Absolute configuration and conformational analysis of brevipolides, bioactive 5,6-dihydro-α-pyrones from Hyptis brevipes

Article abstract of DOI:10.1021/np300740h

The (6′S)-configuration of brevipolides A-J (1-10), isolated from Hyptis brevipes, was established by X-ray diffraction analysis of 9 in conjunction with Mosher's ester analysis of the tetrahydro derivative 11 obtained from both geometric isomers 8 and 9 as well as by chemical correlations. The structure of the new brevipolide J (10) was characterized through NMR and MS data as having the same 6-heptyl-5,6-dihydro-2H-pyran-2-one framework possessing the cyclopropane moiety of all brevipolides but substituted by an isoferuloyl group instead of the p-methoxycinnamoyl moiety found in 8 and 9. Conformational analysis of these cytotoxic 6-heptyl-5,6-dihydro-α- pyrones was carried out on compound 9 by application of a protocol based on comparison between experimental and DFT-calculated vicinal ¹H- ¹H NMR coupling constants. Molecular modeling was used to correlate minimum energy conformers and observed electronic circular dichroism transitions for the isomeric series of brevipolides. Compounds 7-10 exhibited moderate activity (ED₅₀ 0.3-8.0 μg/mL) against a variety of tumor cell lines.

Full text of DOI:10.1021/np300740h

Article
pubs.acs.org/jnp
Absolute Configuration and Conformational Analysis of Brevipolides,
Bioactive 5,6-Dihydro-α-pyrones from Hyptis brevipes
G. Alejandra Suarez-Ortiz,† Carlos M. Cerda-García-Rojas,‡ Adriana Hernandez-Rojas,†
́
́
and Rogelio Pereda-Miranda*,†
†Departamento de Farmacia, Facultad de Química, Universidad Nacional Auton
Universitaria, Mexico City, D. F. 04510 Mexico
‡Departamento de Química y Programa de Posgrado en Farmacología, Centro de Investigacion
Politecnico Nacional, A.P. 14-470, Mexico City, D. F. 07000 Mexico
́
oma de Mex
́
ico, Circuito Exterior Ciudad
́
́
́
y de Estudios Avanzados del Instituto
́
́
́
S
* Supporting Information
ABSTRACT: The (6′S)-configuration of brevipolides A−J
(1−10), isolated from Hyptis brevipes, was established by X-ray
diffraction analysis of 9 in conjunction with Mosher’s ester
analysis of the tetrahydro derivative 11 obtained from both
geometric isomers 8 and 9 as well as by chemical correlations.
The structure of the new brevipolide J (10) was characterized
through NMR and MS data as having the same 6-heptyl-5,6-
dihydro-2H-pyran-2-one framework possessing the cyclo-
propane moiety of all brevipolides but substituted by an isoferuloyl group instead of the p-methoxycinnamoyl moiety found
in 8 and 9. Conformational analysis of these cytotoxic 6-heptyl-5,6-dihydro-α-pyrones was carried out on compound 9 by
1
application of a protocol based on comparison between experimental and DFT-calculated vicinal H−1H NMR coupling
constants. Molecular modeling was used to correlate minimum energy conformers and observed electronic circular dichroism
transitions for the isomeric series of brevipolides. Compounds 7−10 exhibited moderate activity (ED50 0.3−8.0 μg/mL) against a
variety of tumor cell lines.
α,β-Unsaturated δ-lactones, well-known Michael acceptors,
constitute the pharmacophoric group of a broad range of
natural products.1 Many of these compounds display
pharmacologically relevant properties, e.g., antimicrobial,
cytotoxic, and antitumoral activities.2,3 These bioactive
products, occurring in several members of the mint family
(Lamiaceae)4−6 (Figure S1, Supporting Information), comprise
polyacylated-6-heptyl-5,6-dihydro-2H-pyran-2-ones particularly
abundant in the genus Hyptis.3,7,8 They are structurally related
to pironetin (Figure S2, Supporting Information), an anticancer
acetogenin of microbial origin that selectively targets Lys-352 of
α-tubulin.9
Recently, six cytotoxic compounds, brevipolides A−F (1−6,
Figure 1), all of which share a 6-heptyl-5,6-dihydro-2H-pyran-2-
one framework bearing a cyclopropane moiety, were isolated
from Hyptis brevipes collected in Indonesia.10 These products
were structurally related to the skeletal class first characterized
in compound 7 and related compounds 8 and 9 from Lippia
alva (Verbenaceae), which were identified as inhibitors of the
chemokine receptor CCR5 (the principal human immunode-
Figure 1. Brevipolides from Hyptis brevipes.
ficiency virus type 1 co-receptor).11 Compound 7 was also
found to be active in an enzyme-based ELISA NF-κB assay.10
However, the absolute configuration at C-6′ was not
established for any of these 6-heptyl-5,6-dihydro-2H-pyran-2-
related brevipolides, in order to obtain an accurate description
of their three-dimensional properties by applying a protocol
ones. This situation prompted us to undertake a study directed
to the assignment of the absolute configuration and conforma-
tional analysis of compound 9, as a representative model for all
Received: October 26, 2012
Published: January 2, 2013
© 2013 American Chemical Society and
American Society of Pharmacognosy
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1
Table 1. H and 13C NMR Chemical Shifts and Coupling
based on the systematic comparison between DFT theoretical
and experimental vicinal H NMR coupling constants.
12,13
1
a
Constants for 10
In the present investigation, compounds 7−9 and the new
natural product 10 were isolated from a Mexican collection of
H. brevipes and given the trivial names of brevipolides G−J,
respectively. A combination of X-ray diffraction analysis,
chiroptical measurements, chemical correlations, and Mosher
ester derivatization was used to confirm the absolute
configuration of these compounds.
position
δH (J in Hz)
δC
2
164.0
120.8
146.1
23.4
3
6.02 ddd (9.7, 2.2, 0.9)
6.98 ddd (9.7, 6.4, 2.2)
2.70 dddd (18.5, 12.3, 2.2, 2.2)
2.52 dddd (18.5, 6.4, 3.8, 0.9)
4.52 ddd (12.3, 3.8, 3.8)
3.70 dd (6.3, 3.8)
4
5ax
5eq
6
80.7
71.8
26.6
14.5
1′
2′
3′proR
3′proS
4′
5′
6′
7′
1″
2″
3″
4″
5″
6″
7″
8″
9″
OMe
RESULTS AND DISCUSSION
■
1.60 dddd (8.9, 6.3, 6.3, 3.4)
1.12 ddd (8.9, 6.3, 4.0)
1.34 ddd (8.5, 4.0, 3.9)
2.28 ddd (8.5, 3.9, 3.4)
Aerial parts of H. brevipes were powdered and extracted with
CHCl3. The extract was fractionated by column chromatog-
raphy on silica. The fractions containing 5,6-dihydro-2H-pyran-
2-ones were purified by preparative reversed-phase HPLC
through application of the recycling technique.14 This
procedure afforded 7−10, which displayed moderate cytotox-
icity against nasopharyngeal cancer cells (KB) with IC50 values
of 0.3−2.0 μg/mL (Table S7, Supporting Information).
