Labdanes, Withanolides, and Other Constituents from Physalis nicandroides

Article abstract of DOI:10.1021/acs.jnatprod.9b00233

Chemical investigation of the aerial parts (except fruits and calixes) of Physalis nicandroides var. attenuata led to the isolation of a series of new labdane-type diterpenoids, including the closely related compounds 1-3, the labdane glucosides 4 and 5, a mixture of the epimeric alcohols 6 and 7, and one labdanetriol, isolated as its tri-O-acetyl derivative 9. In addition, three new withanolides (14-16) and six known compounds were isolated. The structures of these compounds were elucidated by analysis of their spectroscopic data and chemical transformations, and those of compounds 1, 4, and 16 were confirmed by X-ray diffraction analysis of the natural product (1) and of the corresponding acetyl derivatives 4a and 16a. Fourteen of these compounds were assayed for their in vitro inhibitory activity against yeast α-glucosidase and acetylcholinesterase enzymes. The results were negative in both cases, except for compound 3a that marginally inhibited the activity of acetylcholinesterase with an IC₅₀ value of 64.4 μM.

Full text of DOI:10.1021/acs.jnatprod.9b00233

Article
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
pubs.acs.org/jnp
Labdanes, Withanolides, and Other Constituents from Physalis
nicandroides
Fernando R. Torres, Ana L. Perez-Castorena, Laura Arredondo, Ruben
́ ́
A. Toscano,†
†
†
‡
†
†
,†
Antonio Nieto-Camacho, Mahinda Martínez, and Emma Maldonado*
†
Instituto de Química, Universidad Nacional Auton
́
oma de Mex
́
ico, Circuito Exterior, Ciudad Universitaria, Coyoacan
́
04510, Cd.
Mx, Mexico
‡
Facultad de Ciencias Naturales, Universidad Auton
Queretaro, Mexico
́
oma de Queret
́
aro, Avenida de las Ciencias s/n, Col. Juriquilla, 76230, Qro,
́
*
S Supporting Information
ABSTRACT: Chemical investigation of the aerial parts
except fruits and calixes) of Physalis nicandroides var.
(
attenuata led to the isolation of a series of new labdane-type
diterpenoids, including the closely related compounds 1−3,
the labdane glucosides 4 and 5, a mixture of the epimeric
alcohols 6 and 7, and one labdanetriol, isolated as its tri-O-
acetyl derivative 9. In addition, three new withanolides (14−
16) and six known compounds were isolated. The structures
of these compounds were elucidated by analysis of their
spectroscopic data and chemical transformations, and those of
compounds 1, 4, and 16 were confirmed by X-ray diffraction
analysis of the natural product (1) and of the corresponding
acetyl derivatives 4a and 16a. Fourteen of these compounds were assayed for their in vitro inhibitory activity against yeast α-
glucosidase and acetylcholinesterase enzymes. The results were negative in both cases, except for compound 3a that marginally
inhibited the activity of acetylcholinesterase with an IC50 value of 64.4 μM.
1
0
revision of the literature concerning the Physalis genus
(Solanaceae) showed that the chemistry of these plants is
nicandroides var. attenuata. In continuation of that work, we
now report the isolation and structural elucidation of a series of
11 new labdanes and withanolides and other known
compounds, from the aerial parts of this species, as well as
the results of the evaluation of the α-glucosidase and
acetylcholinesterase inhibitory activities of most of these
compounds. These bioassays were carried out by considering
the use of Physalis species to control the postpandrial blood
A
quite complex and diverse, since although withanolides have
been the constituents most frequently isolated from these
1
−3
plants,
there are species like P. coztomatl in which the co-
occurrence of labdane-type diterpenoids and withanolides was
4
described for the first time or like the well-known producer of
1
,5
withanolides, P. angulata, from which a series of labdanes
14
6
glucose levels of diabetic patients and the proven capacity of
some withanolides to inhibit the activity of acetylcholinester-
were recently isolated. In addition, there are species like P.
7
8
sordida and P. nicandroides whose main metabolites are
labdanes, and no withanolide was detected. Sucrose esters are
another kind of metabolites of Physalis; they are the main
15
ase.
9
,10
RESULTS AND DISCUSSION
constituents of fruits and calyxes of these plants, which also
■
1
1,12
may contain withanolides.
Sterols, flavonoids, and
The MeOH extract of leaves, flowers, and stems of P.
nicandroides var. attenuata was partitioned to afford hexanes
and EtOAc extracts. Each of these extracts was subjected to a
series of column chromatographies to obtain the new labdane-
type diterpenoids 1−7 and 9, together with three new
withanolides (14−16), and several known compounds,
including labdanes, sterols, and flavonoids (Chart 1).
Compound 1 was isolated as colorless crystals with a
molecular formula C H O , determined by the molecular ion
at m/z 306.2560 in the HRFABMS and the C NMR data. Its
1
ceramides have also been described as constituents of Physalis.
In parallel to the advance in the knowledge of the chemistry of
Physalis, different bioassays showed that the isolates from this
genus, mainly the withanolides, possess a variety of
1
−3
bioactivities.
This is not surprising since many of these
investigations have been inspired by the traditional use of some
of these plants to treat a diversity of conditions, including
respiratory and gastrointestinal infections as well as some
chronic degenerative disorders like diabetes and cancer, among
20 34
2
1
3
8
,13
others.
As a part of our studies of the Mexican Physalis we described
the presence of several sucrose esters in the fruits of P.
Received: March 13, 2019
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b00233
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Article
Chart 1
Figure 1. Key COSY, HMBC, and NOESY NMR correlations for compounds 1, 3, 4, 6/7, 14, and 15.
IR spectrum showed absorptions for hydroxy groups (3413,
1a, which showed the corresponding low-field shift of the
NMR signals (Table S1, Supporting Information) of the
−
1
−1
1
13
3
324 cm ) and double bonds (1642 cm ). The H and
C
NMR spectra showed signals for three tertiary (δ 0.88, 0.81
oxymethine (δH 5.14) and oxymethylene protons (δ 4.60).
H
H
and 0.67) and one vinylic (δ 1.70) methyl group; a terminal
The hydroxy groups of compound 1 were located at C-12 and
C-15 by the HMBC cross peaks between the oxymethylene
protons (CH -15) and the olefinic carbons C-13 (δ 142.3)
H
(
δC 149.1, C; δ 4.85 and 4.47, δC 106.5, CH ) and a
H
2
trisubstituted (δ 142.3, C; δH 5.63, δ 123.3, CH) double
C
C
2
C
bond; an oxymethine (δ 4.05 dd, J = 8.5, 3.0 Hz, δ 75.0);
and C-14 (δ 123.3), between the oxymethine proton (CH-
H
C
C
and an oxymethylene (δ 4.22 and 4.19, δ 59.1, CH ). These
12) and C-9 (δ 52.5), C-11 (δ 30.1), C-13, C-14, and C-16
H
C
2
C
C
8(17),13
data were consistent with a Δ
-labdadiene bearing a
primary and a secondary hydroxy group. The presence of both
(δ 12.2), as well as those of H-9 (δ 2.01) with C-8, C-10, C-
C
H
11, C-12, C-17, and C-20, which also confirmed the position of
8
(17)
hydroxy groups was confirmed via the di-O-acetyl derivative
the Δ
-double bond (Figure 1). The E-configuration of the
B
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C-13 double bond was deduced from the NOESY cross peaks
thermore, this correlation also proved that these diterpenoids
belong to the normal labdane series, since compound 3 was
transformed into (−)-pumiloxide (3a), a compound whose
absolute configuration was established by semisynthesis
between H-12 and H-14 as well as those between CH -15 and
2
CH -16 (δH 1.70) (Figure 1). The (12R)-configuration was
3
determined by an X-ray diffraction analysis of 1 (Figure 2), if it
16
starting from manoyl oxide. The identity of 3a was confirmed
by comparison of its specific rotation and NMR data with
16−18
those published.
The transformation of 3 into 3a occurred
during the acquisition of the NMR spectra of 3 (CDCl3
containing traces of DCl). The same result was observed
when an acidified CHCl solution of 3 was left at room
3
temperature for 2 h.
