Withaferin A and withanolide D analogues with dual heat-shock-inducing and cytotoxic activities: Semisynthesis and biological evaluation

Article abstract of DOI:10.1021/acs.jnatprod.7b00918

Withanolides constitute a valuable class of bioactive natural products because some members of the class are known to exhibit cytotoxic activity and also induce a cytoprotective heat-shock response. In order to understand the relationship between their structures and these dual bioactivities of the withanolide scaffold, we obtained 25 analogues of withaferin A (WA) and withanolide D (WD) including 17 new compounds by semisynthesis involving chemical and microbial transformations. Hitherto unknown 16β-hydroxy analogues of WA and WD were prepared by their reaction with triphenylphosphine/iodine, providing unexpected 5β-hydroxy-6α-iodo analogues (iodohydrins) followed by microbial biotransformation with Cunninghamella echinulata and base-catalyzed cyclization of the resulting 16β-hydroxy iodohydrins. Evaluation of these 25 withanolide analogues for their cytotoxicity and heat-shock-inducing activity (HSA) confirmed the known structure-activity relationships for WA-type withanolides and revealed that WD analogues were less active in both assays compared to their corresponding WA analogues. The 5β,6β-epoxide moiety of withanolides contributed to their cytotoxicity but not HSA. Introduction of a 16β-OAc group to 4,27-di-O-acetyl-WA enhanced cytotoxicity and decreased HSA, whereas introduction of the same group to 4-O-acetyl-WD decreased both activities.

Full text of DOI:10.1021/acs.jnatprod.7b00918

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Withaferin A and Withanolide D Analogues with Dual Heat-Shock-
Inducing and Cytotoxic Activities: Semisynthesis and Biological
Evaluation
E. M. Kithsiri Wijeratne,† Maria C. F. Oliveira,†,‡ Jair Mafezoli,†,‡ Ya-Ming Xu,† Sandro Minguzzi,†,§
Pedro H. J. Batista,‡ Otília D. L. Pessoa,‡ Luke Whitesell,*,⊥,∥ and A. A. Leslie Gunatilaka*,†
†Natural Products Center, School of Natural Resources and the Environment, College of Agriculture and Life Sciences, University of
Arizona, 250 E. Valencia Road, Tucson, Arizona 85706, United States
‡Departamento de Química Organ
60455-970, Brazil
̂ ̂ ́
ica e Inorganica, Universidade Federal do Ceara, Campus do Pici, Caixa Postal 6044, Fortaleza-CE,
§Curso de Química, Universidade Estadual de Mato Grosso do Sul, Unidade de Navirai, Rua Emilio Mascoli 275, Navirai-MS,
79950-000, Brazil
⊥Whitehead Institute for Biomedical Research, 455 Main Street, Cambridge, Massachusetts 02142, United States
S
* Supporting Information
ABSTRACT: Withanolides constitute a valuable class of bioactive natural products because some members of the class are
known to exhibit cytotoxic activity and also induce a cytoprotective heat-shock response. In order to understand the relationship
between their structures and these dual bioactivities of the withanolide scaffold, we obtained 25 analogues of withaferin A (WA)
and withanolide D (WD) including 17 new compounds by semisynthesis involving chemical and microbial transformations.
Hitherto unknown 16β-hydroxy analogues of WA and WD were prepared by their reaction with triphenylphosphine/iodine,
providing unexpected 5β-hydroxy-6α-iodo analogues (iodohydrins) followed by microbial biotransformation with Cunning-
hamella echinulata and base-catalyzed cyclization of the resulting 16β-hydroxy iodohydrins. Evaluation of these 25 withanolide
analogues for their cytotoxicity and heat-shock-inducing activity (HSA) confirmed the known structure−activity relationships for
WA-type withanolides and revealed that WD analogues were less active in both assays compared to their corresponding WA
analogues. The 5β,6β-epoxide moiety of withanolides contributed to their cytotoxicity but not HSA. Introduction of a 16β-OAc
group to 4,27-di-O-acetyl-WA enhanced cytotoxicity and decreased HSA, whereas introduction of the same group to 4-O-acetyl-
WD decreased both activities.
analogues have also been found to induce the cytoprotective
lants used in traditional medicines constitute valuable
Presources for the identification of therapeutic leads with a heat-shock response.17,18 Biological activities reported for WD
(2) include cytotoxicity against lymphoma,19 pancreatic
adenocarcinoma,20 and leukemia21 cell lines. Studies in mice
have demonstrated anticancer activity for WA against brain,17
breast,8 prostate,15 and pancreatic14a cancers and immunomo-
dulatory activity for WD.22
Structurally, WA (1) and WD (2) are isomeric withanolides
that feature 2(3)-en-1-one, 4β-hydroxy, and 5β,6β-epoxy
moieties in their A and B rings. They differ from each other
only in the position of the additional hydroxy group present in
their side chain (C-27 in 1 and C-20 in 2) (see Figure 1).
Although 1 has been subjected to a variety of chemical and
variety of structural scaffolds and biological activities.1 Withania
somnifera (L.) Dunal (Indian ginseng, “Ashwagandha” in
Ayurveda) is a prominent plant in traditional Indian medicine,2
with claimed anticancer,3 neuroprotective,4 anti-inflammatory,5
antioxidant,2 and immunomodulatory6 activities. Molecular
pharmacological studies have tied many of these biological
activities of W. somnifera to its constituent withanolides, a group
of steroidal lactones, including withaferin A (1, WA) and
withanolide D (2, WD) (Figure 1).7 WA, the most extensively
studied among withanolides, has been reported to have
cytotoxic activity against breast carcinoma,8 cervical cancer,9
cutaneous melanoma,10 glioma,11 leukemia,12 ovarian carcino-
ma,13 pancreatic cancer,14 prostate adenocarcinoma,15 and renal
carcinoma16 cell lines. Intriguingly, WA and some of its
Received: October 31, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Article
Figure 1. Structures of withaferin A (1), withanolide D (2), their analogues 3−27, and the 4,5-cyclic carbonate derivative (28) of 19.
microbial transformations yielding an array of structural
analogues, similar studies have not been reported for 2.
Significant chemical transformations of WA (1) have involved
(i) conversion of the 2(3)-en-1-one to a 1-one functionality;18
(ii) preparation of 3-substituted 2,3-dihydro analogues by
Michael addition of nucleophiles (e.g., azide and alkyl/aryl
thiols);18,23 (iii) preparation of spiro-pyrrolizidine-indole
adducts across the 2,3-double bond;24 (iv) derivatization of
4β-OH and/or 27-OH groups to their corresponding acetates18
and silyl ethers;25 (v) oxidation of the 4β-OH to a 4-oxo group
followed by reduction leading to the 4α-OH analogue and its
derivatization to the acetate;18 (vi) conversion of the 5β,6β-
epoxy moiety into its corresponding 1,4-oxathiane analogues,26
thiiranes, amino alcohols, and alcohols;27 and (vii) substitution
of the 27-OH group by an azido group.28 When subjected to
microbial biotransformation using the fungus Cunninghamella
echinulata, 1 afforded its 12β-hydroxy (3) and 15β-hydroxy (4)
analogues,18,29 which have been converted to their correspond-
ing acetates.18
on cytotoxic activity. We recently reported evaluation of 24
structural analogues of 1 for cytotoxicity and activation of the
cellular heat-shock response, leading to a conclusion that the
withanolide scaffold is readily amenable to structural
modifications that modulate these activities.18 Herein we report
the preparation and characterization of 25 withanolide
analogues derived from 1 and 2 including 17 new compounds.
Evaluating the influence of structural changes on the cytotoxic
and heat-shock induction activities of these compounds
provided interesting new SAR insights. Certain structural
modifications of 1 and 2 enhanced cytotoxicity over heat-
shock induction activity, while other modifications were found
to have an opposite effect.
RESULTS AND DISCUSSION
■
Withaferin A (1) was obtained from aeroponically grown W.
somnifera.30 Upon microbial biotransformation with C.
echinulata, it provided the expected hydroxylation products,
12β-hydroxywithaferin A (3) and 15β-hydroxywithaferin A
(4).18,29 Other known analogues of WA and their acetates 6, 7,
15, and 16 were obtained as described previously.18 New
analogues 5, 8−10, 12, 13, and 17−27 of WA (1) and WD (2)
Despite the availability of a considerable number of naturally
occurring and semisynthetic analogues of WA (1), only a few of
these have been subjected to any systematic structure−activity
relationship (SAR) studies, and almost all of these have focused
B
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were prepared by the chemical reactions and microbial
biotransformations depicted in Scheme 1. Because 5,6-
pyridine for an extended period of time afforded the diacetate
22 and not the corresponding triacetate, suggesting that the
OH group of 19 resided at C-5 and not C-6. This was
confirmed by the formation of the 4,5-cyclic carbonate (28) on
treatment of 19 with carbonyl diimidazole.34 The orientation of
H-6 in 19 was determined as β based on its 1H NMR data (δH
4.65, dd, J = 13.7, 4.3 Hz)31b and the observed NOE association
between H-6 and CH3-19 (Figure S39, Supporting Informa-
tion). These findings established the structure of 19 as (22R)-
4β,5β,27-trihydroxy-6α-iodo-1-oxowitha-2,24-dienolide. As
noted above, the reaction of steroidal 5β,6β-epoxides with
Ph3P−I2 has been reported to afford 5α-iodo-6β-hydroxy
analogues as major products,33 and formation of these has been
explained as due to the attack by the nucleophile (I−) at the
more substituted position (C-5) of the epoxide ring via a
borderline SN2 mechanism (Scheme 2).35 However, during the
reaction of 1 with Ph3P−I2, it is possible that this reagent first
reacts with the hydroxy group at C-4.36 Introduction of the
bulky Ph3PI moiety at this position would then result in steric
crowding, causing I− to react at the less substituted position (C-
6) of the epoxide with or without participation of a cyclic
dioxyphosphorane that subsequently undergoes ready hydrol-
ysis37 to afford 5β-hydroxy-6α-iodo analogue 19 as the major
product (Scheme 2).
Interestingly, microbial biotransformation of the iodohydrin
19 with C. echinulata afforded 20 (27%) and three minor
products, 21 (8%), 5 (7%), and 4 (3%). The difference in
molecular mass of 16 Da between 19 and its biotransformation
products 20 and 21 suggested that these were monohydroxy-
lated analogues of 19. This was confirmed by the presence of
signals due to hydroxymethine groups in the NMR spectra of
20 [δH 4.26 (ddd, J = 11.8, 7.4, 4.0 Hz); δC 71.2] and 21 [δH
4.17 (t, J = 6.3 Hz); δC 69.1]. The newly introduced OH
groups in 20 and 21 were located at C-16 and C-15,
Scheme 1. Preparation of New Analogues and Derivatives of
a
Withaferin A (1) and Withanolide D (2)
a
Reagents and conditions: (a) Ph3P−I2, CH2Cl2, 25 °C, 2 h; (b)
microbial biotransformation with Cunninghamella echinulata; (c) N,N-
diisopropylethylamine, CH2Cl2, 25 °C, 72 h; (d) Ac2O, pyridine, 25
°C, 16 h; (e) MnO2, CHCl3−EtOAc, 25 °C, 6 h; (f) NaBH4, CeCl3·
7H2O, MeOH, THF, 0 °C, 5 min.
chlorohydrin analogues of WA have been reported to have
potent cytotoxic activity31 and some iodine-containing
compounds have found applications in targeted cancer
therapy,32 it was of interest to prepare and evaluate the new
5,6-iodohydrin analogues of 1 and 2. Steroidal 5β,6β-epoxides
are known to react with phosphine−iodine complexes to yield
their corresponding iodohydrin (5α-iodo-6β-hydroxy) ana-
logues (Scheme 2).33 Treatment of 1 with a Ph3P−I2 complex
in CH2Cl2 at 25 °C afforded its iodohydrin (19) in 93% yield.