21.5
206.7
75.2
5.30 q (7.0)
1.60 d (7.0)
16.1
127.7
113.2
145.9
148.9
110.6
122.1
146.1
114.9
166.7
56.0
7.13 d (1.9)
Compound 7 showed a quasi-molecular ion at m/z 387.1455
[M + H]+ by HRFABMS in the positive mode, consistent with
the molecular formula C21H24O7. The molecular formula of
compound 8 was established as C22H25O7 by HRFABMS with a
quasi-molecular ion peak [M + H]+ at m/z 401.1595.
Compound 9 showed the same ion, thus indicating that 8
and 9 are structural isomers. Methylation of the phenolic group
of 7 with diazomethane afforded a product that was identical to
6.84 d (8.3)
7.03 dd (8.3, 1.9)
7.64 d (15.9)
6.34 d (15.9)
3.92 s
1
compound 8. H and 13C NMR chemical shifts for 7−9 are
a
NMR spectra were acquired in CDCl3 at 25 °C. Chemical shifts (δ)
listed in Table S1 (Supporting Information). All values were
identical to those reported for the compounds isolated from
Lippia alva.11
are in ppm relative to TMS. The spin coupling (J) is given in
parentheses (Hz) and was obtained by nonlinear fit of the
experimental 1H NMR spectrum to the simulated spectrum generated
by iteration of spectral parameters (1H chemical shifts, J-couplings, and
line width).
The molecular formula of the new natural product 10 was
established as C22H24O8 by HRFABMS, showing a quasi-
molecular [M + H]+ ion peak at m/z 416.1467. Compound 10
1
exhibited the characteristic H and 13C NMR signals (Table 1;
Figures S9 and S10, Supporting Information) for the 6-heptyl-
5,6-dihydro-2H-pyran-2-one framework. The α,β-unsaturated
δ-lactone system was identified through the characteristic
resonance for the C-2 carbonyl group (δ 164.0) and the C-3
and C-4 vinylic protons at δ 6.02 and 6.98, respectively, as part
of an AMX2 spin-system with the C-5 methylene group (δ
23.4), which is also coupled to H6 resonating at δ 4.52. The
coupling constants between the methylene protons at C-5 and
H6 (J5ax‑6 = 12.3; J5eq‑6 = 3.8) indicated the pseudoequatorial
orientation of the side chain.4,7 The heptyl side chain was
identified through the C-7′ terminal methyl group (δ 1.60) as
part of an AX3 spin-system with the downshifted methine signal
at C-6′ (δ 5.30), corroborating the substitution of this
Figure 2. X-ray ORTEP drawing of brevipolide I (9).
geometry adopted by this brevipolide, intermolecular hydrogen
bonds (2.06 Å) between the oxygen of the lactone group and
the hydroxy moiety at C-1′ were observed in the crystal packing
(Figure S24, Supporting Information). To ascertain the
absolute configuration of compounds 8 and 9, catalytic
hydrogenation using Pd/C was carried out to reduced both
double bonds, yielding a convergent tetrahydro derivative (11),
followed by application of the Mosher’s ester protocol15
involving its free C-1′ hydroxy group. The hydrogenation
product derived from either 8 or 9 displayed identical physical
3
stereogenic center by an ester group. The observed JCH
correlation of the terminal methyl group with the carbonyl
carbon at δ 206.7 permitted placement of the carbonyl group at
C-5′. The multiplicities of the signals in the upfield region at δ
2.28 (H-4′), 1.60 (H-2′), 1.34 (H-3′proS), and 1.12 (H-3′proR
)
CH
2,3
were attributed to the presence of a cyclopropane ring.
J
correlations from H-6 to H-1′ (δ 3.70) and H-2′ indicated that
the lactone and cyclopropane moieties were connected through
a secondary hydroxy group at C-1′ (δ 71.8). The characteristic
signals for an isoferuloyl group, as the C-6′ substituent, were
also observed (Table 1).
X-ray diffraction analysis was undertaken to establish the
relative configuration of 9 (Figure 2). The crystal parameters
and X-ray coordinates are included in Tables S2−S6
(Supporting Information). Because of the bent U-shaped
1
data and H and 13C NMR spectra (Figures S16 and S17,
Supporting Information), enabling us to assume that the
absolute configurations for all stereogenic centers of both
compounds were the same. The tetrahydro derivative 12 was
similarly prepared from 10. The chemical shift difference values
(Table 2) obtained by comparing the relevant 1H NMR data of
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1
Table 2. H NMR Chemical Shift Data for Diagnostic Signals from the (S)- and (R)-Ester Derivatives of Hydrogenated
Derivative 11
a
proton chemical shifts (ΔδH = δS − δR)
MTPA-ester
H-5ax
1.60
ΔδH
H-5eq
2.19
ΔδH
H-6
4.46
ΔδH
H-2′
ΔδH
H-3′proR
ΔδH
H-3′proS
ΔδH
C-1′ config
S
1.57
0.80
1.10
+0.12
+0.19
+0.09
−0.11
−0.10
−0.03
S
R
1.48
2.00
4.37
1.68
0.90
1.13
a
Data registered in CDCl3 at 300 MHz.
