Methodology for the Absolute Configuration Determination of Epoxythymols Using the Constituents of Piptothrix areolare

Article abstract of DOI:10.1021/acs.jnatprod.0c01113

Since epoxythymols occur in Nature either as scalemic mixtures or as pure enantiomers, the knowledge of their chiral composition and of the absolute configuration (AC) of the dominant enantiomer turns out to be mandatory. This task has already been faced

Full text of DOI:10.1021/acs.jnatprod.0c01113

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Article
Methodology for the Absolute Configuration Determination of
Epoxythymols Using the Constituents of Piptothrix areolare
Héctor M. Arreaga-González, Antonio J. Oliveros-Ortiz, Rosa E. del Río, Gabriela Rodríguez-García,
J. Martín Torres-Valencia, Carlos M. Cerda-García-Rojas,* Pedro Joseph-Nathan,
and Mario A. Gómez-Hurtado*
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ABSTRACT: Since epoxythymols occur in Nature either as scalemic mixtures or as pure enantiomers, the knowledge of their chiral
composition and of the absolute configuration (AC) of the dominant enantiomer turns out to be mandatory. This task has already
1
been faced using 1,1-bis-2-naphthol (BINOL), as a chiral solvating agent in accurate H NMR quantifications to determine the
enantiomeric ratio, and vibrational circular dichroism (VCD) to evidence the AC of the dominant enantiomer. We now explore the
use of electronic circular dichroism (ECD) to determine the AC of an epoxythymol for which time-expensive DFT calculations
would be required unless the AC of a related molecule is already known, from either VCD studies or single-crystal X-ray diffraction
analysis, since one could correlate the ECD Cotton effect with the AC because in ECD only chromophores and their neighborhoods
are evidenced. This method is now applied by using the epoxythymols from Piptothrix areolare. Known areolal (1) and 10-
cinnamoyloxy-8,9-epoxythymol isobutyrate (2) were isolated from the roots, while known 7-acetoxy-10-cinnamoyloxy-8,9-
epoxythymol isobutyrate (3) and 10-cinnamoyloxy-7-hydroxy-8,9-epoxythymol isobutyrate (4), as well as the new enantiopure 7-
acetoxy-10-cinnamoyloxy-6-hydroxy-8,9-epoxythymol isobutyrate (5) and 10-cinnamoyloxy-8,9-epoxy-6-hydroxy-7-northymol
isobutyrate (6), were obtained from the extract of the flowers. Chemical correlation of epoxythymols 1 and 3 was achieved.
Compounds 1−4 were obtained as scalemic mixtures, and 5 and 6 as the pure (8S) enantiomers. In addition, the new 10-
cinnamoyloxy-7-oxo-8,9-dehydrothymol isobutyrate (7) was isolated from the roots. The structures of 5−7 followed from NMR and
1
HRMS data, while enantiomeric compositions of 1−6 were determined by H NMR-BINOL measurements. The AC determination
for 2−6 was done by ECD using a sample of 1 to reference the ECD Cotton effect. In turn, the AC of 1 was determined by VCD
and extensive DFT calculations. The ECD-BINOL methodology turned out to be some 500 times more sensitive than that
1
combining VCD and H NMR-BINOL.
lectronic circular dichroism (ECD) and vibrational
circular dichroism (VCD) are optical methodologies
the molecule. However, the lower sensitivity of VCD, as
compared to ECD, and the need to perform density functional
theory (DFT) calculations, in typical AC determinations, limits
E
that require circularly polarized radiation for the absolute
1
configuration (AC) determination of organic molecules. ECD
3
,4
the methodology.
uses circularly polarized light in the UV region and is highly
sensitive, thus demanding small quantities of sample, but
requires the presence of appropriate chromophores and only
Special Issue: Special Issue in Honor of A. Douglas
Kinghorn
2
provides a limited number of broad absorption bands. In
contrast, VCD is based on the absorption difference of
circularly polarized light by a chiral molecule in the infrared
Received: October 15, 2020
Published: March 8, 2021
(
IR) region, and therefore any chiral organic compound will
provide narrow absorption bands attributed to the chirality of
©
2021 American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.0c01113
J. Nat. Prod. 2021, 84, 707−712
7
07

Journal of Natural Products
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Article
The occurrence of enantiomerically pure, scalemic, or
racemic mixtures of epoxythymols is prevalent in Asteraceae
5
species, and these chiral variations have been related to
6
−8
isolation processes, degradation in plant materials,
metabolic processes.
