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Structure−Activity Relationships for Withanolides as Inducers of theCellular Heat-Shock ResponseE. 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 ofAgriculture and Life Sciences, University of Arizona, 250 East Valencia Road, Tucson, Arizona 85706, United States‡Laboratorio de Oncologia Experimental, Departamento de Fisiologia e Farmacologia, Universidade Federal do Ceara, P.O. Box 3157,Fortaleza, Ceara 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 thestructure and the remarkably diverse bioactivities reported forwithanolides, we obtained withaferin A (WA; 1) and 36analogues (2−37) and compared their cytotoxicity tocytoprotective heat-shock-inducing activity (HSA). By analyz-ing structure−activity relationships for the series, we foundthat 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) andC-15 (30) decreased both activities. Introduction of β-OAc to 4,27-diacetyl-WA (16) at C-15 (37) decreased HSA withoutaffecting cytotoxicity, but at C-12 (36), it had minimal effect. Importantly, acetylation of 27-OH, yielding 15 from 1, 16 from 14,and 35 from 34, enhanced HSA without increasing cytotoxicity. Our findings demonstrate that the withanolide scaffold can bemodified to enhance HSA selectively, thereby assisting development of natural product-inspired drugs to combat proteinaggregation-associated diseases by stimulating cellular defense mechanisms.

■ INTRODUCTIONWithanolides, a class of steroidal lactones structurally based onan ergostane skeleton, are abundant in plants of the familySolanaceae.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 traditionalsystems of medicine practiced throughout Asia and SouthAmerica.2 The beneficial effects of many of these plants havebeen attributed to the presence of withanolides. One of the beststudied of these withanolides is withaferin A (1, WA), a majorconstituent of the plant Withania somnifera (L.) Dunal.3,4

Popularly known as Ashwagandha or Indian ginseng, it hasbeen used in Indian Ayurvedic medicine for over 3000 years.5

Various preparations of Ashwagandha are available as herbaldietary supplements worldwide. The National Center forComplementary and Alternative Medicines (NCCAM) of theU.S. National Institutes of Health has recently recognizedAshwagandha as a high-priority topic for mechanistic research.6

Numerous reports describe anticancer,7 neuroprotective,8−10

anti-inflammatory,11 immunomodulatory,12,13 and antioxidant5

activities for medicinal preparations of W. somnifera and itsconstituent withanolides. Among these, the most extensivelystudied has been the anticancer activity of WA.14−16 For

example, the Developmental Therapeutics Program (DTP) ofthe U.S. National Cancer Institute has tested WA (NSC101088) against its panel of 60 human cancer cell lines andfound a mean 50% growth inhibitory concentration (GI50) of620 nM.17 Other studies have demonstrated significant activityfor WA against human brain,18 prostate,19 pancreatic,20 andbreast cancer21 xenografts in mice.In previous work, we used the heat-shock response (HSR) as

a biosensor to discover potential anticancer compounds thattarget 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 componentof their anticancer activity.18,22 Others have reported inhibitionof cell motility and angiogenesis,23,24 inhibition of NF-κBactivation,25−29 protein kinase C,30 and Notch-1.31 Reports alsodescribe induction of Par-4-dependent apoptosis,19 FOXO3a-and Bim-dependent21 apoptosis, and sensitization to TRAIL-induced apoptosis.32 Despite the great diversity of biologicaleffects reported for natural and semisynthetic withanolides,

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

Article

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© 2014 American Chemical Society 2851 dx.doi.org/10.1021/jm401279n | J. Med. Chem. 2014, 57, 2851−2863