The 13C NMR chemical shift for C-6 (δC 38.6) of 19 indicated
that it is a 5-hydroxy-6-iodo analogue and not the anticipated 5-
iodo-6-hydroxy analogue of WA. Exposure of 19 to Ac2O−
Scheme 2. Proposed Mechanisms for the Reaction of Triphenylphosphine−Iodine Complex with Steroidal 5β,6β-Epoxides and
4β-Hydroxywithanolides, Withaferin A (1), and Withanolide D (2)
C
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Figure 2. Selected NOE correlations for 9, 17, 19, and 26, ROESY correlations for 20, and HMBC correlations for 26.
respectively, by comparison of their 13C NMR data with those
of 19. Thus, the hydroxymethine carbon of 20 was assigned to
C-16 on the basis of the downfield shifts of C-17 (ΔδC 6.0, β-
effect) and C-15 (ΔδC 12.8, β-effect) and upfield shift of C-14
(ΔδC −2.7, γ-effect). The orientation of OH-16 in 20 was
confirmed as β by the upfield shift of C-20 (ΔδC −5.2, γ-gauche
effect) due to the syn-configuration of OH-16 and C-2038 and
the observed ROESY correlation between H-14 (δH 0.97) and
H-16 (Figure 2). Based on this evidence, the structure of 20
was elucidated as (22R)-4β,5β,16β,27-tetrahydroxy-6α-iodo-1-
oxowitha-2,24-dienolide. The hydroxymethine carbon of the
second biotransformation product (21) was assigned to C-15
on the basis of the downfield shifts observed for C-14 (ΔδC 4.4,
β-effect) and C-16 (ΔδC 13.6, β-effect) compared to chemical
shifts observed in the 13C NMR spectrum of 19.35 The
orientation of OH-15 of 21 was determined as β by its
conversion to the known WA analogue 15β-hydroxywithaferin
A (4).18 Thus, the structure of 21 was established as (22R)-
4β,5β,15β,27-tetrahydroxy-6α-iodo-1-oxowitha-2,24-dienolide.
The formation of two minor products, 15β-hydroxywithaferin A
(4)18 and 16β-hydroxywithaferin A (5) (see below), during the
biotransformation reaction of the iodohydrin 19 was suspected
to be due to the elimination of a molecule of HI from 20 and
21 to form their corresponding 5β,6β-epoxides. This
observation was exploited for the chemical conversion of the
biotransformation products, 20 to 4 and 5 to 21. Treatment of
21 with N,N-diisopropylethylamine in CH2Cl2 gave a product
identical to 15β-hydroxywithaferin A (4) previously obtained as
a biotransformation product of WA (1).18,29 Similar treatment
of (22R)-4β,5β,16β,27-tetrahydroxy-6α-iodo-1-oxowitha-2,24-
dienolide (20) gave a product that was identified as 16β-
hydroxywithaferin A (5) by the analysis of its HRMS and NMR
data (Figures S1−S4, Supporting Information). To the best of
our knowledge, this finding constitutes the first report of
microbial biotransformation of a substrate containing an iodine
substituent and the ability of C. echinulata to hydroxylate at C-
16 of a withanolide. As noted above, microbial biotransforma-
tion of WA (1) with C. echinulata has previously afforded 12β-
hydroxywithaferin A (3) and 15β-hydroxywithaferin A (4) as
the only products.21,33 It is possible that the conformational
change caused by the absence of the 5β,6β-epoxide moiety in
iodohydrin 19 of WA allowed the hydroxylating enzyme of C.
echinulata to hydroxylate at C-15 and C-16 instead of C-15 and
C-12.
Withanolide D (2) was obtained from Acnistus arborescens
(L.) Schlecht (Solanaceae)39 and W. somnifera.40 Upon
microbial biotransformation with C. echinulata, it afforded 9
as the only product. The molecular weight difference of 16 Da
between 2 and 9 observed by MS and the presence of signals
due to a hydroxymethine group [δH 4.20 (m); δC 69.5] in the
NMR spectra of 9 suggested that it was a monohydroxylated
product of 2. The downfield shifts of the signals due to C-14
(ΔδC 4.7) and C-16 (ΔδC 12.8) in the 13C NMR spectrum of 9
compared to those of 2 suggested that the microbial
hydroxylation has occurred at C-15.38 The orientation of
OH-15 in 9 was determined as α based on strong NOESY
correlations observed for H-15 (δH 4.20) with H-14 (δH 0.78)
and H-17 (δH 1.54) (see Figure 2 and Figure S15, Supporting
Information). Thus, the structure of 9 was elucidated as 15β-
hydroxywithanolide D.
Semisynthesis of 4-epi-withanolide D (17) was efficiently
achieved by the MnO2 oxidation18,41 of withanolide D (2) to 4-
dehydrowithanolide D (14) followed by regio- and stereo-
selective reduction of the carbonyl group at C-4 with NaBH4−
18
CeCl3 (Scheme 1). The NOE data for 17 confirmed an α-
orientation for the OH group at C-4 (Figure S32, Supporting
Information). The treatment of WD (2) with Ph3P−I2 in
CH2Cl2 at 25 °C afforded 25. Comparison of its NMR data
with those of 19 suggested that the opening of the 5β,6β-
epoxide ring of 2 resulted in the formation of an iodohydrin
(5β-hydroxy-6α-iodo analogue) similar to the product formed
from WA (1) upon treatment with Ph3P−I2 (Scheme 2).
Analysis of its HRMS and NMR data (Figures S61−S64,
Supporting Information) identified 25 as (22R)-4β,5β,20-
trihydroxy-6α-iodo-1-oxowitha-2,24-dienolide. Microbial bio-
transformation of 25 with C. echinulata afforded 26. The
molecular weight difference of 16 Da between 25 and 26 in
their mass spectra and the presence of an oxygen-bearing
carbon at δC 73.6 in the 13C NMR spectrum of 26 suggested it
to be a monohydroxylated product of 25. The proton of this
D
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Figure 3. Heat-shock-inducing and cytotoxic activities of withaferin A (1), withanolide D (2), and their analogues, 5−27. (A) Heat-shock reporter
induction in confluent 293T cells after overnight exposure to the indicated compounds at 1.0 and 5.0 μM. (B) Inhibition of 293T cell growth and
survival as measured by resazurin dye reduction after 72 h of exposure to the indicated compounds at 1.0 and 5.0 μM. In both panels, the mean of
two biological replicates for each determination is depicted.
newly introduced OH group of 26 (δH 4.21) showed HMBC
correlations with C-15 (δC 36.1), C-16 (δC 73.6), and C-17 (δC
58.0) locating the OH group at C-16 (Figure S69, Supporting
Information). The orientation of this OH group was
determined to be β based on the observed NOE correlation
between H-17 (δH 1.27) and H-16 (Figure S70, Supporting
Information). Thus, the biotransformation product of 25 was
identified as (22R)-4β,5β,16β,20-tetrahydroxy-6α-iodo-1-oxo-
witha-2,24-dienolide (26). As expected, treatment of 26 with
N,N-diisopropylethylamine in CH2Cl2 afforded 16β-hydrox-
ywithanolide D (10) as the only product. The O-acetyl
analogues 6−8, 11−13, 16, 18, 22−24, and 27 were obtained
by the acetylation (Ac2O−pyridine) of the corresponding
hydroxywithanolides (see Experimental Section).
We have previously used the heat-shock response (HSR) as a
biosensor to discover potential anticancer agents targeting
protein homeostasis. Using this approach, it was found that WA
(1) and several of its analogues strongly activated the heat-
shock factor 1 (HSF1)-dependent stress response as a
prominent component of their anticancer activity.17,18 Because
WA and its analogues have also been reported to have potent
cytotoxic activity,18,25,42 it was of interest to investigate the
specific structural determinants that might contribute to these
distinctive bioactivities for withanolides. Thus, WA (1), WD
(2), and their analogues 5−27 were evaluated for their ability to
activate the HSR using a cell-based reporter system, and their
cytotoxic activity was measured in the same reporter cell line
(293T), initially at screening concentrations of 1.0 and 5.0 μM.
WA (1) was used as a positive control and internal standard for
comparison between assays because it possesses both these
activities. WA analogues 3 and 4 were previously shown to have
no heat-shock-inducing activity (HSA) and therefore were not
evaluated in this study.18 To measure concentration-dependent
activation of the HSR of 1, 2, and 5−27, serial dilutions of each
compound were applied in a 384-well format to human 293T
reporter cells stably expressing a green fluorescent protein−
firefly luciferase fusion protein under transcriptional control of
classical heat-shock promoter elements.43 After overnight
incubation, luciferase activity was determined as a quantitative
measure of relative heat-shock activation. Depicted in Figure 3A
is Log2 of the maximal HSA (fold induction) for each of the
compounds when tested at concentrations of 1.0 and 5.0 μM.
These data suggested that WA analogues were more active than
their corresponding WD analogues. WA analogues 6−8, 16,
and 23 exhibited enhanced heat-shock activation compared to
WA (1) at both concentrations, whereas 15 showed higher
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Figure 4. Concentration-dependent inhibition of growth and proliferation of 293T cells (A and B) and CHP-100 cells (C and D) by WA (1), WD
(2), and their active analogues, 6−8, 11, 13, 15, 16, and 23, at 10 concentrations. All determinations were performed in 384-well format using
quadruplicate replicates.
activity than 1 at 5.0 μM. Although WD (2) was less active, its
analogues 11, 13, and 14 showed the same or higher HSA in
comparison to WA (1) at a concentration of 1.0 μM.
Concentration-dependent HSA of WA (1), WD (2), and
their analogues 6−8, 11, 13, 15, 16, and 23 was determined by
incubating heat-shock reporter 293T cells overnight in a 384-
well format followed by measurement of luciferase activity on a
microplate luminometer. Heat-shock-inducing activity for 1, 2,
6−8, 11, 13, 15, 16, and 23 as depicted in Figure 5
demonstrates that reporter response was not a monotonic
function but rather peaked over a relatively limited concen-
tration range for each active compound and then declined,
presumably as toxicity compromised the ability of cells to
respond. Presented in Table 1 is the area under the curve
(AUC in arbitrary units) as a measure of relative activity for
each of these withanolides together with those for the classical
heat-shock activators MG-132 (proteasome inhibitor) and
STA-9090 (Ganetespib) (HSP90 inhibitor) (Figure S78,
Supporting Information). These compounds were included
because the proteasome and HSP90 have been reported as
potential targets for WA (1).14a
To determine whether the dual heat-shock induction and
cytotoxic activities of withanolides 1, 2, 6−8, 11, 13, 15, 16,
and 23 could be dissected on the basis of their structural
features, potential correlation of the two activities was examined
across these analogues. The ratio of AUC for heat-shock
induction divided by cytotoxicity (IC50) was calculated for each
analogue when applied to the 293T reporter line (Table 1).
Analogues 6−8, 11, 16, and 23 enhanced heat-shock-inducing
potential compared to WA (1) in the order 16 > 7 > 8 > 6 > 23
> 11. To better visualize the relationship between activities for
these compounds, a scatter plot is provided (Figure 6). The
absence of a linear correlation between activities, especially for
WA analogues, supports the reported conclusion that heat-
shock induction by withanolides is not a simple consequence of
cytotoxicity nor solely a result of impairment of the
proteasome, HSP90, or any other single mediator of protein
homeostasis. As is evident in Figure 6, there was actually
Next, the cytotoxicity of WA (1), WD (2), and their
analogues 5−27 was determined using the same reporter cell
line (293T) at 1.0 and 5.0 μM concentrations. Relative cell
proliferation and survival were measured by resazurin dye
reduction after 72 h of compound exposure.44 As evident in
Figure 3B, the cytotoxic activity of WA analogues was more
pronounced than that of the corresponding WD analogues. WA
analogues 6−8, 15, 16, 19, and 23, as well as WD (2) and its
analogue 11, were found to be more cytotoxic than WA (1). In
follow-up experiments, the concentrations resulting in 50%
inhibition (IC50’s) were determined for those cytotoxic
compounds (6−8, 11, 15, 16, and 23) that exhibited enhanced
HSA compared to 1. WA (1) and WD (2) were included for
comparison purposes. The IC50’s of these withanolides for the
human Ewing’s sarcoma cell line CHP-100 were also
determined using the same resazurin dye reduction assay.44
This rapidly proliferating cell line was chosen to maximize
sensitivity over a short period (24 h) of compound exposure
and for the purpose of comparing data obtained for WA
analogues in our previous studies.17,18 Concentration-depend-
ent growth inhibition curves for these withanolides when
applied to the 293T and CHP-100 cell lines are presented in
Figure 4. IC50 data derived from these curves are summarized in
Table 1. These data established the following order of cytotoxic
potency for withanolide analogues included in this study. For
the CHP-100 cell line it was 8 > 7 > 16 > 6 > 11 > 15 > 2 > 1 >
23, whereas for the 293T cell line the order of potency was
found to be 8 > 7 > 11 > 16 > 6 > 15 > 1 > 23 > 2, suggesting
some degree of cell-line-selective cytotoxicity of these
compounds by mechanism(s) that remain to be defined in
future work.