Compound 4 was isolated as colorless crystals. Its IR
−
1
spectrum showed bands for double bonds (1642 cm ), ester
−
1
−1
(
1726 cm ), and hydroxy groups (3596, 3415 cm ). The
+
HRFABMS showed a [M + Na] peak at m/z 575.3550,
indicative of a molecular formula C H O , which was in
3
1
52
8
1
3
accordance with the 31 carbon signals observed in the
C
NMR spectrum. Inspection of the NMR spectra revealed that,
like compounds 1 and 3, compound 4 was an (E)-labda-
8(17),13-diene with oxygenated functions at C-12 and C-15
(
Tables 3 and 4). Moreover, a set of signals between δ 3.2
H
1
Figure 2. ORTEP projection of nicandrodiol (1).
and 4.0 in the H NMR spectrum, together with the signals for
an anomeric methine (δH 4.16, δC 99.3), indicated the
presence of a sugar moiety. The sugar was identified as β-D-
glucopyranose by the J values of its proton signals (J1′,2′ = 7.5
is assumed that compound 1 belongs to the normal labdane
series, as will be demonstrated later. Thus, the structure of
nicandrodiol (1) was assigned as (12R,E)-labda-8(17),13-
diene-12,15-diol.
Hz; J2 = J3′,4′= J4′,5′= 9.0 Hz) and the specific rotation of the
sugar obtained by acid hydrolysis of 4. This reaction also
afforded a complex mixture, from which the original aglycone
could not be isolated. The sugar was located at C-12 by the
′,3′
19
Compound 2 also showed a molecular formula C H O
2
0
34
2
+
(
HRFABMS: m/z 329.2464 [M + Na] ) and IR absorptions
HMBC interactions of H-14, H -16, and the anomeric proton
−
1
3
for hydroxy groups and double bonds (3330, 1644 cm ).
Analysis of the NMR spectra indicated that it has the same 2D
structure as compound 1. Some differences were observed for
(
H-1′) with C-12 and those of H-12 with C-11, C-14, C-16,
and C-1′ (Figure 1). The presence of an isovaleryl group was
evident from the NMR signals at δ 173.0 (C-1″), δ 2.19 d, δ
C
H
C
the H-9, C-11−C-14, and C-16 signals that in 1 appeared at δ
H
4
3.5 (CH −2″), δ 2.10 hept, δ 25.7 (CH-3″), δ 0.96 d, and
2
H
C
H
2
.01, δ 30.1, 75.0, 142.3, 123.3, and 12.2, respectively, while
C
δ 22.5 (CH -4″, CH -5″). This group was located at C-15 by
the chemical shift of the CH -15 protons (δ 4.67 and 4.60)
C
3
3
in compound 2, the signals were observed at δ 1.47, δ 28.6,
H
C
2
H
7
7.2, 140.2, 126.6, and 10.6, respectively. Similar differences
were observed between their di-O-acetyl derivatives 1a and 2a
Tables S1 and S2, Supporting Information), suggesting that
and their connectivities, in the HMBC spectrum, with C-13, C-
14, and C-1″. In order to determine the C-12 configuration,
(
the tetra-O-acetyl derivative of 4 was synthesized (Tables 3
and 4). This crystalline derivative (4a) was subjected to an X-
ray diffraction analysis whose results (Figure 3) confirmed the
proposed structure and showed a (12S) absolute configuration.
Thus, the structure of nicandroside A (4) was determined to
compounds 1 and 2 are C-12-epimers. The isolation of the
related compound 3 and its correlation with 1 and 2 permitted
clarification of this issue. The ion at m/z 302.2560 [M] in the
+
HRFABMS indicated the molecular formula of 3 as C H O .
2
0
32
2
Its 1D and 2D NMR spectra indicated that it is a labdane,
which differs from 1 and 2 by the presence of an α,β-
unsaturated carbonyl moiety in the side chain. This was
−
1
evident from the IR band at 1672 cm , as well as from the
carbon signals at δ 201.1 for the ketocarbonyl and δ 137.7
C
C
and 138.8 for the α and β carbons. These carbon signals were
assigned to C-12, C-13, and C-14 by the correlations of H-9,
H-11a, H-11b, H-14, and H-16 with C-12, and those of the
hydroxymethylene protons (δ 4.45, 2H, CH −15) with C-13
H
2
and C-14, which were observed in the HMBC spectrum. NOE
cross peaks between CH −15 and CH -16 (δ 1.76)
2
3
H
established the E-configuration of the C-13 double bond
(
Figure 1). Thus, the structure of nicandrone (3) was defined
as (E)-15-hydroxylabda-8(17),13-dien-12-one.
The relationship between compounds 1, 2, and 3 was
established by reduction of 3 with NaBH , which afforded the
4
labdanes 1 and 2, thus confirming the epimeric relationship of
these diols and, therefore, a (12S)-configuration for compound
2
, which was named 12-epi-nicandrodiol and its structure
established as (12S,E)-labda-8(17),13-diene-12,15-diol. Fur-
Figure 3. ORTEP projection of tetra-O-acetylnicandroside A (4a).
C
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be (12S,13E)-15-O-isovaleryllabda-8(17),13-diene-12,15-diol
acetyl derivatives 6a/7a; thus, a (12R) configuration was
proposed. This was confirmed by chemical correlation between
1 and 6/7 through the photo-oxidation of compound 1, which
1
2-O-β-D-glucopyranoside.
Compound 5 was characterized as the deisovaleryl derivative
of 4, since, in addition to the absence of the IR absorption for
the ester carbonyl, it showed an [M + Na] ion at m/z
gave a mixture of 6/7, that upon NaIO oxidation afforded
compound 8. The semisynthetic and natural compounds
4
+
4
91.2969, which is in accordance with the molecular formula
showed the same R values (TLC) and the same sets of signals
f
1
13
C H O . The NMR data (Tables 3 and 4) of 4 and 5 were
quite similar, except those of CH -15, which in compound 5
appeared at higher field (δ 4.31 and 3.97; δ 58.3) compared
to those of compound 4, thus supporting the presence of a
hydroxy group connected to CH -15 in 5. The penta-O-acetyl
derivative 5a obtained after acetylation proved the presence of
this group. Compounds 4 and 5 were chemically correlated
through the base hydrolysis of 4 that produced 5, which was
indistinguishable from the natural product (R , H NMR, and
in the H and C NMR spectra (Figures S43, S44, and S50,
Supporting Information). Compounds 6 and 7 were identified
as the C-14 epimers of (12R)-labda-8(17),13(16)-diene-
12,14,15-triol and were named nicantriol and 14-epi-nicantriol.
Compound 9 was isolated after acetylating a complex
mixture. The spectroscopic data of this tri-O-acetyl derivative
indicated the structure of an 8(17),13-labdadiene with acetoxy
2
6
44
7
2
H
C
2
20
1
groups at C-12, C-15, and C-16, as deduced from the H NMR
1
signals at δ 5.23 (dd, J = 11.0, 2.0 Hz; H-12), 4.72 (dd, J =
f
H
2
0
[
α]D ). Thus, the structure of nicandroside B (5) was defined
14.0, 6.5 Hz; H-15a), 4.70 (dd, J = 14.0, 6.5 Hz; H-15b), 4.70
(d, J = 13.0 Hz; H-16a), and 4.66 (d, J = 13.0 Hz; H-16b).
This was confirmed by the long-range HMBC connectivities of
H-14 (δ 5.82 td, J = 6.5, 0.5 Hz) with C-12 (δ 74.5), C-15
as (12S,13E)-labda-8(17),13-diene-12,15-diol 12-O-β-D-gluco-
pyranoside.
