Structure−activity relationships for withanolides as inducers of the cellular heatshock response

Structure

−

Activity Relationships for Withanolides as Inducers of the

Cellular Heat-Shock Response

E. M. Kithsiri Wijeratne,

†,#

Ya-Ming Xu,

†,#

Ruth Scherz-Shouval,

§

Marilyn T. Marron,

†

Danilo D. Rocha,

†,‡

Manping X. Liu,

†

Leticia V. Costa-Lotufo,

‡

Sandro Santagata,

§

Susan Lindquist,

§,∥,⊥

Luke Whitesell,

*

,§

_{and A. A. Leslie Gunatilaka}

*

,†

†

SW Center for Natural Products Research and Commercialization, School of Natural Resources and the Environment, College of

Agriculture and Life Sciences, University of Arizona, 250 East Valencia Road, Tucson, Arizona 85706, United States

‡

Laboratório de Oncologia Experimental, Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceara

́, P.O. Box 3157,

Fortaleza, Ceará

60430-270, Brazil

§

Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, Massachusetts 02142, United States

∥

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, United States

⊥

Howard Hughes Medical Institute, Cambridge, Massachusetts 02142, United States

*

S Supporting Information

ABSTRACT:

To understand the relationship between the

structure and the remarkably diverse bioactivities reported for

withanolides, we obtained withaferin A (WA;

1

) and 36

analogues (

2

−

37

) and compared their cytotoxicity to

cytoprotective heat-shock-inducing activity (HSA). By

analyz-ing structure

−

activity relationships for the series, we found

that the ring A enone is essential for both bioactivities.

Acetylation of 27-OH of 4-

epi

-WA (

28

) to

33

enhanced both activities, whereas introduction of

β-OH to WA at C-12 (

29

) and

C-15 (

30

) decreased both activities. Introduction of

β-OAc to 4,27-diacetyl-WA (

16

) at C-15 (

37

) decreased HSA without

a

ff

_{ecting cytotoxicity, but at C-12 (}

₃₆

_{), it had minimal e}

ff

_{ect. Importantly, acetylation of 27-OH, yielding}

₁₅

_from

₁

_,

₁₆

_from

₁₄

_,

and

35

from

34

, enhanced HSA without increasing cytotoxicity. Our

fi

_{ndings demonstrate that the withanolide sca}

ff

_{old can be}

modi

fi

_{ed to enhance HSA selectively, thereby assisting development of natural product-inspired drugs to combat protein}

aggregation-associated diseases by stimulating cellular defense mechanisms.

■

INTRODUCTION

Withanolides, a class of steroidal lactones structurally based on

an ergostane skeleton, are abundant in plants of the family

Solanaceae.

1

_{Plants of this family belonging to genera}

Withania

,

Acnistus

, and

Physalis

have been extensively investigated, which,

in large part, is because they are used in many of the traditional

systems of medicine practiced throughout Asia and South

America.

2

_{The bene}

_fi

_{cial e}

_ff

_{ects of many of these plants have}

been attributed to the presence of withanolides. One of the best

studied of these withanolides is withaferin A (

1

, WA), a major

constituent of the plant

Withania somnifera

(L.) Dunal.

3,4

Popularly known as Ashwagandha or Indian ginseng, it has

been used in Indian Ayurvedic medicine for over 3000 years.

5

Various preparations of Ashwagandha are available as herbal

dietary supplements worldwide. The National Center for

Complementary and Alternative Medicines (NCCAM) of the

U.S. National Institutes of Health has recently recognized

Ashwagandha as a high-priority topic for mechanistic research.

6

Numerous reports describe anticancer,

7

neuroprotective,

8−10

anti-in

fl

_ammatory,

11

_{immunomodulatory,}

12,13

_{and antioxidant}

5

activities for medicinal preparations of

W. somnifera

and its

constituent withanolides. Among these, the most extensively

studied has been the anticancer activity of WA.

14−16

_For

example, the Developmental Therapeutics Program (DTP) of

the U.S. National Cancer Institute has tested WA (NSC

101088) against its panel of 60 human cancer cell lines and

found a mean 50% growth inhibitory concentration (GI

50

) of

620 nM.

17

Other studies have demonstrated signi

fi

_{cant activity}

for WA against human brain,

18

prostate,

19

pancreatic,

20

and

breast cancer

21

xenografts in mice.

In previous work, we used the heat-shock response (HSR) as

a biosensor to discover potential anticancer compounds that

target protein homeostasis. We found that WA and other

thiol-reactive natural products activate the heat shock factor 1

(HSF1)-dependent stress response as a prominent component

of their anticancer activity.

18,22

Others have reported inhibition

of cell motility and angiogenesis,

23,24

inhibition of NF-κB

activation,

25−29

_{protein kinase C,}

30

_{and Notch-1.}

31

_{Reports also}

describe induction of Par-4-dependent apoptosis,

19

FOXO3a-and Bim-dependent

21

apoptosis, and sensitization to

TRAIL-induced apoptosis.

32

Despite the great diversity of biological

e

ff

_{ects reported for natural and semisynthetic withanolides,}

Received: August 19, 2013 Published: March 13, 2014

structure

−

activity relationship studies have relied almost

exclusively on cytotoxicity as their end point for activity.

To begin probing how cytoprotective heat-shock-inducing

activity relates to the cytotoxic activity of withanolides, we

isolated

1

and natural withanolides

2

−

13

using the biomass

derived from aeroponically cultivated

W. somnifera

and

prepared 24 structurally related analogues,

14

−

37

, by

perform-ing chemical and microbial transformations of

1

that had been

isolated from the same material, providing a total of 36

analogues. We evaluated these compounds for their ability to

activate the heat-shock response using cell-based reporter

systems, whereas antiproliferative activity was measured in a

reporter cell line as well as two other cancer cell lines. This

approach allowed us to identify relatively modest structural

modi

fi

_{cations that alter the chemical reactivity of analogues}

toward thiols and selectively enhance heat-shock-inducing

activity over cytotoxicity and vice versa. Importantly, we

con

fi

_{rmed these reporter-based cell culture results through}

exploratory pharmacodynamic studies in mice. Our

fi

_ndings

suggest that reporter assay-guided tuning of the withanolide

sca

ff

_{old provides a useful approach to improving the}

therapeutic potential of this class and perhaps other

thiol-reactive natural products as anticancer or neuroprotective

agents.

Figure 1.Withanolides1−13obtained from aeroponically grownWithania somniferaand their derivatives14−26.

■

RESULTS AND DISCUSSION

Isolation and Semisynthesis of Withanolides 1

−

37.

We investigated the e

ff

_{ects of various substituents, their}

position and stereochemistry, and their lipophilicity on

antiproliferative versus heat-shock-induction activity for 37

withanolides. The panel of compounds used in this study

included natural withanolides

1

−

13

obtained from

aeroponi-cally grown

W. somnifera

22,33

and their derivatives

14

−

26

(Figure 1). Analogues

27

−

31

were obtained from WA by

chemical and microbial transformations. These compounds

were further derivatized to yield compounds

32

−

37

(Figure 2).

Semisynthesis of 4-

epi

-withaferin A (

28

) was e

ffi

_ciently

achieved by the MnO2

oxidation of

1

to 4-dehydrowithaferin

A (

27

)

3

followed by regio- and stereoselective reduction of its

C-4 carbonyl group with NaBH

4

/CeCl

3

. The use of lanthanoid

cations (such as Ce

3+

_{) in reactions of enones with NaBH}

4

is

known to cause 1,2-reduction of the carbonyl group with high

selectivity compared to 1,4-reduction caused by NaBH

4

in the

absence of these cations,

34

with the ratio of epimeric alcohols

formed being determined by steric factors.

35

_{Conversion of}

27

to

28

, however, constitutes the

fi

_{rst report of a steroselective}

reduction of only one carbonyl group of an ene-dione with

NaBH

4

/CeCl

3

/MeOH/THF. The high degree of regio- and

stereoselectivity observed for

27

yielding

28

may be explained

as being due to the chelation of the boron atom of the reducing

species [NaBH

4−n

(OMe)

n

]

34

to the oxygen atom of the ring-B

oxirane of

27

, delivering the hydride from the

β-phase. The

structure of

28

was elucidated by the analysis of its

1

_H,

13

_{C, and}

2D NMR spectroscopic data including HMBC. The

α-orientation of the 4-OH was con

fi

_{rmed by NOE experiments}

(see Supporting Information Figure S17). The two O-sulfated

analogues of

1

, withaferin A-27-sulfate (

18

) and withaferin

A-4,27-disulfate (

19

), were prepared by the reaction of WA with

SO3-pyridine.