or
9
,10
Thus, an easy methodology to
establish the enantiomeric purity of natural epoxythymols by
using 1,1-bis-2-naphtol (BINOL) as a chiral solvating agent for
1
9
precise H NMR quantification was recently described, while
the AC determination was done using VCD. This stereo-
11
chemical aspect becomes relevant since antiproliferative,
antibacterial, anti-inflammatory, antioxidant, antiprotozoal,
5
cytotoxic, piscicidal, and allelopathic activities have been
described for epoxythymols.
Application of an ECD-based method could be an
alternative strategy for the AC determination of epoxythymols
due to the presence of an aromatic chromophore. Its feasibility
is herein reported using the constituents of Piptothrix areolare.
Thus, known areolal (1) and 10-cinnamoyloxy-8,9-epoxythy-
mol isobutyrate (2) from roots and known 7-acetoxy-10-
cinnamoyloxy-8,9-epoxythymol isobutyrate (3) and 10-cinna-
moyloxy-7-hydroxy-8,9-epoxythymol isobutyrate (4) as well as
the new epoxthymols (8S)-7-acetoxy-10-cinnamoyloxy-6-hy-
droxy-8,9-epoxythymol isobutyrate (5) and (8S)-10-cinnamoy-
loxy-8,9-epoxy-6-hydroxy-7-northymol isobutyrate (6) from
flowers (Figure 1) were studied by ECD. Also, the new 10-
1
Figure 2. Comparison of the H NMR spectra of epoxythymols 1−4
before (lower traces) and after (upper traces) (S)-BINOL addition.
Enantiomeric proportions are (a) 1, 91:9 S/R, (b) 2, 62:38 S/R, (c)
3
, 92:8 S/R, and (d) 4, 81:19 S/R.
10
product (1) was isolated as scalemic mixtures. These
variations encouraged us to study the AC of the dominant
enantiomer, (−)-(8S)-areolal, using VCD measurements in
10
combination with DFT calculations. The NMR data of 1−4
12−14
were similar to the reported values.
The HRESIMS data of the epoxythymol analogue 5 showed
+
Figure 1. Formulas of epoxythymols 1−7 from Piptothrix areolare.
an [M + H] ion at m/z 455.1703 (calcd as 455.1700 for
+
1
C H O + H ). Its H NMR spectrum displayed two singlets
2
5
26
8
at δ 7.03 and 6.92 for H-2 and H-5, respectively, indicative of a
1,2,4,5-tetrasubstituted aromatic ring. The typical resonances
of a trans-cinnamate moiety were observed at δ 7.59 and 6.33
as doublets (J = 16.0 Hz) and the aromatic protons in the δ
7.44−7.31 range. An isobutyrate moiety was also revealed by
the δ 2.78 (1H, sept, J = 7.0) and 1.25 (6H, d, J = 7.0 Hz)
resonances. The signals of these two ester moieties are in close
analogy with those of 1−4, as are two pairs of methylene
protons at δ 4.56 and 4.30 (J = 12.2 Hz) and at δ 3.01 and 2.77
(J = 5.2 Hz) assigned to CH -10 and CH -9, respectively.
cinnamoyloxy-7-oxo-8,9-dehydrothymol isobutyrate (7) was
isolated from the roots. The enantiomeric purities of 1−6 were
1
determined by H NMR-BINOL experiments as shown in
Figure 2 for 1−4, while epoxythymols 5 and 6 were
enantiomerically pure compounds. UV and ECD data of 1−
6
(Figure 3) revealed similar spectra for all compounds. Since
10
the AC of (−)-(8S)-areolal (1) is known, the AC of the
dominant enantiomer in the 2−4 scalemic mixtures and of
enantiopure 5 and 6 also followed to be (8S). Chemical
correlation between 1 and 3 revealed structural similarity of
epoxythymols from the roots and flowers of P. areolare.