Labdanes 6 and 7 were isolated as a ∼3:2 mixture. The
H
C
molecular formula of each of these compounds was determined
(δ 60.3), and C-16 (δ 59.5). The (13E)-configuration was
C
C
+
as C H O by HRFABMS (m/z 323.2584 [M + H] ). The
determined via the NOESY interactions of H-12 with H-14
2
0
34
3
1
3
C NMR spectrum showed a single set of signals for the
and H -16, while the (12R)-configuration was defined based
2
carbons of the decalin (C-1 to C-10) and for C-18−C-20 of
on the chemical shift of H-9 at δ 1.68. Compound 9 was
H
1
both compounds (Table 2). The H NMR spectrum also
named triacetylisonicantriol and its structure determined as the
tri-O-acetyl derivative of (12R,13E)-labda-8(17),13-diene-
12,15,16-triol.
exhibited a single set of signals for the protons associated to
these carbons (Table 1). Comparison of these signals with
those of 1 showed a close similarity, thus indicating the same
substitution in the decalin residue. On the contrary, each of the
components of the 6/7 mixture generated a set of NMR signals
for the side chain that resemble each other. These signals
The molecular formula of compound 14 was determined as
+
C
H
40
O
by the [M + Na] ion at m/z 551.2628 in the
30
8
HRESIMS. It was identified as a withanolide by the
characteristic NMR signals for a 2-en-1-one moiety in ring A
1
3(16)
indicated the presence of a terminal Δ
-double bond (δC
(δ
CH-3; and δ
unsaturated-δ-lactone (δ 80.94, δ 4.24, CH-22; δ 31.6, δ
H
C
203.8, C-1; δ
C
128.8, δ
H
6.00, CH-2; δ
C
145.0, δ
H
6.86,
1
51.1/151.6, C-13; δ 111.9/112.5, δ 5.20 and 5.14/5.17,
C
33.0, δ
H
2.98 and 1.94, CH
−4); an α,β-
2
C
H
CH -16) and three hydroxy groups located at C-12 (δ 72.1/
2
C
C
H
C
1.6, δ 4.20/4.24 dd, J = 10.0, 1.5 Hz; CH-12), C-14 (δC
2.37 and 2.05, CH −23; δ 148.5, C-24; δ 122.2, C-25 and δ
H
2
C
C
C
3.3/73.2, δ 4.38/4.40 dd, J = 7.0, 4.0 Hz; CH-14), and C-15
165.5, C-26); and for two tertiary, one secondary, and two
vinylic methyl groups (Tables 5 and 6). The presence of a
5β,6β-epoxy group was deduced from the chemical shift of the
C-5 (δ 61.3) and CH-6 (δ 63.8, δ 3.21 d, J = 2.5 Hz)
H
C H H
2
C
C
H
3
,21,22
signals.
One acetoxy (δ 169.7 and 21.6, δ 1.99 s) and
C H
−
1
two hydroxy groups were present in 14 (IR 1730, 3413 cm ).
The chemical shifts of C-20 (δ 75.3) and CH -21 (δ 21.2, δ
C
3
C
H
1.44), the multiplicity of the H-22 signal (dd), and the HMBC
correlation of the OH proton with C-17 (δ 54.1) permitted
C
location of the first OH-group (δ 3.06 br s) at C-20. The
H
second hydroxy group was connected to C-14 as indicated by
the chemical shift of C-14 (δ 82.8). The chemical shift of H-
C
8
15 (δ 4.99 d, J = 6.5 Hz) and its HMBC correlations with C-
H
relationship at C-14 was confirmed when a single product,
the aldehyde 8, was obtained by oxidation of 6/7 with NaIO4.
Compound 8 showed a molecular formula C H O
13 (δC 48.3), C-14, C-17, and with the acetoxy carbonyl
(Figure 1), located this ester group at C-15. The NOESY
correlation between CH -18 and CH -21 and a W-coupling
1
9
30
2
3
3
+
(
HRESIMS: m/z 291.23146 [M + H] ), NMR signals for a
between the HO-20 proton and H-17, observed in the COSY
spectrum, established the β-orientation of the side chain.
NOESY correlations of 14 were not conclusive to determine
the orientation of HO-14 and AcO-15 groups. They were
proposed as β- and α-oriented, respectively, by the close
similarity of the NMR signals of ring D to those of
conjugated formyl moiety (δ 194.7, δ 9.58, CH-14; δ 153.1,
C
H
C
C-13; δC 133.6, δH 6.48 and 6.05, CH −16), and a
2
hydroxymethine moiety (δ 68.0, δ 4.53 br d, J = 10.0 Hz;
C
H
1
CH-12). Examination of the H NMR data of several 12-
hydroxylabdanes showed that the chemical shift of H-9 signal
is affected by the configuration at C-12. Thus, in the (12R)
21
withaneomexolides B-D. Configurations of C-20 and C-22
were assumed as R, but this will be confirmed later. Thus, the
structure of physanicandrolide A (14) was defined as
(20R,22R)-15α-acetoxy-5β,6β-epoxy-14β,20-dihydroxy-1-oxo-
witha-2,24-dienolide.
4
compounds nicandrodiol (1) and physacoztomatin, H-9
resonates at ∼δ 2.05, while in their 12-O-acetyl derivatives
H
7
it was observed at ∼δ 1.70. When the C-12 configuration is S
H
as in 2, H-9 appeared at ∼δ 1.5 and at ∼δ 1.40 in its acetyl
H
H
+
13
and glucosyl derivatives (2a, 4, 4a, 5, and 5a). In compounds
The HRESIMS [M + Na] ion at m/z 583.2855 and the C
NMR data of compound 15 indicated a molecular formula of
6
, 7, and 8, H-9 resonates at ∼δ 2.05 and at ∼δ 1.70 in the
H
H
D
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Table 2. 13C NMR Data of Compounds 1−3 and 6−9 (CDCl , 125 MHz)
3
c
position
1
2
3
6/7
39.1
19.3
42.1
33.6
55.5
24.4
38.3
8
9
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
CH2
CH2
CH2
C
39.0
19.3
42.1
33.6
55.5
24.4
38.3
149.1
52.5
39.3
30.1
75.0
142.3
123.3
59.1
12.2
106.5
33.6
21.7
14.6
39.0
19.4
42.1
33.6
55.6
24.4
38.3
149.1
53.2
39.5
28.6
77.2
140.2
126.6
59.2
10.6
106.7
33.5
21.7
14.6
39.2
19.3
42.0
33.5
55.1
24.0
38.3
149.6
51.6
39.0
32.9
201.1
137.7
138.8
60.2
11.9
106.1
33.6
21.8
14.8
39.0
19.3
42.1
33.6
55.5
24.4
38.3
148.5
52.1
39.3
30.9
68.0
153.1
194.7
39.1
19.3
42.1
33.6
55.6
24.3
38.2
148.2
52.6
39.3
28.9
74.5
138.4
126.7
60.3
59.5
106.6
33.5
21.7
14.6
CH
CH2
CH2
C
CH
0 C
1 CH2
2 CH
3 C
4 CH
5 CH2
6 CH3
7 CH2
8 CH3
9 CH3
149.1/149.0
52.5/52.6
39.4
31.0/30.6
72.1/71.6
151.1/151.6
73.3/73.2
66.2/66.7
111.9/112.5
106.4/106.6
33.6
a
b
b
b
133.6
107.1
33.6
21.7
14.6
21.7
14.6
0 CH3
a
b
c
C. CH . Ac: δ 170.7, 170.3, 170.5, 21.2, 20.9, 20.8.
2
C
C H O . The NMR data were similar to those of 14, except
for the signals concerning the A-ring, which were in accordance
with the presence of a 3-methoxycyclohexan-1-one moiety, as
concerning to ring B, which indicated the presence of a 5α-
chloro-6β-hydroxywithanolide. This was deduced from the
3
1
44
9
chemical shifts of H-4β (δ 3.56 dt, J = 20.5, 2.5 Hz), C-5 (δC
H
deduced from the signals for a ketocarbonyl at δ 210.9 (C-1),
80.9), and CH-6 (δ 4.10 br s; δ 74.5), as well as from the
C
H
C
an α-methylene (δ 42.7, δ 2.77 dd, J = 14.0, 6.0 Hz and δ
small coupling constants of H-6, which were in accordance
C
H
H
22
2
7
.67 ddd, J = 14.0, 4.5, 1.0 Hz; CH -2), an oxymethine (δC
with its equatorial orientation. This was confirmed by the
2
2.9, δ 3.75 br s; CH-3), another methylene (δ 36.2, δ 2.22
NOESY cross peak between the α-equatorial H-4 (δ 2.54 dd,
H
C
H
H
dd, J = 14.5, 4.0 Hz and δ 1.59 m; CH -4), and a methoxy
J = 20.5, 5.0 Hz) and H-6. Finally, the structure and the
absolute configuration of 16, especially those of D-ring and
side chain were confirmed as (14S,15S,17S,20R,22R) by X-ray
diffraction analysis of its O-acetyl derivative 16a (Figure 4),
using the anomalous dispersion of the chlorine atom [Flack
parameter 0.00 (4)]. These results can be extended to 14 and
15 by the similarity of their NMR data. Thus, the structure of
physanicandrolide C (16) was defined as (20R,22R)-15α-
acetoxy-5α-chloro-6β,14β,20-trihydroxy-1-oxowitha-2,24-dien-
olide.