36

3-Azido-2,3-dihydrowithaferin A (

31

) was

obtained by treating

1

with NaN3/Et3N.

37

Microbial biotransformation of

1

with the fungus

Cunning-hamella echinulata

a

ff

_{orded 12β-hydroxywithaferin A (}

₂₉

_{) and}

15β-hydroxywithaferin A (

30

).

38

Controlled acetylation

(Ac2O/pyridine) of

1

yielded 27-acetylwithaferin A (

15

) and

4,27-diacetylwithaferin A (

16

). Acetyl analogues

21

−

26

,

32

,

and

35

−

37

were obtained by the standard acetylation of their

corresponding alcohols using Ac

2

O/pyridine. In contrast,

preparation of the 4-acetyl analogues, 4-acetylwithaferin A

(

14

) and 4-acetyl-4-

epi

-withaferin A (

34

), of

1

and

28

,

respectively, required protection of their more reactive

27-OH groups as

tert

-butyldimethylsilyl (TBDMS) ether

deriva-tives

39

(Scheme 1) and

42

(Scheme 2). Treatment of

39

and

42

with Ac2O/pyridine followed by deprotection (HCl/THF/

MeOH) a

ff

_orded

₁₄

_and

₃₄

_{, respectively.}

Scheme 1. Conversion of 1 to 14

a

a_{Reagents and conditions: (a) TBDMS-Cl, 4-PP, DMF, 60°C; (b) Ac}

2O, pyridine, 25°C; (c) 2 N HCl, THF, MeOH, 0°C.

Scheme 2. Conversion of 1 to 28 and Its Derivatives

a

a_{Reagents and conditions: (a) MnO}

2, CHCl3, EtOAc, 25 °C; (b) NaBH4, CeCl3·7H2O, MeOH, THF, 0°C; (c) Ac2O, pyridine, 25°C; (d)

Cytotoxicity.

As an initial screen, we measured the acute

cytotoxicity of

1

and its analogues

2

−

37

at a single

concentration (4.0

μM) using the human Ewing’s sarcoma

cell line CHP-100 (Supporting Information, Figure S29). We

chose this very rapidly proliferating cell line to maximize

sensitivity over a short period of compound exposure. Cells

were incubated for 24 h with compounds, and the relative

viable cell number was measured by a standard dye-reduction

assay.

39

Doxorubicin and DMSO were used as positive and

negative controls, respectively. WA and analogues

2

,

5

,

14

−

17

,

21

,

22

,

24

,

28

, and

31

−

37

inhibited the overall proliferation

and survival of CHP-100 cells over this short time interval by

>80%. In a follow-up experiment, we determined the IC

50

for

each of these cytotoxic compounds using the same

method-ology (Table 1). Intriguingly, the most potent analogues,

namely,

15

and

33

, contained a 27-OAc in addition to the 4α/

β-hydroxy-2(3)-en-1-one moiety in ring A. These data are

consistent with previous reports that the 2(3)-en-1-one moiety

in ring A of withanolides is essential for their cytotoxic

activity.

40−47

The absence of cytotoxic activity for 2,3-dihydrowithaferin

A-3β-

O

-sulfate (

4

) was expected on the basis of our previous

fi

_{nding that the conversion of}

₄

_to

₁

_{in cell culture media is a}

slow process requiring longer than the 24 h incubation period

used for these experiments.

22

In contrast, acetyl derivatives of

4

,

namely,

21

and

22

, were found to be highly active, suggesting

that acetylation of the OH groups of

4

assists in ready

conversion of the 1-oxo-3β-

O

-sulfate to a 2(3)-en-1-one system

in cell culture medium. Analogues

5

and

31

, which contain a

masked 2(3)-en-1-one system capable of generating this moiety

under physiological conditions, also exhibited cytotoxic activity

comparable to

1

. 3-Azido-1-ones such as

31

are known to

undergo elimination of HN

3

under mildly basic conditions

(e.g., Al

2

O

3

) to produce their corresponding 2(3)-en-1-ones.

48

A recent report, however, suggested that the nature of the

substituent at C-3 rather than the 2(3)-en-1-one group is

essential for the enhanced cytotoxicity of a series of

3-substituted withanolides including

31

.

37

This report prompted

us to investigate the possibility that

31

could undergo

elimination of HN

3

to produce

1

under physiological

conditions. Thus,

31

was incubated in cell culture medium

consisting of Dulbecco/Vogt modi

fi

_{ed Eagle’s minimal essential}

medium (DMEM) supplemented with 10% fetal bovine serum

(FBS), and its conversion to

1

was monitored by HPLC.

22

Over the course of 24 h at 37

°

C, nearly all of

31

disappeared

from the culture medium with the corresponding de novo

appearance of

1

(Supporting Information, Figure S30),

suggesting that the cytotoxic activity exhibited by

31

may be

partly due to its conversion to

1

under the experimental

conditions. Cytotoxic activity exhibited by

3β-uracyl-2,3-dihydrowithaferin A (

5

) may also be explained as a result of

its conversion to

1

in cell culture medium because the uracyl

anion is known to be a good leaving group.

49

Our data indicate that the nature of the substituent at C-4

has a major e

ff

_{ect on the antiproliferative activity of}

withanolides, at least the ones we examined. An

electron-withdrawing carbonyl group at C-4, as in

27

, reduced activity

when compared to

1

, supporting our previous observation that

the reactivity of the 2(3)-en-1-one moiety of withanolides

determines their ability to adduct thiols and their consequent

cytotoxicity.

18

4-Dehydrowithaferin A (

27

) has been previously

reported to be more cytotoxic than

1

,

47

but di

ff

_{erent conditions}

and cell lines used for cytotoxicity assays could easily account

for such discrepancy.

Comparing the potencies of

1

and

28

indicates that the

orientation of the 4-OH has very little e

ff

_{ect on cytotoxicity}

toward CHP-100 cells. In our previous study of the

antiproliferative activities of

28

and

1

against pancreatic cancer

cell lines MIAPaCa-2 and BxPC-3, both withanolides also had

similar activities. In pancreatic cancer cell line PANC-1,

however,

1

demonstrated ca. 4-fold higher potency than

28

for reasons that are unclear, but this could be due to their

di

ff

_{erences in cellular uptake and/or metabolism.}

16

_{Several SAR}

studies have noted the importance of the 5β,6β-epoxy group in

ring B for the cytotoxicity of withanolides.

41,46

When this group

is replaced with a double bond. as in

24

, acute cytotoxicity was

retained against CHP-100 cells (Table 1 and Supporting

Information, Figure S29). This

fi

_{nding indicates that the}

5β,6β-epoxy group is not required but can enhance the cytotoxicity of

withanolides. Our

fi

_{ndings with}

₂

_and

₁₅

_{agree with previous}

reports that the presence of an OH group at C-27 of

withanolides leads to a reduction in their antiproliferative

activity.

46,47

This

fi

_{nding together with the enhancement of}

activity observed for acetyl derivatives

15

,

16

,

21

,

22

,

24

,

32

,

34

, and

35

−

37

as compared to their parent alcohols suggests

that increased lipophilicity for substituents at C-4, C-12, C-15,

or C-27 tends to enhance the cytotoxicity of withanolides.

However, our results using an alternative cell line (H929

myeloma cells) indicated that acetylation of the OH at C-27 of

1

,

14

, and

34

to

15

,

16

, and

35

, respectively, had very little

e

ff

_{ect on cytotoxicity in a more typical 3 day drug-exposure}

design, but C-27 acetylation of

28

to provide

33

yielded over a

5-fold enhancement of cytotoxic activity under these conditions

(Table 2).

Heat-Shock Induction.

The heat-shock response plays a

critical role in maintaining protein homeostasis and helps cells

to cope with a wide range of proteotoxic insults.