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structure−activity relationship studies have relied almostexclusively on cytotoxicity as their end point for activity.To begin probing how cytoprotective heat-shock-inducing

activity relates to the cytotoxic activity of withanolides, weisolated 1 and natural withanolides 2−13 using the biomassderived from aeroponically cultivated W. somnifera andprepared 24 structurally related analogues, 14−37, by perform-ing chemical and microbial transformations of 1 that had beenisolated from the same material, providing a total of 36analogues. We evaluated these compounds for their ability toactivate the heat-shock response using cell-based reportersystems, whereas antiproliferative activity was measured in a

reporter cell line as well as two other cancer cell lines. Thisapproach allowed us to identify relatively modest structuralmodifications that alter the chemical reactivity of analoguestoward thiols and selectively enhance heat-shock-inducingactivity over cytotoxicity and vice versa. Importantly, weconfirmed these reporter-based cell culture results throughexploratory pharmacodynamic studies in mice. Our findingssuggest that reporter assay-guided tuning of the withanolidescaffold provides a useful approach to improving thetherapeutic potential of this class and perhaps other thiol-reactive natural products as anticancer or neuroprotectiveagents.

Figure 1. Withanolides 1−13 obtained from aeroponically grown Withania somnifera and their derivatives 14−26.

Figure 2. Analogues 27−31 obtained by chemical and microbiological transformations of withaferin A, their derivatives 32−37, and withaferin A N-acetyl cysteine adduct 38.

Journal of Medicinal Chemistry Article

dx.doi.org/10.1021/jm401279n | J. Med. Chem. 2014, 57, 2851−28632852

■ RESULTS AND DISCUSSION

Isolation and Semisynthesis of Withanolides 1−37.We investigated the effects of various substituents, theirposition and stereochemistry, and their lipophilicity onantiproliferative versus heat-shock-induction activity for 37withanolides. The panel of compounds used in this studyincluded natural withanolides 1−13 obtained from aeroponi-cally grown W. somnifera22,33 and their derivatives 14−26(Figure 1). Analogues 27−31 were obtained from WA bychemical and microbial transformations. These compoundswere further derivatized to yield compounds 32−37 (Figure 2).Semisynthesis of 4-epi-withaferin A (28) was efficientlyachieved by the MnO2 oxidation of 1 to 4-dehydrowithaferinA (27)3 followed by regio- and stereoselective reduction of itsC-4 carbonyl group with NaBH4/CeCl3. The use of lanthanoidcations (such as Ce3+) in reactions of enones with NaBH4 isknown to cause 1,2-reduction of the carbonyl group with highselectivity compared to 1,4-reduction caused by NaBH4 in theabsence of these cations,34 with the ratio of epimeric alcoholsformed being determined by steric factors.35 Conversion of 27to 28, however, constitutes the first report of a steroselectivereduction of only one carbonyl group of an ene-dione withNaBH4/CeCl3/MeOH/THF. The high degree of regio- andstereoselectivity observed for 27 yielding 28 may be explainedas being due to the chelation of the boron atom of the reducing

species [NaBH4−n(OMe)n]34 to the oxygen atom of the ring-B

oxirane of 27, delivering the hydride from the β-phase. Thestructure of 28 was elucidated by the analysis of its 1H, 13C, and2D NMR spectroscopic data including HMBC. The α-orientation of the 4-OH was confirmed by NOE experiments(see Supporting Information Figure S17). The two O-sulfatedanalogues of 1, withaferin A-27-sulfate (18) and withaferin A-4,27-disulfate (19), were prepared by the reaction of WA withSO3-pyridine.

36 3-Azido-2,3-dihydrowithaferin A (31) wasobtained by treating 1 with NaN3/Et3N.

37

Microbial biotransformation of 1 with the fungus Cunning-hamella echinulata afforded 12β-hydroxywithaferin A (29) and15β-hydroxywithaferin A (30).38 Controlled acetylation(Ac2O/pyridine) of 1 yielded 27-acetylwithaferin A (15) and4,27-diacetylwithaferin A (16). Acetyl analogues 21−26, 32,and 35−37 were obtained by the standard acetylation of theircorresponding alcohols using Ac2O/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 and42 with Ac2O/pyridine followed by deprotection (HCl/THF/MeOH) afforded 14 and 34, respectively.

Scheme 1. Conversion of 1 to 14a

aReagents and conditions: (a) TBDMS-Cl, 4-PP, DMF, 60 °C; (b) Ac2O, pyridine, 25 °C; (c) 2 N HCl, THF, MeOH, 0 °C.