H
2
group (δ 55.9, δ 3.27). The NOESY interactions of CH -19
C
H
3
with H-2a (δH 2.77), H-4a (δH 2.22), and H-8 (δH 1.83)
established the β-axial orientation of these protons, which was
confirmed by the W-coupling (J = 1.0 Hz) between the
equatorial H-2b (δH 2.67) and H-4b (δH 1.59) protons,
observed in the COSY spectrum (Figure 1). The magnitude of
the coupling constants of H-3 was consistent with an eq−ax
relationship with H-2a (J = 6.0 Hz), an eq−eq relationship
with 2b (J = 4.5 Hz), and an eq−ax relationship with H-4a (J =
4
.0 Hz), indicating an equatorial disposition of H-3. The
In addition to the new compounds, the investigation of P.
nicandroides led to the isolation of the known labdanes:
(−)-(13E)-labd-13-ene-8α,15-diol (12),(+)-(Z)-labda-8-
(17),13-diene-15,16-diol (13), and the mixture of physanican-
triol and 14-epi-physanicantriol (10/11), previously isolated
observed strong NOESY cross-peaks of H-3 with the vicinal β-
axial protons and its weak interactions with the α-equatorial H-
2
b and H-4b protons established the α-orientation of the
3
CH O-3 group (Figure 1). The H−H distances measured in
8
the energy-minimized 3D structure of 15 (Figure S76 and
Table S3, Supporting Information) together with the similarity
of the NOESY correlations and J values of the ring A protons
of 15 and those of the 3α-methoxywithanolide, physangulatin
I, whose structure was determined by X-ray analysis, gave
from P. nicandroides. The flavonol 3,7-di-O-methylquercetin
2
5,26
(18)
and the known phytosterols β-sitosterol/stigmasterol
27
and physalindicanol A (17) were also isolated.
The acetylcholinesterase inhibitory activity of compounds
1−5, 3a, 4a, 10−17, and 16a was evaluated. At the highest
used concentration (100 μM) most of these compounds
increased the enzymatic activity in a range of 6.7 to 96% except
compounds 3a, 13, and 17 that moderately inhibited the
activity in 57.5, 33.9, and 24%, respectively (Figure S77 and
Table S4, Supporting Information). The IC50 of the most
active compound 3a was determined, but it resulted 4 orders of
magnitude higher (IC50 = 64.4 μM) than that of the AChE
inhibitor, physostigmine (IC50 = 3.0 nM). The ability of
compounds 1-5, 4a, 10-17, and 16a to inhibit the activity of
yeast α-glucosidase was also evaluated. In this case, a slight
activation of the enzyme by the labdanes (from 1.1 to 29.4%,
23
further support to this assumption. Therefore, the structure
of physanicandrolide B (15) was determined as (20R,22R)-
1
5α-acetoxy-5β,6β-epoxy-14β,20-dihydroxy-3α-methoxy-1-
oxowith-24-enolide. This compound could be an artifact
produced by Michael addition of MeOH to compound 14,
however, this must be proven since the natural or artificial
11,24
origin of 3-OH and 3-alkoxy withanolides is controversial.
Compound 16 was isolated as colorless crystals with a
+
molecular formula C H ClO , determined by the [M + H]
30
41
8
1
13
ion at m/z 565.2562 in the HRFABMS. The H and C NMR
signals of 16 were closely related to those of 14, except those
F
DOI: 10.1021/acs.jnatprod.9b00233
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 3. H NMR Data of Compounds 4, 4a, 5, and 5a (500 MHz, CDCl )
3
position
1
4
4a
5
5a
1.70 m
.86 m
1.55 br dd (13.5, 13.5)
.48 m
1.39 m
.15 m
1.69 m
0.85 m
1.72 m
0.86 m
1.69 m
0.85 m
0
2
3
1.52 br dd (13.5, 13.5)
1.45 m
1.37 br t (13.0)
1.14 ddd (13.5, 13.5, 4.0)
0.99 m
1.56 br dd (13.5, 13.5)
1.49 m
1.55 br dd (13.5, 13.5)
1.45 m
1
1.37 m
1.17 m
1.37 m
1
1.14 ddd (13.0, 13.0, 4.0)
0.99 m
5
6
1.01 dd (12.0, 2.5)
1.70 m
1.04 dd (12.0, 2.5)
1.70 m
1.69 m
1.69 m
1
.29 dddd (13.0, 13.0, 13.0, 4.0)
2.35 br d (12.5)
.85 ddd (13.0,12.5, 4.5)
1.29 dddd (13.0, 13.0, 13.0, 4.0)
2.34 ddd (13.0, 4.0, 2.5)
1.84 ddd (13.0,13.0, 4.5)
1.32 m
1.28 m
2.36 br d (12.0)
1.87 m
1.37 m
1.79 m
1.29 dddd (13.0,13.0,13.0, 4.0)
2.34 ddd (13.0, 4.0, 2.5)
1.84 ddd (13.0,13.0, 4.5)
1.32 m
7
1
9
1.37 m
1.76 m
1
1
1.70 m
1.70 m
1
.65 m
1.70 m
1.70 m
1.70 m
1
1
1
2
4
5
4.24 dd (11.0, 4.0)
5.38 br t (7.0)
4.67 dd (13.0, 7.0)
4.19 dd (9.5, 7.5)
5.38 br t (7.0)
4.65 dd (13.0, 7.0)
4.61 dd (13.0, 7.0)
1.58 s
4.17 dd (10.5, 3.0)
5.50 br t (7.0)
4.31 dd (12.0, 8.0)
3.97 dd (12.0, 4.0)
1.64 s
4.19 dd (9.5, 7.5)
5.38 br t (7.0)
4.65 dd (13.0, 7.0)
4.61 dd (13.0, 7.0)
1.57 s
4
.60 dd (13.0, 7.0)
1
1
6
7
1.66 s
4.83 br s
4.82 br s
4.85 br s
4.82 br s
4
.65 br s
4.63 br s
4.68 br s
4.63 br s
1
1
2
1
2
3
4
5
6
8
9
0
′
′
′
′
′
′
0.86 s
0.79 s
0.68 s
4.16 d (7.5)
3.39 dd (9.0, 7.5)
3.48 dd (9.0, 9.0)
3.61 dd (9.0, 9.0)
3.21 br d (9.0)
3.83 dd (12.5, 3.0)
0.85 s
0.78 s
0.67 s
4.35 d (8.0)
4.98 dd (9.5, 8.0)
5.15 dd (9.5, 9.5)
5.05 dd (9.5, 9.5)
3.61 ddd (9.5, 5.5, 2.5)
4.23 dd (12.0, 5.5)
4.10 dd (12.0, 2.5)
2.21 d (7.0)
0.87 s
0.80 s
0.70 s
4.24 d (8.0)
3.32 br t (8.5)
3.47 t (9.0)
3.50 t (9.0)
3.29 m
0.85 s
0.78 s
0.67 s
4.35 d (8.0)
4.98 dd (9.5, 8.0)
5.15 dd (9.5, 9.5)
5.05 dd (9.5, 9.5)
3.61 ddd (9.5, 5.5, 2.5)
4.23 dd (12.0, 5.5)
4.10 dd (12.0, 2.5)
3.79 m
3.79 m
3
.80 dd (12.5, 3.0)
2
3
4
′′
′′
′′, 5′′
2.19 d (7.0)
2.10 br hept (7.0)
0.96 d (7.0, 6H)
a
a
2.11 br hept (7.0)
0.98 d (7.0, 6H)
Ac
2.08, 2.01 (6H), 1.99
2.08, 2.01 (6H), 1.99
a
hept = heptuplet.
1
00 μM) was observed, except for 4a and 13 which, like the
atoms, except those bonded to oxygen atoms, were included at
calculated positions and were not refined.
Plant Material. Physalis nicandroides var. attenuata Waterf. was
steroidal compounds 14-17, and 16a showed poor inhibition.
collected in Tlayacapan, State of Morelos, Mexico, in August 2004. A
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points (uncor-
rected) were determined on a Fisher-Johns melting point apparatus
Fisher Scientific, U.S.A.). Optical rotations were measured on a
́
■
voucher specimen of the plant (M. Martinez s/n) was identified by
́
Dr. Mahinda Martinez and deposited at the Herbarium of the
Universidad Autonoma de Queretaro.