50

As a result,

the ability of WA and other electrophilic natural products to

activate this response could provide a valuable approach to

combating protein aggregation-associated neurodegenerative

disorders such as Parkinson’s disease and Alzheimer’s

disease.

51,52

Previous SAR studies on the withanolide sca

ff

_old,

however, have focused on cytotoxicity as their end point, not

the heat-shock response. To begin de

fi

_{ning structural features}

contributing to heat-shock induction, we measured

concen-tration-dependent activation of the response by withanolides

1

−

37

. We applied serial dilutions of each compound in a

384-Table 1. Cytotoxicity of 1 and Its Active Analogues against

Ewing

’

s Sarcoma Cell Line CHP-100

withanolide IC50a withanolide IC50a

1 0.97±0.01 24 0.53±0.05

2 0.64±0.27 28 0.93±0.08

5 0.96±0.01 31 0.81±0.06

14 0.78±0.15 32 0.39±0.02

15 0.23±0.01 33 0.22±0.02

16 0.35±0.00 34 0.83±0.08

17 0.76±0.01 35 0.35±0.01

21 0.73±0.03 36 0.28±0.07

22 0.39±0.03 37 0.31±0.04

a_{Concentration required to inhibit cell proliferation/survival by 50%}

after 24 h of compound exposure, with each measured in octuplicate. IC50 values were determined from dose−response curves using

well format to transduced reporter cells stably expressing a

green

fl

_{uorescent protein}

₋

fi

_re

fl

_{y luciferase fusion protein under}

transcriptional control of classical heat-shock promoter

elements.

53

After overnight incubation, luciferase activity was

determined as a quantitative measure of relative heat-shock

activation. As evident in Figure 3, for

1

and its

fi

_{ve most active}

analogues (

15

,

16

,

33

,

35

, and

36

), reporter response was not a

monotonic function but rather peaked over a limited

concentration range for each active compound and then

declined, presumably as toxicity compromised the ability of

cells to respond. To quantitate this type of concentration

dependence in a way that would capture overall

heat-shock-inducing potential, we de

fi

_{ned a heat-shock index (HSI) for}

each compound calculated as Log

2

of the maximal response

(fold induction) divided by the concentration required to

stimulate this response (Table 2). Interestingly, all

fi

_ve

withanolide analogues (

15

,

16

,

33

,

35

, and

36

) that

demonstrated a HSI more than 2-fold of that of

1

contained

acetyl substituents at C-12 and/or C-27 in addition to the

ring-A 2(3)-en-1-one and ring-B 5β,6β-oxirane moieties. Greater

heat-shock-induction activity for analogues

33

and

35

as

compared to WA was con

fi

_{rmed using an alternate heat-shock}

reporter cell line, as previously described.

18

Using this reporter

system in nontransformed cells instead of cancer cells and

fl

_{uorescence instead of luciferase activity as an end point,}

heat-shock indices for

1

,

33

, and

35

were calculated as 2.1, 4.7, and

5.2, respectively (data not shown). Conservation of the rank

order for these compounds under di

ff

_{erent assay conditions}

indicates that their heat-shock induction activity is an intrinsic

property, not an artifact of the particular system used to

measure it.

Heat-Shock Induction vs Cytotoxicity.

Next, to

determine whether the heat-shock-inducing activity and

cytotoxicity of withanolides

1

−

37

could be dissected on the

basis of structural features, we examined the correlation of these

two activities across all 37 compounds (Figure 4). To monitor

cytotoxicity in the most sensitive manner possible, we

incubated H929 myeloma cells for 3 days with serial dilutions

of each compound in a 384-well format. This human cell line,

as with most myeloma cell lines, is particularly sensitive to

agents that disrupt protein homeostasis, especially proteasome

inhibitors such as MG-132.

54

If heat-shock induction was solely

a consequence of cytotoxicity arising from impairment of the

proteasome and HSP90 (previously reported targets for WA

20

)

or other mediators of protein homeostasis, then we would have

expected to see a consistent correlation between these activities

across all analogues tested. Instead, we found a relatively poor

correlation (

r

2

= 0.62), primarily because of a group of outlying

analogues (

15

,

16

,

35

, and

36

) that displayed greater

heat-shock induction than

1

at approximately the same level of

toxicity. In contrast, withanolide

33

was both more cytotoxic

and more heat-shock active than

1

, and the proteasome

inhibitor MG-132 used as a positive control showed greater

Table 2. Heat-Shock Index and Cytotoxicity of 1 and Its

Analogues 2

−

37

withanolide heat-shock indexa _cytotoxicityb

1 1.35 0.25 (0.239−0.255)

2 0.52 0.88 (0.826−0.941)

3 0.00 >10

4 0.00 5.25 (4.689−5.881)

5 0.59 0.54 (0.447−0.657)

6 0.00 0.61 (0.564−0.656)

7 0.00 8.74 (8.147−9.372)

8 0.00 >10

9 0.00 >10

10 0.00 5.09 (4.430−5.841)

11 0.00 >10

12 0.00 >10

13 0.00 4.96 (4.486−5.489)

14 1.73 0.25 (0.242−0.261)

15 3.81 0.25 (0.243−0.266)

16 3.44 0.22 (0.202−0.232)

17 1.53 0.43 (0.387−0.475)

18 0.20 3.32 (3.115−3.528)

19 0.00 >10

20 0.00 >10

21 0.36 1.12 (1.076−1.163)

22 0.78 1.04 (0.933−1.642)

23 0.00 >10

24 0.90 1.63 (1.567−1.703)

25 0.74 2.29 (2.161−2.430)

26 0.00 >10

27 0.79 1.16 (0.914−1.475)

28 1.68 0.28 (0.268−0.298)

29 0.00 4.30 (3.950−4.686)

30 0.00 3.83 (3.085−4.755)

31 0.00 1.65 (1.580−1.715)

32 0.92 0.46 (0.408−0.516)

33 3.69 0.05 (0.044−0.054)

34 1.64 0.25 (0.222−0.272)

35 3.74 0.27 (0.255−0.287)

36 3.31 0.18 (0.156−0.200)

37 1.65 0.24 (0.221−0.252)

MG-132 0.71 0.17 (0.164−0.184)

a_{Measured in the 293T reporter line and calculated as [Log}

2(treated/

control]/concentration (μM). b_{Concentration resulting in 50%}

reduction in relative viable myeloma (H929) cell number after 72 h exposure based on a nonlinear curvefi_{t of the dose−response data in} Prism 5.0 software (95% confi_{dence interval).}

toxicity than heat-shock induction compared to

1

. As noted

earlier, analogues with the greatest heat-shock activity all

contained acetyl substituents at C-4 and/or C-27 in addition to

the ring-A 2(3)-en-1-one and ring-B 5β,6β-oxirane moieties.

Withanolide

33

was acetylated at only C-27, whereas

35

carried

acetyl substituents at both C-4 and C-27.

Given the surprising nature of these

fi

_{ndings, we veri}

fi

_{ed the}

results by repeat testing of analogues

33

and

35

in a

low-throughput format.

1

and MG-132 were included for reference

(Figure 5). Although the absolute magnitude of e

ff

_{ects changed}

with the alternate format, relative relationships were preserved.

Again,

35

demonstrated greater heat-shock-inducing activity

(Figure 5a) with less cytotoxicity. This was the case both in the

cell line used for the reporter assays (293T cells, Figure 5b) and

in H929 cells, the line used for our initial correlation analysis

(Figure 5c). MG-132 was more cytotoxic for H929 cells than

293T cells, consistent with the known hypersensitivity of

myeloma cells to proteasome inhibition. It is noteworthy that

the more potent cytotoxicity of

33

in these assays was

consistent with results from the acute toxicity assay we used to

generate the data summarized in Table 1. Here, using CHP-100

cells, 24 h compound exposure, and MTT dye reduction as the

end point,

33

(IC

50

= 0.22

±

0.02) was also signi

fi

cantly more

potent than withanolide

35

(IC50

= 0.35

±

0.01).

Thiol Reactivity and Biological Activity.

Compound

1

contains three electrophilic sites that could play important roles

in its biological activity, including HSA and cytotoxicity. These

are C-3 of the ring-A 2(3)-en-1-one moiety, C-5/C-6 of the

ring-B 5β,6β-oxirane moiety, and C-24 of the ring-E

unsaturated lactone moiety. We have previously shown that

coincubation of our heat-shock reporter cells with

N

-acetylcysteine (NAC) and

1

leads to a near complete

suppression of heat-shock activation caused by

1

.