F
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Table 1. Concentration-Dependent Heat-Shock Induction
and Cytotoxicity Data for WA (1), WD (2), and Their
Analogues 6−8, 11, 15, 16, and 23
cell line
CHP-100
293T
heat-shock
induction
ratio (heat-
shock
induction/
cytotoxicity)
cytotoxicity, cytotoxicity,
(reporter
a
b
c
compound
IC50 (nM)
IC50 (nM)
AUC)
1
424
324
232
120
59
299
710
180
116
88
68
0.23
0.02
1.95
3.61
2.23
0.50
0.18
4.45
0.56
2
13
6
351
419
196
71
7
Figure 6. Correlation of heat-shock-inducing activity with cytotoxicity
for WA (1), WD (2), and their analogues, 6−8, 11, 13, 15, 16, and 23.
The dashed line depicts an idealized linear relationship between
bioactivities. Heat-shock induction and cytotoxic activities were
determined in 293T cells as described in Table 1.
8
11
15
16
23
292
311
142
466
141
250
154
417
44
685
235
332
59
d
f
f
MG-132
ND
ND
e
f
f
STA-9090
ND
ND
a
particularly interesting that the 5,6-iodohydrin (23) of 4,27-di-
O-acetylwithaferin A (6) showed 3.5-fold higher HSA, but 1.4-
fold less cytotoxicity compared to WA (1). The remaining
analogues 7 and 8 with higher HSA were found to be more
cytotoxic than 1. Comparison of HSA and cytotoxic potencies
of WA analogues 6 and 23 suggested that the presence of the
5β,6β-epoxide moiety in 6 may be responsible for its enhanced
cytotoxic activity.
Concentration resulting in 50% reduction in relative viable cell
number after 24 h of compound exposure based on four-parameter
curve fit of dose−response data using GraphPad Prism 6 software; R2
for all curve fits ≥ 0.95; all determinations were performed in 384-well
b
format using quadruplicate replicates. Concentration resulting in 50%
reduction in relative viable cell number after 72 h of compound
exposure based on four-parameter curve fit of dose−response data
using GraphPad Prism 6 software; R2 for all curve fits ≥ 0.95. All
determinations were performed in 384-well format using quadruplicate
c
replicates. Heat-shock reporter 293T cells were incubated in serial
In summary, while confirming the previous SAR data for the
cytotoxic activity and HSA of some analogues of WA (1), we
now show that (i) analogues of WD (2) exhibit reduced
cytotoxicity and heat-shock induction activity compared to their
corresponding WA analogues; (ii) the 5β,6β-epoxide moiety of
withanolides contribute to their cytotoxicity but not HSA; and
(iii) introduction of a 16β-OAc group to 4,27-di-O-acetyl-WA
enhances cytotoxicity and decreases HSA, whereas introduction
of a 16β-OAc group to 4-O-acetyl-WD decreases both activities.
From these SAR data and those from the previous studies,18 it
may be concluded that the withanolide scaffold is amenable to
structural modifications, leading to modulation of one or both
of its dual heat-shock induction and cytotoxic activities. The
more potent bioactivities found for withanolide analogues 7, 8,
and 16 suggest that further translational investigation and/or in
vivo evaluation of these compounds should be considered.
dilutions of the indicated compounds overnight in 384-well format
followed by measurement of luciferase activity on a microplate
luminometer; data were plotted over a 4-Log10 concentration range as
the mean ratio of treated to control, with all determinations performed
in quadruplicate wells. The area under the curve (AUC in arbitrary
units) for each plot was calculated using GraphPad Prism 6 software.
d
e
f
Proteasome inhibitor. HSP90 inhibitor. ND = not determined.
relatively poor correlation between heat-shock induction and
cytotoxic activities across the panel of bioactive withanolides.
Indeed, analogues 16, 23, and 6−8 all showed substantially
higher HSA than WA (1) while demonstrating varying degrees
of cytotoxic activity. Our previous studies have shown that 4,27-
di-O-acetyl-4-epi-withaferin A (16) was capable of inducing the
heat-shock response in cell culture at subcytotoxic concen-
trations and in mice at systemically tolerable doses.18 It is
Figure 5. Concentration-dependent induction of heat-shock reporter activity by WA (1), WD (2), and their active analogues, 6−8, 11, 13, 15, 16,
and 23, and the controls, MG-132 (proteasome inhibitor) and STA-9090 (HSP90 inhibitor). Heat-shock reporter cells were incubated with the
indicated compound overnight in a 384-well format followed by measurement of luciferase activity on a microplate luminometer. Data are plotted as
the mean ratio of treated to control, with all determinations performed in quadruplicate wells. Error bars, SD.
G
DOI: 10.1021/acs.jnatprod.7b00918
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
60.9 (CH, C-14), 55.0 (CH, C-17), 47.8 (C, C-10), 44.2 (CH, C-9),
41.9 (C, C-13), 40.6 (CH2, C-12), 34.6 (CH2, C-16), 31.1 (CH2, C-
23), 30.5 (CH2, C-7), 25.2 (CH, C-8), 21.3 (CH2, C-11), 20.5 (CH3,
C-28), 20.3 (CH3, C-21), 17.0 (CH3, C-19), 16.2 (CH3, C-18), 12.4
(CH3, C-27); HRESIMS m/z 509.2510 [M + Na]+ (calcd for
C28H38NaO7, 509.2547).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined in capillary tubes using an Electrothermal melting point
apparatus and are uncorrected. Optical rotations were measured with a
JASCO Dip-370 digital polarimeter using CHCl3 or MeOH as solvent.
1D and 2D NMR spectra were recorded in CDCl3 with a Bruker
Preparation of 4-Dehydrowithanolide D (14). To a solution of
withanolide D (2, 10.0 mg) in CHCl3−EtOAc (75:25, 3.0 mL) was
added MnO2 (18.5 mg), and the mixture was stirred at 25 °C. After 16
h (TLC control), the reaction mixture was filtered, the filtrate was
evaporated under reduced pressure, and the residue was subjected to
silica gel (1.0 g) CC using CH2Cl2−MeOH (96:4) to afford 4-
dehydrowithanolide D (14) (9.6 mg, 96%) as a white powder: mp
273−274 °C (lit.44 272−275 °C); [α]D25 +112 (c 0.8, MeOH) [lit.44
1
AVANCE III instrument at 400 MHz for H NMR and 100 MHz for
13C NMR using residual CHCl3 as the internal standard. The chemical
shift (δ) values are given in parts per million (ppm), and the coupling
constants (J values) are in Hz. Low-resolution mass spectra were
obtained using a Shimadzu LCMS-QP8000α mass spectrometer,
whereas high-resolution mass spectra were recorded on Shimadzu
LCMS-IT-TOF (225-07100-34 equipped with ESI and APCI as
ionization sources) and Agilent G6224A TOF mass spectrometers. All
reagents for chemical and microbial transformations were purchased
from Sigma-Aldrich (St. Louis, MO, USA) and Fisher Scientific
(Pittsburgh, PA, USA). All solvents used were distilled before use. The
progress of all reactions was monitored by TLC on silica gel 60 F254
plates (Merck, Darmstadt, Germany), and spots were visualized under
UV and using a solution of anisaldehyde in H2SO4 and HOAc
followed by heating. Column chromatographic separations were
performed on silica gel 40 μm flash chromatography packing (J. T.
Baker, Jackson, TN, USA) or Sephadex LH-20 (25−100 μm; GE
Healthcare AB, Uppsala, Sweden). Analytical and preparative TLC
were performed on precoated 0.20 mm thick plates of silica gel 60 F254
(Merck, Darmstadt, Germany). Preparative HPLC was performed on a
Waters Delta Prep 4000 system equipped with a Waters 996
photodiode array detector and a Waters Prep LC controller utilizing
Empower Pro software and using a reversed-phase (RP) column
(Phenomenex, Luna 5 μm, C18, 100 Å, 250 × 10 mm); chromatograms
were acquired at 254 and 270 nm.
1
+113.5 (c 0.22)]; APCI-MS (+) m/z 469 [M + 1]+; H NMR data
were consistent with those reported.45
Preparation of 4-Epi-withanolide D (17). To a stirred solution of
4-dehydrowithanolide D (14) (9.0 mg) in MeOH (0.5 mL) and THF
(0.5 mL) was added CeCl3·7H2O (71.7 mg). The reaction mixture was
cooled to 0 °C in an ice bath, and NaBH4 (1.0 mg) was added. The
reaction was monitored by TLC, and after the disappearance of the
starting material ice-cold water (5.0 mL) was added, concentrated
under reduced pressure, and extracted with EtOAc (3 × 10 mL). The
combined EtOAc extracts were washed with H2O, died over
anhydrous Na2SO4, and evaporated under reduced pressure to provide
the crude product mixture. Purification by RP-HPLC (MeOH−H2O,
65:35) yielded 17 (6.2 mg, 69%, tR 22.2 min) as a white powder: mp
210−212 °C; [α]2D5 +65 (c 0.1, CHCl3); 1H NMR (CDCl3, 400 MHz)
δ 6.83 (1H, dd, J = 10.1, 1.8 Hz, H-3), 6.00 (1H, dd, J = 10.1, 2.5 Hz,
H-2), 4.69 (1H, dd, J = 2.5, 1.8 Hz, H-4), 4.17 (1H, dd, J = 13.1, 3.9
Hz, H-22), 3.65 (1H, brd, J = 2.1 Hz, H-6), 2.41, (1H, dd, J = 15.2,
13.1 Hz, H-23a), 2.16 (1H, m, H-7a), 2.06 (1H, dd, J = 15.2, 3.9 Hz,
H-23b), 1.96 (1H, m, H-12a), 1.93 (3H, s, H3-28), 1.92 (2H, m, H-11a
and H-16a), 1.86 (3H, s, H3-27), 1.62 (1H, m, H-15a), 1.51 (1H, m,
H-8), 1.50 (1H, m, H-11b), 1.43 (1H, m, H-17), 1.24 (3H, s, H3-21),
1.22 (1H, m, H-7b), 1.20 (3H, s, H3-19), 1.17 (1H, m, H-15b), 1.16
(1H, dd, J = 12.6, 5.5 Hz, H-12b), 0.90 (1H, dd, J = 10.1, 6.9 Hz, H-
14), 0.88 (1H, dd, J = 5.2, 2.4 Hz, H-9), 0.84 (3H, s, H3-18); 13C
NMR (CDCl3, 100 MHz) δ 201.1 (C, C-1), 166.0 (C, C-26), 148.9
(C, C-24), 147.4 (CH, C-3), 128.6 (CH, C-2), 122.0 (C, C-25), 80.9
(CH, C-22), 77.1 (C, C-20), 65.6 (C, C-5), 64.9 (CH, C-4), 56.4
(CH, C-14), 55.2 (CH, C-6), 54.6 (CH, C-17), 47.5 (C, C-10), 45.5
(CH, C-9), 42.8 (C, C-13), 39.8 (CH2, C-12), 31.6 (CH2, C-23), 30.6
(CH2, C-7), 29.2 (CH, C-8), 23.7 (CH2, C-15), 22.9 (CH2, C-16),
21.9 (CH2, C-11), 20.9 (CH3, C-21), 20.6 (CH3, C-28), 13.6 (CH3, C-
18), 13.5 (CH3, C-19), 12.5 (CH3, C-27); HRESIMS m/z 493.2547
[M + Na]+ (calcd for C28H38NaO6, 493.2561).