Scheme 2. Conversion of 1 to 28 and Its Derivativesa

aReagents and conditions: (a) MnO2, CHCl3, EtOAc, 25 °C; (b) NaBH4, CeCl3·7H2O, MeOH, THF, 0 °C; (c) Ac2O, pyridine, 25 °C; (d)TBDMSCl, 4-PP, DMF, 60 °C; (e) 2 N HCl, THF, MeOH, 0 °C.



Cytotoxicity. As an initial screen, we measured the acutecytotoxicity of 1 and its analogues 2−37 at a singleconcentration (4.0 μM) using the human Ewing’s sarcomacell line CHP-100 (Supporting Information, Figure S29). Wechose this very rapidly proliferating cell line to maximizesensitivity over a short period of compound exposure. Cellswere incubated for 24 h with compounds, and the relativeviable cell number was measured by a standard dye-reductionassay.39 Doxorubicin and DMSO were used as positive andnegative controls, respectively. WA and analogues 2, 5, 14−17,21, 22, 24, 28, and 31−37 inhibited the overall proliferationand survival of CHP-100 cells over this short time interval by>80%. In a follow-up experiment, we determined the IC50 foreach 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 areconsistent with previous reports that the 2(3)-en-1-one moietyin ring A of withanolides is essential for their cytotoxicactivity.40−47

The absence of cytotoxic activity for 2,3-dihydrowithaferin A-3β-O-sulfate (4) was expected on the basis of our previousfinding that the conversion of 4 to 1 in cell culture media is aslow process requiring longer than the 24 h incubation periodused for these experiments.22 In contrast, acetyl derivatives of 4,namely, 21 and 22, were found to be highly active, suggestingthat acetylation of the OH groups of 4 assists in readyconversion of the 1-oxo-3β-O-sulfate to a 2(3)-en-1-one systemin cell culture medium. Analogues 5 and 31, which contain amasked 2(3)-en-1-one system capable of generating this moietyunder physiological conditions, also exhibited cytotoxic activitycomparable to 1. 3-Azido-1-ones such as 31 are known toundergo elimination of HN3 under mildly basic conditions(e.g., Al2O3) to produce their corresponding 2(3)-en-1-ones.48

A recent report, however, suggested that the nature of thesubstituent at C-3 rather than the 2(3)-en-1-one group isessential for the enhanced cytotoxicity of a series of 3-substituted withanolides including 31.37 This report promptedus to investigate the possibility that 31 could undergoelimination of HN3 to produce 1 under physiologicalconditions. Thus, 31 was incubated in cell culture mediumconsisting of Dulbecco/Vogt modified Eagle’s minimal essentialmedium (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 disappearedfrom the culture medium with the corresponding de novoappearance of 1 (Supporting Information, Figure S30),suggesting that the cytotoxic activity exhibited by 31 may bepartly due to its conversion to 1 under the experimentalconditions. Cytotoxic activity exhibited by 3β-uracyl-2,3-dihydrowithaferin A (5) may also be explained as a result ofits conversion to 1 in cell culture medium because the uracylanion is known to be a good leaving group.49

Our data indicate that the nature of the substituent at C-4has a major effect on the antiproliferative activity ofwithanolides, at least the ones we examined. An electron-withdrawing carbonyl group at C-4, as in 27, reduced activitywhen compared to 1, supporting our previous observation thatthe reactivity of the 2(3)-en-1-one moiety of withanolidesdetermines their ability to adduct thiols and their consequentcytotoxicity.18 4-Dehydrowithaferin A (27) has been previouslyreported to be more cytotoxic than 1,47 but different conditionsand cell lines used for cytotoxicity assays could easily accountfor such discrepancy.Comparing the potencies of 1 and 28 indicates that the