18

Thus, it

was of interest to assess the chemical reactivity of

1

compared

to informative analogues using NAC as a representative thiol

nucleophile. Treatment of

1

with NAC a

ff

_{orded the product of}

Michael addition at C-3 (

38

) in 66% yield. No addition was

observed at C-5, C-6, or C-24 by

1

_{H NMR and HPLC. As}

expected, 2,3-dihydrowithaferin A (

20

) was unreactive under

these conditions. Because

20

is devoid of both HSA and

cytotoxic activity, the presence of the ring-A 2(3)-en-1-one

moiety appears to be essential for many, if not all, of WA’s

biological activities. To probe the relationship between the thiol

reactivity of this enone moiety and HSA further, we monitored

reaction of

1

with 1 equiv of NAC in DMSO-

d

6

and, in parallel,

reaction of NAC with

35

, our analogue with the highest

heat-shock index. The progress of these reactions was assessed by

1

_H

NMR using the disappearance of the signal from H-2 of the

ring-A 2(3)-en-1-one moiety as the end point (Supporting

Information, Figure S31). The ratio of the starting material to

product was used to determine the Gibb’s free energy (Δ

G

) of

the thiol addition/elimination processes.

55

Reactions of

1

and

Figure 4. Correlation of heat-shock induction with cytotoxicity for

compounds1−37.The proteasome inhibitor MG-132 is included as a mechanistically and structurally distinct control compound. The solid line depicts a linear curve fi_{t for all data points (r}2 _{= 0.62), as} performed in Microsoft Excel software. The heat-shock-active analogues lying furthest off _{the curve} fi_{t are circled in red.} Heat-shock index (determined in 293T reporter cells) was calculated as [Log2(treated/control)]/concentration. Cytotoxicity (determined in

H929 myeloma cells) was calculated as Log2 IC50, where IC50 is

concentration (μM) resulting in 50% reduction in the relative viable cell number.

35

with NAC showed typical second-order reaction curves,

with calculated

Δ

G

values of

−

7.0 and

−

9.3 kcal/mol,

respectively, indicating that analogue

35

reacts more readily

with NAC than

1

. Therefore, in regards to the group appended

at C-4 of the withanolide sca

ff

_{old, its identity (OH or OAc),}

orientation (α

or

β), or a combination of these factors can alter

the chemical reactivity of the ring-A 2(3)-en-1-one system. The

more reactive analogue,

35

, demonstrated greater HSA, but

additional experimentation will be required to de

fi

_{ne the}

relationship between the relative chemical reactivity of this

enone system and HSA fully. We can conclude, however, that

orientation of the group at C-4 appears to exert little or no

e

ff

_{ect on acute cytotoxicity, as the pairs of analogues}

₁

_and

₂₈

_,

15

and

33

,

16

and

35

, and

17

and

34

showed very similar IC

50

values in limiting the growth and survival of CHP-100 cells

(Table 1).

Heat-Shock Induction In Vivo.

To determine whether the

heat-shock activity of withanolides demonstrated in cell culture

would translate to whole animals, we performed exploratory

pharmacodynamic studies in mice (Figure 6). Establishing a

biocompatible cyclodextrin-based formulation for these very

poorly water-soluble compounds, we compared the

dose-dependent ability of

1

and

35

to activate a systemic heat-shock

response after parenteral administration. Single doses up to 50

mg/kg were tolerated without overt acute toxicity. Choosing

spleen as a sentinel organ representative of the hematopoietic

compartment, we assayed lysates for upregulation of heat shock

protein 72 (HSP72), the most highly heat-inducible isoform of

the HSP70 family of molecular chaperones. At a dose of 25

mg/kg, several mice receiving

35

responded with a robust

increase in relative HSP72 level, leading to a highly signi

fi

_cant

increase in variance for this group. In contrast,

1

caused a much

smaller, nonsigni

fi

_{cant e}

ff

_{ect on variance, indicative of little}

treatment e

ff

_{ect under these conditions. Increasing the dose of}

35

to 40 mg/kg produced more uniform induction across the

treatment group and a signi

fi

_{cant di}

ff

_{erence in the mean HSP72}

level compared to the vehicle control group. Although much

work beyond the scope of this initial report obviously remains,

these exploratory

fi

_{ndings con}

fi

_{rm that withanolides can}

activate the heat-shock response in mice at systemically

tolerable exposures. Whether activation of the heat-shock

response per se will prove a key determinant of the therapeutic

bene

fi

_{ts ascribed to withanolides in diverse human diseases}

remains to be determined. Equally important may be their

ability to form thiol adducts with a range of important

electrophile sensors in cells that are among the

fi

_rst-line

defenses for launching adaptive transcriptional and

post-transcriptional responses.

56,57

Nevertheless,

fi

_{ndings presented}

here indicate that heat-shock induction can serve as a useful

biomarker for their activity in vivo.

■

CONCLUSIONS

same relative level of cytotoxicity, it does appear possible to

discriminate between these activities through speci

fi

_{c structural}

modi

fi

_{cations to the withanolide core. Conversely, other}

modi

fi

_{cations increased bioactivity in general, both heat-shock}

activation and cytotoxicity, perhaps by enhancing cellular

uptake or limiting metabolic inactivation.

From our results, we conclude that the withanolide sca

ff

_old

can be modi

fi

_{ed to shift its spectrum of bioactivity while}

preserving potency. Enhancing heat-shock response while

minimizing cytotoxicity could provide a better therapeutic

index in pursuit of compounds that activate intrinsic cellular

defense mechanisms to combat protein aggregation-associated

neurodegenerative disorders. Conversely, minimizing activation

of the cytoprotective heat-shock response while maintaining

antiproliferative activity could provide more e

ff

_{ective anticancer}

agents. Furthermore, reporter assay-guided tuning of the

withanolide core appears to provide a practical route to

realizing the full therapeutic potential of this versatile sca

ff

_old.

■

MATERIALS AND METHODS

General Procedures.Melting points were determined in capillary tubes using a Mel-Temp apparatus and are uncorrected. Optical rotations were measured in MeOH or CHCl3 with a Jasco DIP-370

digital polarimeter. UV spectra were determined in MeOH on a Shimadzu UV-1601 spectrometer. One-dimensional and 2D NMR spectra were recorded in CDCl3, unless otherwise stated, using

residual solvents as internal standards on Bruker DRX-500, DRX-600, and Avance III 400 spectrometers at 500, 600, and 400 MHz for1_H

NMR and 125, 150, and 100 MHz for13_{C NMR. The chemical shift}

values (δ) are given in parts per million (ppm), and the coupling constants (Jvalues) are in Hz. LR−MS and HR−MS were recorded using Shimadzu LCMS 8000QPαand JEOL HX110A spectrometers, respectively. Analytical thin-layer chromatography was carried out on silica gel 60 F254aluminum-backed TLC plates (Merck). Preparative

thin-layer chromatography was performed on Analtech silica gel 500

μm glass plates. Compounds were visualized with short-wavelength UV and by spraying with anisaldehyde-sulfuric acid spray reagent and heating until the spots appeared. Silica gelfl_{ash chromatography was} accomplished using 230−400 mesh silica gel. All yields refer to yields of isolated compounds. Unless otherwise stated, chemicals and solvents were of reagent grade and used as obtained from commercial sources without further purifi_{cation. Purity of all}fi_{nal compounds was} determined to be≥95% by HPLC and1_{H NMR analysis.}

Isolation of Naturally Occurring Withanolides 1−13. With-aferin A (1), 27-deoxywithaferin A (2), viscosalactone B (3), 2,3-dihydrowithaferin A-3β-O-sulfate (4), 3α -(uracil-1-yl)-2,3-dihydrowi-thaferin A (5), 3β-(adenin-9-yl)-2,3-dihydrowithaferin A (6), 3β-O -butyl-2,3-dihydrowithaferin A (7), withanolide A (8), 27-hydroxywi-thanolide B (9), 4β,27-dihydroxy-1-oxo-22R-witha-2,5,24-trienolide (10), 2,3-didehydrosomnifericin (11), jaborasalactone D (12), and pubesenolide (13) were obtained from aeroponically grown W. somniferaas described previously.22,33

General Procedure for Acetylation of Withanolides. To a solution of the withanolide (2.0 mg) in anhydrous pyridine (0.5 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). The reaction mixture was poured into ice/water (10.0 mL), and the resulting solution was passed through a short column of RP (C18) silica gel (0.2 g). The column was washed with

water (30.0 mL) followed by elution with MeOH (10.0 mL). The MeOH fraction after evaporation was subjected to preparative TLC (silica gel) to yield the corresponding acetyl derivative.