2
2
1
3
These and other data are summarized in Table 1. The
C
NMR spectrum showed 23 signals including three carbonyl
carbon signals at δ 175.6 (C-1′), 173.6 (Ac), and 166.3 (C-1″)
and 10 aromatic carbon signals in the δ 153.2−118.1 range, of
which the C-6′/C8′ (δ 128.9) and C-5″/C-9″ (δ 128.1) pairs
of signals are more intense. The isobutyrate signals appeared at
δ 34.1 (CH-2′), 19.0 (CH -3′), and 18.9 (CH -4′), while a
RESULTS AND DISCUSSION
■
Chromatography of the root extract of P. areolare afforded
thymol derivatives 1, 2, and 7, while the flower extract yielded
epoxythymols 3−6 (Figure 1). Areolal (1) was originally
3
3
12
described as racemic crystals, and, recently, this natural
signal at δ 20.9 was attributed to the acetoxy methyl group
7
08
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(
Table 1). There is again a significant similarity of signals
between 5 and 1−4. Carbons attached to hydrogen atoms were
verified by a HETCOR experiment, while quaternary carbons
and positional assignments were done by an HMBC
experiment, where the correlation between H -7 and the
2
acetoxy carbonyl carbon is evident. The data of 5 particularly
12
resembled those of 3, but are indicative of an additional
hydroxy group at C-6.
The HRESIMS data of 6 showed an [M + H] ion at m/z
+
+
1
383.1492 (calcd as 383.1489 for C H O + H ). Its H NMR
22 22 6
spectrum showed doublets at δ 6.90 (J = 3.0 Hz) and 6.84 (J =
.7 Hz) and a doublet of doublets at δ 6.72 (J = 8.7, 3.0 Hz),
8
assigned to H-5, H-2, and H-1, respectively, evidencing a
trisubstituted aromatic ring. Methylene protons at δ 4.59 and
4.32 (J = 12.2 Hz) as well as at δ 3.02 and 2.78 (J = 5.2 Hz)
were assigned to CH -10 and CH -9, respectively. In addition,
2
2
cinnamate and isobutyrate moieties were evidenced as in the
1
3
previous case (Table 1). The C NMR spectrum showed 20
signals, of which two carbonyl carbon signals appeared at δ
1
75.8 (C-1′) and 166.4 (C-1″). The aromatic carbon signals
appeared in the δ 153.5−115.4 range, and the cinnamate and
isobutyrate signals resemble those of 1−5 (Table 1). The
HMBC plot showed correlations of H-1 with C-3 (δ 141.9), C-
5
(δ 115.4), and C-6 (δ 153.5), while H-2 correlated with C-3,
C-4 (δ 130.0), and C-6, demonstrating the structure of the nor-
epoxythymol derivative 6.
+
The HRESIMS data of 7 showed an [M + Na] ion at m/z
+
1
4
01.1360 (calcd as 401.1359 for C H O + Na ). Its H
23 5
22
12
Figure 3. Comparation of (a) UV and (b) ECD spectra of
epoxythymols 1−6 and transition assignments according to published
NMR spectrum was similar to that of 1, but showed doublets
at δ 5.50 (J = 1.0, H-9a) and 5.26 (J = 1.0, H-9b), indicating a
vinylic methylene instead of the epoxide AB spin system. The
21,24
data for analogue moieties.