orientation of the 4-OH has very little effect on cytotoxicitytoward CHP-100 cells. In our previous study of theantiproliferative activities of 28 and 1 against pancreatic cancercell lines MIAPaCa-2 and BxPC-3, both withanolides also hadsimilar activities. In pancreatic cancer cell line PANC-1,however, 1 demonstrated ca. 4-fold higher potency than 28for reasons that are unclear, but this could be due to theirdifferences in cellular uptake and/or metabolism.16 Several SARstudies have noted the importance of the 5β,6β-epoxy group inring B for the cytotoxicity of withanolides.41,46 When this groupis replaced with a double bond. as in 24, acute cytotoxicity wasretained against CHP-100 cells (Table 1 and SupportingInformation, Figure S29). This finding indicates that the 5β,6β-epoxy group is not required but can enhance the cytotoxicity ofwithanolides. Our findings with 2 and 15 agree with previousreports that the presence of an OH group at C-27 ofwithanolides leads to a reduction in their antiproliferativeactivity.46,47 This finding together with the enhancement ofactivity observed for acetyl derivatives 15, 16, 21, 22, 24, 32,34, and 35−37 as compared to their parent alcohols suggeststhat 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 (H929myeloma cells) indicated that acetylation of the OH at C-27 of1, 14, and 34 to 15, 16, and 35, respectively, had very littleeffect on cytotoxicity in a more typical 3 day drug-exposuredesign, but C-27 acetylation of 28 to provide 33 yielded over a5-fold enhancement of cytotoxic activity under these conditions(Table 2).

Heat-Shock Induction. The heat-shock response plays acritical role in maintaining protein homeostasis and helps cellsto cope with a wide range of proteotoxic insults.50 As a result,the ability of WA and other electrophilic natural products toactivate this response could provide a valuable approach tocombating protein aggregation-associated neurodegenerativedisorders such as Parkinson’s disease and Alzheimer’sdisease.51,52 Previous SAR studies on the withanolide scaffold,however, have focused on cytotoxicity as their end point, notthe heat-shock response. To begin defining structural featurescontributing to heat-shock induction, we measured concen-tration-dependent activation of the response by withanolides1−37. We applied serial dilutions of each compound in a 384-

Table 1. Cytotoxicity of 1 and Its Active Analogues againstEwing’s Sarcoma Cell Line CHP-100

withanolide IC50a withanolide IC50

a

1 0.97 ± 0.01 24 0.53 ± 0.052 0.64 ± 0.27 28 0.93 ± 0.085 0.96 ± 0.01 31 0.81 ± 0.0614 0.78 ± 0.15 32 0.39 ± 0.0215 0.23 ± 0.01 33 0.22 ± 0.0216 0.35 ± 0.00 34 0.83 ± 0.0817 0.76 ± 0.01 35 0.35 ± 0.0121 0.73 ± 0.03 36 0.28 ± 0.0722 0.39 ± 0.03 37 0.31 ± 0.04

aConcentration 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 usingMicrosoft Excel software; ± refers to standard deviation.



well format to transduced reporter cells stably expressing agreen fluorescent protein−firefly luciferase fusion protein undertranscriptional control of classical heat-shock promoterelements.53 After overnight incubation, luciferase activity wasdetermined as a quantitative measure of relative heat-shockactivation. As evident in Figure 3, for 1 and its five most activeanalogues (15, 16, 33, 35, and 36), reporter response was not amonotonic function but rather peaked over a limitedconcentration range for each active compound and thendeclined, presumably as toxicity compromised the ability ofcells to respond. To quantitate this type of concentrationdependence in a way that would capture overall heat-shock-inducing potential, we defined a heat-shock index (HSI) foreach compound calculated as Log2 of the maximal response(fold induction) divided by the concentration required to

stimulate this response (Table 2). Interestingly, all fivewithanolide analogues (15, 16, 33, 35, and 36) thatdemonstrated a HSI more than 2-fold of that of 1 containedacetyl 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. Greaterheat-shock-induction activity for analogues 33 and 35 ascompared to WA was confirmed using an alternate heat-shockreporter cell line, as previously described.18 Using this reportersystem in nontransformed cells instead of cancer cells andfluorescence instead of luciferase activity as an end point, heat-shock indices for 1, 33, and 35 were calculated as 2.1, 4.7, and5.2, respectively (data not shown). Conservation of the rankorder for these compounds under different assay conditionsindicates that their heat-shock induction activity is an intrinsicproperty, not an artifact of the particular system used tomeasure it.