́
́
(
Extraction and Isolation. Dried and ground aerial parts of the
PerkinElmer 343 polarimeter. IR spectra were measured on a Nicolet
FTIR-Magna 750 or Bruker Tensor 27 spectrophotometers. NMR
plant, without fruits and calices (3.3 kg), were extracted with MeOH.
The extract (578 g) was dissolved in MeOH/H O (4:1, 1 L) and
2
1
spectra were recorded on a Varian 500 spectrometer ( H at 500 MHz;
partitioned with hexanes to obtain a hexanes-soluble extract (197.2 g).
After removal of MeOH from the aq MeOH solution and addition of
13
C at 125 MHz), with TMS as internal standard. HRMS were
measured on a JEOL JMS-SX102A, JEOL MStation JMS-700, Bruker
micrOTOF II or Jeol AccuTOF JMS-T100LC mass spectrometers.
Vacuum column chromatography (VCC) was performed on silica gel
800 mL of H
EtOAc-soluble (81.47 g) and a H
2
O, this fraction was extracted with EtOAc to obtain an
O-soluble (297.0 g) extract. The
2
hexanes-soluble extract was fractioned by silica gel VCC, eluted with
hexanes-acetone mixtures of increasing polarity to obtain fractions:
A1-A6 (1:0), A7-A22 (19:1), A23-A37 (9:1), A38-A45 (17:3), A46-
A51 (4:1), A52-A60 (7:3), A61-A63 (3:2), A64-A74 (2:3), A75-A76
(1:4), and A77 (0:1). Fractions A8−A25 (63.0 g) were discolored
with activated charcoal and fractioned by silica gel VCC with a
gradient of hexanes−EtOAc to give fractions B1−B11 (39:1), B12−
B17 (95:5), B18−22 (9:1), B23−B29 (17:3), B30−B34 (4:1), and
B35−B37 (3:1). A mixture of β-sitosterol and stigmasterol (3.51g)
was obtained from fractions B5−B10. Fractions B11−B37 (13.64 g)
were purified by VCC (hexanes−EtOAc 19:1 to 4:1) to obtain
fractions B′1−B′44. Repeated VCC of fractions B′10−B′17 (7.73 g)
6
0 G (Macherey-Nagel). Analytical TLC was carried out on
precoated Alugram Sil G/UV254 plates. Preparative TLC was
performed on precoated Sil G-200 UV254 plates (Macherey-Nagel).
Reversed-phase HPLC analysis was performed on a Waters 600
apparatus (Macrosphere C , 250 × 2.1 mm, 5 μm) equipped with a
1
8
photodiode array detector (λ = 200−650 nm). X-ray crystallographic
analysis was carried out on a Bruker Smart Apex CCD diffractometer
with graphite-monochromated Mo Kα radiation (λ 0.71073 Å). The
structures were solved by direct methods using the SHELXS
2
8
program. Non-hydrogen atoms were refined with anisotropic
28
displacement parameters using the SHELXL program. Hydrogen
G
DOI: 10.1021/acs.jnatprod.9b00233
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
3
preparative TLC (hexanes−CHCl 1:3) to afford 3 (43 mg). Fractions
Table 4. C NMR Data of Compounds 4, 4a, 5, and 5a (125
MHz, CDCl3)
3
F9−F32 (12.2 g) were discolored with activated charcoal and
fractioned by VCC eluted with hexanes−acetone to obtain fractions
G1−G44 (9:1) and G45−G80 (17:3). Compound 1 was obtained
from fractions G26−G40. Mother liquors of 1 and fractions G14−
G25 and G41−G68 were mixed and subjected to VCC eluted with
hexanes−EtOAc 7:3 to obtain fractions H1−H88. Fractions H18−
H26 gave 1 (1.43 g). Fractions H41−H43 gave 93.4 mg of 2, and
fractions H51−H54 afforded 121.2 mg of 13. Fractions F33−F39
a
b
position
4
4a
5
5a
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
1
2
3
4
5
6
1
2
3
4
CH2
CH2
CH2
C
CH
CH2
39.0
19.4
42.1
33.6
55.6
24.3
38.2
148.2
52.4
39.3
26.8
82.2
138.6
125.1
60.4
10.4
106.9
33.5
21.7
14.7
99.3
73.3
76.6
69.8
75.3
61.8
173.0
43.5
25.7
22.5
39.0
19.4
42.1
33.6
55.6
24.3
38.2
148.0
52.4
39.3
26.9
82.9
138.6
124.9
60.2
10.1
106.9
33.5
21.7
14.5
97.8
71.3
73.1
68.8
71.7
62.3
172.8
43.5
25.7
22.5
39.1
19.5
42.1
33.5
55.5
24.4
38.3
148.2
52.4
39.4
26.9
83.0
136.5
130.0
58.3
10.5
107.0
33.6
21.8
14.7
99.2
73.3
76.5
70.1
75.3
61.9
39.0
19.3
42.1
33.5
55.7
24.3
38.2
148.0
52.4
39.3
26.9
82.8
138.5
124.8
60.6
10.1
106.9
33.5
21.7
14.5
97.8
71.3
73.0
68.8
71.6
62.3
(6.92 g) were subjected to VCC eluted with mixtures of hexanes−
EtOAc 7:3 to 1:1 to afford fractions I1−I98. Crystallization of fraction
I9−I11 (EtOAc-hexanes) gave 18 (17.4 g). Compound 14 was
obtained from fractions I15−I31. Mother liquors of 14 and 18 were
mixed with fractions I12−I14 and purified by VCC using a gradient of
hexanes−EtOAc (7:3 to 1:1) to give fractions J1−J25 (7:3), J26−J33
(3:2), J34−J38 (1:1). Fractions J31−J38 (3.94 g) were purified by
VCC eluted with hexanes−EtOAc 7:3 to afford fractions K1−K60.
Fractions K30−K40 gave 14 (2.84 g). Fractions J10−J30 and K10−
K24 were combined and purified by two successive VCCs (hexanes−
CH2
C
CH
0 C
1 CH2
2 CH
3 C
4 CH
5 CH2
6 CH3
7 CH3
8 CH3
EtOAc 7:3; CHCl −acetone 9:1) to give 246 mg of a mixture of 6/7.
3
Fractions I32−I65 were subjected to VCC (CHCl −acetone 9:1) to
3
give fractions L1−L54. Compound 15 (47 mg) was obtained from
fractions L9−L18 after repeated VCC (CHCl −acetone 9:1).
3
Fractions L19−L30 were purified by VCC (CHCl −acetone 17:3)
3
9 CH3
to obtain compound 16 (63 mg). Compounds contained in fractions
I88−I93 could not be purified; therefore, a portion (48.9 mg) was
acetylated, and the reaction mixture was purified by preparative TLC
(hexanes−EtOAc 17:3) to obtain 9 (11.9 mg). Fractions F40−F68
and I66−I94 contained compound 4; therefore, they were combined
0 CH3
′ CH
′ CH
′ CH
′ CH
′ CH
′ CH2
′′ C
(42.60 g), discolored with activated charcoal and purified by three
consecutive silica gel VCC’s (hexanes−acetone 7:3; hexanes−acetone
4
:1; benzene−EtOAc 4:1) to give two fractions enriched in
compound 4: M1 (25.07 g, 32%) and M2 (14.15 g, 48%), indicating
a yield of 14.95 g of 4 in these fractions. The percentages were
′′ CH2
′′ CH
′′, 5′′ CH3
determined by reversed-phase HPLC eluted with MeOH−H O 7:3 to
2
19:1. Fractions F69−F91 (13.82 g) were discolored with activated
charcoal and purified by VCC eluted with EtOAc to obtain fractions
N1−N11. Fractions N3−N7 were subjected to VCC using mixtures
of hexanes−EtOAc 17:3 to obtain fractions O1−O80. Fractions
O55−O76 (2.27 g) contained crude 5, and 71.2 mg of these fractions
were purified by preparative TLC (benzene−MeOH 9:1, × 3) to
obtain pure 5 (29.6 mg). Fractions of columns I to O containing 10/
a
Ac signals of 4a: δ 170.6, 170.3, 169.4, 169.2, 20.74, 20.69, 20.64,
0.62. Ac signals of 5a: δ 170.7, 170.6, 170.3, 169.4, 169.2, 20.9,
0.7, 20.63, 20.59, 20.57.