1
3
C NMR spectrum showed 20 signals (Table 1), with the
Table 1. NMR Data of 5−7 in CDCl (δ in ppm, J in Hz)
3
5
6
7
position
δC
δH (J in Hz)
HMBC
3, 6, 7
δC
δH (J in Hz)
HMBC
3, 5
3, 4, 6
δC
δH (J in Hz)
HMBC
1
122.6
125.7
141.6
131.8
118.1
153.2
62.4
116.4
123.5
141.9
130.0
115.4
153.5
6.72 dd (8.7, 3.0)
6.84 d (8.7)
137.0
130.8
148.8
138.9
123.6
127.2
190.8
139.8
118.9
2
3
4
5
6
7
8
9
9
1
1
1
2
3
4
1
2
3
4
5
6
7
7.03 s
7.43 m
1, 4, 6, 7
6.92 s
4.99 s
3, 6, 8
6.90 d (3.0)
3
7.52 d (1.5)
7.69 dd (7.8, 1.5)
9.92 s
1, 2, 7
1, 2, 6, Ac (C)
56.8
50.9
57.0
51.0
a
b
0a
0b
′
′
′
′
″
″
″
″
3.01 d (5.2)
2.77 d (5.2)
4.56 d (12.2)
4.30 d (12.2)
8, 9, 10
4, 8, 10
8, 9, 1″
4, 8, 9, 1″
3.02 d (5.2)
2.78 d (5.2)
4.59 d (12.2)
4.32 d (12.2)
4, 8
4, 8
4, 8, 9, 1″
4, 8, 9, 1″
5.50 d (1.0)
5.26 d (1.0)
4.9 s
8, 10
8, 10
8, 9, 1″
65.2
65.0
65.8
175.6
34.1
19.0
175.8
34.1
19.0
175.1
34.1
18.8
2.78 sept (7.0)
1.25 d (7.0)
1.25 d (7.0)
1′, 3′, 4′
1′, 2′, 4′
1′, 2′, 3′
2.78 sept (7.0)
1.25 d (7.0)
1.25 d (7.0)
1′, 3′, 4′
1′, 2′,4′
1′, 2′, 3′
2.75 sept (7.0)
1.24 d (7.0)
1.24 d (7.0)
1′, 3′, 4′
1′, 2′, 4′
1′, 2′, 3′
18.9
18.9
18.8
166.3
145.6
117.1
134.1
128.1
128.9
130.4
173.6
20.9
166.4
145.7
117.1
134.1
128.1
128.9
130.5
166.3
145.5
117.4
134.2
128.1
128.9
130.5
7.59 d (16.0)
6.33 d (16.0)
1″, 5″, 8″
1″, 4″
7.58 d (16.0)
6.32 d (16.0)
7.60 d (16.0)
6.34 d (16.0)
1″
4″
1″, 4″
″, 9″
″, 8″
″
7.31 m
7.44 m
7.31 m
4″
7″
7.31 m
7.43 m
7.31 m
7.31 m
7.43 m
7.31 m
4″, 7″
4″, 5″, 9″
Ac (C)
Ac CH3)
2.03 s
Ac (C)
7
09
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vinylic carbons resonating at δ 139.8 (C-8) and 118.9 (C-9).
Comparison of the structures of 1 and 7 readily suggested that
271 nm, which were assigned to π−π* transitions of the
20 21−23
benzaldehyde and cinnamate moieties,
respectively. In
7
could be the biosynthetic precursor of 1.
Once the identification of the known compounds and the
D structure elucidation of the new metabolites was
the ECD spectrum, negative Cotton effects at 258, 263, 272,
and 298 nm were observed (Table 2). The first two negative
2
completed, attention was turned to the determination of the
AC of 2−6. It should be kept in mind that areolal (1) was
originally isolated as achiral crystals. To confirm its racemic
Table 2. Maximum UV Absorptions and Main ECD
Transitions of Epoxythymols 1−6
a
a
1
compound
λ/nm (log ε)
λ/nm (Δε)
nature, two H NMR doublets of equal intensity were
1
2
1
248 (4.91)
258 (−52.2)
263 (−42.4)
observed for H-10b in the presence of a chiral europium
shift reagent. In contrast, for the quantification of enantiomers
significantly milder conditions were used since (S)-BINOL
acts as a chiral solvating agent, which is equivalent to using a
chiral solvent. The (S)-BINOL methodology was used for
2
71 (4.98)
2
72 (−49.9)
2
98 (1.3)
2
3
4
5
6
273 (4.92)
275 (4.87)
275 (4.88)
274 (4.99)
274 (4.99)
259 (−21.5)
265 (−17.7)
9
,10
recent
epoxythymol studies; thus, its validity to define
2
72 (−22.8)
enantiomeric compositions of this type of molecule is well
established. In addition, the enantiomeric mixture of
epoxythymols can be recovered by column chromatography
separation after NMR-BINOL quantifications.