the R- and S-MTPA esters (Figure S22, Supporting
Information) of compound 11 indicated the absolute
configuration of C-1′ to be S. Therefore, the absolute
configuration for C-6, C-1′, C-2′, C-4′, and particularly the
previously unassigned C-6′10,11 was confirmed as 6R, 1′S, 2′S,
4′S, and 6′S according to the relative configuration obtained
from X-ray diffraction analysis of 9 (Figure 2). Additionally, in
order to validate that all stereogenic centers at the 6-heptyl-5,6-
dihydro-2H-pyran-2-one core of brevipolides were identical,
saponification of compounds 11 and 12 was conducted,
providing the same optically active functionalized dodecanoic
acid methyl ester 13.
kcal mol−1, offering evidence for the flexibility of this
molecule.12,13 The magnetic shielding tensors were calculated
with the gauge-including atomic orbital method, followed by
theoretical calculation of the NMR spin−spin coupling
constants at the B3LYP/DGDZVP level. These values were
Boltzmann-averaged to yield DFT-calculated coupling con-
stants according to protocols previously described.12,13 The
theoretical values showed good correlation with the exper-
imentally registered JH−H for 9 (Table 3), reflecting the
conformational behavior of the brevipolide core in solution.
The solid-state conformation (Figure 2) is different from all the
minimum energy conformers (Figure 3), perhaps as a result of
the replacement of the intermolecular hydrogen bonding
(Figure S24, Supporting Information) with a C1−O1···H−
O1′−C1′ intramolecular bond. The release of the crystal-packing
constraints promotes the presence of multiple conformational
arrangements that fulfill the entropic requirements for this
flexible system as predicted by DFT modeling (Table 3). Also,
the most relevant conformers 9a−9f, accounting for 88% of the
conformational population, were useful to rationalize the
observed NOESY correlations as shown in Figure 3. The
global minimum 9a is responsible for the observed correlations
between H6−H1′, H1′−H3′proR, and H4′−H6′, while the second
minimum 9b produces the complementary interactions
between H5eq−H2′ and H1′−H4′. The subsequent conformers
also contribute to enhance the above-mentioned effects. The
NMR features are consistent along the brevipolide series,
indicating that the conformational behavior in solution for the
6-heptyl-5,6-dihydro-2H-pyran-2-one core in compounds 1−10
Chiroptical measurements of 7−10 were undertaken to
explore the electronic circular dichroism (ECD) spectra of the
brevipolide series. A positive n→π* Cotton effect for the α,β-
unsaturated δ-lactone was observed for the four compounds
centered at λmax 259−264 nm, confirming that the stereogenic
center at C-6 is R (Figure S23, Supporting Information), as it
has been described in all 6-substituted 5,6-dihydro-α-pyrones
from the mint familiy.4 ECD for the chiral trans-cinnamoyl
derivatives 7, 8, and 10 exhibited a positive Cotton effect
centered around 300 nm, in contrast with the cis-compound 9,
which showed a negative Cotton effect at 319 nm (Δε = −1.3).
It is important to note that this effect was not previously
reported10,11 because of the fact that brevipolides were isolated
as mixtures in different degrees of both geometric isomers
where the positive Cotton effect for the trans-isomer over-
lapped the negative one for the cis-compound.
A molecular model for compound 9 was generated to
correlate the 3D structure with its spectroscopic and chiroptical
properties. The starting geometry was modeled by taking into
account the configuration and conformation of the 6-heptyl-
5,6-dihydro-2H-pyran-2-one framework from the X-ray dif-
fraction data (Figure 2). A systematic search afforded 108
conformers even though several conformational arrangements
were discarded because of the presence of hindering steric
effects, partial atomic overlapping, or a high MMFF16 energy.
The 64 contributing conformers were geometrically optimized
at the B3LYP/DGDZVP level.17 Table S2 lists the relative free
energies as well as the Boltzmann distribution for the most
relevant conformers within a ΔG° range of between 0 and 3.3
3
is essentially the same, e.g., JHH (Table S1, Supporting
Information) and observed NOEs for the new compound 10
(Figure S15, Supporting Information).
The molecular models for the most stable conformers of 9
(Figure 3) were also useful to provide validation for the
observed lowest energy negative Cotton effect in the ECD
spectra of cis-isomer 9. TDDFT calculations18 were carried out
to corroborate the sign for the n→π* transition corresponding
to the chirally substituted 4-methoxy-cis-cinnamoyl moiety.19
Negative values for the main conformers 9a−9f were obtained
ranging from λ = 316 to 324 nm with Rvelocity between −1.3 and
−136.9 × 10−40 erg·esu·cm·Gauss−1 (Table 4), in agreement
with the experimental value (Figure S23, Supporting
Information). Additionally, the same theoretical protocol was
applied to calculate the n→π* transitions for the hypothetical
C6′-(R)-epimer of brevipolide I. The main conformers of this
structure (C6′R-9a to C6′R-9a) showed positive values for the
lowest energy transition, within the range from λ = 313 to 328
nm with Rvelocity between 42.3 and 128.1 × 10−40
erg·esu·cm·Gauss−1 (Table 4).