Preparation of 27-Acetylwithaferin A (15_{) and}

4,27-Diacetylwi-thaferin A (16_{).To a solution of1(10.0 mg) in pyridine (0.1 mL) was}

added Ac2O (2.4μL), and the mixture was stirred at 25°C. After 2 h,

EtOH (15.0 mL) was added to the reaction mixture and evaporated under reduced pressure. The residue thus obtained was separated by

preparative TLC (silica gel) using 6% MeOH in CH2Cl2as eluant to

give15(2.1 mg, 19%) and16(8.5 mg, 72%).

27-Acetylwithaferin A (15_{).White solid; mp 218}−220°C; [α]D25

+128 (c0.8, CHCl3);1H NMR (500 MHz, CDCl3)δ6.90 (dd,J= 9.9,

5.8 Hz, 1H, H-3), 6.18 (d,J= 9.9 Hz, 1H, H-2), 4.88 (d,J= 11.8 Hz, 1H, H-27a), 4.84 (d,J= 11.8 Hz, 1H, H-27b), 4.38 (dt,J= 13.6, 3.3 Hz, 1H, H-22), 3.74 (dd,J= 5.8, 2.1 Hz, 1H, H-6), 3.22 (s, 1H, H-4), 2.51 (dd,J= 13.2, 10.9 Hz, 2H), 2.12 (ddd,J= 14.9, 6.3, 2.6, 1H, H-7a), 2.05 (s, 3H, H3-28), 2.04 (s, 3H, OAc), 1.96 (m, 2H), 1.93 (dt,J=

9.6, 3.3 Hz, 1H), 1.82 (dt,J= 14.2, 3.6 Hz, 1H), 1.69−1.59 (m, 2H), 1.53−1.43 (m, 2H), 1.39 (s, 3H, H3-18), 1.25 (m, 3H), 1.18−1.01 (m,

2H), 0.98 (d,J= 6.6 Hz, 3H, H3-21), 0.91−0.82 (m, 2H), 0.69 (s, 3H,

H3-19); HRMS (ESI): [M + H]+calcd for C30H41O7, 513.2847; found,

513.2850.

4,27-Diacetylwithaferin A (16_{).White solid; mp 232}−234°C;1_H

NMR data were consistent with those reported;3_{APCI-MS (+)m/z:}

[M + 1]+_555.

Sulfation of Withaferin A.To a stirred solution1(10.0 mg) in pyridine (0.5 mL) was added SO3-pyridine complex (5.0 mg). and the

reaction mixture was heated at 80°C. After 1 h (TLC control), the reaction mixture was poured to ice water (30 mL), and the resulting solution was introduced to a short column of RP (C18) silica gel (5.0

g) and washed with water (50 mL) followed by elution with 40% MeOH(aq). The crude product obtained by evaporation of MeOH-(aq) eluents was fi_{nally puri}fi_{ed by C18 preparative TLC (40%} MeOH(aq)) to aff_{ord18(8.3 mg) and19(3.6 mg).}

Withaferin A 27-Sulfate (18_{). Amorphous colorless solid; [α]D}20

+55.9 (c0.78, MeOH);1_{H NMR (400 MHz, pyridine-d5)δ0.53 (s,}

3H), 0.94 (d,J= 7.0 Hz, 3H), 1.84 (s, 3H), 2.09 (s, 3H), 2.30 (dd,J= 13.5, 17.5 Hz, 1H), 3.59 (brs, 1H), 4.02 (d,J= 6.5 Hz, 1H), 4.42 (brd,

J= 13.0 Hz, 1H), 5.31 (d,J= 11.0 Hz, 1H), 5.44 (d,J= 11.0 Hz, 1H), 6.41 (d,J= 9.5 Hz, 1H), 7.22 (dd,J= 6.5, 9.5 Hz, 1H);13_{C NMR}

(125 MHz, pyridine-d5)δ11.7, 13.5, 17.3, 20.5, 21.8, 24.6, 27.3, 30.1, 30.4, 31.8, 39.1, 39.6, 42.7, 44.7, 48.6, 52.0, 56.1, 60.2, 61.2, 64.6, 70.4, 78.5, 122.7, 132.4, 145.2 158.2, 165.8, 202.6.

Withaferin A 4,27-Disulfate (19_{).Amorphous colorless solid; [α]D}20

+158.5 (c0.2, MeOH);1_{H NMR (400 MHz, CD}

3OD)δ0.74 (s, 3H),

1.00 (d,J= 6.5 Hz, 3H), 1.36 (s, 3H), 2.13 (s, 3H), 2.54 (dd,J= 13.0, 18.0 Hz, 1H), 3.29 (brs, 1H), 4.28 (d,J= 6.0 Hz, 1H), 4.45 (dt,J= 3.5, 13.0 Hz, 1H), 4.76 (d,J= 11.0 Hz, 1H), 4.85 (overlap with H2O

peak, 1H), 6.23 (d,J= 10.0 Hz, 1H), 7.17 (dd,J= 6.0, 10.0 Hz, 1H);

13_{C NMR (125 MHz, CD}

3OD)δ 10.5, 12.2, 14.7, 19.3, 20.9, 24.0,

26.8, 29.5, 29.6, 31.0, 38.9, 39.1, 42.2, 44.2, 51.6, 55.8, 59.8, 60.8, 61.3, 75.6, 78.6, 121.2, 132.4, 134.6, 142.1, 156.9, 166.4, 201.7.

2,3-Dihydrowithaferin A (20_{). To a solution of 1(15.0 mg) in}

EtOH (1.0 mL) were added Et3N (60μL) and 10% Pd/C (1.0 mg),

and the mixture was stirred under an atmosphere of H2for 1 h. The

reaction mixture wasfi_{ltered, and the} fi_{ltrate was evaporated under} reduced pressure to give 2,3-dihydrowithaferin A (20) as a white solid (14.7 mg, 98%); mp 267−269°C;1_{H NMR data were consistent with}

those reported;3APCI-MS (+)m/z: [M + 1]+_473.

27-Acetyl-2,3-dihydrowithaferin A 4-Sulfate (21_{).Acetylation of4}

by the general procedure aff_{orded21as an amorphous colorless solid;} [α]D20 +27.7 (c 0.33, MeOH); 1H NMR (400 MHz, pyridine-d5) δ

0.51(s, 3H), 0.97 (d,J= 6.0 Hz, 3H), 1.67 (s, 3H), 2.00 (d,J= 1.5 Hz, 3H), 2.02 (s, 3H), 2.41 (dd,J= 13.0, 16.0 Hz, 1H), 3.27 (dd,J= 6.0, 18.0 Hz, 1H), 3.60 (dd,J= 10.5, 14.0 Hz, 1H), 4.42 (brd,J= 13.0 Hz, 1H), 4.50 (brs, 1H), 5.12 (d,J= 12.0 Hz, 1H), 5.22 (d,J= 12.0 Hz, 1H), 5.59 (dd,J= 6.5, 8.0 Hz, 1H);13_{C NMR (125 MHz, pyridine-d5)}

δ11.5, 13.5, 15.4, 20.3, 20.8, 21.3, 24.5, 27.3, 30.0, 30.2, 31.5, 39.2 (×2), 41.6, 42.8 (×2), 49.5, 52.0, 56.0, 56.8, 58.6, 64.7, 72.6, 76.4, 78.4, 122.0, 157.9, 165.3, 170.7, 209.1; HRMS (ESI): [M−H]−

calcd for C30H41O11S, 609.2375; found, 609.2365.

4,27-Diacetyl-2,3-dihydrowithaferin A 3β-O-Sulfate (22_).