Heat-Shock Induction vs Cytotoxicity. Next, todetermine whether the heat-shock-inducing activity andcytotoxicity of withanolides 1−37 could be dissected on thebasis of structural features, we examined the correlation of thesetwo activities across all 37 compounds (Figure 4). To monitorcytotoxicity in the most sensitive manner possible, weincubated H929 myeloma cells for 3 days with serial dilutionsof each compound in a 384-well format. This human cell line,as with most myeloma cell lines, is particularly sensitive toagents that disrupt protein homeostasis, especially proteasomeinhibitors such as MG-132.54 If heat-shock induction was solelya consequence of cytotoxicity arising from impairment of theproteasome and HSP90 (previously reported targets for WA20)or other mediators of protein homeostasis, then we would haveexpected to see a consistent correlation between these activitiesacross all analogues tested. Instead, we found a relatively poorcorrelation (r2 = 0.62), primarily because of a group of outlyinganalogues (15, 16, 35, and 36) that displayed greater heat-shock induction than 1 at approximately the same level oftoxicity. In contrast, withanolide 33 was both more cytotoxicand more heat-shock active than 1, and the proteasomeinhibitor MG-132 used as a positive control showed greater

Table 2. Heat-Shock Index and Cytotoxicity of 1 and ItsAnalogues 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 >104 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 >109 0.00 >1010 0.00 5.09 (4.430−5.841)11 0.00 >1012 0.00 >1013 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 >1020 0.00 >1021 0.36 1.12 (1.076−1.163)22 0.78 1.04 (0.933−1.642)23 0.00 >1024 0.90 1.63 (1.567−1.703)25 0.74 2.29 (2.161−2.430)26 0.00 >1027 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)

aMeasured in the 293T reporter line and calculated as [Log2(treated/control]/concentration (μM). bConcentration resulting in 50%reduction in relative viable myeloma (H929) cell number after 72 hexposure based on a nonlinear curve fit of the dose−response data inPrism 5.0 software (95% confidence interval).

Figure 3. Concentration-dependent induction of heat-shock reporteractivity by WA and its five most active analogues. Heat-shock reportercells were incubated with the indicated compounds overnight in a 384-well format followed by measurement of luciferase activity on amicroplate luminometer. 1 refers to withaferin A isolated fromaeroponically grown biomass for this study. WA refers to a commercialsample of the compound (Chromadex, Irvine, CA) that was assayedindependently in this experiment to verify the reproducibility of thereporter assay. Data are plotted as the mean ratio of treated to control,with all determinations performed in quadruplicate wells. Error bars,SD.



toxicity than heat-shock induction compared to 1. As notedearlier, analogues with the greatest heat-shock activity allcontained acetyl substituents at C-4 and/or C-27 in addition tothe ring-A 2(3)-en-1-one and ring-B 5β,6β-oxirane moieties.Withanolide 33 was acetylated at only C-27, whereas 35 carriedacetyl substituents at both C-4 and C-27.Given the surprising nature of these findings, we verified 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 effects changedwith 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 thecell line used for the reporter assays (293T cells, Figure 5b) andin H929 cells, the line used for our initial correlation analysis(Figure 5c). MG-132 was more cytotoxic for H929 cells than293T cells, consistent with the known hypersensitivity ofmyeloma cells to proteasome inhibition. It is noteworthy thatthe more potent cytotoxicity of 33 in these assays wasconsistent with results from the acute toxicity assay we used togenerate the data summarized in Table 1. Here, using CHP-100cells, 24 h compound exposure, and MTT dye reduction as theend point, 33 (IC50 = 0.22 ± 0.02) was also significantly morepotent than withanolide 35 (IC50 = 0.35 ± 0.01).Thiol Reactivity and Biological Activity. Compound 1

contains three electrophilic sites that could play important rolesin its biological activity, including HSA and cytotoxicity. Theseare C-3 of the ring-A 2(3)-en-1-one moiety, C-5/C-6 of thering-B 5β,6β-oxirane moiety, and C-24 of the ring-Eunsaturated lactone moiety. We have previously shown thatcoincubation of our heat-shock reporter cells with N-acetylcysteine (NAC) and 1 leads to a near completesuppression of heat-shock activation caused by 1.18 Thus, itwas of interest to assess the chemical reactivity of 1 compared

