hydrogen
atoms were treated anisotropically, and the hydrogen atoms, included in
the structure factor calculation, were refined isotropically. The final R
indices were [I > 2σ(I)] R1 = 3.3% and wR2 = 8.3%. Largest difference
peak and hole were 0.144 and −0.133 e Å3. The Olex2 v1.1.5 software37
permitted calculation of the Flack34 x = −0.0(3) and the Hooft35
y = 0.1(1) parameters. For the inverted structure these parameters were
x = 1.0(3) and y = 1.0(1), respectively. Crystallographic data (excluding
structure factors) have been deposited (No. 1470326) at the Cambridge
Crystallographic Data Centre. Copies of the data can be obtained free
of charge on application to the CCDC, 12 Union Road, Cambridge CB2
IEZ, UK. Fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk.
Vibrational Circular Dichroism. VCD and IR measurements were
done on a BioTools dualPEM ChiralIR FT spectrophotometer. Samples
of 8 and 13 (4 and 10 mg, respectively) were dissolved in CDCl3
(150 μL) and placed in BaF2 cells with a path length of 100 μm. Data for
8 and 13 were acquired at a resolution of 4 cm−1 for 7 and 6 h,
respectively. The baseline was provided by subtracting the spectrum of
the solvent acquired under the same conditions. The stability of samples
was monitored by 1H NMR measurements immediately before and after
VCD measurements.
1
by H NMR in D2O and a specific rotation of +48 (c 0.4, H2O) in
comparison with an authentic sample.
Acid Hydrolysis of 3. A solution of 3 (60 mg) in 2% HCl
(3 mL) was treated as done for 1. The reaction residue was column-
chromatographed with silica gel and hexanes−EtOAc mixtures.
Fractions 4−9 (hexanes−EtOAc, 3:2) afforded 12 (8 mg, 26%), frac-
tions 11−14 (hexanes−EtOAc, 1:1) yielded 11 (7 mg, 23%), and frac-
tions 16−19 (hexanes−EtOAc, 2:3) gave 10 (15 mg, 49%). The sugar
identity was established as D-glucose following the same procedure,
as described above, for the acid hydrolysis of 1.
Preparation of Acetonide Derivatives. Solutions of 5 (25 mg),
6 (25 mg), or 12 (20 mg) in acetone (2 mL) were treated with
p-toluenesulfonic acid (8 mg). The reaction mixtures were stirred for 2 h
at room temperature and subsequently extracted with CH2Cl2. The
organic layers were washed with H2O, dried, filtered, and evaporated to
yield 7 (20 mg, 65%), 9 (19 mg, 62%), and 13 (11 mg, 45%).
(+)-(1R,4S,5R,6R)-1,5,6-Trihydroxy-2-menthene (5): colorless oil;
[α]589 +33, [α]578 +34, [α]546 +38, [α]436 + 64 (c 0.3, CHCl3); IR νmax
3605, 2964, 2875, 1635, 1051 cm−1; 1H NMR (400 MHz, CDCl3) and
13C NMR (100 MHz, CDCl3) data, see Tables 3 and 4; HRESIMS m/z
+
186.1256 [M]+ (calcd for C10H18O3 , 186.1251).
VCD Calculations. For the calculated VCD and IR spectra, Monte
Carlo search protocols were carried out for 8 and 13 using the Merck
Molecular Force Field (MMFF94) as implemented in the Spartan’04
program. An energy cutoff of 10 kcal/mol was considered, which yielded
nine conformers for 8 and six for 13. All conformers were examined
to discard duplicates. The single-point energy of each conformer was
calculated with the DFT B3LYP/6-31G(d) level of theory in the
Spartan’04 program, giving three low-energy conformers for 8 and
five for 13. These structures were geometry optimized with DFT at
the B3LYP/DGDZVP level of theory employing the Gaussian 03W
program. The minimized structures in the first 3 kcal/mol, three for each
compound (Table 5), were used to calculate the thermochemical
parameters and the IR and VCD frequencies at 298 K and 1 atm. The
six minimum energy structures were verified for the presence of no
imaginary frequencies, and their relative free energies were employed
to calculate their Boltzmann population. The Boltzmann-weighted
IR and VCD spectra for each compound were calculated considering
Lorentzian bands with half-widths of 6 cm−1. Molecular visualization
was carried out with the GaussView 3.0 program. Geometry optimiza-
tion and vibrational calculations required some 12 h computational time
per conformer when using a laptop computer operating at 2.20 GHz
with 8 Gb RAM.
(+)-(1S,4S,5R,6R)-1,5,6-Trihydroxy-2-menthene (6): colorless oil;
[α]589 +64, [α]578 +66, [α]546 +75 (c 0.7, CHCl3); IR νmax 3416, 2963,
2875, 1625 cm−1; 1H NMR (400 MHz, CDCl3) and 13C NMR
(100 MHz, CDCl3) data, see Tables 3 and 4; HRESI/APCIMS m/z
209.1156 [M + Na]+ (calcd for C10H18O3 + Na+, 209.1148).