Amor-phous colorless solid; [α]D20 +42.4 (c 0.38, MeOH);1H NMR (400

8.5 Hz, 1H), 5.86 (brs, 1H);13_{C NMR (125 MHz, pyridine-d5)δ11.5,}

13.5, 15.1, 20.3, 20.7, 20.8, 21.4, 24.4, 27.2, 29.6, 30.1, 31.0, 39.08, 39.13, 41.7, 42.7, 42.8, 50.0, 51.9, 55.9, 57.7, 58.6, 62.1, 69.7, 78.2, 78.4, 122.1, 157.9, 165.3, 170.4, 170.7, 208.2; HRMS (ESI): [M + H]+

calcd for C32H45O12S, 653.2626; found, 653.2626.

4,6,27-Triacetyl-2,3-didehydrosomnifericin (25_{). Amorphous}

col-orless solid; [α]D20 +203.2 (c 0.03, MeOH); 1H NMR (400 MHz,

CDCl3)δ0.68 (s, 3H), 0.96 (d,J= 6.0 Hz, 3H), 1.28 (s, 3H), 2.02 (s,

3H), 2.04 (s, 3H), 2.06 (s, 3H), 2.18 (s, 3H), 2.49 (dd,J= 13.5, 17.6 Hz, 1H), 4.38 (dt,J= 3.4, 13.2 Hz, 1H), 4.85 (d,J= 19.0 Hz, 1H), 4.88 (d,J= 19.0 Hz, 1H), 5.06 (dd,J= 5.0, 12.1 Hz, 1H), 6.06 (dd,J= 2.2, 10.3 Hz, 1H), 6.29 (dd,J= 2.2, 10.3 Hz, 1H), 6.35 (t,J= 2.2 Hz, 1H); APCI-MSm/z: [M + H]+_615.

4-Dehydrowithaferin A (27_{). To a solution of 1 (30 mg) in}

CHCl3/EtOAc (5:7, 2.0 mL) was added freshly prepared manganese

dioxide (300 mg), and the mixture was stirred at 25°C. After 16 h, the reaction mixture wasfi_{ltered, the}fi_{ltrate was evaporated under reduced} pressure, and the residue was purifi_{ed by preparative TLC (silica gel)} using 8% MeOH in CH2Cl2as eluant to give27(18.4 mg, 62%) as a

white powder; mp 273−275°C (lit.3_{272−275°C); [α]}

D

25_{+143 (c0.8,}

MeOH) [lit.3+147 (c0.83, MeOH)]; APCI-MS (+)m/z: [M + 1]+

469;1_{H NMR data were consistent with those reported.}3

27-Acetyl-4-dehydrowithaferin A (32_{).To a solution of27 (5.0}

mg) in pyridine (0.1 mL) was added Ac2O (0.05 mL), and the mixture

was stirred at 25°C for 18 h. The reaction mixture was evaporated under reduced pressure and by adding EtOH, and the residue was purifi_{ed by preparative TLC (silica gel) using 4% MeOH in CH2Cl2as} eluant to give32(5.25 mg, 96%) as a white solid; mp 173−175°C;1_H

NMR (500 MHz, CDCl3)δ6.85 (d,J= 10.3 Hz, 1H, H-3), 6.82 (d,J

= 10.3 Hz, 1H, H-2), 4.88 (d,J= 11.8 Hz, 1H, H-27a), 4.85 (d,J= 11.8 Hz, 1H, H-27b), 4.39 (dt,J= 13.2, 3.4 Hz, 1H, H-22), 3.41 (d,J

= 2.3 Hz, 1H, H-6), 2.51 (dd,J= 18.4, 13.2 Hz, 1H, H-23a), 2.15 (dt,J

= 15.2, 3.4 Hz, 1H, H-7a), 2.06 (s, 3H, H3-28), 2.04 (s, 3H, OAc),

2.03−1.95 (m, 3H), 1.70−1.57 (m, 5H), 1.47−1.39 (m, 2H), 1.35−

1.07 (m, 5H), 1.37 (s, 3H, H3-18), 1.00 (d,J= 6.7 Hz, 3H, H3-21),

0.71 (s, 3H, H3-19); APCI-MS (+)m/z: [M + 1]+511.

Withaferin A 27-tert-Butyldimethylsilyl Ether (39_{).To a solution}

of1(25 mg) in DMF (1.5 mL) were addedt-BDMS-Cl (63 mg) and 4-PP (78 mg), and the mixture was stirred at 60°C for 3 h, after which the reaction mixture was diluted with EtOAc, washed with brine, dried over anhydrous Na2SO4, and evaporated under reduced pressure, and

the residue was separated on preparative TLC (silica gel) using 3% MeOH in CH2Cl2as eluant to give39as a white solid (28 mg, 90%);

mp 178−180°C;1_{H NMR (500 MHz, CDCl}

3)δ6.90 (dd,J= 9.9, 5.8

Hz, 1H, H-3), 6.18 (d,J= 9.9 Hz, 1H, H-2), 4.48 (d,J= 11.6 Hz, 1H, H-27a), 4.37 (d,J= 11.6 Hz, 1H, H-27b), 4.36 (dt,J= 16.6, 3.3 Hz, 1H, H-22), 3.73 (d,J= 5.8 Hz, 1H, H-4), 3.21 (s, 1H, H-6), 2.44 (dd,

J= 17.4, 13.5 Hz, 1H, H-23a), 2.13 (m, 1H, H-7a), 2.04 (s, 3H, H3

-28), 1.97−1.91 (m, 2H), 1.80 (dq,J= 14.2, 3.6 Hz, 1H), 1.68−1.58 (m, 3H), 1.52−1.43 (m, 2H), 1.39 (s, 3H, H3-18), 1.37−0.99 (m,

11H), 0.95 (d,J= 8.8 Hz, 3H, H3-21), 0.87 (s, 9H, 3 x CH3), 0.68 (s,

3H, H3-19), 0.07 (s, 3H, SiCH3), 0.06 (s, 3H, SiCH3); APCI-MS (+) m/z: [M + 1]+_585.

4-Acetylwithaferin A 27-tert-Butyldimethylsilyl Ether (40_).

Acetylation of39(20.0 mg) by the usual procedure (Ac2O/pyridine)

aff_{orded40as a white solid (21.0 mg, 98%); APCI-MS (+)m/z: [M +} 1]+_627.

4-Acetylwithaferin A (14_{). Deprotection of 40 (19.0 mg) was}

carried out by treating a solution of it in THF (0.3 mL) and MeOH (0.05 mL) with 2 N HCl (0.05 mL) at 0°C for 1 h. The reaction mixture was diluted with H2O, evaporated under reduced pressure,

and extracted with EtOAc. The EtOAc layer was evaporated under reduced pressure, and the residue was purifi_{ed by preparative TLC} (silica gel) using 5% MeOH in CH2Cl2as eluant to give14as a white

solid (15 mg, 96%); mp 192−194°C;1_{H NMR data were consistent}

with those reported;58 _{HRMS (ESI): [M + H]}+_{calcd for C} 30H41O7,

513.2847; found, 513.2852.

4-epi-Withaferin A (28_{).To a stirred solution of 27(6.0 mg) in}

MeOH (1.0 mL) and THF (0.5 mL) was added CeCl3·7H2O (17 mg).

The reaction mixture was cooled to 0°C in an ice bath, and NaBH4

(2.0 mg) was added. After 30 min at 0°C, the reaction mixture was evaporated, and the residue was separated on preparative TLC (silica gel) using 6% MeOH in CH2Cl2as eluant to give28as a white solid

(4.2 mg, 70%); mp 227−228 °C; [α]D25 +29.9 (c 1.0, CHCl3);1H

NMR (500 MHz, CDCl3+ CD3OD)δ6.80 (dd,J= 10.1, 1.5 Hz, 1H,

H-3), 5.97 (dd,J= 10.1, 2.5 Hz, 1H, H-2), 4.64 (brs, 1H, H-4), 4.37 (dt,J= 13.5, 3.3 Hz, 1H, H-22), 4.32 (d,J= 12.5 Hz, 1H, H-27a), 4.27 (d,J= 12.5 Hz, 1H, H-27b), 3.65 (brs, 1H, H-6), 2.45 (dd,J= 13.6, 7.2 Hz, 1H, H-23a), 2.10 (brd, 1H, H-7a), 2.00 (s, 3H, H3-28),1.96−

1.89 (m, 4H), 1.78 (brs, 1H), 1.67−1.58 (m, 2H), 1.49−1.42 (m, 2H), 1.31 (m, 1H), 1.18 (s, 3H, H3-18), 1.15−1.00 (m, 4H), 0.94 (d,J= 6.6

Hz, 3H, H3-21), 0.88 (m, 1H), 0.66 (s, 3H, H3-19);13C NMR (125

MHz, CDCl3+ CD3OD)δ201.4, 167.1, 153.3, 148.0, 128.4, 125.6,

78.7, 65.8, 64.4, 57.0, 55.9, 55.4, 51.9, 47.6, 45.5, 42.6, 39.4, 38.8, 30.7, 29.8, 27.2, 24.2, 22.2, 20.0, 13.8, 13.2, 12.0; HRMS (ESI): [M + H]+

calcd for C28H39O6, 471.2747; found, 471.2764.