General Procedure for Preparation of 5β-Hydroxy-6α-
iodowithanolides. To a stirred solution of the withanolide (1 or
2, 50.0 mg) in CH2Cl2 (1.0 mL) at 0 °C was added a solution of Ph3P
(31.0 mg, 1.1 mol equiv) and I2 (30.0 mg, 1.1 mol equiv) in CH2Cl2.
The ice bath was removed, and the reaction mixture was stirred at 25
°C and monitored by TLC. After disappearance of the starting
material, the reaction mixture was diluted with CH2Cl2 (25 mL),
washed with aqueous Na2S2O3 solution and H2O, dried over
anhydrous Na2SO4, and evaporated under reduced pressure to yield
the crude product mixture. The crude product mixtures derived from 1
(95.1 mg) and 2 (98.3 mg) on purification by silica gel (3 g) CC
(Et2O−MeOH, 97:3) afforded 19 (59.1 mg, 93%) and 25 (61.0 mg,
96%), respectively.
Isolation of Withaferin A (1) and Withanolide D (2).
Withaferin A (1) was obtained from the aerial parts of aeroponically
grown W. somnifera,30 whereas withanolide D (2) was isolated from
the leaves of soil-cultivated A. arborescens39 and aerial parts of
aeroponically grown W. somnifera,40 as reported previously.
Microbial Biotransformation of Withanolide D (2). A spore
solution (1.0 mL, OD600 0.6) of C. echinulata (ATCC 10028B) was
transferred to four Erlenmeyer flasks (250 mL), each holding 50 mL of
soybean meal−glucose medium. After shaking for 48 h (28 °C and 160
rpm), the mycelium in each flask was separated from the liquid
medium by filtration, washed twice with 10 mL portions of phosphate
buffer solution (PBS, pH 7), and transferred to Erlenmeyer flasks (4 ×
250 mL) each holding 50 mL of PBS (pH 7) and withanolide D (2)
(5.0 mg in 0.5 mL of dimethylformamide (DMF)). After shaking for
48 h (28 °C and 160 rpm), mycelium was separated by filtration and
the combined filtrate was extracted with EtOAc (3 × 1 L). The
resulting organic layer was washed with H2O (3 × 500 mL), dried over
anhydrous Na2SO4, and evaporated under reduced pressure, providing
the crude product (28.0 mg). This was separated by preparative RP-
TLC (RP-18) and elution with MeOH−H2O (70:30) to give partially
pure product (7.8 mg), which was further purified by RP-HPLC
[MeOH−H2O (75:25)], affording 15β-hydroxywithanolide D (9) (5.9
mg, 16%, tR 6.0 min) as a white powder: mp 268−270 °C; [α]2D5 +148
(c 0.1, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 6.92 (1H, dd, J = 9.9,
6.3 Hz, H-3), 6.18 (1H, d, J = 9.9 Hz, H-2), 4.20 (1H, m, H-15), 4.16
(1H, dd, J = 13.3, 3.5 Hz, H-22), 3.70 (1H, d, J = 6.3 Hz, H-4), 3.23
(1H, brs, H-6), 2.47 (1H, dd, J = 17.1, 13.3 Hz, H-23a), 2.37 (1H, dt, J
= 14.8, 3.3 Hz, H-7a), 2.13 (1H, ddd, J = 14.8, 8.6, 2.0 Hz, H-16a),
2.07 (1H, dd, J = 17.1, 3.5 Hz, H-23b), 1.91 (3H, s, H3-28), 1.90 (1H,
m, H-8), 1.89 (1H, m, H-16b), 1.84 (3H, s, H3-27), 1.83 (1H, m, H-
12a), 1.74 (1H, brd, J = 14.7, 4.5 Hz, H-11a), 1.54 (1H, dd, J = 11.4,
8.6 Hz, H-17), 1.42 (1H, m, H-11b), 1.40 (3H, s, H3-19), 1.35 (1H,
dd, J = 14.8, 5.0 Hz, H-7b), 1.21 (3H, s, H3-21), 1.14 (1H, dt, J = 12.6,
4.0 Hz, H-12b), 1.01 (3H, s, H3-18), 0.96 (1H, dd, J = 11.7, 5.0 Hz, H-
9), 0.78 (1H, dd, J = 11.7, 5.0 Hz, H-14); 13C NMR (CDCl3, 100
MHz) δ 201.3 (C, C-1), 166.0 (C, C-26), 149.2 (C, C-24), 142.1 (CH,
C-3), 132.6 (CH, C-2), 121.8 (C, C-25), 80.6 (CH, C-22), 74.7 (C, C-
20), 69.9 (C, C-4), 69.5 (CH, C-15), 64.3 (CH, C-5), 61.8 (CH, C-6),
(22R)-4β,5β,27-Trihydroxy-6α-iodo-1-oxowitha-2,24-dienolide
(19): white powder; mp >220 °C dec; [α]2D5 +64 (c 0.2, CHCl3); H
1
NMR (CDCl3, 400 MHz) δ 6.45 (1H, dd, J = 10.5, 2.3 Hz, H-3), 5.99
(1H, dd, J = 10.5, 1.6 Hz, H-2), 4.95 (1H, m, H-4), 4.65 (1H, dd, J =
13.7, 4.3 Hz, H-6), 4.39 (1H, dd, J = 13.3, 3.6 Hz, H-22), 4.37 (1H, dd,
J = 12.5, 6.2 Hz, H-27a), 4.32 (1H, dd, J = 12.5, 6.2 Hz, H-27b), 2.56
(1H, dt, J = 13.9, 4.4 Hz, H-7a), 3.05 (1H, d, J = 5.9 Hz, D2O
exchangeable, OH-4), 2.46, (1H, dd, J = 17.5, 13.4 Hz, H-23a), 2.82
(1H, t, J = 6.2 Hz, D2O exchangeable, OH-27), 2.02 (3H, s, H3-28),
1.99 (1H, m, H-7b), 1.96 (1H, m, H-20), 1.95 (1H, m, H-23b), 1.89
H
DOI: 10.1021/acs.jnatprod.7b00918
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(1H, dt, J = 12.1, 3.3 Hz, H-12a), 1.71 (1H, m, H-8), 1.67 (1H, m, H-
16a), 1.63 (1H, m, H-15a), 1.36 (1H, m, Hb −16), 1.32 (1H, m, H-9),
1.31 (1H, m, H-11a), 1.25 (3H, s, H3-19), 1.17 (1H, m, H-15b), 1.10−
1.00 (3H, m, H-12b, H-14, H-17), 0.95 (3H, d, J = 6.9, H3-21), 0.87
(1H, m, H-11b), 0.65 (3H, s, H3-18); 13C NMR (CDCl3, 100 MHz) δ
200.3 (C, C-1), 166.9 (C, C-26), 152.3 (C, C-24), 142.7 (CH, C-3),
127.6 (CH, C-2), 125.8 (C, C-25), 78.6 (CH, C-22), 77.5 (C, C-5),
66.5 (CH, C-4), 57.5 (CH2, C-27), 56.9 (C, C-10), 55.1 (CH, C-14),
51.7 (CH, C-17), 46.3 (CH, C-9), 43.3 (CH2, C-7), 43.1 (C, C-13),
38.9 (CH2, C-12), 38.7 (CH, C-20), 38.6 (CH, C-6), 37.4 (CH, C-8),
29.8 (CH2, C-23), 27.2 (CH2, C-16), 23.9 (CH2, C-15), 22.9 (CH2, C-
11), 20.0 (CH3, C-28), 13.3 (CH3, C-21), 11.8 (CH3, C-18), 9.6
(CH3, C-19); HRESIMS m/z 621.1673 [M + Na]+ (calcd for
C28H39NaIO6, 621.1684).
13.4, 4.2 Hz, H-22), 4.38 (1H, d, J = 12.7 Hz, H-27a), 4.32 (1H, d, J =
12.7 Hz, H-27b), 4.26 (1H, ddd, J = 11.8, 7.4, 4.0 Hz, H-16), 3.05
(1H, d, J = 5.1 Hz, D2O exchangeable, OH-4), 2.55 (1H, dt, J = 13.4,
4.2 Hz, H-7a), 2.47 (1H, dd, J = 17.2, 12.3 Hz, H-23a), 2.46 (1H, m,
H-20), 2.26 (1H, dt, J = 12.9, 7.6 Hz, H-15a), 2.12 (1H, m, H-23b),
2.03 (1H, m, H-7b), 2.02 (3H, s, H3-28), 1.89 (1H, dt, J = 12.8, 3.2
Hz, H-12a), 1.75 (1H, m, H-8), 1.33 (1H, m, H-9), 1.31 (1H, m, Ha-
11), 1.26 (3H, s, H3-19), 1.23 (1H, m, H-15b), 1.02 (1H, m, H-12b),
1.00 (1H, m, H-17), 0.99 (3H, d, J = 6.4 Hz, H3-21), 0.97 (1H, m, H-
14), 0.87 (3H, s, H3-18), 0.88 (1H, m, H-11b); 13C NMR (CDCl3,
100 MHz) δ 200.2 (C, C-1), 166.6 (C, C-26), 152.6 (C, C-24), 142.8
(CH, C-3), 127.7 (CH, C-2), 125.8 (C, C-25), 79.3 (CH, C-22), 77.5
(C, C-5), 71.2 (CH, C-16), 66.5 (CH, C-4), 57.7 (CH, C-17), 57.4
(CH2, C-27), 56.9 (C, C-10), 53.4 (CH, C-14), 46.3 (CH, C-9), 43.3
(CH2, C-7), 43.0 (C, C-13), 39.3 (CH2, C-12), 38.3 (CH, C-6), 36.9
(CH, C-8), 36.7 (CH2, C-15), 33.5 (CH, C-20), 31.6 (CH2, C-23),
22.5 (CH2, C-11), 20.1 (CH3, C-28), 13.4 (CH3, C-18), 13.0 (CH3, C-
21), 9.8 (CH3, C-19); HRESIMS m/z 615.1833 [M + H]+ (calcd for
C28H39IO7, 615.1774).
(22R)-4β,5β,20-Trihydroxy-6α-iodo-1-oxowitha-2,24-dienolide
1
(25): white powder; mp >164 °C dec; [α]2D5 +51 (c 0.8, CHCl3); H
NMR (CDCl3, 400 MHz) δ 6.44 (1H, dd, J = 10.5, 3.2 Hz, H-3), 5.97
(1H, dd, J = 10.5, 1.9 Hz, H-2), 4.94 (1H, m, H-4), 4.63 (1H, dd, J =
13.4, 5.0 Hz, H-6), 4.16 (1H, dd, J = 13.0, 3.6 Hz, H-22), 3.10 (1H, d,
J = 5.8 Hz, D2O exchangeable, OH-4), 2.54 (1H, dt, J = 13.4, 4.2 Hz,
H-7a), 2.43 (1H, brt, J = 13.0 Hz, H-23a), 2.05 (1H, m, H-23b), 1.99
(1H, m, H-7b), 1.91 (3H, s, H3-28), 1.87 (1H, m, H-12a), 1.84 (3H, s,
H3-27), 1.65 (1H, m, H-15a), 1.62 (1H, m, H-8), 1.56 (1H, m, H-17),
1.55 (1H, m, H-11a), 1.34−1.31 (3H, m, H-9, H-11b, H-16a), 1.24
(3H, s, H3-19), 1.19 (3H, s, H3-21), 1.18 (1H, m, H-12b), 1.15 (1H,
m, H-15b), 1.08 (1H, ddd, J = 12.5, 10.3, 6.3 Hz, H-14), 0.85 (1H, m,
H-16b), 0.78 (3H, s, H3-18); 13C NMR (CDCl3, 100 MHz) δ 200.4
(C, C-1), 166.0 (C, C-26), 149.0 (C, C-24), 142.8 (CH, C-3), 127.6
(CH, C-2), 121.9 (C, C-25), 80.6 (CH, C-22), 77.6 (C, C-5), 74.9 (C,
C-20), 66.4 (CH, C-4), 56.9 (C, C-10), 55.5 (CH, C-14), 54.4 (CH,
C-17), 46.2 (CH, C-9), 43.2 (C, C-13), 43.0 (CH2, C-7), 39.1 (CH2,
C-12), 38.6 (CH, C-6), 36.8 (CH, C-8), 31.2 (CH2, C-23), 23.4 (CH2,
C-15), 22.7 (CH2, C-16), 21.9 (CH2, C-11), 20.6 (CH3, C-28), 20.4
(CH3, C-21), 13.7 (CH3, C-18), 12.4 (CH3, C-27), 9.8 (CH3, C-18);
HRESIMS m/z 621.1711 [M + Na]+ (calcd for C28H39NaIO6,
621.1684).