2
99 (3.2)
259 (−20.3)
2
2
2
63 (−17.5)
72 (−22.3)
98 (2.3)
The technique provides enantioresolution of some proton
signals close to the epoxythymol stereocenter, producing
1
260 (−45.4)
264 (−41.7)
sufficient H NMR signal splitting for the determination of
15,16
enantiomeric excess.
The enantiomeric excess quantifica-
2
73 (−57.0)
tion is also done by CH -10 signal intensity measurements
2
2
98 (3.1)
from spectra determined under good magnetic homogeneity
conditions, since the peak separations are too close for routine
257 (−16.4)
263 (−12.7)
271 (−19.4)
298 (3.1)
9
,10
integration
8
(Figure 2), where 1 showed a 91:9 proportion,
9
2% ee, as seen in traces (a). Its chemical shift splitting was
10
similar to that reported. Compound 2 exhibited a 62:38
24% ee) proportion (traces b), 3 showed a 92:8 (84% ee)
ratio (traces c), and 4 gave an 81:19 (62% ee) proportion
258 (−24.6)
(
2
2
63 (−20.3)
72 (−30.5)
(
traces d).
For all 1H NMR-BINOL measurements, the dominant
298 (2.5)
a
Data for ε are given in L mol−1 cm−1.
enantiomer showed the H-9a and H-10b proton signals
significantly shielded, in contrast to the respective H-9b and
H-10a resonances. The signal splitting magnitudes of H-9a
were Δδ = 0.008, 0.017, 0.009, and 0.010, while those for H-
Cotton effects were associated with the π−π* transition of the
24
epoxystyrene moiety, while the negative Cotton effect at 272
nm was assigned to the π−π* transition of the cinnamate
functionality. Finally, the weak positive Cotton effect at 298
10b were Δδ = 0.017, 0.041, 0.019, and 0.020 for 1−4,
respectively. Quantification of the scalemic mixtures was
feasible in spite of the fact that the signal of H-10a in 4
appeared close to the OH signal at δ 4.58 (Figure 2d, upper
trace), or the H-9b signals of 1−4 overlap with the methine
proton of the isobutyrate moiety. This is because an advantage
of this methodology is that the scalemic proportion can be
determined from at least one visible signal, H-10b for instance.
In the enantiopure analyses of 5 and 6, no splitting was
observed after addition of BINOL, revealing only one set of
slightly shielded signals.
21
nm was related to the n−π* cinnamate transition.
Once the UV and ECD spectra of (8S)-1 were assigned, the
AC of epoxythymols 2−6 became plausible, since, as seen in
Figure 3b, the ECD spectra of 1−6 are quite similar and
further reveal a qualitative absorption dependence associated
with the enantiomeric excess of the studied samples. All the
UV spectra showed λmax in the 273−275 nm range associated
with π−π* transitions of the cinnamate moiety, as in 1 (Figure
3
2
a). All ECD spectra showed negative Cotton effects in the
57−260 and 263−265 nm regions associated with π−π*
After enantiopurity determination of 1−6, UV and ECD
data were recorded. These analyses were not explored
previously for the AC determination of epoxythymols despite
the higher sensitivity of the method, faster measurement times,
and the presence of suitable chromophores close to the
stereogenic center. ECD has classically been used in organic
chemistry and in natural product studies for the AC
transitions of the epoxystyrene unit, while the π−π* transitions
of the cinnamate moieties were related to the negative Cotton
effects in the 272−273 nm region. All spectra also showed
weak positive Cotton effects at 298−299 nm for the n−π*
transition of the cinnamate moieties (Table 2). On the basis of
these results, the AC of the major enantiomers in 2−4 and
those for the enantiomeric pure epoxythymols 5 and 6 are
17
determination by data comparison using model compounds,
(
8S), thus completing the structure and AC determination of
although with the advent of contemporary computational
resources, the required time-dependent DFT calculations for
the new epoxythymols 5 and 6.