4,27-Diacetyl-4-epi-withaferin A (35_{).Acetylation of28(1.0 mg)}

by the usual procedure (Ac2O/pyridine) followed by purification on

preparative TLC (silica gel) using 6% MeOH in CH2Cl2 as eluant

aff_{orded 35 (1.1 mg, 93%); mp 214−216} °C; [α]D25 +36.8 (c 1.1,

CHCl3);1H NMR (600 MHz, CDCl3)δ6.66 (dd,J= 10.1, 1.5 Hz,

1H, H-3), 6.05 (dd,J= 10.1, 2.4 Hz, 1H, H-2), 5.87 (brs, 1H, H-4), 4.88 (d,J= 11.8 Hz, 1H, H-27a), 4.84 (d,J= 11.8 Hz, 1H, H-27b), 4.38 (dt,J= 13.1, 3.3 Hz, 1H, H-22), 3.53 (brs, 1H, H-6), 2.50 (dd,J= 17.6, 13.3 Hz, 1H, H-23a), 2.09 (s, 3H, OAc), 2.05 (s, 3H, H3-28),

2.03 (s, 3H, OAc), 2.01−1.92 (m, 4H), 1.67−1.33(m, 6H), 1.28 (s, 3H, H3-18), 1.23−1.01 (m, 4H), 0.98 (d, J = 6.6 Hz, 3H, H3-21),

0.94−0.81 (m, 2H), 0.70 (s, 3H, H3-19); 13C NMR (100 MHz,

CDCl3)δ201.1, 170.9, 169.8, 165.3, 157.0, 144.9, 129.4, 121.9, 78.2,

65.8, 63.3, 58.0, 55.9, 55.8, 52.0, 48.1, 45.5, 42.5, 39.5, 38.8, 34.6, 31.8, 30.8, 30.1, 29.6, 27.3, 24.2, 22.6, 20.9, 20.8, 20.5, 14.6, 14.1, 13.3, 11.7; HRMS (ESI): [M + H]+ _{calcd for C}

32H43O8, 555.2952; found,

555.2950.

27-Acetyl-4-epi-withaferin A (33_{).To a stirred solution of32(3.0}

mg) in THF (0.2 mL) and MeOH (0.2 mL) at 0 °C were added CeCl3·7H2O (65 mg) and NaBH4 (small portion), and the mixture

was stirred at 0°C. After 10 min, a small ice cube was added to the reaction mixture, the solvent and water were evaporated under reduced pressure, and the residue was partition between H2O and

EtOAc. The EtOAc layer was dried over anhydrous Na2SO4 and

evaporated under reduced pressure, and the residue was separated on preparative TLC (silica gel) using 2% MeOH in CH2Cl2as eluant to

give33(2.5 mg, 70%) as a white solid; mp 188−190°C; [α]D25+ 42.4

(c1.0, CHCl3);1H NMR (500 MHz, CDCl3)δ6.83 (dd,J= 10.2, 1.4

Hz, 1H, H-3), 6.00 (d,J= 10.2, 2.5 Hz, 1H, H-2), 4.88 (d,J= 11.9 Hz, 1H, H-27a), 4.85 (d,J= 11.9 Hz, 1H, H-27b), 4.71 (s, 1H, H-4), 4.38 (dt,J= 13.2, 3.3 Hz, 1H, H-22), 3.63 (s, 1H, H-6), 2.50 (dd,J= 17.6, 14.5 Hz, 1H, H-23a), 2.12 (m, 1H, H-7a), 2.05 (s, 3H, H3-28), 2.03 (s,

3H, OAc), 1.99 (dd,J= 13.2, 3.3 Hz, 1H), 1.93 (brd,J= 9.9 Hz, 1H), 1.68−1.45 (m, 4H), 1.34 (m, 1H), 1.28−1.22 (m, 3H), 1.21 (s, 3H, H3-18), 1.18−1.03 (m, 4H), 0.98 (d,J= 6.7 Hz, 3H, H3-21), 0.94−

0.81 (m, 2H), 0.69 (s, 3H, H3-19);13C NMR (100 MHz, CDCl3) δ

201.2, 170.9, 165.3, 156.9, 147.6, 141.5, 129.0, 121.9, 78.2, 65.6, 64.6, 58.0, 55.3, 52.0, 47.6, 45.8, 42.5, 35.4, 38.8, 30.8, 30.1, 27.7, 27.3, 24.3, 22.6, 22.2, 20.9, 20.6, 14.1, 13.7, 13.3, 11.8, 11.6; HRMS (ESI): [M + H]+_{calcd for C}

30H41O7, 513.2847; found, 513.2854.

4-Dehydrowithaferin A 27-tert-Butyldimethylsilyl Ether (41_{).To a}

solution of27(11.3 mg) in DMF (0.5 mL) were addedt-BDMS-Cl (36.4 mg) and 4-pp (42.9 mg), and the mixture was stirred under an atmosphere of N2for 1 h at 60°C. The reaction mixture was then

diluted with EtOAc, washed with brine, and evaporated under reduced pressure, and the residue was separated on preparative TLC using CH2Cl2as eluant to give41(9.5 mg). APCI-MS (+)m/z: [M + 1]+

583.

4-epi-Withaferin A 27-tert-Butyldimethylsilyl Ether (42_{). To a}

solution of41(9.5 mg) in THF (0.2 mL) and MeOH (0.2 mL) at 0

°C was added CeCl3·7H2O (125 mg), and the mixture was stirred at 0 °C for 5 min. To this solution was then added NaBH4(small portion),

under reduced pressure, and the residue was partition between H2O

and EtOAc. The EtOAc layer was dried over anhydrous Na2SO4and

evaporated under reduced pressure, and the residue was separated on preparative TLC (silica gel) using 2% MeOH in CH2Cl2as eluant to

give42(7.5 mg, 70%) as a white solid; APCI-MS (+)m/z: [M + 1]+

585.

4-Acetyl-4-epi-withaferin A 27-tert-Butyldimethylsilyl Ether (43_).

A solution of42(7.5 mg) in pyridine (0.3 mL) and Ac2O (0.2 mL)

was stirred at 25°C for 4 h. The reaction mixture was evaporated under reduced pressure to give43(8.0 mg) as a white solid; APCI-MS (+)m/z: [M + 1]+_627.