to informative analogues using NAC as a representative thiolnucleophile. Treatment of 1 with NAC afforded the product ofMichael addition at C-3 (38) in 66% yield. No addition wasobserved at C-5, C-6, or C-24 by 1H NMR and HPLC. Asexpected, 2,3-dihydrowithaferin A (20) was unreactive underthese conditions. Because 20 is devoid of both HSA andcytotoxic activity, the presence of the ring-A 2(3)-en-1-onemoiety appears to be essential for many, if not all, of WA’sbiological activities. To probe the relationship between the thiolreactivity of this enone moiety and HSA further, we monitoredreaction of 1 with 1 equiv of NAC in DMSO-d6 and, in parallel,reaction of NAC with 35, our analogue with the highest heat-shock index. The progress of these reactions was assessed by 1HNMR using the disappearance of the signal from H-2 of thering-A 2(3)-en-1-one moiety as the end point (SupportingInformation, Figure S31). The ratio of the starting material toproduct was used to determine the Gibb’s free energy (ΔG) ofthe thiol addition/elimination processes.55 Reactions of 1 and

Figure 4. Correlation of heat-shock induction with cytotoxicity forcompounds 1−37. The proteasome inhibitor MG-132 is included as amechanistically and structurally distinct control compound. The solidline depicts a linear curve fit for all data points (r2 = 0.62), asperformed in Microsoft Excel software. The heat-shock-activeanalogues lying furthest off the curve fit are circled in red. Heat-shock index (determined in 293T reporter cells) was calculated as[Log2(treated/control)]/concentration. Cytotoxicity (determined inH929 myeloma cells) was calculated as Log2 IC50, where IC50 isconcentration (μM) resulting in 50% reduction in the relative viablecell number.

Figure 5. Verification of WA analogues found as outliers for their heat-shock-inducing activity by correlation analysis: 33 was most cytotoxic,whereas 35 was least toxic. In each panel, the mean percent comparedto vehicle-treated control cells is plotted, with all determinationsperformed in quadruplicate wells. Error bars, SD. (a) Heat-shockreporter induction in confluent 293T cells after overnight exposure tocompounds, (b) inhibition of 293T cell growth and survival asmeasured by resazurin dye reduction after 72 h, and (c) inhibition ofH929 cell growth and survival as measured by resazurin dye reductionafter 72 h.



35 with NAC showed typical second-order reaction curves,with calculated ΔG values of −7.0 and −9.3 kcal/mol,respectively, indicating that analogue 35 reacts more readilywith NAC than 1. Therefore, in regards to the group appendedat C-4 of the withanolide scaffold, its identity (OH or OAc),orientation (α or β), or a combination of these factors can alterthe chemical reactivity of the ring-A 2(3)-en-1-one system. Themore reactive analogue, 35, demonstrated greater HSA, butadditional experimentation will be required to define therelationship between the relative chemical reactivity of thisenone system and HSA fully. We can conclude, however, thatorientation of the group at C-4 appears to exert little or noeffect on acute cytotoxicity, as the pairs of analogues 1 and 28,15 and 33, 16 and 35, and 17 and 34 showed very similar IC50

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 culturewould translate to whole animals, we performed exploratorypharmacodynamic studies in mice (Figure 6). Establishing abiocompatible cyclodextrin-based formulation for these verypoorly water-soluble compounds, we compared the dose-dependent ability of 1 and 35 to activate a systemic heat-shockresponse after parenteral administration. Single doses up to 50mg/kg were tolerated without overt acute toxicity. Choosingspleen as a sentinel organ representative of the hematopoieticcompartment, we assayed lysates for upregulation of heat shockprotein 72 (HSP72), the most highly heat-inducible isoform ofthe HSP70 family of molecular chaperones. At a dose of 25mg/kg, several mice receiving 35 responded with a robustincrease in relative HSP72 level, leading to a highly significantincrease in variance for this group. In contrast, 1 caused a muchsmaller, nonsignificant effect on variance, indicative of little