C
b
2
2
C
using hexanes−iPrOH 98:2 as eluent, afforded compound 3 (3.33 g).
Fractions B′18−B′26 (1.83 g) were combined and purified by
repeated VCC eluted with hexanes−EtOAc 9:1 to obtain 36.7 mg of
1
1 were combined (4.87 g) and purified by VCC eluted with CHCl −
3
acetone 17:3 to afford 126.7 mg of 16 and a mixture of 10/11 which
was subjected to VCC eluted with hexanes−acetone 3:1 to obtain
1
7. Fractions A26−A52 were combined (24.26 g), discolored with
3.24 g of 10/11.
activated charcoal, and fractioned by VCC eluted with hexanes−
acetone gradient (9:1 to 7:3) to give fractions C1−C23 (9:1), C24−
C29 (17:3), C30−C32 (4:1), and C33−C37 (7:3). Fractions C4−
C26 (18.30 g) were subjected to a VCC using mixtures of hexanes−
EtOAc 3:1 as eluent to afford fractions D1−D39. Compound 1 was
obtained from fractions D3−D17 by crystallization from EtOAc−
hexanes. Mother liquors of 1 were combined with fractions D18−D33
Nicandrodiol (1): colorless crystals (EtOAc-hexanes); mp 130−
20
132 °C; [α]
+52 (c 0.2, CHCl ); IR (film) ν 3413, 3324, 1642
D
3 max
−
1
1
13
cm ; H NMR and C NMR (CDCl ) see Tables 1 and 2;
HRFABMS m/z 306.2560 [M] (calcd for C H O , 306.2559).
X-ray Crystallographic Data of Nicandrodiol (1). The data were
collected from a colorless prism (0.452 × 0.182 × 0.072 mm) 298(2)
K: C H O , M 306.47; monoclinic, space group P2 ; a =
3
+
2
0
34
2
2
9
2
0
34
2
r
1
(
11.37 g) and subjected to repeated VCC (benzene−EtOAc 4:1) and
1
9.3184(15) Å, b = 7.2915(6) Å, c = 22.0305(17) Å; α = 90°, β =
crystallization (EtOAc−hexanes) to obtain 1 (total yield 4.17 g). Two
successive VCCs of the mother liquors of 1 (hexanes−acetone 4:1
and hexanes−EtOAc 3:1) gave compound 12 (898 mg). The
combined fractions A53−A77 (74.5 g) were discolored with activated
charcoal to afford 53.7 g of residue which was fractioned by VCC
using a gradient of hexanes−EtOAc−MeOH to give fractions E1−
E47 (60:40:0.6), E48−E52 (55:45:0.6), E53−E55 (50:50:0.6), and
E56−E58 (40:60:0.6). Fractions E17−E56 were repeatedly subjected
to VCC (benzene−EtOAc 3:2) and crystallization (hexanes) to
obtain 4 (15.95 g).
3
113.7375 (17)°, γ = 90°, V = 2840.7(4) Å ; Z = 6; Dcalc= 1.075 g/
3
cm ; F(000) = 1020.0. Reflections collected 9948; independent
reflections 5673 for I ≥ 2σ (I).
1
2-epi-Nicandrodiol (2): colorless crystals (EtOAc−hexanes); mp
20
1
36−138 °C; [α] −9 (c 0.10, CHCl ); IR (film) ν 3330, 1644
D
3
max
−
1
1
13
cm ; H and C NMR (CDCl ) see Tables 1 and 2; HRFABMS m/
3
+
z 329.2464 [M + Na] (calcd for C H O Na, 306.2457).
20 34
2
2
0
D
Nicandrone (3): pale yellow oil; [α]
−8 (c 0.3, CHCl ); UV
3
(MeOH) λ (log ε) 215 (3.88) nm; IR (film) ν 3443, 1672, 1645
max max
−
1
1
13
cm ; H NMR and C NMR (CDCl ) see Tables 1 and 2;
HRFABMS m/z 304.2560 [M] (calcd for C H O , 304.2559).
The EtOAc-soluble extract (81.47 g) was fractioned by VCC and
eluted with hexanes−acetone mixtures of increasing polarity to obtain
fractions: F1−F6 (19:1), F7−F18 (9:1), F19−F26 (17:3), F27−F32
4:1), F33−F47 (7:3), F48−F65 (6:4), F66−F81 (1:1), F82−F95
2:3), F96−F108 (1:4), and F109−F113 (0:1). Fractions F4−F8
2.84 g) were subjected to repeated VCC (hexanes−EtOAc 9:1) and
3
+
20 32
2
2
0
Nicandroside A (4): colorless crystals; mp 90−94 °C; [α]
−24
D
−
1
1
(
(
(
(c 0.2, CHCl
3
); IR (film) νmax 3596, 3415, 1726, 1642 cm ; H and
1
3
C NMR (CDCl
) see Tables 3 and 4; HRFABMS m/z 575.3550 [M
3
+
+ Na] (calcd for C H O Na, 575. 3560).
3
1
52
8
H
DOI: 10.1021/acs.jnatprod.9b00233
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 5. H NMR Data of Compounds 14−16 and 16a (500 MHz, CDCl )
3
a
b
position
2
14
15
16
16a
5.94 dd (10.0, 2.5)
6.00 dd (10.0, 2.5)
2.77 dd (14.0, 6.0)
5.91 dd (10.0, 2.5)
2
.67 ddd (14.0, 4.5, 1.0)
3
4
6.86 ddd (10.0, 6.0, 2.5)
2.98 dt (19.0, 2.5)
3.75 br s
2.22 dd (14.5, 4.0)
1.59 m
3.26 br s
2.53 dt (14.5, 3.0)
1.50 m
6.67 ddd (10.0, 5.0, 2.0)
3.56 dt (20.5, 2.5)
2.54 dd (20.5, 5.0)
4.10 br s
2.10 m
2.10 m
6.62 ddd (10.0, 5.0, 2.0)
3.02 dt (20.0, 2.5)
2.56 dd (20.0, 5.0)
5.28 t (3.0)
2.12 m
1
.94 dd (19.0, 6.0)
6
7
3.21 d (2.5)
2.50 br dt (14.0, 2.5)
1
.44 m
2.12 m
8
9
1
1.97 m
1.97 m
1.98 m
1.83 td (12.0, 3.5)
2.01 m
1.38 m
1.35 m
1.92 m
1.48 m
5.01 d (6.5)
2.39 m
1.62 m
1.81 br t (9.0)
1.31 s
1.18 s
1.43 s
4.25 dd (13.5, 3.5)
2.39 m
2.02 m
2.12 m
2.00 m
2.77 td (12.0, 3.5)
2.29 ddd (13.5, 7.0, 3.5)
1.34 m
2.80 td (12.5, 3.5)
2.31 ddd (13.5, 7.0, 3.5)
1.33 m
2.09 m
1.59 m
5.05 d (6.5)
2.41 ddd (15.0, 7.5, 3.5)
1.64 dd (15.0, 9.0)
1.86 m
1.40 s
1.34 s
1
2
1
.52 m
1.98 m
.52 m
1
2.09 m
1
1.53 dt (10.0, 3.0)
5.07 d (6.5)
2.42 ddd (15.0, 7.0, 7.0)
1.64 dd (15.0, 9.5)
1.84 br t (8.5)
1.392 s
1.385 s
1.44 s
4.28 dd (13.5, 3.5)
2.36 br t (13.5)
2.07 m
1
1
5
6
4.99 d (6.5)
2.37 ddd (15.0, 8.5, 6.5)
1
.58 dd (15.0, 9.5)
1
1
1
2
2
2
7
8
9
1
2
3
1.82 br t (9.0)
1.35 s
1.26 s
1.44 s
4.24 dd (13.5, 3.5)
2.37 m
1.46 s
4.27 dd (13.5, 3.5)
2.37 m
2
.05 br dd (13.5, 3.5)
2.09 m
2
2
1
2
1
7
8
1.89 s
1.96 s
3.59 br s
3.06 br s
1.99 s
1.89 s
1.96 s
3.69 br s
3.11 br s
2.05 s
1.88 s
1.97 s
4.35 br s
4.00 br s
1.89 s
1.97 s
3.65 br s
3.10 d (2.5)
2.07 s
4-OH
0-OH
5-OAc
2.07 s
a
b
3
-OMe: δH 3.27 s. 6-OAc: δ 2.13 s.