18,19
Compound 3 was previously isolated as a natural product
from P. areolare, but its chiroptical properties and absolute
new groups of molecules are currently feasible,
quite expensive in terms of computation time.
although
25
configuration were not examined at that time. The 92:8 S/R
A sample of enantiomerically enriched (−)-(8S)-areolal (1)
scalemic mixture of 3 isolated herein showed a levorotatory
enabled ECD data comparison for the AC determination of 2−
specific rotation [α]2
2 °C
−5.7 (c 1.1, CHCl ), while the 91:9
6
. The UV spectrum of 1 (Figure 3a) showed λ at 248 and
max
589
3
7
10
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Journal of Natural Products
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Article
S/R mixture of areolal (1) showed [α] 25 82 9° C −5.4 (c 1.0,
using a hexanes−EtOAc mixture in ascending polarity afforded 3 (73
mg, 0.04%) and 6 (20 mg, 0.01%) with a 7:3 mixture and 5 (8 mg,
CHCl ). In order to collect independent evidence for the
3
0
.004%) and 4 (12 mg, 0.006%) with a 3:2 mixture.
Determination of Scalemic Ratios Using (S)-BINOL. The
absolute configuration of 3, a sample of areolal (1), enriched in
the dextrorotatory (8R) enantiomer of [α]2
2 °C
5
89
+ 0.5, [α]578
enantiomeric ratio for each epoxythymol was determined from the
1
+
0.5, [α]546 +0.6, and [α]436 +2.6 (c 2.17, CHCl ), was
3
12
height of the signals from H NMR measurements obtained under
converted into 3 by using a described sequence. Areolal (1)
good magnet homogeneity (better than 0.3 Hz), considering that the
width at half-height of the evaluated signals in both enantiomers is the
same, by using 6 mg of 1 and 2, 3 mg of 3−6, and in each case 30 mg
was treated with NaBH in MeOH at room temperature, and
4
the reduction product was immediately acetylated with Ac O
2
in pyridine. The hemisynthetic sample of 3 showed identical
of (−)-(S)-BINOL in 0.7 mL of CDCl .
3
1
13
ECD Spectroscopic Measurements. The measurements were
performed in the 220 to 420 nm region using solutions containing 10
μg of individual epoxythymols 1−6 in 1 mL of EtOH at 25 °C. The
ECD spectra were baseline corrected by subtracting the spectrum of
the solvent acquired under identical instrument conditions.
H and C NMR spectra to those of natural 3, but
dextrorotatory values of [α]2
2 °C
5
89
+0.7, [α]578 +0.7, [α]546
+
0.8, [α]436 +2.1, and [α]365 +6.2 (c 3.9, CHCl ), thus
3
reinforcing the specific rotation sign/absolute configuration
relationship for (−)-(8S)-3 and (+)-(8R)-3. It also follows that
the chemical changes at C-7 in 1−4 or the presence of a
phenolic hydroxy group at C-6 in 5 and 6 is not influencing the
ECD spectra. As more epoxythymols become available for this
type of study, there will be more data to compare and assign
the AC of this kind of compound, many of which presumably
occur as scalemic mixtures.
(8S)-7-Acetoxy-10-cinnamoyloxy-6-hydroxy-8,9-epoxythymol
2
2 °C
isobutyrate (5): pale yellow oil; [α]
+2.4, [α] +2.7, [α] +2.4,
578 546
5
89
1
13
[α]436 −2.7, [α]
−27.2 (c 0.29, CHCl ); H and + C NMR
365
3
(CDCl ) see Table 1; HRESIMS m/z 455.1703 [M + H] (calcd for
3
+
C
H O + H , 455.1700).