treatment effect under these conditions. Increasing the dose of35 to 40 mg/kg produced more uniform induction across thetreatment group and a significant difference in the mean HSP72level compared to the vehicle control group. Although muchwork beyond the scope of this initial report obviously remains,these exploratory findings confirm that withanolides canactivate the heat-shock response in mice at systemicallytolerable exposures. Whether activation of the heat-shockresponse per se will prove a key determinant of the therapeuticbenefits ascribed to withanolides in diverse human diseasesremains to be determined. Equally important may be theirability to form thiol adducts with a range of importantelectrophile sensors in cells that are among the first-linedefenses for launching adaptive transcriptional and post-transcriptional responses.56,57 Nevertheless, findings presentedhere indicate that heat-shock induction can serve as a usefulbiomarker for their activity in vivo.

■ CONCLUSIONS

To identify structural features responsible for the divergentbiological activities ascribed to withaferin A (WA), weexamined a total of 37 compounds (1−37) consisting ofnatural withanolides, chemical and microbial transformationproducts of 1, and their derivatives. We focused on heat-shockactivation (HSA) and cytotoxicity as distinct activities because,contrary to common assumption, HSA is not a generalconsequence of cytotoxicity. Rather, it is a specific transcrip-tional response to the disruption of protein homeostasis withincells. Many highly cytotoxic chemotherapeutics do not inducethe heat-shock response, and its activation is not a conservedfeature of either apoptotic or necrotic cell death pathways.Because we were able to identify withanolides (e.g., 35) thatpossess greater heat-shock-inducing potential than 1 at the

Figure 6. Induction of systemic heat-shock response by WA (1) and 4,27-diacetyl-4-epi-withaferin A (35). (A) Compounds were formulated incyclodextrin vehicle and administered by subcutaneous injection (25 mg/kg, five mice per treatment group). Spleens were harvested the followingday, and lysate from each animal was loaded in a separate lane for gel electrophoresis followed by immunoblotting for HSP72, a highly inducibleisoform of the HSP70 family. Actin was blotted as a loading control. Integrated band intensity for HSP72 normalized to actin for each sample isplotted on the right. The median with interquartile range is indicated by horizontal bars for each treatment group. F test was used to comparevariance between vehicle and withanolide 35, p < 0.005, and between vehicle and WA, not significant. (B) Witanolide 35 administered at higher dose(40 mg/kg, three mice per group) yields more consistent heat-shock induction. Immunoblotting was performed, and analysis is depicted as in panelA. Student’s t test comparing vehicle to compound treatment, p = 0.01.



same relative level of cytotoxicity, it does appear possible todiscriminate between these activities through specific structuralmodifications to the withanolide core. Conversely, othermodifications increased bioactivity in general, both heat-shockactivation and cytotoxicity, perhaps by enhancing cellularuptake or limiting metabolic inactivation.From our results, we conclude that the withanolide scaffold

can be modified to shift its spectrum of bioactivity whilepreserving potency. Enhancing heat-shock response whileminimizing cytotoxicity could provide a better therapeuticindex in pursuit of compounds that activate intrinsic cellulardefense mechanisms to combat protein aggregation-associatedneurodegenerative disorders. Conversely, minimizing activationof the cytoprotective heat-shock response while maintainingantiproliferative activity could provide more effective anticanceragents. Furthermore, reporter assay-guided tuning of thewithanolide core appears to provide a practical route torealizing the full therapeutic potential of this versatile scaffold.