(22R)-4β,5β,15β,27-Tetrahydroxy-6α-iodo-1-oxowitha-2,24-dien-
olide (21): white powder; mp >224 °C dec; [α]2D5 +156 (c 0.1,
1
CHCl3); H NMR (CDCl3, 400 MHz) δ 6.46 (1H, dd, J = 10.4, 2.6
Hz, H-3), 5.97 (1H, dd, J = 10.4, 1.8 Hz, H-2), 4.93 (1H, dd, J = 2.6,
1.8 Hz, H-4), 4.68 (1H, dd, J = 13.9, 4.8 Hz, H-6), 4.35 (1H, dt, J =
13.4, 3.6 Hz, H-22), 4.34 (1H, d, J = 12.3 Hz, H-27a), 4.28 (1H, d, J =
12.3 Hz, H-27b), 4.17 (1H, t, J = 6.3 Hz, H-15), 3.35 (1H, d, J = 5.8
Hz, D2O exchangeable, OH-4), 2.87 (1H, dt, J = 13.8, 4.4 Hz, H-7a),
2.44 (1H, dd, J = 17.1, 13.4 Hz, H-23a), 2.22 (1H, dt, J = 14.2, 9.0 Hz,
H-16a), 2.08 (1H, m, H-8), 2.05 (1H, m, H-7b), 2.02 (1H, m, H-20),
2.03 (1H, m, H-7b), 2.01 (3H, s, H3-28), 1.90 (1H, dd, J = 17.1, 3.6
Hz, H-23b), 1.84 (1H, dt, J = 12.7, 3.1 Hz, H-12a), 1.42 (1H, m, H-
16b), 1.35 (1H, m, H-11a), 1.34 (1H, m, H-9), 1.26 (3H, s, H3-19),
1.01 (1H, m, H-14), 1.00 (1H, m, H-12b), 0.94 (3H, d, J = 6.7 Hz, H3-
21), 0.92 (3H, s, H3-18), 0.86 (1H, m, H-17), 0.85 (1H, m, Hb-11);
13C NMR (CDCl3, 100 MHz) δ 200.8 (C, C-1), 167.0 (C, C-26),
153.1 (C, C-24), 143.2 (CH, C-3), 127.4 (CH, C-2), 125.8 (C, C-25),
78.6 (CH, C-22), 77.0 (C, C-5), 69.1 (CH, C-15), 66.3 (CH, C-4),
59.5 (2 x CH, C-10,C-14), 57.0 (CH2, C-27), 52.2 (CH, C-17), 46.7
(CH, C-9), 43.0 (C, C-13), 42.1 (CH2, C-7), 40.9 (CH2, C-16), 40.3
(CH2, C-12), 38.5 (2 x CH, C-6, C-8), 33.6 (CH, C-20), 30.0 (CH2,
C-23), 22.9 (CH2, C-11), 20.1 (CH3, C-28), 14.3 (CH3, C-18), 13.4
(CH3, C-21), 9.9 (CH3, C-19); HRESIMS m/z 613.1676 [M − H]−
(calcd for C28H38IO7, 613.1662).
General Procedure for Microbial Biotransformations of
Withanolide Iodohydrins 19 and 25. Small-scale fermentation of
C. echinulata (ATCC 10028B) was performed in an Erlenmeyer flask
(125 mL) containing soybean meal−glucose medium (25 mL) on a
rotary shaker operating at 160 rpm at 28 °C for 24 h.
Biotransformation was performed under the same conditions in
eight Erlenmeyer flasks (250 mL) each holding 50 mL of soybean
meal−glucose medium and inoculated with 5.0 mL of the above small-
scale culture of C. echinulata. A solution of the substrate (5 mg in 0.5
mL of DMF; a total amount of 40 mg of 19 or 25) was added to each
flask containing the inoculum (72 h old for 19 and 24 h old for 25).
After disappearance of the substrate in the culture broth (TLC
control), fermentation broths resulting from each substrate were
combined, mycelium was filtered off and washed with H2O (100 mL),
and the washings were combined with the filtrate and extracted with
EtOAc (3 × 300 mL). The combined organic extracts was washed with
H2O (3 × 200 mL), dried over anhydrous Na2SO4, and evaporated
under reduced pressure to give the crude extract. Gel-permeation
chromatography of the EtOAc extract (48.5 mg, derived from the
biotransformation of 19) over a column of Sephadex LH-20 (1.5 g)
followed by RP-HPLC [MeOH−H2O (60:40)] purification afforded
20 (11.2 mg, 27%, tR 14.5 min) and a mixture of three compounds
with the same retention time (tR 11.3 min). This mixture was
separated by preparative TLC (silica gel) using Et2O−MeOH (92:8)
to give 4 (0.8 mg, 3%, Rf 0.29), 5 (2.1 mg, 7%, Rf 0.43), and 21 (3.1
mg, 8%, Rf 0.64). Gel-permeation chromatography of the EtOAc
extract (42.3 mg) derived from the biotransformation of 25 over a
column of Sephadex LH-20 (1.5 g) followed by RP-HPLC [MeOH−
H2O (65:35)] purification afforded 26 (3.8 mg, 10%, tR 14.5 min).
(22R)-4β,5β,16β,27-Tetrahydroxy-6α-iodo-1-oxowitha-2,24-dien-
olide (20): white powder; mp >212 °C dec; [α]2D5 +148 (c 0.1,
(22R)-4β,5β,16β,20-Tetrahydroxy-6α-iodo-1-oxowitha-2,24-dien-
olide (26): white powder; mp >228 °C dec; [α]2D5 +152 (c 0.1,
1
CHCl3); H NMR (CDCl3, 400 MHz) δ 6.44 (1H, dd, J = 10.2, 1.9
Hz, H-3), 5.97 (1H, dd, J = 10.0, 2.8 Hz, H-2), 4.93 (1H, m, H-4),
4.65 (1H, dd, J = 13.4, 4.5 Hz, H-6), 4.60 (1H, m, H-16), 4.47 (1H,
dd, J = 13.0, 3.4 Hz, H-22), 4.21 (1H, d, J = 4.5 Hz, D2O exchangeable,
OH-16), 4.05 (1H, brs, D2O exchangeable, OH-20), 3.10 (1H, d, J =
5.6 Hz, D2O exchangeable, 1H, OH-4), 2.55 (2H, m, Ha-7, H-23a),
2.20 (1H, dt, J = 13.3, 7.6 Hz, H-15a), 2.12 (1H, dd, J = 17.4, 3.5 Hz,
H-23b), 2.00 (1H, m, H-12a), 1.99 (1H, dd, J = 13.6, 3.5 Hz, H-7b),
1.93 (1H, s, 1H, H3-28), 1.84 (3H, s, H3-27), 1.75 (1H, dq, J = 10.2,
4.3 Hz, H-8), 1.40 (1H, m, H-11a), 1.39 (1H, m, H-15b), 1.33 (1H, dt,
J = 13.9, 2.8 Hz, H-9), 1.28 (3H, s, H3-19), 1.27 (3H, s, H3-21), 1.27
(1H, m, H-17), 1.10 (1H, m, H-12b), 1.08 (3H, s, H3-18), 0.88 (2H,
m, H-11b, H-14); 13C NMR (CDCl3, 100 MHz) δ 200.3 (C, C-1),
165.9 (C, C-26), 149.3 (C, C-24), 142.9 (CH, C-3), 127.6 (CH, C-2),
121.8 (C, C-25), 81.9 (CH, C-22), 77.5 (C, C-5), 77.4 (C, C-20), 73.6
(CH, C-16), 66.5 (CH, C-4), 58.0 (CH, C-17), 56.8 (C, C-10), 53.0
(CH, C-14), 46.3 (CH, C-9), 43.8 (C, C-13), 42.9 (CH2, C-7), 39.7
(CH2, C-12), 38.2 (CH, C-6), 36.4 (CH, C-8), 36.1 (CH2, C-15), 31.3
(CH2, C-23), 22.6 (CH2, C-11), 22.8 (CH3, C-21), 20.7 (CH3, C-28),
14.8 (CH3, C-18), 12.4 (CH3, C-27), 9.8 (CH3, C-19); HRESIMS m/
z 637.1646 [M + Na]+ (calcd for C28H39NaIO7, 637.1633).
Reaction of Withanolide Iodohydrins with N,N-Diisopropylethyl-
amine. To separate solutions of the iodohydrin 20 (2.0 mg), 21 (1.9
mg), or 26 (2.5 mg) in CH2Cl2 (0.2 mL) was added N,N-
diisopropylethylamine (0.03 mL), and the mixtures were stirred at
1
CHCl3); H NMR (CDCl3, 400 MHz) δ 6.45 (1H, dd, J = 10.4, 2.6
Hz, H-3), 5.92 (1H, dd, J = 10.4, 1.9 Hz, H-2), 4.94 (1H, dd, J = 2.6,
1.9 Hz, H-4), 4.65 (1H, dd, J = 13.6, 4.5 Hz, H-6), 4.62 (1H, dt, J =
I
DOI: 10.1021/acs.jnatprod.7b00918
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
25 °C. The reaction was monitored by TLC, and after the
disappearance of the starting material (120 h), the solvent was
evaporated under reduced pressure and the resulting crude products
were purified by RP-HPLC (MeOH−H2O, 65:35) to afford epoxides
5 (1.6 mg, 98%), 4 (1.5 mg, 99%), and 10 (1.9 mg, 96%), respectively.
16β-Hydroxywithaferin A (5): white powder; mp 256−258 °C;
iodo-1-oxowitha-2,24-dienolide (23), (22R)-4β,15β,27-triacetoxy-5β-
hydroxy-6α-iodo-1-oxowitha-2,24-dienolide (24), and (22R)-4β-ace-
toxy-5β,20-dihydroxy-6α-iodo-1-oxowitha-2,24-dienolide (27).
4,16,27-Tri-O-acetyl-16β-hydroxywithaferin A (8): white powder;
1
mp 168−170 °C; [α]2D5 +108 (c 0.1, CHCl3); H NMR (CDCl3, 400
MHz) δ 7.02 (1H, dd, J = 9.9, 6.1 Hz, H-3), 6.22 (1H, d, J = 9.9 Hz,
H-2), 4.95 (1H, ddd, J = 11.8,7.5, 4.1 Hz, H-16), 4.88 (1H, dd, J =
11.8 Hz, Ha-27), 4.81 (1H, dd, J = 11.8 Hz, Hb-27), 4.65 (1H, d, J =
6.1 Hz, H-4), 4.23 (1H, dt, J = 13.2, 3.2 Hz, H-22), 3.20 (1H, brs, H-
6), 2.53 (1H, dd, J = 17.5, 13.2 Hz, H-23a), 2.50 (1H, m, H-20), 2.41
(1H, dt, J = 13.8, 7.4 Hz, H-15a), 2.11 (1H, m, H-7), 2.04 (9H, s, 3 x
OAc), 2.02 (3H, s, H3-28), 1.97 (1H, dd, J = 17.5, 3.3 Hz, H-23b),
1.95 (1H, m, H-12a), 1.71 (1H, dt, J = 10.7, 4.0 Hz, H-11a), 1.53 (1H,
m, H-8), 1.49 (1H, m, H-11b), 1.38 (3H, s, H3-19), 1.26 (1H, dd, J =
14.4, 11.1 Hz, H-7b), 1.13 (1H, m, H-17), 1.12 (1H, m, H-15b), 1.05
(1H, m, H-12b), 1.02 (3H, d, J = 6.8 Hz, H3-21), 0.89 (3H, s, H3-18),
0.85 (1H, m, H-14); 13C NMR (CDCl3, 100 MHz) δ 201.1 (C, C-1),
170.9 (C, OAc), 170.8 (C, OAc), 170.1 (C, OAc), 165.1 (C, C-26),
156.6 (C, C-24), 140.0 (CH, C-3), 133.8 (CH, C-2), 122.0 (C, C-25),
77.2 (CH, C-22), 77.1 (C, C-5), 74.1 (CH, C-16), 72.0 (CH, C-4),
60.2 (CH, C-6), 57.9 (CH2, C-27), 55.0 (CH, C-17), 54.4 (CH, C-
14), 48.1 (C, C-10), 44.1 (CH, C-9), 42.8 (C, C-13), 39.2 (CH2, C-
12), 35.3 (CH2, C-15), 33.5 (CH, C-20), 31.0 (CH2, C-23), 30.4
(CH2, C-7), 29.2 (CH, C-8), 21.4 (CH3, C-28), 21.1 (CH2, C-11),
20.9 (CH3, OAc), 20.8 (CH3, OAc), 20.7 (CH3, OAc), 15.8 (CH3, C-
19), 12.9 (CH3, C-21), 12.2 (CH3, C-18); HRESIMS m/z 635.2833
[M + Na]+ (calcd for C34H44NaO10, 635.2832).