H
2
0
Nicandroside B (5): colorless crystals; mp 121−122 °C; [α]
dried with Na SO , and purified by preparative TLC [(hexanes−
D
2
4
−
1
1
−
39 (c 0.1, CHCl ); IR (film) ν 3360, 1643 cm ; H and 13C
NMR (CDCl ) see Tables 3 and 4; HRESIMS m/z 491.2969 [M +
EtOAc 7:3)−MeOH 98:2, × 3] to afford 3.9 mg of 1 and 5.4 mg of 2.
3
max
Treatment of Nicandrenone (3) with Acid. Concentrated HCl
3
+
Na] (calcd for C H O Na, 491.2979).
(0.1 mL) was added to a solution of 3 in CHCl
was stirred by 2 h at room temperature, washed with NaHCO
water, dried over anhydrous Na SO , and purified by crystallization
3
(5 mL). The mixture
26
44
7
Nicantriol/14-epi-nicantriol (6/7): colorless oil; IR (film) ν
3
and
max
−
1
1
13
3
379, 1642 cm ; H and C NMR (CDCl ) see Tables 1 and 2;
HRFABMS m/z 323.2584 [M + H] (calcd for C H O , 323.2586).
2
4
3
+
from EtOAc−hexanes to afford 16 mg of pumiloxide (3a): colorless
2
0
35
3
2
0
13
2
0
crystals; mp 86−90 °C; [α] −21 (c 0.2, CHCl ) [lit. mp 87−89
Tri-O-acetylisonicantriol (9): colorless oil; [α]
+43 (c 0.3,
D
3
D
2
0
−
1
1
13
°C; [α] −20 (c 0.8, CHCl )].
CHCl ); IR (film) ν 1744, 1643 cm ; H and C NMR (CDCl3)
see Tables 1 and 2; FABMS m/z 471 [M + Na] (C H O Na), 449
M + H] , 389 [449 − AcOH] , 329 [449 − 2AcOH] , 269 [449 −
+
D
3
3
max
+
Acetylation of Compounds 1, 2, 4−7, and 16. Compounds 1
(48.3 mg), 2 (16.8 mg), 4 (433.5 mg), 5 (114.1 mg), 6/7 (41.8 mg),
and 16 (32.2 mg) were acetylated with pyridine−acetic anhydride at
room temperature (16 at 45 °C). After completion, the reaction
mixtures were treated in the usual way to obtain crudes 1a, 2a, 4a−7a,
and 16a, respectively. These derivatives were purified by different
techniques (1a:VCC hexanes−EtOAc 19:1; 2a: preparative TLC
hexanes−EtOAc 98:2; 4a: VCC hexanes−EtOAc 88:12 and
crystallization from EtOAc−hexanes; 5a: VCC hexanes−EtOAc 4:1
and preparative TLC hexanes−EtOAc 7:3; 6a/7a: VCC hexanes−
EtOAc 95:5; 16: crystallization from EtOAc−hexanes). Yield: 52.4
mg of 1a, 13.2 mg of 2a, 487 mg of 4a, 88 mg of 5a, 53.5 mg of 6a/7a,
2
6
40
6
+
+
+
[
3
AcOH] (38).
Physanicandrolide A (14): colorless crystals (acetone−hexanes);
2
0
D
mp 167−168 °C; [α] +58 (c 0. 1, CHCl ); UV (MeOH) λ (log
3
max
−
1 1
ε) 225 (4.18) nm; IR (film) ν 3413, 1730 sh, 1708, 1670 cm ; H
max
and 1 C NMR see Tables 5 and 6; HRFABMS 551.2628 [M + Na]
3
+
(
calcd for C H O Na, 551.2621).
30
40
8
Physanicandrolide B (15): colorless crystals; mp 166−169 °C;
α] +14 (c 0.2, CHCl ); IR (film) ν 3409, 1710 cm ; H and
C NMR, see Tables 5 and 6; HRESIMS 583.2855 [M + Na] (calcd
20
−1 1
[
D 3 max
13
+
for C H O Na, 583.2878).
31
44
9
and 30.7 mg of 16.
Physanicandrolide C (16): colorless crystals; mp 208−210 °C;
α] +68 (c 0.2, CHCl ); UV (MeOH) λ (log ε) 227 (3.91) nm;
IR (film) νmax 3413, 1725 sh, 1689 cm ; H and C NMR see
Tables 5 and 6; HRFABMS 565.2562 [M + H] (calcd for
20
1
2,15-Di-O-acetylnicandrodiol (1a): colorless oil; [α]
+43 (c
D
20
D
[
−1
1
13
3
max
0.2, CHCl ); IR (CHCl ) ν 1731, 1642 cm ; H and C NMR
3 3 max
−
1
1
13
see Tables S1 and S2 (Supporting Information); HRFABMS m/z
+
+
3
91.2839 [M + H] (calcd for C H O , 391.2848).
24 39 4
2
0
C H ClO , 565.2568).
3
0
42
8
12,15-Di-O-acetyl-12-epi-nicandrodiol (2a): colorless oil; [α]
D
−
1
1
13
Reduction of Nicandrone (3). Sodium borohydride (85.3 mg) was
+
2 (c 0.2, CHCl ); IR (film) ν 1741, 1644 cm ; H and C NMR
3 max
stepwise added to a stirred solution of 3 (45.3 mg) in EtOH (5 mL)
at 0 °C. The reaction mixture was stirred at room temperature for 4 h,
neutralized with HOAc, dried with an air stream, and partitioned
between EtOAc and water. The organic layer was washed with water,
see Tables S1 and S2 (Supporting Information); HRFABMS m/z
+
391.2843 [M + H] (calcd for C H O , 391.2848).
2
4
39
4
Acid Hydrolysis of Compound 4. Compound 4 in dioxane (3 mL)
was hydrolyzed with 2 N HCl at 78 °C for 2 h. The reaction mixture
I
DOI: 10.1021/acs.jnatprod.9b00233
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
2
0
1
3
Basic Hydrolysis of Compound 4. Aqueous NaOH (5% w/v, 3.2
mL) was added dropwise (0.4 mL/each h) to a stirred solution of 4
Table 6. C NMR Data of Compounds 14−16 and 16a (125
MHz, CDCl )
3
(
89.4 mg) in MeOH−CH Cl 3:2 (5 mL). The reaction mixture was
2
2
a
c
position
14
203.8
128.8
145.0
33.0
61.3
63.8
26.3
33.5
38.9
48.6
22.7
40.1
48.3
82.8
80.88
31.2
54.1
19.1
14.9
75.3
21.2
80.94
31.6
148.5
122.2
165.5
12.4
15
16
201.5
128.5
141.8
37.3
80.9
74.5
28.7
33.6
36.1
53.1
21.9
40.2
49.0
83.2
80.4
31.3
54.1
19.7
15.6
75.1
21.2
81.4
31.6
149.0
122.1
166.4
12.4
20.6
170.2, 21.7
16a
200.6
allowed to stand overnight (16 h). TLC (CHCl −MeOH 9:1)
3
showed partial hydrolysis of 4. Then a NaOH solution (0.5 mL) was
added. After 4 h, no progress of the reaction was observed; therefore,
the reaction mixture was diluted with CH Cl (15 mL) and washed
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
1
C
CH
CH
CH2
C
CH
CH2
CH
210.9
b
42.7
72.9
36.2
61.4
62.1
26.2
32.9
37.1
52.1
21.2
39.6
48.4
82.9
81.1
31.1
54.1
19.0
13.8
75.3
21.2
80.9
31.6
148.4
122.2
165.5
12.4
20.5
128.7
140.7
36.7
78.6
74.8
25.7
34.6
35.9
53.0
22.2
40.7
48.7
83.0
80.2
31.4
54.1
19.6
15.0
75.4
21.3
81.0
31.7
148.6
122.2
165.5
12.5
20.6
2
2
with a satd solution of NH Cl. The CH Cl layer was separated and
the aqueous layer re-extracted with CH Cl . The combined CH Cl
layers were washed with H O, dried with Na SO , and purified by
VCC (CHCl −MeOH 12:1) to obtain 53 mg of the starting
4
2
2
2
2
2
2
2
2
4
3
compound 4 and 27.1 mg of 5.