(+)-(1S,4S,5R,6R)-1,5,6-Trihydroxy-2-menthene 1,6-acetonide (7):
colorless oil; [α]589 +34, [α]578 +35, [α]546 +38, [α]436 +69 (c 0.1,
CHCl3); IR νmax 3585, 2958, 2854, 1098, 1003 cm−1; 1H NMR
(400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data, see
Tables 3 and 4; HRESIMS m/z 244.1908 [M + NH4]+ (calcd for
+
C13H22O3 + NH4 , 244.1907).
(+)-(1S,4S,5R,6R)-5-Acetyloxy-1,6-dihydroxy-2-menthene 1,6-ace-
tonide (8): colorless oil; [α]589 +62, [α]578 +64, [α]546 +73, [α]436 +124
(c 0.6, CHCl3); IR νmax 2958, 2925, 2869, 2851, 1730, 1465 cm−1;
1H NMR (300 MHz, CDCl3) and 13C NMR (75.4 MHz, CDCl3) data,
see Tables 3 and 4; HRESI/APCIMS m/z 286.2001 [M + NH4]+ (calcd
+
for C15H24O4 + NH4 , 286.2013).
(+)-(1R,4S,5R,6R)-1,5,6-Trihydroxy-2-menthene 5,6-acetonide (9):
colorless oil; [α]589 +15, [α]578 +16, [α]546 +18 (c 0.3, CHCl3); IR νmax
3596, 2962, 2876, 1655, 1060 cm−1; 1H NMR (300 MHz, CDCl3) and
13C NMR (75.4 MHz, CDCl3) data, see Tables 3 and 4; HRESI/
APCIMS m/z 233.1733 [M + Li]+ (calcd for C13H22O3 + Li+, 233.1723).
I
DOI: 10.1021/acs.jnatprod.6b00491
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
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Biotechnol. 1986, 24, 24−30.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jnatprod.6b00491.
■
S
́
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Bellido, I. S. Phytochemistry 1983, 22, 2749−2751.
(24) Joseph-Nathan, P.; Gordillo-Roman, B. Vibrational Circular
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1H, 13C, COSY, NOESY, HSQC or HETCOR, and
HMBC spectra of compounds 1−13 and overlay plots of
experimental and VCD spectra of 8 and 13 (PDF)
Dichroism Absolute Configuration Determination of Natural Products
In Progress in the Chemistry of Organic Natural Products; Kinghorn, A. D.,
Falk, H., Kobayashi, J., Eds.; Springer International Publishing:
Switzerland, 2015; Vol. 100, pp 311−451.
(25) Burgueno-Tapia, E.; Joseph-Nathan, P. Nat. Prod. Commun. 2015,
̃
AUTHOR INFORMATION
Corresponding Authors
*E-mail (C. M. Cerda-García-Rojas): ccerda@cinvestav.mx.
Tel: +52 55 5747 4035. Fax: +52 55 5747 7137.
*E-mail (R. E. del Río): ndelrio@umich.mx. Tel: +52 443 326
5788. Fax: +52 443 326 5790.
10, 1785−1795.
■
(26) Holtje, H. D.; Folkers, G. In Molecular Modeling. Basic Principles
̈
and Applications; Mannhold, R.; Kubinyi, H.; Timmerman, H., Eds.;
Methods and Principles in Medicinal Chemistry; VCH: Weinheim,
1996; Vol. 5, Chapter 2, p 29.
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Chem. 1992, 70, 560−571. (b) Andzelm, J.; Wimmer, E. J. Chem. Phys.
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Notes
(28) Debie, E.; Gussem, E. D.; Dukor, R. K.; Herrebout, W.; Nafie, L.
A.; Bultinck, P. ChemPhysChem 2011, 12, 1542−1549.
The authors declare no competing financial interest.
(29) Bustos-Brito, C.; San
J. S.; Calzada, F.; Yep
Quijano, L. J. Nat. Prod. 2015, 78, 2580−2587.
(30) Gordillo-Roman, B.; Camacho-Ruiz, J.; Bucio, M. A.; Joseph-
Nathan, P. Chirality 2012, 24, 147−154.
(31) Gordillo-Roman, B.; Camacho-Ruiz, J.; Bucio, M. A.; Joseph-
Nathan, P. Chirality 2013, 25, 939−951.
(32) García-Sanchez, E.; Ramírez-Lopez, C. B.; Talavera-Aleman
Leon-Hernandez, A.; Martínez-Munoz, R. E.; Martínez-Pacheco, M. M.;
Gomez-Hurtado, M. A.; Cerda-García-Rojas, C. M.; Joseph-Nathan, P.;
del Río, R. E. J. Nat. Prod. 2014, 77, 1005−1012.
́
(33) Gutierrez-Nicolas, F.; Gordillo-Roman, B.; Oberti, J. C.; Estevez-
́ ́
chez-Castellanos, M.; Esquivel, B.; Calderon,
ez-Mulia, L.; Joseph-Nathan, P.; Cuevas, G.;
ACKNOWLEDGMENTS
■
́
We thank CIC-UMSNH for financial support. J.C.P.N. and
H.M.A.G. are grateful to CONACYT-Mexico for scholarships
268710 and 295638, respectively. We are grateful to Prof.
J. Rzedowski, Instituto de Ecología, A. C., Centro Regional del
́
́
Bajío, Pat
material.
́ ́
zcuaro, Michoacan, Mexico, for identifying the plant
́
́
́
, A.;
́
́
̃
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