[α]2D5 +78 (c 0.1, CHCl3); H NMR (CDCl3, 400 MHz) δ 6.91 (1H,
1
dd, J = 10.0, 5.7 Hz, H-3), 6.18 (1H, d, J = 10.0 Hz, H-2), 4.67 (1H, dt,
J = 13.4, 4.2 Hz, H-22), 4.37 (1H, dd, J = 12.9, 6.3 Hz, H-27a), 4.32
(1H, dd, J = 12.9, 6.3 Hz, H-27b), 4.22 (1H, m, H-16), 3.74 (1H, d, J
= 5.7 Hz, H-4), 3.22 (1H, brs, H-6), 2.86 (1H, t, J = 6.3 Hz, D2O
exchangeable, OH-27), 2.54 (1H, brd, J = 2.9 Hz, D2O exchangeable,
OH-16), 2.51 (1H, dd, J = 17.0, 4.1 Hz, H-23a), 2.49 (1H, m, H-20),
2.26 (1H, dt, J = 13.3, 8.2 Hz, H-15a), 2.13 (1H, ddd, J = 14.8, 6.5, 3.4
Hz, H-7a), 2.09 (1H, dd, J = 17.0, 13.4 Hz, H-23b), 2.02 (3H, s, H3-
28), 1.93 (1H, dt, J = 12.8, 3.7 Hz, H-12a), 1.80 (1H, m, H-11a), 1.61
(1H, dd, J = 14.8, 5.1 Hz, H-8), 1.46 (1H, dd, J = 13.0, 3.7 Hz, H-11b),
1.40 (3H, s, H3-19), 1.24 (1H, m, H-7b), 1.18 (1H, dd, J = 13.3, 4.2
Hz, H-15b), 1.07 (1H, dd, J = 12.8, 3.7 Hz, H-12b), 1.02 (3H, d, J =
6.8 Hz, H3-21), 1.02−0.95 (2H, m, H-9, H-14), 0.90 (3H, s, H3-18),
0.80 (1H, m, H-17); 13C NMR (CDCl3, 100 MHz) δ 202.3 (C, C-1),
166.8 (C, C-26), 152.7 (C, C-24), 142.9 (CH, C-3), 132.2 (CH, C-2),
125.7 (C, C-25), 79.2 (CH, C-22), 77.7 (C, C-5), 71.6 (CH, C-16),
69.8 (CH, C-4), 62.6 (CH, C-6), 57.7 (CH, C-14), 57.5 (CH2, C-27),
54.2 (CH, C-17), 47.7 (C, C-10), 44.1 (CH, C-9), 42.6 (C, C-13),
39.7 (CH2, C-12), 37.3 (CH2, C-15), 33.5 (CH, C-20), 31.4 (CH2, C-
23), 31.1 (CH2, C-7), 29.4 (CH, C-8), 21.9 (CH2, C-11), 20.0 (CH3,
C-28), 17.5 (CH3, C-19), 13.3 (CH3, C-21), 12.9 (CH3, C-18);
HRESIMS m/z 487.2648 [M + H]+ (calcd for C28H38O7, 487.2690).
16β-Hydroxywithanolide D (10): white powder; mp 278−280 °C;
[α]2D5 +136 (c 0.1, CHCl3); 1H NMR (CDCl3, 400 MHz) δ 6.92 (1H,
dd, J = 9.9, 6.3 Hz, H-3), 6.17 (1H, dd, J = 9.9, 2.4 Hz, H-2), 4.59 (1H,
ddd, J = 12.6, 7.8, 4.7 Hz, H-16), 4.52 (1H, dd, J = 13.0, 3.5 Hz, H-22),
4.25 (1H, d, J = 2.4 Hz, D2O exchangeable, OH-16), 4.06 (1H, s, D2O
exchangeable, OH-20), 3.75 (1H, dd, J = 6.1, 2.4 Hz, H-4), 3.23 (1H,
brs, H-6), 2.57 (1H, dd, J = 17.1, 13.0 Hz, H-23a), 2.50 (1H, d, J = 2.4
Hz, D2O exchangeable, OH-4), 2.20 (1H, dt, J = 14.6, 5.5 Hz, H-7a),
2.19 (1H, m, H-15a), 2.13 (1H, dd, J = 17.1, 3.5 Hz, H-23b), 2.06
(1H, dt, J = 12.7, 3.5 Hz, H-12a), 1.94 (3H, s, H3-28), 1.87 (3H, s, H3-
27), 1.83 (1H, dd, J = 11.4, 4.4 Hz, H-11a), 1.62 (1H, dd, J = 11.2, 3.5
Hz, H-8), 1.51 (1H, m, H-11b), 1.41 (3H, s, H3-19), 1.37 (1H, dd, J =
14.6, 5.0 Hz, H-7b), 1.36 (1H, dd, J = 13.7, 5.2 Hz, H-15b),1.31 (3H,
s, H3-21), 1.24 (1H, m, H-17), 1.12 (3H, s, H3-18), 1.08 (1H, m, H-
12b), 0.99 (1H, dt, J = 11.7, 4.4 Hz, H-9), 0.73 (1H, m, H-14); 13C
NMR (CDCl3, 100 MHz) δ 202.3 (C, C-1), 165.9 (C, C-26), 149.2
(C, C-24), 142.0 (CH, C-3), 132.2 (CH, C-2), 122.1 (C, C-25), 83.8
(C, C-5), 82.0 (CH, C-22), 77.7 (C, C-20), 73.8 (CH, C-16), 69.8
(CH, C-4), 62.7 (CH, C-6), 58.3 (CH, C-17), 54.1 (CH, C-14), 47.7
(C, C-10), 44.1 (CH, C-9), 43.2 (C, C-13), 40.2 (CH2, C-12), 36.7
(CH2, C-15), 31.4 (CH2, C-23), 31.0 (CH2, C-7), 28.9 (CH, C-8),
22.8 (CH3, C-21), 22.0 (CH2, C-11), 20.6 (CH3, C-28), 17.5 (CH3, C-
19), 14.7 (CH3, C-18), 12.4 (CH3, C-27); HRESIMS m/z 509.2540
[M + Na]+ (calcd for C28H38NaO7, 509.2510).
4,15-Di-O-acetyl-15β-hydroxywithanolide D (12): white powder;
mp 236−238 °C; [α]2D5 +116 (c 0.1, CHCl3); H NMR (CDCl3, 400
1
MHz) δ 7.00 (1H, dd, J = 9.9, 6.1 Hz, H-3), 6.23 (1H, d, J = 9.9 Hz,
H-2), 5.05 (1H, ddd, J = 8.0, 5.6, 2.5 Hz, H-15), 4.67 (1H, d, J = 6.1
Hz, H-4), 4.14 (1H, dd, J = 13.2, 3.3 Hz, H-22), 3.21 (1H, brt, J = 1.8
Hz, H-6), 2.40 (1H, t, J = 15.0 Hz, H-23a), 2.22 (1H, dt, J = 15.2, 6.9
Hz, H-16a), 2.10 (1H, m, H-7a), 2.04 (1H, m H-23b), 1.95 (1H, m,
H-12a), 1.92 (3H, s, H3-28), 1.91 (1H, m, Hb-16), 1.85 (3H, s, H3-
27), 1.83 (1H, dd, J = 11.3, 4.2 Hz, H-8), 1.71 (1H, ddd, J = 14.2, 6.9,
3.3, Hz, H-11a), 1.49 (1H, m, H-11b), 1.44 (1H, m, H-17), 1.40 (3H,
s, H3-19), 1.31 (1H, m, H-7b), 1.25 (3H, s, H3-21), 1.15 (1H, dt, J =
11.3, 6.3 Hz, H-12b), 1.03 (3H, s, H3-18), 0.96 (1H, dd, J = 11.3, 6.3
Hz, H-14), 0.88 (1H, dd, J = 6.9, 3.7 Hz, H-9); 13C NMR (CDCl3, 100
MHz) δ 201.1 (C, C-1), 170.9 (C, OAc), 170.0 (C, OAc), 165.8 (C,
C-26), 148.7 (C, C-24), 139.9 (CH, C-3), 133.8 (CH, C-2), 122.1 (C,
C-25), 80.7 (CH, C-22), 76.8 (C, C-20), 72.0 (CH, C-15), 71.9 (C, C-
4), 61.1 (CH, C-5), 60.3 (CH, C-6), 59.2 (CH, C-14), 54.7 (CH, C-
17), 48.1 (C, C-10), 44.4 (CH, C-9), 42.8 (C, C-13), 40.5 (CH2, C-
12), 33.3 (CH2, C-16), 31.4 (CH2, C-23), 30.2 (CH2, C-7), 25.4 (CH,
C-8), 21.3 (CH2, C-11), 20.5 (CH3, C-28), 20.8 (CH3, C-21), 15.4
(CH3, C-19), 15.3 (CH3, C-18), 12.4 (CH3, C-27); HRESIMS m/z
593.2723 [M + Na]+ (calcd for C32H42NaO9, 593.2727).