In conclusion, the present investigation exemplified a
protocol for stereochemical analysis by application of a
combined theoretical and experimental methodology.13 The
absolute configuration and conformation of the brevipolide
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Journal of Natural Products
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a
b
Table 3. DFT B3LYP/DGDZVP Relative Free Energies, Population, and Comparison between DFT and Experimental
c
1H−1H Coupling Constants for the 20 Lowest Energy Conformers of Brevipolide I (9)
a
b
conformer
ΔG
P
J3−4
J4−5eq
J4−5ax
J5eq‑6
J5ax‑6
J6−1′
J1′‑2′
J2′‑3′proS
J2′‑3′proR
J2′‑4′
J3′proS‑4′
J3′proR‑4′
9a
9b
9c
9d
9e
9f
0.000
0.100
0.307
0.671
1.029
1.060
1.349
1.474
1.669
1.682
2.184
2.334
2.391
2.397
2.566
2.610
2.679
2.698
2.905
3.545
weighted
28.2
23.8
16.8
9.1
5.0
4.7
2.9
2.3
1.7
1.6
0.7
0.5
0.5
0.5
0.4
0.3
0.3
0.3
0.2
0.1
9.75
9.81
9.74
9.80
9.81
9.79
9.79
9.79
9.73
9.81
9.82
9.80
9.78
9.80
9.80
9.81
9.82
9.81
9.82
9.73
9.78
7.16
6.95
7.14
6.85
6.73
6.97
6.97
6.83
6.71
6.96
6.82
6.81
6.88
6.81
6.82
6.82
6.79
6.81
7.00
6.92
7.01
2.41
2.34
2.42
2.27
2.31
2.36
2.36
2.26
2.29
2.35
2.30
2.26
2.36
2.26
2.26
2.27
2.30
2.26
2.45
2.30
2.36
3.94
3.98
4.00
3.67
3.56
4.01
4.01
3.69
3.55
3.97
3.39
3.82
3.90
3.83
3.78
3.82
3.46
3.88
3.16
3.59
3.90
12.82
12.62
12.81
12.35
13.65
12.72
12.72
12.36
14.05
12.61
13.75
12.38
12.73
12.38
12.40
12.39
13.78
12.39
13.87
12.96
12.77
2.54
2.74
2.47
8.65
8.04
2.63
2.66
8.60
2.45
2.63
2.94
8.78
1.44
8.76
8.74
8.75
2.89
8.77
3.89
1.21
3.68
1.20
10.38
10.27
9.36
1.12
9.24
8.99
9.55
9.81
10.53
1.63
2.19
10.81
2.20
2.35
2.40
1.52
2.02
2.90
11.18
6.83
9.70
9.02
9.71
9.68
8.88
9.59
9.59
9.54
8.99
8.98
9.51
9.53
9.10
9.54
9.52
9.50
9.51
9.56
9.55
9.12
9.45
6.26
6.61
6.07
6.08
6.28
6.02
6.04
5.74
5.54
5.52
5.90
6.21
5.84
6.22
6.18
6.17
5.90
6.23
5.60
5.18
6.23
3.19
2.91
3.19
2.68
3.23
3.81
3.86
3.10
3.17
2.70
3.94
3.75
3.06
3.74
3.66
3.68
3.86
3.70
3.25
3.34
3.13
3.92
3.99
3.93
4.71
3.55
3.43
3.43
3.66
5.21
3.87
3.58
3.96
3.46
3.98
4.13
4.10
3.60
3.97
4.46
3.80
3.97
8.23
8.81
8.20
7.67
8.73
9.77
9.87
9.41
7.34
8.12
9.57
8.90
9.15
8.87
8.70
8.73
9.49
8.86
7.76
9.44
8.50
9g
9h
9i
9j
9k
9l
9m
9n
9o
9p
9q
9r
9s
9t
cd
,
values
experimental
9.70
6.50
2.20
3.80
12.30
3.80
6.30
8.90
6.00
3.40
3.90
8.10
e
values
a
b
c
In kcal/mol. In percent from ΔG° values at 298 K and 1 atm, Gglobal = −1379.11433 kcal/mol. In Hz calculated from the B3LYP/DGDZVP
d
e
structures. ∑i Ji × Pi, where Ji is the coupling constant value for each conformer and Pi is the population for the ith conformation. Couplings
obtained by spectral simulation.
Table 4. Calculated Energy, Wavelength, and Oscillator and
Rotatory Strengths for the Lowest Energy n→π* Transition
of Brevipolide I (9) and Its Hypothetical C6′-(R)-Epimer at
the B3LYP/DGDZVP Level of Theory
a
b
c
d
e
f
conformer (P)
ΔE
λmax
f
Rvelocity
Rlength
9a (28.2)
9b (23.8)
9c (16.8)
9d (9.1)
9e (5.0)
9f (4.7)
3.929
3.899
3.886
3.827
3.906
3.896
3.915
3.774
3.942
3.859
3.920
3.966
316
318
319
324
317
318
317
328
315
321
316
313
0.662
0.661
0.572
0.565
0.660
0.670
0.692
0.577
0.667
0.573
0.696
0.748
−1.26
−136.87
−4.55
−88.65
−108.73
−127.95
82.15
−2.30
−134.59
−4.03
−89.75
−107.05
−126.58
79.61
C6′R-9a (39.2)
C6′R-9b (29.5)
C6′R-9c (11.5)
C6′R-9d (8.0)
C6′R-9e (3.6)
C6′R-9f (2.7)
128.06
60.00
127.54
57.20
123.14
55.14
119.62
55.82
42.27
42.77
a
b
e
Conformational population in percentage is given in parentheses.
c
d
Excitation energy in eV. Wavelength in nm. Oscillator strength.
Rotatory strength in velocity form (×10−40 erg·esu·cm·Gauss−1).
Rotatory strength in length form (×10−40 erg·esu·cm·Gauss−1).
f
series were established by the systematic comparison between
theoretical and experimental vicinal 1H NMR coupling
constants and DFT ECD calculations. These results provided
support for the (6′S)-configuration in this class of bioactive
compounds from the mint family.
Figure 3. The most relevant conformers of brevipolide I (9)
accounting for 88% of the conformational population. NOESY
correlations are indicated with arrows in the global minimum (9a)
and the second lowest energy minimum (9b).
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were
determined on a Fisher-Johns apparatus and are uncorrected. Optical
■
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rotations were measured with a Perkin-Elmer model 341 polarimeter.
1H (400 and 300 MHz) and 13C (100 and 75 MHz) NMR
experiments were registered on a Varian Inova instrument. Positive-ion
HRFABMS were recorded using a matrix of 3-nitrobenzyl alcohol on a
Thermo DFS spectrometer. Analytical and semipreparative HPLC
analysis were carried out on Waters equipment (Millipore Corp.,
Waters Chromatography Division, Milford, MA, USA), which was
composed of a 600E multisolvent delivery system equipped with a 996
photodiode array detector. Empower 2 software was used to control
equipment and for data acquisition, processing, and management of
the chromatographic information.
see Table S1 (Supporting Information); HRFABMS m/z 401.1595 [M
+ H]+ (calcd for C22H25O7, 401.1522).
Brevipolide I (9): colorless prisms; mp 124−125 °C; ORD (c 0.15,
CHCl3) [α]589 +36,[α]578 +37.3, [α]546 +42, [α]436 +59; ECD (c 2.2 ×
10−5 M, MeCN) λmax (Δε) 229 (−0.91), 264 (+2.20), 319 (−1.26);
1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz,CDCl3) data
(Figures S7 and S8, Supporting Information), see Table S1
(Supporting Information); HRFABMS m/z 401.1595 [M + H]+
(calcd for C22H25O7, 401.1522).