4-Acetyl-4-epi-withaferin A (34_{).To a solution of43(8.0 mg) in}

THF (0.5 mL) and MeOH (0.3 mL) at 0°C was added 2 N HCl (0.15 mL), and the mixture was stirred at 0°C. After 1 h, the reaction mixture was diluted with H2O, MeOH and THF were evaporated

under reduced pressure and extracted with EtOAc (3×15 mL), the combined EtOAc layer was washed with H2O, dried over anhydrous

Na2SO4, and evaporated under reduced pressure, and the residue was

separated on preparative TLC (silica gel) using 5% MeOH in CH2Cl2

as eluant to give34as a white solid (5.3 mg, 70%); mp 236−38°C; [α]D25+ 29.7 (c1.2, CHCl3);1H NMR (600 MHz, CDCl3)δ6.66 (dd, J= 10.4, 1.5 Hz, 1H, H-3), 6.05 (dd,J= 10.4, 2.4 Hz, 1H, H-2), 5.87 (brs, 1H, H-4), 4.39 (brd,J= 13.4, 3.3 Hz, 1H, H-22), 4.37 (d,J= 12.5 Hz, 1H, H-27a), 4.32 (d,J= 12.5 Hz, 1H, 27b), 3.53 (brs, 1H, H-6), 2.48 (dd,J= 16.2, 13.9 Hz, 1H, H-23a), 2.11 (brd, 1H, H-7a), 2.09 (s, 3H, OAc), 2.01 (s, 3H, H3-28), 1.97−1.93 (m, 4H), 1.54−1.45 (m,

2H), 1.34 (m, 1H), 1.28 (s, 3H, H3-18), 1.23−1.00 (m, 6H), 0.98 (d,J

= 6.6 Hz, 3H, H3-21), 0.94−0.84 (m, 2H), 0.69 (s, 3H, H3-19);13C

NMR (100 MHz, CDCl3)δ201.1, 169.9, 166.0, 152.8, 144.8, 129.4,

125.8, 78.7, 65.8, 63.3, 57.5, 55.9, 55.8, 52.0, 48.1, 45.5, 42.6, 39.5, 38.8, 30.6, 29.9, 27.3, 24.2, 22.9, 20.8, 20.0, 14.5, 13.3, 11.6; HRMS (ESI): [M + H]+_{calcd for C}

30H41O7, 513.2847; found, 513.2850. Microbial Biotransformation of 1.Small-scale fermentation of

Cunninghamella echinulata (ATCC 10028B) was performed in an Erlenmeyerfl_{ask (125 mL) containing soybean meal-glucose medium} (25 mL) on a rotary shaker operating at 220 rpm at 28°C for 24 h. Large-scale fermentation was performed under the same conditions in Erlenmeyerfl_{asks (3}×250 mL) holding 50 mL of the medium in each fl_{ask, which was inoculated with 15% of the 1 day old inoculum. A} solution of 1(5 mg in 0.5 mL of DMF) was added to each fl_ask containing 24 h old second cultivation. After 72 h, fermentation broths were combined, and mycelium wasfi_{ltered o}ff_{and washed with H2O} (100 mL), which was combined withfi_{ltrate and extracted with EtOAc} (3×200 mL). The combined organic layer was washed with H2O (2 ×200 mL), dried over anhydrous Na2SO4, and evaporated to give

EtOAc extract (42 mg). Gel-permeation chromatography of this extract over a column of Sephadex LH-20 (3.0 g) followed by preparative TLC (silica gel) gave 12β-hydroxywithaferin A (29, 3.8 mg, 24%) and 15β-hydroxywithaferin A (30, 4.9 mg, 32%).

12β-Hydroxywithaferin A (29_{).mp 120−121} °C (lit.38 119−120

°C);1_{H NMR data were consistent with those reported.}38

15β-Hydroxywithaferin A (30_{).mp 271}−273 °C (lit.38 _270−274

°C);1_{H NMR data were consistent with those reported.}38

12β-Acetoxy-4,27-diacetylwithaferin A (36_{).To a solution of 29}

(1.0 mg) in pyridine (0.05 mL) was added Ac2O (0.05 mL), and the

mixture was stirred at 25 °C for 18 h. The reaction mixture was evaporated under reduced pressure and by adding EtOH, and the residue was purifi_{ed on preparative TLC (silica gel) using 4% MeOH} in DCM as eluant to give36(1.2 mg, 95%) as a white amorphous solid.1_{H NMR data were consistent with those reported.}38

15β-Acetoxy-4,27-diacetylwithaferin A (37_{).To a solution of 30}

(1.0 mg) in pyridine (0.05 mL) was added Ac2O (0.05 mL), and the

mixture was stirred at 25 °C for 18 h. The reaction mixture was evaporated under reduced pressure and by adding EtOH, and the residue was purifi_{ed on preparative TLC (silica gel) using 4% MeOH} in DCM as eluant to give37(1.2 mg, 95%) as a white amorphous solid.1_{H NMR data were consistent with those reported.}38

Withaferin A N-Acetylcysteine Adduct (38_{). To a solution of1}

(40.0 mg) in MeOH (4.0 mL) was addedN-acetylcysteine (NAC) (80.0 mg), and the solution was stirred at 25°C. After 48 h, MeOH

was removed under reduced pressure, and the residue was separated by HPLC [Phenomenex Luna C18 (5μ) 10 ×250 mm, gradient solvent system from 60 to 80% in 20 min, 3 mL/min fl_{ow rate,} detection at 230 nm] to aff_{ord38(22.0 mg; 66% based on recovered}

1) (tR = 13.0 min) and 1(9.4 mg) (tR = 14.8 min). 38: off-white

amorphous solid;1_{H NMR (400 MHz, CDCl}

3)δ4.36 (1H, brd,J=

12.8 Hz, H-22), 4.32 and 4.27 (1H each, d,J= 12.4 Hz, H-27), 3.43 (1H, brs, H-4), 3.29 (1H, brs, H-6), 3.00 (2H, brs, Cys-CH2), 2.76 and

2.40 (1H each, m, H2-2), 2.00 (6H, s, Cys-CH3and H3-28), 1.23 (3H,

s, H3-19), 0.93 (3H, d,J= 6.4 Hz, H3-21), 0.61 (3H, s, H3-18);13C

NMR (100 MHz, pyridine-d5) δ 209.8 (C-1), 174.3 (Cys-CON), 170.6 (Cys-CO2H), 166.7 26), 154.2 3), 128.9 2), 121.9

(C-25), 88.1 (C-17), 80.9 (C-14), 79.5 (C-22), 78.9 (C-20), 65.3 (C-18), 64.3 (C-24), 127.8 (C-25), 78.7 (C-22), 77.2 (C-4), 65.4 (C-5), 59.5 (C-6), 56.6 (C-27), 56.3 (C-14), 54.0 (Cys-CH), 52.3 (C-17), 51.4 (C-10), 44.8 (C-3), 43.8 (C-9), 43.0 (C-13), 41.1 (C-2), 39.6 (C-16), 39.5 (C-20), 34.9 (Cys-CH2), 31.9 (C-23), 30.4 (C-8), 30.3 (C-7),

27.6 (C-12), 24.8 (C-15), 23.4 (Cys-CH3), 22.1 (C-11) 20.5 (C-28),

16.0 (C-19), 13.9 (C-21), 11.9 (C-18); LR-APCIMS (+):m/z[M + Na]+_{656, [M + H]}+_{634, [M + H-NAC]}+_471.

Cytotoxicity Assays. Ewing’s sarcoma cell line CHP-100 and myeloma cell line H929 were cultured at 37°C under 6% CO2 in

RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), and 293T cells were grown in DMEM supplemented with 10% FBS. All cell lines were tested and found to be 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 infl_{at-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 dye-reduction assay using the substrate [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide] (MTT). Cytotoxicity over 3 days against H929 and 293T cells was measured in 384-well format (3000 cells/well), and the relative viable cell number was determined by reduction of the dye resazurin (Alamar Blue) as previously described.40

Heat-Shock Reporter Assays.Reporter cells were generated by infecting 293T cells (American Type Culture Collection) with a previously reported lentiviral vector encoding a fusion protein consisting of enhanced GFP fused tofi_refl_{y luciferase under control} ofHSP70B′promoter elements.54 _{The plasmid encoding the fusion}

protein was generously provided by Khalid Shah (Massachusetts General Hospital, Boston, MA, USA). To isolate a homogeneous population of high responding cells, a transduced culture was heat-shocked at 42°C for 1 h and then processed 8 h later byfl uorescence-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 was continued overnight. Measurement of relative luciferase activity was achieved using an Envision plate luminometer (PerkinElmer) and Steady-Glo reagent (Promega) per the manufacturer’s recommendations. As a con-fi_{rmatory assay for some compounds, 3T3-Y9-B12 reporter cells were} seeded in blackfl_{at-bottom 96-well plates (20 000/well) and allowed} to adhere overnight as previously reported.23 Cells were then incubated for 24 h in the presence of WA or analogues (1, 2, or 4

μM). After washing with PBS, fl_{uorescence was quanti}fi_{ed using an} Analyst AD (LJL Biosystems) plate reader with excitation and emissionfi_{lters set at 485 and 530 nm, respectively.}