Tetra-O-acetylnicandroside A (4a): colorless crystals; mp 110−
2
0
D
−1
CH
1
13 °C; [α] −31 (c 0.3, CHCl ); IR (film) ν 1755, 1643 cm ;
3
max
1
13
0 C
1 CH2
2 CH2
3 C
4 C
5 CH
H and C NMR see Tables S1 and S2, Supporting Information;
+
HRFABMS m/z 743.3979 [M + Na] (calcd for C H O Na,
39
60 12
743.3982).
X-ray Crystallographic Data of Tetra-O-acetylnicandroside A
4a). The data were collected from a colorless needle (0.446 ×
.136 × 0.032 mm) 298(2) K: C H O , M 720.87; monoclinic,
space group P2 ; a = 10.8266(14) Å, b = 7.7886(11) Å, c = 23.976(3)
Å; α = 90°, β = 90.913°, γ = 90°, V = 2021.5(5) Å ; Z = 2; D
.184 g/cm ; F(000) = 780. Reflections collected 6812; independent
2
9
(
0
3
9
60 12
r
1
6 CH2
3
=
calc
7 CH
3
1
8 CH3
9 CH3
0 C
reflections 4555 for I ≥ 2σ (I).
20
Penta-O-acetylnicandroside B (5a): colorless oil; [α]
.2, CHCl ); IR (film) ν 1756, 1643 cm ; H and C NMR see
−20 (c
D
−1
1
13
0
3 max
1 CH3
Tables S1 and S2 (Supporting Information); FABMS m/z 701 [M +
+
+
2 CH
3 CH2
4 C
5 C
6 C
Na] (C H O Na), 679 [M + H] (C H O ).
36 54 12 36 55 12
Tri-O-acetylnicantriol/tri-O-acetyl-14-epi-nicantriol (6a/7a): col-
orless oil; IR (film) νmax 1746, 1646 cm ; H and C NMR see
Tables S1 and S2 (Supporting Information); HRFABMS m/z
−
1
1
13
+
4
49.2906 [M + H] (calcd for C H O 449.2903).
26 41
6
6
-O-Acetylphysanicandrolide C (16a): colorless crystals; mp 263−
64 °C; [α] +98 (c 0.2, CHCl ); UV (MeOH) λ (log ε) 226
4.47) nm; IR (film) νmax: 3526, 1742, 1714, 1691 cm ; H and
NMR, see Tables 5 and 6; EIMS m/z 607 [M + H] (2), 564 (3), 511
(4), 265 (61); 169 (63); 125 (94), 43 (100).
7 CH3
20
2
D
3
max
8 CH3
20.6
169.7, 21.6
−1
1
13
(
C
5-OAc
169.7, 21.5
170.0, 21.7
+
a
b
c
OMe signal: δ 55.9. CH . 6-OAc signals: δ 169.4, 21.3.
C
2
C
X-ray Crystallographic Data of 6-O-Acetylphysanicandrolide C
2
9
(
16a). The data were collected from a colorless prism (0.278 ×
0
.098 × 0.058 mm) at 173(2) K: C H ClO , C H O M 695.21;
32 43 9 4 8 2 r
monoclinic, space group P2 ; a = 14.5187(14) Å, b = 7.7890(8) Å, c =
1
3
15.6480(15) Å; α = 90°, β = 91.811 (2)°, γ = 90°, V = 1768.7(3) Å ;
3
Z = 2; Dcalc= 1.305 g/cm ; F(000) = 7.44. Reflections collected 6442;
independent reflections 5139 for I ≥ 2σ (I).
Photooxidation of Compound 1. Methylene blue (5.2 mg) was
added to a solution of compound 1 (96.4 mg) in acetone (20 mL) at
0
°C. The mixture was stirred under a 100 W lamp for 22 h
(temperature was allowed to rise to 40 °C). Acetone was removed
with an air stream, and then the residue was dissolved in MeOH (10
mL) and NaBH (52 mg) was added. The mixture was stirred for 0.5
4
h, quenched with glacial HOAc (0.5 mL), concentrated, diluted with
H O (∼15 mL), and extracted with EtOAc (4 × 5 mL). The organic
2
fraction was washed with H O, dried with anhyd Na SO ,
2
2
4
concentrated, and purified by VCC (hexanes−EtOAc gradient 4:1−
0
2
:1) to give 17 mg of a mixture of semisynthetic compounds 6/7 and
2 mg of the starting material (1).
Oxidation of Compounds 6/7. Si gel supported NaIO
4
30
reagent (170 mg) was added to a solution of the mixture of 6/7
74.7 mg) in CH Cl (3 mL). The suspension was stirred at room
(
2
2
temperature for 3 h, and the solids were filtered off. The filtrate was
purified by preparative TLC eluted with hexanes−EtOAc 4:1 to give
2
0
the aldehyde 8 (19.7 mg) as a pale yellow gum: [α] +32 (c 0.4,
D
Figure 4. ORTEP projection of 6-O-acetylphysanicandrolide C (16a)
crystallographic numbering).
−
1
1
13
CHCl ); IR (CHCl ) ν 3442, 1690, 1642 cm ; H and C NMR
(
3
3
max
+
see Tables 1 and 2; HRESIMS m/z 291.23146 [M + H] (calcd for
C H O , 291.23240).
1
9
31
2
Oxidation of Semisynthetic Compounds 6/7. Si gel supported
3
0
was extracted with EtOAc. The aqueous phase was concentrated and
purified by flash CC eluted with EtOAc−MeOH 3:1 to afford 35.3 mg
of D-glucose: [α]20 +36 (c 0.2, H O).
NaIO reagent (36.4 mg) was added to a solution of the mixture of
4
6/7 (13.1 mg) in CH Cl (3 mL). The suspension was stirred at
room temperature for 2.5 h, and the solids were filtered off. The
2
2
D
2
J
DOI: 10.1021/acs.jnatprod.9b00233
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
filtrate was purified by flash CC hexanes−acetone 19:1 to give the
REFERENCES
2
0
■
aldehyde 8 (2.4 mg): [α] +30 (c 0.1, CHCl ).
D
3
(
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2
3
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́
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presented as the mean ± standard deviation.
(
(
́
(
́
Acetylcholinesterase Assay. Inhibition of acetylcholinesterase
(
1
(
(
AChE from Electrophorus electricus) activity was determined using
32 33
Ellman’s colorimetric method with some modifications. Briefly, a
solution (50 μL) of test compound in 2% MeCN in PBS (11.3 mM,
́
ez-Castorena,
−
1
pH 7.4) was added to an enzyme solution (50 μL, 0.195 U mL ) and
incubated for 30 min at 25 °C. The reaction was started by addition of
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̈
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ATChI) in PBS (15.85 mM, pH 7.5). The formation of the colored
product was measured in a Synergy HT Bio Tek microplate reader at
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(
4
Physostigmine was used as a positive control. The percentages of
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Significant differences between groups were determined at p ≤ 0.05
and p ≤ 0.01
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ASSOCIATED CONTENT
■
(
16) Cambie, R. C.; Moratti, S. C.; Rutledge, P. S.; Weston, R. J.;
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*
S
Supporting Information
(
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00233.
(
́
, R.; Zacchino, S.;
̃
Tables S1 and S2 containing the H and 13C NMR data
of derivatives 1a, 2a, and 6a/7a; 1D and 2D NMR
spectra of compounds 1−7, 9, 14−16, and 5a, and 1D
NMR spectra of their derivatives as well as the results of
the AChE inhibition assay (PDF)
1
́
́
̃
2
(
(
(
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Corresponding Author
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*
Tel: 525556224412. Fax: 525556162217. E-mail:
emmaldon@unam.mx.
2
(
ORCID
Emma Maldonado: 0000-0002-2971-4029
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
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■
́
We are grateful to H. Rios, I. Chav
́
ez, B. Quiroz, R. Gavin
a, and E. Huerta for the NMR spectra; R. Patino for the IR
ez, and L. Triana for the
quez for the HPLC analysis. This
work was supported by CONACyT (Project 34993-N).
̃
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(
(
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29) Crystallographic data for the structures reported in this paper
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have been deposited with the Cambridge Crystallographic Data
Centre: CCDC 1850443 (1), CCDC 1850444 (4a), and CCDC
1850445 (16a). Copies of these data can be obtained free of charge
́
K
DOI: 10.1021/acs.jnatprod.9b00233
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