Brevipolide J. (10): colorless oil; ORD (c 0.17, CHCl3) [α]589
+147.0, [α]578 +156.5, [α]546 +183.5, [α]436 +392.9; ECD (c 8.6 ×
10−5 M, MeCN) λmax (Δε) 229 (−4.11), 262 (+6.11), 294 (+5.83);
1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz,CDCl3) data,
see Table 1 (Figures S9 and S10, Supporting Information);
HRFABMS m/z 416.1467 [M + H]+ (calcd for C22H25O7, 416.1471).
Methylation of Compound 7. A MeOH solution of 7 (3 mg/100
μL) was treated with CH2N2 in Et2O (1 mL) at 0 °C. The solution
was monitored over 1 h by TLC until reaction completion and
dissipation of the yellow color. The solvent was evaporated under
reduced pressure inside a fume-hood to afford a product that was
identical to compound 8 (3 mg).
Hydrogenation of Compounds 8−10. A solution of compounds
8 and 9 (15 mg each) in THF was treated with Pd/C (5% mol,
0.045g) under a N2 atmosphere during 15 min. The solution was
transferred to a 45 mL stainless steel Parr reactor and pressurized with
H2 (200 psi) at room temperature during 24 h.20 Then, the excess of
gas was released. The residue was filtered, concentrated under vacuum,
and purified by HPLC on a Symmetry C18 column (Waters; 7 μm, 19
× 300 mm) with an isocratic elution of MeCN−H2O and a flow rate
of 9.0 mL/min (sample injection, 500 μL; concentration, 0.06 mg/
μL). These procedures afforded pure compound 11 (25 mg) as the
only reaction product. The same process was individually applied to
each pure natural product 8 and 9 (3 mg) to afford a convergent
derivative (2.5 mg) that was chromatographically (HLPC coelution
experiments, tR 30.68 min) and spectroscopically (NMR) identical
with compound 11. Compound 10 (5 mg) was also subjected to the
same hydrogenation protocol to generate the tetrahydro derivative 12.
Tetrahydro derivative 11: colorless oil; ORD (c 0.36, CHCl3)
[α]589 +46.4, [α]578 +48.3, [α]546 +56.7, [α]436 +115.8, [α]365 +242.8;
1H NMR (300 MHz, CDC13) δ 7.11 (d, J = 8.7 Hz, H-2″ and H-6″),
6.82 (d, J = 8.7 Hz, H-3″ and H-5″), 5.16 (q, J = 7.1 Hz, H-6′), 4.39
(ddd, J = 10.8, 3.2, 3.2 Hz, H-6), 3.78 (s, OCH3), 3.54 (dd, J = 6.1, 3.2
Hz, H-1′), 2.90 (t, J = 7.4 Hz, H-8″), 2.68 (t, J = 7.4 Hz, H-7″), 2.67
(m, H-3ax), 2.47 (m, H-3eq), 2.10 (ddd, J = 8.5, 3.9, 3.4 Hz, H-4′),
1.98−1.75 (4m, H-4ax, H-4eq, H-5ax, and H-5eq), 1.54 (dddd, J = 8.9,
6.3, 6.3, 3.4 Hz, H-2′), 1.43 (d, J = 7.1 Hz, Me-7′), 1.27 (ddd, J = 8.5,
4.0, 3.9 Hz, H-3′proS), 1.02 (ddd, J = 8.9, 6.3, 4.0 Hz, H-3′proR) (Figure
S16, Supporting Information); 13C NMR (75 MHz, CDC13) δ 206.5
(C-5′), 173.1 (C-9″), 171.7 (C-2), 158.1 (C-4″), 132.3 (C-1″), 129.3
(C-2″ and C-6″), 113.9 (C-3″ and C-5″), 83.3 (C-6), 75.2 (C-6′), 72.5
(C-1′), 55.3 (OCH3), 35.8 (C-8″), 29.9 (C-7″), 29.8 (C-3), 26.4 (C-
2′), 21.6 (C-5), 21.3(C-4′), 18.4 (C-4), 15.6 (C-7′), 14.3 (C-3′)
(Figure S17, Supporting Information); HRFABMS m/z 405.1875 [M
+ H]+ (calcd for C22H29O7, 405.1908).
Plant Material. Aerial parts of Hyptis brevipes were collected in
́
Dos Rıos, Municipio de Emiliano Zapata, Veracruz, Mexico, in
November 2009. The plant material was identified by A.H.-R., and a
voucher specimen has been deposited in the herbarium of the Instituto
́
de Ecologıa, Xalapa, Veracruz, Mexico (accession number
XAL0000247). Also, a voucher specimen was archived at the Botanical
Collection of Facultad de Ciencias, Universidad Nacional Auton
́
oma
de Mexico (voucher 127321).
́
Extraction and Isolation. Aerial parts (2 kg) were powdered and
extracted exhaustively by maceration at room temperature with CHCl3
to afford, after removal of the solvent, a dark brown syrup (50 g). The
extract (50 g) was fractionated by open column chromatography over
silica gel (1 kg), using a gradient of hexanes−CH2Cl2, followed by
CH2Cl2−acetone and acetone−MeOH in several proportions.
Altogether, 100 eluates (300 mL each one) were collected and
combined in 20 fractions. Fraction 12 (2.3 g, eluted with CH2Cl2−
acetone, 4:1) was fractionated by passage on silica gel (230−400
mesh) and using a gradient of increasing polarity of hexanes−EtOAc,
EtOAc−acetone, and acetone−MeOH. A total of 10 pooled
subfractions were collected. Subfraction VII (200 mg, eluted with
EtOAc−acetone, 9:1) was resolved by HPLC on a Symmetry C18
column (Waters; 7 μm, 19 × 300 mm) with an isocratic elution of
MeCN−H2O (2:3) and a flow rate of 7.5 mL/min (sample injection,
500 μL; concentration, 0.1 mg/μL). Eluates across the peaks with tR
values of 37.60 min (peak I) and 40.37 min (peak II) were collected by
the technique of heart cutting and independently reinjected in the
apparatus operating in the recycle mode to achieve total homogeneity
after 5−10 consecutive cycles and employing the same reversed-phase
column and the instrumental conditions as described above. These
techniques afforded pure compounds 8 (30 mg) from peak I and 9 (15
mg) from peak II. Compound 9 was recrystallized from hexanes−
CH2Cl2 (4:1).