26 8
25
(8S)-10-Cinnamoyloxy-8,9-epoxy-6-hydroxy-7-northymol isobu-
2
2 °C
tyrate (6): pale yellow oil; [α]
−3.8, [α] −4.2, [α] −5.4,
5
89
578
546
1
13
In conclusion, the AC determination of epoxythymols using
ECD spectroscopy requires a fraction of a milligram compared
to several milligrams for VCD data acquisition. The sample
amount ratio for enantiomerically pure epoxythymols seems to
be around 1:500 in favor of ECD. Furthermore, inspection of
the ECD spectra of 1−6 showed that relatively small
enantiomeric excesses would be sufficient to define the
chirality of the dominant enantiomer in scalemic mixtures.
This is an additional advantage over VCD determinations, in
which, due to the inherent lower sensitivity, samples having
[α]436 −17.6 (c 5.34, CHCl ); H and C NMR (CDCl ) see Table
3
3
+
+
1; HRESIMS m/z 383.1492 [M] (calcd for C22
H
O
22
+ H ,
6
3
83.1489).
1
0-Cinnamoyloxy-7-oxo-8,9-dehydrothymol isobutyrate (7):
1
13
pale yellow oil; H and C NMR (CDCl ) see Table 1; HRESIMS
m/z 401.1360 [M] (calcd for C H O + Na , 401.1359).
3
+
+
2
3
22
5
Chemical Correlation of Epoxythymols 1 and 3. A solution of
2 °C
3
60 mg of areolal (1) of [α]2
+0.5, [α]578 +0.5, [α]546 +0.6, and
589
[
α]436 +2.6 (c 2.17, CHCl ) in 15 mL of MeOH was stirred in the
3
presence of 70 mg of NaBH at room temperature for 30 min. The
4
9
reaction mixture was extracted with CH Cl (2 × 20 mL), and the
2
2
some 50% ee seem to be a practical limit. In addition, since
combined extracts were washed with 50 mL of H O, dried over
2
VCD provided the AC of a sample of 1, its ECD spectrum can
now be used as a reference for the AC determination of related
epoxythymols, thereby precluding the need to perform
expensive computer TDDFT calculations.
anhydrous Na SO , filtered, and evaporated to yield 70 mg of 4. A
2
4
portion of the residue (49 mg) was dissolved in 5 mL of CH Cl and
2
2
0
.5 mL of pyridine and treated with 0.5 mL of Ac O at room
2
temperature for 1 h. After extraction and isolation, the oily residue
2
5
2 °C
(50 mg) showed [α]
+0.7, [α]578 +0.7, [α]546 +0.8, [α]436 +2.1,
89
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
recorded on a PerkinElmer 341 polarimeter. UV and ECD spectra
were obtained on a stand-alone JASCO CD-2095 circular dichroism
■
and [α]365 + 6.2 (c 3.9, CHCl ). EIMS m/z (rel int) 389 (0.1), 290
3
1
13
(
33), 232 (35), 220 (100), 204 (94). The H and C NMR spectra
12
were identical with those of natural 3.
1
detector. 1D and 2D NMR spectra were measured at 300 MHz for H
and 75.4 for 13C on a Varian Mercury 300 NMR spectrometer in
ASSOCIATED CONTENT
■
CDCl using TMS as the internal reference. Chemical shift values are
3
*
sı Supporting Information
reported in ppm, and coupling constants (J) are given in Hz.
HRESIMS data were acquired on a Waters Synapt G2 spectrometer at
the Department of Biochemistry, University of Colorado, Boulder,
CO, USA. Silica gel 230−400 mesh (Merck) was used for column
chromatography.
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01113.
1H and 13C NMR spectra of 5−7 (PDF)
Plant Material. Piptothrix areolare (DC.) R.M. King & H. Rob.
specimens were collected during the flowering stage, in September
and October 2019, near km 35 of the Tiripetıo
N 19°20.645′, W 101°21.348′) at 2509 m above sea level. A voucher
specimen (246342) is deposited at the Herbarium of Instituto de
Ecologıa, A. C., Centro Regional del Bajıo, Patzcuaro, Michoacan
Mexico.