4,16-Di-O-acetyl-16β-hydroxywithanolide D (13): white powder;
1
mp 234−236 °C; [α]2D5 +166 (c 0.1, CHCl3); H NMR (CDCl3, 400
MHz) δ 7.02 (1H, dd, J = 10.0, 6.0 Hz, H-3), 6.21 (1H, d, J = 10.0 Hz,
H-2), 5.39 (1H, ddd, J = 11.8, 7.6, 4.4 Hz, H-16), 4.63 (1H, dd, J = 6.0
Hz, H-4), 4.18 (1H, dd, J = 12.8, 3.5 Hz, H-22), 3.20 (1H, brs, H-6),
2.41 (1H, dt, J = 13.7, 6.9 Hz, H-23a), 2.14−2.07 (4H, m, H-7, H-12a,
H-15a, H-23a), 2.05 (3H, s, OAc), 2.03 (3H, s, OAc), 1.92 (3H, s, H3-
28), 1.85 (3H, s, H3-27), 1.68 (1H, dd, J = 14.9, 4.2 Hz, H-11a), 1.62
(1H, dd, J = 11.2, 3.5 Hz, H-8), 1.51 (1H, m, H-11b), 1.44 (1H, m, H-
15b), 1.36 (3H, s, H3-19), 1.31 (3H, s, H3-21), 1.24 (1H, m, H-17),
1.25 (1H, m, H-7b), 1.16 (1H, dd, J = 13.0, 4.2 Hz, H-12b), 1.03 (3H,
s, H3-18), 0.96−0.80 (2H, m, H-9, H-14); 13C NMR (CDCl3, 100
MHz) δ 200.9 (C, C-1), 170.0 (C, OAc), 169.9 (C, OAc), 165.6 (C,
C-26), 149.0 (C, C-24), 139.9 (CH, C-3), 133.8 (CH, C-2), 121.8 (C,
C-25), 81.3 (CH, C-22), 76.4 (C, C-20), 75.5 (CH, C-16), 72.1(CH,
C-4), 61.0 (C, C-5), 60.1 (CH, C-6), 57.9 (CH, C-17), 54.5 (CH, C-
14), 48.1 (C, C-10), 43.9 (CH, C-9), 42.8 (C, C-13), 39.1 (CH2, C-
12), 34.8 (CH2, C-15), 30.8 (CH2, C-23), 30.7 (CH2, C-7), 28.8 (CH,
C-8), 22.5 (CH3, OAc), 21.6 (CH2, C-11), 20.8 (CH3, C-21), 20.7
(CH3, OAc), 20.6 (CH3, C-28), 15.6 (CH3, C-19), 14.7 (CH3, C-18),
General Procedure for the Preparation of O-Acetylwithano-
lides 6−8, 11−13, 16, 18, 22−24, and 27. To a solution of the
withanolide (2.0 mg) in anhydrous pyridine (0.2 mL) was added Ac2O
(0.5 mL), and the mixture was stirred at 25 °C until the reaction was
complete (judged by the disappearance of the starting material by
TLC). Excess EtOH was added to the reaction mixture and evaporated
under reduced pressure. The crude product was purified by preparative
TLC (CH2Cl2−MeOH, 96:4) or RP-HPLC (MeOH−H2O, 75:25) to
yield the corresponding O-acetyl derivative. Application of this
procedure afforded the known acetates 4,27-di-O-acetylwithaferin A
(6),18 4,12,27-tri-O-acetyl-12β-hydroxywithaferin A (7),18 4-O-acetyl-
withanolide D (11),46 and 4,27-di-O-acetyl-4-epi-withaferin A (16)18
and the new O-acetyl derivatives 4,16,27-tri-O-acetyl-16β-hydroxywi-
thaferin A (8), 4,15-di-O-acetyl-15β-hydroxywithanolide D (12), 4,16-
di-O-acetyl-16β-hydroxywithanolide D (13), 4-O-acetyl-4-epi-with-
anolide D (18), (22R)-4β,27-diacetoxy-5β-hydroxy-6α-iodo-1-oxowi-
tha-2,24-dienolide (22), (22R)-4β,16β,27-triacetoxy-5β-hydroxy-6α-
J
DOI: 10.1021/acs.jnatprod.7b00918
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
12.4 (CH3, C-27); HRESIMS m/z 593.2725 [M + Na]+ (calcd for
C32H42NaO9, 593.2727).
19); HRESIMS m/z 763.1956 [M + Na]+ (calcd for C34H45INaO10,
763.1955).
4-O-Acetyl-4-epi-withanolide D (18): white powder; mp 184−186
(22R)-4β,15β,27-Triacetoxy-5β-hydroxy-6α-iodo-1-oxowitha-
2,24-dienolide (24): white powder; mp >145 °C dec; [α]2D5 +98 (c 0.1,
°C; [α]2D5 +73 (c 0.1, CHCl3); H NMR (CDCl3, 400 MHz) δ 6.65
1
1
CHCl3); H NMR (CDCl3, 400 MHz) δ 6.28 (1H, dd, J = 10.1, 2.1
(1H, dd, J = 10.2, 1.8 Hz, H-3), 6.05 (1H, dd, J = 10.2, 2.6 Hz, H-2),
5.86 (1H, dd, J = 2.6, 1.8 Hz, H-4), 4.17 (1H, dd, J = 13.4, 3.4 Hz, H-
22), 3.53 (1H, brd, J = 2.7 Hz, H-6), 2.42 (1H, brt, J = 15.1 Hz, H-
23a), 2.11 (1H, m, H-7a), 2.09 (3H, s, OAc), 2.04 (1H, m, H-23b),
1.98−1.93 (3H, m, H-11a, Ha-12, H-16a), 1.92 (3H, s, H3-28), 1.86
(3H, s, H3-27), 1.62 (1H, m, H-15a), 1.52−1.45 (4H, m, H-8, H-11b,
H-16b, H-17), 1.27 (3H, s, H3-21), 1.23 (3H, s, H3-19), 1.19−1.12
(3H, m, H-7b, H-12b, H-15b), 0.98−0.88 (2H, m, H-9, H-14), 0.84
(3H, s, H3-18); 13C NMR (CDCl3, 100 MHz) δ 200.9 (C, C-1), 169.7
(C, OAc), 166.0 (C, C-26), 148.8 (C, C-24), 144.8 (CH, C-3), 129.4
(CH, C-2), 122.0 (C, C-25), 80.9 (CH, C-22), 77.7 (C, C-20), 75.1
(CH, C-4), 65.8 (C, C-5), 56.4 (CH, C-14), 55.8 (CH, C-6), 54.7
(CH, C-17), 48.1 (C, C-10), 45.4 (CH, C-9), 42.8 (C, C-13), 39.8
(CH2, C-12), 31.5 (CH2, C-23), 30.6 (CH2, C-7), 29.1 (CH, C-8),
23.8 (CH2, C-15), 22.5 (CH2, C-16), 21.9 (CH2, C-11), 20.8 (CH3, C-
21), 20.7 (CH3, C-28), 20.5 (CH3, OAc), 14.1 (CH3, C-18), 13.6
(CH3, C-19), 12.5 (CH3, C-27); HRESIMS m/z 535.2678 [M + Na]+
(calcd for C30H40NaO7, 535.2672).
Hz, H-3), 6.10 (1H, dd, J = 2.1, 1.9 Hz, H-4), 6.05 (1H, dd, J = 10.1,
2.0 Hz, H-2), 4.91 (1H, m, H-15), 4.88 (1H, d, J = 11.8 Hz, H-27a),
4.83 (1H, d, J = 11.8 Hz, H-27b), 4.63 (1H, dd, J = 13.2, 4.7 Hz, H-6),
4.33 (1H, dt, J = 13.1, 3.4 Hz, H-22), 2.49 (1H, dd, J = 17.7, 13.1 Hz,
H-23a), 2.42 (1H, m, H-7a), 2.40 (1H, m, H-16a), 2.20 (3H, s, OAc),
2.06 (3H, s, H3-28), 2.08−2.02 (3H, m, H-7b, H-8, H-20), 2.05 (3H, s,
OAc), 2.04 (3H, s, OAc), 1.94 (1H, dd, J = 17.7, 3.4 Hz, H-23b), 1.90
(1H, m, H-12a), 1.45 (1H, m, H-9), 1.38 (1H, m, H-11a), 1.37 (1H,
m, H-16b), 1.30 (3H, s, H3-19), 1.09 (3H, m, H-12b, H-14, H-17),
0.96 (3H, d, J = 6.5 Hz, H3-21), 0.94 (1H, m, H-11b), 0.88 (3H, s, H3-
18); 13C NMR (CDCl3, 100 MHz) δ 199.1 (C, C-1), 171.0 (C, OAc),
170.8 (C, OAc), 170.7 (C, OAc), 165.0 (C, C-26), 156.5 (C, C-24),
140.4 (CH, C-3), 128.1 (CH, C-2), 122.2 (C, C-25), 77.7 (CH, C-22),
77.6 (C, C-5), 72.7 (CH, C-15), 67.7 (CH, C-4), 57.9 (CH2, C-27),
57.4 (CH, C-14), 57.0 (C, C-10), 51.8 (CH, C-17), 46.6 (CH, C-9),
43.6 (C, C-13), 41.1 (2 x CH2, C-7, C-16), 40.2 (CH2, C-12), 38.2
(CH, C-8), 33.9 (CH, C-8), 33.8 (CH, C-20), 30.1 (CH2, C-23), 23.0
(CH2, C-11), 22.4 (CH3, OAc), 21.3 (CH3, OAc), 20.9 (CH3, OAc),
20.6 (CH3, C-28), 13.8 (CH3, C-18), 13.3 (CH3, C-21), 9.5 (CH3, C-
19); HRESIMS m/z 763.1956 [M + Na]+ (calcd for C34H45INaO10,
763.1955).
(22R)-4β,27-Diacetoxy-5β-hydroxy-6α-iodo-1-oxowitha-2,24-di-
enolide (22): white powder; mp >179 °C dec; [α]2D5 +76 (c 0.2,
1
CHCl3); H NMR (CDCl3, 400 MHz) δ 6.46 (1H, dd, J = 10.5, 2.3
Hz, H-3), 6.11 (1H, dd, J = 2.3, 2.0 Hz, H-4), 6.03 (1H, dd, J = 10.5,
2.0 Hz, H-2), 4.61 (1H, dd, J = 13.4, 4.2 Hz, H-6), 4.87 (1H, d, J =
12.0 Hz, H-27a), 4.83 (1H, d, J = 12.0, H-27b), 4.37 (1H, dt, J = 13.1,
3.5 Hz, H-22), 2.44 (1H, dt, J = 13.9, 4.4 Hz, H-7a), 2.44 (1H, dd, J =
17.7, 13.1 Hz, H-23a), 2.19 (3H, s, OAc), 2.06 (3H, s, H3-28), 2.02
(3H, s, OAc), 2.01 (1H, m, H-7b), 1.98 (1H, m, H-23b), 1.96 (1H,m,
H-20), 1.88 (1H, dt, J = 12.8, 3.1 Hz, H-12a), 1.69 (1H, m, H-16a),
1.64 (1H, 1H, m, H-8), 1.61 (1H, m, H-15a), 1.39 (1H, m, H-9), 1.35
(1H, m, H-16b), 1.31 (1H, m, H-11a), 1.25 (3H, s, H3-19), 1.13 (1H,
m, H-15b), 1.14 (1H, m, H-14), 1.08 (1H, m, H-17), 1.03 (1H, dd, J =
12.8, 3.7 Hz, H-12b), 0.94 (3H, d, J = 6.8, H3-21), 0.90 (1H, m, H-
11b), 0.65 (3H, s, H3-18); 13C NMR (CDCl3, 100 MHz) δ 199.3 (C,
C-1), 170.9 (C, OAc), 170.1 (C, OAc), 165.2 (C, C-26), 156.9 (C, C-
24), 140.4 (CH, C-3), 128.0 (CH, C-2), 121.9 (C, C-25), 78.0 (CH,
C-22), 77.6 (C, C-5), 67.8 (CH, C-4), 58.4 (C, C-10), 58.0 (CH2, C-
27), 54.8 (CH, C-14), 51.7 (CH, C-17), 46.1 (CH, C-9), 43.3 (C, C-
13), 42.1 (CH2, C-7), 39.0 (CH2, C-12), 38.7 (CH, C-20), 37.6 (CH,
C-8), 34.7 (CH, C-6), 30.0 (CH2, C-23), 27.2 (CH2, C-16), 23.8
(CH2, C-15), 23.0 (CH2, C-11), 22.4 (CH3, OAc), 20.9 (CH3, OAc),
20.6 (CH3, C-28), 13.2 (CH3, C-21), 11.8 (CH3, C-18), 9.3 (CH3, C-
19); HRESIMS m/z 705.1894 [M + Na]+ (calcd for C32H43INaO8,
705.1900).