Fraction 15 (9.0 g, eluted with CH2Cl2−acetone, 7:3) was
submitted to column chromatography on silica gel (230−400 mesh)
using a gradient of increasing polarity of hexanes−EtOAc, EtOAc−
acetone, and acetone−MeOH. In total, eigth pooled subfractions were
collected. Subfraction VI (100 mg, eluted with EtOAc−acetone) was
resolved by HPLC on a Symmetry C18 column (Waters; 7 μm, 19 ×
300 mm) with an isocratic elution of MeCN−H2O (7:3) and a flow
rate of 9.0 mL/min (sample injection, 500 μL; concentration, 0.1 mg/
μL). Eluates across the peaks with tR values of 31.04 min (peak III)
and 34.79 min (peak IV) were collected by the technique of heart
cutting and independently reinjected (sample injection, 500 μL;
concentration, 0.1 mg/μL) in the apparatus operating in the recycle
mode to achieve total homogeneity after 10 cycles. These techniques
afforded pure compound 7 (15 mg) from peak III and compound 10
(12 mg) from peak IV.
Brevipolide G (7): colorless oil; ORD (c 0.08, CHCl3) [α]589
+138.7, [α]578 +142.5, [α]546 +167.5, [α]436 +361.2; ECD (c 5.5 ×
10−5 M, MeCN) λmax (Δε) 230 (−2.99), 260 (+4.22), 301 (+4.02);
1H NMR (300 MHz, CDCl3) and 13C NMR (75 MHz, CDCl3) data
(Figures S3 and S4, Supporting Information), see Table S1
(Supporting Information); HRFABMS m/z 387.1455 [M + H]+
(calcd for C22H23O7, 387.1438).
Tetrahydro Derivative 12: colorless oil; ORD (c 0.12, CHCl3)
[α]589 +39.2, [α]578 +40.0, [α]546 +46.7, [α]436 +95.0, [α]365 +190.8;
1H NMR (300 MHz, CDC13) δ 7.06 (d, J = 1.9 Hz, H-2″), 6.80 (d, J =
8.2 Hz, H-5″), 6.71 (dd, J = 8.2, 1.9 Hz, H-6″), 5.20 (q, J = 7.1 Hz, H-
6′), 4.42 (ddd, J = 10.7, 3.1, 3.1 Hz, H-6), 3.89 (s, OCH3), 3.54 (dd, J
= 6.3, 3.3 Hz, H-1′), 2.90 (t, J = 7.1 Hz, H-8″), 2.71 (t, J = 7.1 Hz, H-
7″), 2.70−2.45 (2m, H-3ax and H-3eq), 2.13 (ddd, J = 8.5, 3.9, 3.4 Hz,
H-4′), 2.10−1.80 (4m, H-4ax, H-4eq, H-5ax, and H-5eq), 1.57 (dddd,
J = 8.9, 6.3, 6.3, 3.4 Hz, H-2′), 1.47 (d, J = 7.1 Hz, H-7′), 1.32 (ddd, J
= 8.5, 4.0, 3.9 H-3′proS), 1.04 (ddd, J = 8.9, 6.3, 4.0 Hz, H-3′proR
)
(Figure S18, Supporting Information); 13C NMR (75 MHz, CDC13) δ
206.6 (C-5′), 172.5 (C-9″), 171.5 (C-2), 145.5 (C-3″) 145.1 (C-4″),
133.6 (C-1″), 119.7 (C-6″), 114.6 (C-2″), 110.8 (C-5″), 83.2 (C-6),
75.3 (C-6′), 72.6 (C-1′), 56.0 (OCH3), 35.6 (C-8″), 30.1 (C-7″), 29.8
(C-3), 26.5 (C-2′), 21.6 (C-5), 21.3 (C-4′), 18.4 (C-4), 15.9 (C-7′),
Brevipolide H (8): colorless solid; mp 114−116 °C; ORD (c 0.24,
CHCl3) [α]589 +157.5, [α]578 +165, [α]546 +195, [α]436 +421, [α]365
+1067.9; ECD (c 2.8 × 10−5 M, MeCN) λmax (Δε) 230 (−3.71), 259
1
(+3.83), 305 (+8.54); H NMR (400 MHz, CDCl3) and 13C NMR
(100 MHz,CDCl3) data (Figures S5 and S6, Supporting Information),
76
dx.doi.org/10.1021/np300740h | J. Nat. Prod. 2013, 76, 72−78

Journal of Natural Products
Article
14.3 (C-3′) (Figure S19, Supporting Information); HRFABMS m/z
420.1779 [M ]+ (calcd for C22H28O8, 420.1784).
Crystallographic Data Centre (CCDC 914932). Copies of the data can
be obtained free of charge on application to the Director, CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0)1223-336033 or
e-mail: deposit@ccdc.cam.ac.uk).
Preparation of R- and S-MTPA Ester Derivatives of 11. A
solution of compound 11 (3.0 mg) in CDCl3 (0.75 mL) was
transferred into a clean NMR tube containing DMPA (0.5 mg) in
pyridine-d5 (0.10 mL). (S)- or (R)-α-Methoxy-α-(trifluoromethyl)-
phenylacetyl chloride (S- or R-MTPA-Cl) (10 μL) was immediately
added into the NMR tube under a N2 stream.21 The 1H NMR
spectrum of the reaction mixture for each derivative (S- or R-MTPA-
ester) was obtained directly from the reaction tube, which was
permitted to stand at room temperature and monitored every 1 h. The
reaction was found to be completed after 4 h.