́
-Eren
́
dira federal road
AUTHOR INFORMATION
■
(
Corresponding Authors
́
́
́
́
,
Carlos M. Cerda-García-Rojas − Departamento de Química,
Centro de Investigación y de Estudios Avanzados del Instituto
Politécnico Nacional, Mexico City 07000, Mexico;
orcid.org/0000-0002-5590-7908; Phone: +52 55 5747
́
Extraction and Isolation of Epoxythymols. Fresh roots (2 kg)
of P. areolare were macerated in hexanes (3 × 3 L) at room
temperature for 3 days. Filtration and solvent evaporation gave 5 g
4
7
035; Email: ccerda@cinvestav.mx; Fax: +52 55 5747
137
(
0.3%) of a yellowish oil. Column chromatography of the extract,
using hexanes−EtOAc mixtures in ascending polarity, afforded 7 (20
mg 0.001%) with 9:1 to 7:3 mixtures, 1 (22 mg, 0.001%) with a 7:3
mixture, and 2 (60 mg, 0.003%) with a 3:2 mixture.
Mario A. Gómez-Hurtado − Instituto de Investigaciones
Químico Biológicas, Universidad Michoacana de San Nicolás
de Hidalgo, Michoacán 58030, Mexico; orcid.org/0000-
0002-2386-4819; Phone: +52 443 326 5788;
Dried flowers (200 g) were macerated in CH Cl (3 × 1 L) at
2
2
room temperature for 3 days. Filtration and solvent evaporation gave
10 g (5%) of a yellowish oil. Column chromatography of a 5 g aliquot
Email: magomez@umich.mx; Fax: +52 443 326 5790
7
11
https://dx.doi.org/10.1021/acs.jnatprod.0c01113
J. Nat. Prod. 2021, 84, 707−712

Journal of Natural Products
pubs.acs.org/jnp
Article
Authors
Cerda-García-Rojas, C. M.; Joseph-Nathan, P.; Gómez-Hurtado, M.
A. J. Nat. Prod. 2018, 81, 63−71.
Héctor M. Arreaga-González − Departamento de Química,
Centro de Investigación y de Estudios Avanzados del Instituto
Politécnico Nacional, Mexico City 07000, Mexico;
orcid.org/0000-0002-3746-0137
Antonio J. Oliveros-Ortiz − Instituto de Investigaciones
Químico Biológicas, Universidad Michoacana de San Nicolás
de Hidalgo, Michoacán 58030, Mexico
Rosa E. del Río − Instituto de Investigaciones Químico
Biológicas, Universidad Michoacana de San Nicolás de
Hidalgo, Michoacán 58030, Mexico; orcid.org/0000-
(10) Arreaga-González, H. M.; Rodríguez-García, G.; del Río, R. E.;
Ferreira-Sereno, J. A.; García-Gutiérrez, H. A.; Cerda-García-Rojas, C.
M.; Joseph-Nathan, P.; Gómez-Hurtado, M. A. J. Nat. Prod. 2019, 82,
3
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Universidad Autónoma del Estado de Hidalgo, Hidalgo
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2184, Mexico; orcid.org/0000-0001-6426-7562
Pedro Joseph-Nathan − Departamento de Química, Centro de
Investigación y de Estudios Avanzados del Instituto
Politécnico Nacional, Mexico City 07000, Mexico;
orcid.org/0000-0003-3347-3990
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c01113
(22) Ravindram, B.; Madhurambal, G.; Mariappan, M.; Ramamurthi,
K.; Mojumdar, S. C. J. Therm. Anal. Calorim. 2011, 104, 909−914.
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Notes
(
The authors declare no competing financial interest.
(
2
ACKNOWLEDGMENTS
■
2
We thank CIC-UMSNH and CONACYT-Mexico (Grant No.
84194) for partial financial support. H.M.A.G. and A.J.O.O.
are grateful to CONACYT-Mexico for scholarships 295638
and 956977, respectively.
DEDICATION
■
Dedicated to Professor Dr. A. Douglas Kinghorn, The Ohio
State University, for his pioneering work on bioactive natural
products.
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