(22R)-4β-Acetoxy-5β,20-dihydroxy-6α-iodo-1-oxowitha-2,24-di-
enolide (27): white powder; mp 147−149 °C; [α]2D5 +63 (c 0.2,
1
CHCl3); H NMR (CDCl3, 400 MHz) δ 6.26 (1H, dd, J = 10.5, 2.3
Hz, H-3), 6.12 (1H, dd, J = 2.3, 1.9 Hz, H-4), 6.03 (1H, dd, J = 10.5,
1.9 Hz, H-2), 4.63 (1H, dd, J = 13.6, 4.5 Hz, H-6), 4.16 (1H, dd, J =
13.2, 3.3 Hz, H-22), 2.46 (1H, dt, J = 13.7, 4.3 Hz, H-7a), 2.36 (1H,
brt, J = 16.9 Hz, H-23a), 2.20 (3H, s, OAc), 2.07 (1H, m, H-7b), 2.06
(1H, m, H-23b), 1.99 (1H, m, H-11a), 1.96 (1H, m, H-12), 1.93 (3H,
s, H3-28), 1.87 (3H, s, H3-27), 1.65 (1H, m, H-8), 1.62 (1H, m, H-
15a), 1.55 (1H, dd, J = 12.7, 3.6 Hz, H-11b), 1.49 (1H, m, H-17), 1.40
(1H, m, H-9), 1.25 (3H, s, H3-21), 1.33 (1H, dd, J = 12.7, 3.6 Hz, H-
16a), 1.22 (3H, s, H3-19), 1.17 (2H, m, H-14, H-15b), 1.16 (1H, m,
H-12b), 0.91 (1H, m, H-16b), 0.81 (3H, s, H3-18); 13C NMR (CDCl3,
100 MHz) δ 199.4 (C, C-1), 170.0 (C, OAc), 166.0 (C, C-26), 148.8
(C, C-24), 140.4 (CH, C-3), 128.1(CH, C-2), 122.0 (C, C-25), 80.8
(CH, C-22), 77.5 (C, C-5), 75.0 (C, C-20), 67.7 (CH, C-4), 57.1 (C,
C-10), 55.4 (CH, C-14), 54.4 (CH, C-17), 46.2 (CH, C-9), 43.5 (C,
C-13), 42.1 (CH2, C-7), 39.4 (CH2, C-12), 37.0 (CH, C-8), 34.8 (CH,
C-6), 31.5 (CH2, C-23), 23.4 (CH2, C-15), 22.7 (CH2, C-16), 22.5
(CH3, OAc), 21.8 (CH2, C-11), 20.8 (CH3, C-21), 20.6 (CH3, C-28),
13.7 (CH3, C-18), 12.5 (CH3, C-27), 9.3 (CH3, C-19); HRESIMS m/
z 663.1769 [M + Na]+ (calcd for C30H41INaO7, 663.1795).
(22R)-4β,16β,27-Triacetoxy-5β-hydroxy-6α-iodo-1-oxowitha-
2,24-dienolide (23): white powder; mp >168 °C dec; [α]2D5 +127 (c
(22R)-4β,5β,27-Trihydroxy-6α-iodo-1-oxowitha-2,24-dienolide
4,5-carbonate (28). To a stirred solution of 19 (5.0 mg, 8.4 μmol) in
dry CH2Cl2 (0.5 mL) was added 1,1′-carbonyldiimidazole (3.25 mg,
20.2 μmol), and the mixture was stirred at 25 °C. After stirring for 16
h, the mixture was concentrated and the residue was partially purified
using a short column of silica gel and elution with EtOAc to provide a
crude product. This was further purified by silica gel preparative TLC
[CH2Cl2−MeOH (95:5)] to afford a white solid (2.6 mg, 43%, Rf
0.63), which on hydrolysis with LiOH (0.5 mg) in THF (0.4 mL) and
H2O (0.02 mL) at 0 °C for 3 h followed by usual workup afforded a
crude product. This was purified by silica gel preparative TLC
[CH2Cl2−MeOH (95:5)] to afford 28 (2.0 mg, Rf 0.57) as a white
1
0.1, CHCl3); H NMR (CDCl3, 400 MHz) δ 6.27 (1H, dd, J = 10.4,
2.7 Hz, H-3), 6.09 (1H, dd, J = 2.7, 2.0 Hz, H-4), 6.04 (1H, dd, J =
10.4, 2.0 Hz, H-2), 4.95 (1H, ddd, J = 11.1, 7.4, 3.4 Hz, H-16), 4.88
(1H, d, J = 12.0 Hz, H-27a), 4.81 (1H, d, J = 12.0 Hz, H-27b), 4.59
(1H, dd, J = 13.2, 4.2 Hz, H-6), 4.23 (1H, dt, J = 13.2, 3.2 Hz, H-22),
2.51 (1H, m, H-23a), 2.50 (1H, m, H-20), 2.43 (1H, m, H-15a), 2.42
(1H, m, H-7a), 2.20 (3H, s, OAc), 2.05 (3H, s, H3-28), 2.04 (3H, s,
OAc), 2.02 (3H, s, OAc), 1.99 (1H, m, H-23b), 1.92 (1H, dd, J = 13.0,
3.0 Hz, H-7b), 1.91 (1H, m, H-12a), 1.73 (1H, m, H-8), 1.41 (1H, m,
H-9), 1.38 (1H, m, H-11a), 1.26 (3H, s, H3-19), 1.17 (1H, dd, J =
12.4, 7.1 Hz, H-15b), 1.16 (1H, m, H-14), 1.07 (1H, m, H-12b), 1.10
(1H, m, H-17), 0.94 (1H, m, H-11b), 0.86 (3H, s, H3-18); 13C NMR
(CDCl3, 100 MHz) δ 199.1 (C, C-1), 170.8 (C, OAc), 170.7 (C,
OAc), 170.1 (C, OAc), 165.0 (C, C-26), 156.6 (C, C-24), 140.4 (CH,
C-3), 128.1 (CH, C-2), 122.0 (C, C-25), 77.6 (C, C-5), 76.9 (CH, C-
22), 73.8 (CH, C-16), 67.4 (CH, C-4), 57.9 (CH2, C-27), 54.8 (CH,
C-14), 57.0 (C, C-10), 53.1 (CH, C-17), 46.1 (CH, C-9), 41.9 (CH2,
C-7), 43.5 (C, C-13), 38.9 (CH2, C-12), 37.1 (CH, C-8), 34.9 (CH2,
C-15), 33.8 (CH, C-6), 33.4 (CH, C-20), 30.3 (CH2, C-23), 22.7
(CH2, C-11), 22.4 (CH3, OAc), 21.3 (CH3, OAc), 20.8 (CH3, OAc),
20.7 (CH3, C-28), 12.8 (CH3, C-21), 12.5 (CH3, C-18), 9.4 (CH3, C-
1
solid: H NMR (400 MHz, CDCl3) δ 6.70 (1H, dd, J = 10.4, 3.7 Hz,
H-3), 6.20 (1H, d, J = 10.4 Hz, H-2), 5.16 (1H, d, J = 3.7 Hz, H-4),
4.69 (1H, dd, J = 13.5, 4.5 Hz, H-6), 4.38 (1H, dt, J = 17.7, 13.5 Hz,
H-22), 4.37 (1H, d, J = 13.0 Hz, H-27a), 4.32 (1H, d, J = 13.0 Hz, H-
27b), 2.58 (1H, dt, J = 13.5, 4.2 Hz, H-7a), 2.45 (1H, dd, J = 13.5, 3.6
Hz, H-23a), 2.02 (3H, s, H3-28), 1.97 (1H, m, H-20), 1.93 (1H, m, H-
23b), 1.91 (1H, m, H-12a), 1.79 (1H, m, H-7b), 1.68 (1H, m, H-16a),
1.65 (1H, m, H-15a), 1.59 (1H, m, H-8), 1.37 (1H, m, H-16b), 1.34
(1H, m, H-11a), 1.32 (3H, s, H3-19), 1.25 (1H, m, H-9), 1.18 (1H, m,
H-15b), 1.09 (1H, m, H-17), 1.06 (1H, m, H-12b), 0.96 (1H, m, H-
K
DOI: 10.1021/acs.jnatprod.7b00918
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
14), 0.95 (3H, d, J = 6.4 Hz, H3-21), 0.86 (1H, m, Hb-11), 0.66 (3H, s,
H3-18); 13C NMR (100 MHz, CDCl3) δ 197.3 (C, C-1), 166.8(C, C-
26), 152.5 (C, C-24), 151.4 [C, O(C = O)O], 135.6 (CH, C-3), 129.3
(CH, C-2), 125.8 (C, C-25), 87.6 (C, C-5), 78.5 (CH, C-22), 72.9
(CH, C-4), 57.5 (CH2, C-27), 54.7 (CH, C-14), 54.6 (C, C-10), 51.7
(CH, C-17), 48.5 (CH, C-9), 43.1 (CH2, C-7), 42.7 (C, C-13), 38.6
(CH2, CH, C-12 and C-20), 36.9 (CH, C-8), 29.9 (CH2, C-23), 28.2
(CH, C-6), 27.1 (CH2, C-16), 23.9 (CH2, C-15), 22.3 (CH2, C-11),
20.0 (CH3, C-28), 13.3 (CH3, C-21), 11.7 (CH3, C-18), 10.2 (CH3, C-
19); HRESIMS m/z 647.1474 [M + Na]+ (calcd for C29H37INaO7,
647.1482).
*Tel: (520) 621-9932. Fax: (520) 621-8378. E-mail: leslieg1@
email.arizona.edu.
ORCID
A. A. Leslie Gunatilaka: 0000-0001-9663-3600
Present Address
∥Department of Molecular Genetics, University of Toronto,
Toronto, ON M6G 2W5, Canada.
Notes
The authors declare no competing financial interest.
Cytotoxicity Assays. Ewing’s sarcoma cell line CHP-100 was
cultured at 37 °C under 6% CO2 in RPMI 1640 media supplemented
with 10% fetal bovine serum (FBS), while 293T (American Type
Culture Collection) kidney cancer cells were grown in Dulbecco’s
modified Eagle’s medium supplemented with 10% FBS. All cell lines
were tested and found negative for Mycoplasma contamination.
Cultures were passaged twice weekly, and cells in exponential growth
were used for experiments. Stock solutions of compounds were
formulated in DMSO and maintained at −20 °C protected from light.
To measure acute toxicity, CHP-100 cells were seeded in flat-bottom
96-well plates (7500 cells/well) and allowed to adhere overnight.
Serial dilutions of compounds or DMSO vehicle control (not
exceeding 0.2%) were added, and the relative viable cell number was
determined 24 h later by the dye reduction assay using resazurin
(Alamar Blue) as previously described.18 Cytotoxicity over 3 days
against 293T cells was measured in 384-well format (3000 cells/well),
and the relative viable cell number was determined by the same
(Alamar Blue) assay.18
ACKNOWLEDGMENTS
■
Financial support for this work by the Arizona Biomedical
Research Commission (Contract No. 0804), College of
Agriculture and Life Sciences and School of Natural Resources
and the Environment of the University of Arizona, and CNPq
of Brazil [fellowship awards to M.C.F.O. (Process: 233171/
2012-6), J.M. (Process: 236451/2012-0), and S.M. (Process:
246314/2012-5), and Research Grants to M.C.F.O. (Process:
303365/2014-5) and M.C.F.O. and A.A.L.G. (Process:
405001/2013-4)] is gratefully acknowledged. We thank Prof.
L. Xuan of Center for Modernization of TCM, Shanghai
Institute of Materia Medica, PR China, for obtaining HRESIMS
data for some withanolide analogues and Dr. K. Shah,
Massachusetts General Hospital, Boston, Massachusetts, USA,
for providing plasmid encoding the fusion protein.
Heat-Shock Reporter Assays. Reporter cells were generated by
infecting 293T cells with a reported lentiviral vector encoding a fusion
protein consisting of enhanced green fluorescent protein (GFP) fused
to firefly luciferase under control of HSP70B′ promoter elements.47
The plasmid encoding the fusion protein was generously provided by
Khalid Shah (Massachusetts General Hospital, Boston, Massachusetts,
USA). To isolate a homogeneous population of high-responding cells,
a transduced culture was heat shocked at 42 °C for 1 h and processed
8 h later by fluorescence activated cell sorting (FACS). Prior to use,
cells were reverse selected by FACS to eliminate a minority population
of cells constitutively expressing the reporter in the absence of
induction. To evaluate compounds, cells were seeded in white 384-well
plates (20 000 cells/well). The following day, serial compound
dilutions were added to quadruplicate wells and incubation continued
overnight. Measurement of relative luciferase activity was achieved
using an Envision plate luminometer (PerkinElmer) and Steady-Glo
reagent (Promega) per manufacturer’s recommendations. As a
complementary assay for some compounds, 3T3-Y9-B12 reporter
cells were seeded in black flat-bottom 96-well plates (20 000/well) and
allowed to adhere overnight as previously reported.30a Cells were
incubated for 24 h in the presence of withanolide analogues (1.0 or 5.0
μM). After washing with PBS, fluorescence was quantified using an
Analyst AD (LJL Biosystems) plate reader with excitation and
emission filters set at 485 and 530 nm, respectively.
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