Molecular Modeling Calculations. Molecular building and
conformational search for isomers 8 and 9 and the corresponding
C6′-(R)-epimer were carried out in the Spartan’04 program23 using the
MMFF94 force-field calculation on a Windows operating system
machine. A systematic search protocol was performed in which the
torsion angles C(5)−C(6)−C(1′)−C(2′), C(1′)−C(2′)−C(4′)−
C(5′), and C(2′)−C(4′)−C(5′)−C(6′) in the side chain were varied
by 120°, starting at 60° for each central bond, and the torsion angles of
the C(4′)−C(5′)−C(6′)−C(7′) dihedral angles were rotated in steps
of 180°. The fragment of cinnamic acid was restricted to its most
stable conformation, generating a total of 108 initial conformers for
each stereoisomer. The cinnamoyl moiety began at H−Csp3−O−Csp2
and Csp3−O−CO dihedral angles ca. 0°, and conformational
explorations for this group were achieved within dihedral angle ranges
of +60° and −60°. All structures were minimized to a rmsd gradient of
1 × 10−6 kcal/mol on the potential energy surface. An energy cutoff of
10 kcal/mol was selected in order to have a wide frame of conformers
in the Boltzmann distribution. All structures inside the cutoff window
were geometrically optimized using the hybrid DFT method B3LYP
and basis set DGDZVP (B3LYP/DGDZVP). The optimized
structures were used to calculate the thermochemical parameters
estimated at 298 K and 1 atm. Magnetic shielding tensors were
calculated with the gauge-invariant atomic orbital (GIAO) method.
Total NMR spin−spin coupling constants (SSCC, J (Hz)) were
calculated as the summation of the Fermi contact, diamagnetic spin−
orbit, spin-dipolar, and paramagnetic spin−orbit, which were
calculated from B3LYP/DGDZVP-optimized structures by using the
spin−spin option during the NMR jobs. All quantum mechanical
NMR calculations were carried out using the Gaussian 03 program on
a Linux operating system in the KanBalam cluster, which includes
1368 AMD Opteron processors at 2.6 GHz and a RAM memory of 3
terabytes. For each job, a maximum of four processors was used and
each conformer required four different DFT jobs: geometric
optimizations, frequency calculations, SSCC estimations, and TD
ECD calculations. The total cpu time consumed in this work was 1.33
× 103 h. The free energy equation (ΔG = −RT ln K) was used to
obtain the conformational population, taking into account a cyclic
equilibrium at 298 K between the selected conformers of 9 within a
0.0−3.3 kcal/mol window with respect to the global minimum. The
free energy values ΔG° were obtained from the vibrational frequency
calculations as the sum of electronic and thermal free energies.
Cytotoxicity Assay. Nasopharyngeal (KB), laryngeal (Hep-2),
colon (HCT-15), cervix (HeLa), breast (MCF-7),24 and prostate
carcinoma (PC-3) cell lines were maintained in RPMI 1640 medium
supplemented with 10% fetal bovine serum and were cultured at 37 °C
in an atmosphere of 5% CO2 in air (100% humidity). The cytotoxicity
was determined using the SRB assay.25 The cells were harvested at log
phase of their growth cycle, treated in triplicate with various
concentrations of the test samples (0.16−20 μg/mL), and incubated
for 72 h at 37 °C as described above. Results are expressed as the
concentration that inhibits 50% control growth after the incubation
period (IC50). The values were estimated from a semilog plot of the
drug concentration (μg/mL) against the percentage of growth
inhibition. Vinblastine was included as a positive control drug.
1
R-MTPA ester of 11: H NMR δ 7.03 (d, J = 8.6 Hz, H-2″ and H-
6″), 6.73 (d, J = 8.6 Hz, H-3″ and H-5″), 5.00 (q, J = 7.1, H-6′), 4.70
(dd, J = 9.5, 2.9 Hz, H-1′), 4.37 (ddd, J = 11.0, 3.2, 3.2 Hz, H-6), 2.80
(t, J = 7.4 Hz, H-8″), 2.58 (m, H-3ax), 2.55 (t, J = 7.4 Hz, H-7″), 2.37
(ddd, J = 14.0, 4.0, 4.0 Hz, H-3eq), 2.18 (ddd, J = 8.5, 3.9, 3.4 Hz, H-
4′), 2.00 (ddd, J = 14.0, 10.0, 6.0 Hz, H-5eq), 1.83−1.60 (2m, H-4ax
and H-4eq), 1.68 (m, H-2′), 1.48 (dddd, J = 14.0, 12.0, 12.0, 3.0 Hz,
H-5ax), 1.25 (d, J = 7.1 Hz, Me-7′), 1.13 (ddd, J = 8.5, 4.0, 3.9 Hz, H-
3′proS), 0.90 (ddd, J = 8.9, 6.3, 4.0 Hz, H-3′proR) (Figure S22,
Supporting Information).
1
S-MTPA ester of 11: H NMR δ 7.03 (d, J = 8.6 Hz, H-2″ and H-
6″), 6.72 (d, J = 8.6 Hz, H-3″ and H-5″), 5.00 (q, J = 7.1 Hz, H-6′),
4.77 (dd, J = 8.8, 2.6 H-1′), 4.46 (ddd, J = 11.0, 3.1, 3.1 Hz, H-6), 2.82
(t, J = 7.4 Hz, H-8″), 2.62 (m, H-3ax), 2.62 (t, J = 7.4 Hz, H-7″), 2.48
(ddd, J = 14.0, 4.0, 4.0 Hz, H-3eq), 2.19 (ddd, J = 14.0, 10.0, 6.0 Hz,
H-5eq), 2.17 (ddd, J = 8.5, 3.9, 3.4 Hz, H-4′), 1.90−1.60 (2m, H-4ax
and H-4eq), 1.60 (dddd, J = 14.0, 12.0, 12.0, 3.0 Hz, H-5ax), 1.57 (m,
H-2′), 1.26 (d, J = 7.1 Hz, Me-7′), 1.10 (ddd, J = 8.5, 4.0, 3.9 Hz, H-
3′proS), 0.80 (ddd, J = 8.9, 6.3, 4.0 Hz, H-3′proR) (Figure S22,
Supporting Information).
Hydrolysis of Compounds 11 and 12. Compound 11 (21.7 mg)
was dissolved in MeOH (400 μL), and NaOMe (3.6 mg) was added.
The reaction mixture was stirred at room temperature for 1 h.
Saturated aqueous NH4Cl solution was added, and the mixture was
diluted with CH2Cl2.22 The organic layer was dried, filtered, and
concentrated under reduced pressure. The residue was purified by
HPLC on a Symmetry C18 column (Waters; 5 μm, 4.6 × 250 mm)
with an isocratic elution of MeOH−MeCN (7:3) and a flow rate of 0.3
mL/min (sample injection, 20 μL; concentration, 0.05 mg/μL) to
afford pure compound 13 (6 mg). Derivative 12 was also hydrolyzed,
and the reaction residue was purified using the same procedures as
described above to yield compound 13.
Compound 13: colorless oil; ORD (c 0.07, CHCl3) [α]589 +88.6,
1
[α]578 +92.9, [α]546 +107.1, [α]436 +207.1, [α]365 +412.9; H NMR
(300 MHz, CDC13) δ 4.49 (q, J = 7.0 Hz, H-11), 3.74 (m, H-5), 3.71
(s, OCH3), 3.24 (dd, J = 7.4, 3.5 Hz, H-6), 2.42 (t, J = 7.1 Hz, H-2a
and H-2b), 2.13 (ddd, J = 8.5, 3.9, 3.4 Hz, H-9), 1.87 (m, H-3a), 1.74
(2m, H-3b and H-7), 1.59 (2m, H-4a and H-4b), 1.47 (d, J = 7.0 Hz,
Me-12), 1.37 (ddd, J = 8.5, 4.0, 3.9 H-8proS), 1.10 (ddd, J = 8.9, 6.3, 4.0
Hz, H-8proR) (Figure S20, Supporting Information); 13C NMR (75
MHz, CDC13) δ 211.9.5 (C-10), 174.4 (C-1), 75.6 (C-6), 73.9 (C-5),
73.1 (C-11), 51.7 (OCH3), 33.6 (C-2), 31.1 (C-4), 26.9 (C-7), 21.4
(C-9), 21.0 (C-3), 19.8 (C-12), 15.5 (C-8) (Figure S21, Supporting
Information); EIMS m/z 256 [M − H2O]+ (1), 230 (4), 197 (11), 179
(19), 151 (21), 132 (28), 131 (44), 100 (55), 99 (100), 97 (32), 95
(16), 87 (29), 83 (27), 71 (46), 55 (48); FABMS m/z 297 [M + Na]+.
Single-Crystal X-ray Diffraction Analysis of 9. Single-crystal X-
ray diffraction analysis was collected on a Bruker SMART APEX CCD
diffractometer with graphite-monochromated Mo Kα radiation (λ =
0.71073 Å). Crystal data for 9 were C22H24O7, M = 400.41,
monoclinic, space group P21, a = 8.9649(12) Å, b = 7.7800(11) Å, c
= 14.731(2) Å, β = 98.250(2)°, V = 1016.9(1) Å3, Z = 2, ρ = 1.31 mg/
mm3, λ(Mo Kα) = 0.71073 Å, total reflections = 7442, independent
reflections 2617 (Rint 0.04%), final R indices [I > 2σ(I)], R1 = 4.20%,
wR2 = 5.25%. For the structural refinement, the non-hydrogen atoms
were treated anisotropically, and the hydrogen atoms included in the
structure factor calculations were refined isotropically. Crystallographic
data reported in this paper have been deposited with the Cambridge
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds 7−13 and DEPTQ-
135 and 2D NMR spectra including H−1H COSY, HSQC,
1
HMBC, and NOESY of compound 10. Crystal parameters and
X-ray coordinates of 9. CD spectra for 7−10. Table of
cytotoxicity for 7−10. This material is available free of charge
via the Internet at http://pubs.acs.org.
77
dx.doi.org/10.1021/np300740h | J. Nat. Prod. 2013, 76, 72−78

Journal of Natural Products
Article
(12) Mendoza-Espinoza, J. A.; Lop
́
ez-Vallejo, F.; Fragoso-Serrano,
AUTHOR INFORMATION
■
M.; Pereda-Miranda, R.; Cerda-García-Rojas, C. M. J. Nat. Prod. 2009,
72, 700−708.
Corresponding Author
*Tel: +52 55 5622-5288. Fax: +52 55 5622 5329. E-mail:
pereda@unam.mx.
(13) Lop
Hernan
R. J. Org. Chem. 2011, 76, 6057−6066.
(14) Pereda-Miranda, R.; Hernandez-Carlos, B. Tetrahedron 2002,
́ ́
ez-Vallejo, F.; Fragoso-Serrano, M.; Suarez-Ortiz, G. A.;
dez-Rojas, A. C.; Cerda-García-Rojas, C. M.; Pereda-Miranda,
́
Notes
́
The authors declare no competing financial interest.
58, 3145−3154.
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ACKNOWLEDGMENTS
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Quinoa, E.; Riguera, R. Chem. Rev. 2004, 104, 17−117.
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Financial support was provided by Consejo Nacional de
(16) (a) Halgren, T. J. Comput. Chem. 1996, 17, 490−519.
(b) Halgren, T. J. Comput. Chem. 1996, 17, 520−552. (c) Halgren,
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́
Ciencia y Tecnologıa (CONACyT, grant 104887). G.A.S.O. is
also grateful to CONACyT for a graduate student scholarship
(226766). Geometry optimizations and coupling constant
calculations were performed in the HP Cluster Platform 4000
(17) (a) Godbout, N.; Salahub, D. R.; Andzelm, J.; Wimmer, E. Can.
J. Chem. 1992, 70, 560−571. (b) Andzelm, J.; Wimmer, E. J. Chem.
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́ ́
(KanBalam) at Departamento de Supercomputo, Direccion
General de Servicios de Com
are due to G. Duarte and M. Guzman
́
puto Academ
́
ico, UNAM. Thanks
(USAI, Facultad de
́
(18) (a) Cerda-García-Rojas, C. M.; García-Gutier
́
rez, H. A.;
́
Quımica, UNAM) for recording of the mass spectra. The
authors also acknowledge Dr. A. Toscano for the X-ray
crystallographic analysis. We also wish to thank Dr. M. Fragoso-
Hernandez-Hernandez, J. D.; Roman-Marín, L. U.; Joseph-Nathan,
́
́
́
P. J. Nat. Prod. 2007, 70, 1167−1172. (b) Ding, Y.; Li, X.-C.; Ferreira,
D. J. Nat. Prod. 2009, 72, 327−335.
(19) Choudhary, M. I.; Naheed, N.; Abbaskhan, A.; Ali, S.; Atta-ur-
Rahman.. Phytochemistry 2009, 70, 1467−1473.
́
Serrano (Facultad de Quımica, UNAM) for HPLC assistance
and cytotoxicity evaluations.
(20) Tolmacheva, N. A.; Gerus, I. I.; Dolovanyuk, V. G.; Kondratov,
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