■ MATERIALS AND METHODSGeneral Procedures. Melting points were determined in capillary

tubes using a Mel-Temp apparatus and are uncorrected. Opticalrotations were measured in MeOH or CHCl3 with a Jasco DIP-370digital polarimeter. UV spectra were determined in MeOH on aShimadzu UV-1601 spectrometer. One-dimensional and 2D NMRspectra were recorded in CDCl3, unless otherwise stated, usingresidual solvents as internal standards on Bruker DRX-500, DRX-600,and Avance III 400 spectrometers at 500, 600, and 400 MHz for 1HNMR and 125, 150, and 100 MHz for 13C NMR. The chemical shiftvalues (δ) are given in parts per million (ppm), and the couplingconstants (J values) are in Hz. LR−MS and HR−MS were recordedusing Shimadzu LCMS 8000QPα and JEOL HX110A spectrometers,respectively. Analytical thin-layer chromatography was carried out onsilica gel 60 F254 aluminum-backed TLC plates (Merck). Preparativethin-layer chromatography was performed on Analtech silica gel 500μm glass plates. Compounds were visualized with short-wavelengthUV and by spraying with anisaldehyde-sulfuric acid spray reagent andheating until the spots appeared. Silica gel flash chromatography wasaccomplished using 230−400 mesh silica gel. All yields refer to yieldsof isolated compounds. Unless otherwise stated, chemicals andsolvents were of reagent grade and used as obtained from commercialsources without further purification. Purity of all final compounds wasdetermined to be ≥95% by HPLC and 1H 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), andpubesenolide (13) were obtained from aeroponically grown W.somnifera as described previously.22,33

General Procedure for Acetylation of Withanolides. To asolution 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 untilthe reaction was complete (judged by the disappearance of the startingmaterial by TLC). The reaction mixture was poured into ice/water(10.0 mL), and the resulting solution was passed through a shortcolumn of RP (C18) silica gel (0.2 g). The column was washed withwater (30.0 mL) followed by elution with MeOH (10.0 mL). TheMeOH 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 of 1 (10.0 mg) in pyridine (0.1 mL) wasadded Ac2O (2.4 μL), and the mixture was stirred at 25 °C. After 2 h,EtOH (15.0 mL) was added to the reaction mixture and evaporatedunder reduced pressure. The residue thus obtained was separated by

preparative TLC (silica gel) using 6% MeOH in CH2Cl2 as eluant togive 15 (2.1 mg, 19%) and 16 (8.5 mg, 72%).

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

+128 (c 0.8, CHCl3);1H NMR (500 MHz, CDCl3) δ 6.90 (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, 3.3Hz, 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; 1HNMR data were consistent with those reported;3 APCI-MS (+) m/z:[M + 1]+ 555.

Sulfation of Withaferin A. To a stirred solution 1 (10.0 mg) inpyridine (0.5 mL) was added SO3-pyridine complex (5.0 mg). and thereaction mixture was heated at 80 °C. After 1 h (TLC control), thereaction mixture was poured to ice water (30 mL), and the resultingsolution was introduced to a short column of RP (C18) silica gel (5.0g) and washed with water (50 mL) followed by elution with 40%MeOH(aq). The crude product obtained by evaporation of MeOH-(aq) eluents was finally purified by C18 preparative TLC (40%MeOH(aq)) to afford 18 (8.3 mg) and 19 (3.6 mg).

Withaferin A 27-Sulfate (18). Amorphous colorless solid; [α]D20

+55.9 (c 0.78, MeOH); 1H NMR (400 MHz, pyridine-d5) δ 0.53 (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); 13C NMR(125 MHz, pyridine-d5) δ 11.7, 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]D20

+158.5 (c 0.2, MeOH); 1H NMR (400 MHz, CD3OD) δ 0.74 (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 H2Opeak, 1H), 6.23 (d, J = 10.0 Hz, 1H), 7.17 (dd, J = 6.0, 10.0 Hz, 1H);13C NMR (125 MHz, CD3OD) 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) inEtOH (1.0 mL) were added Et3N (60 μL) and 10% Pd/C (1.0 mg),and the mixture was stirred under an atmosphere of H2 for 1 h. Thereaction mixture was filtered, and the filtrate was evaporated underreduced pressure to give 2,3-dihydrowithaferin A (20) as a white solid(14.7 mg, 98%); mp 267−269 °C; 1H NMR data were consistent withthose reported;3 APCI-MS (+) m/z: [M + 1]+ 473.

27-Acetyl-2,3-dihydrowithaferin A 4-Sulfate (21). Acetylation of 4by the general procedure afforded 21 as an amorphous colorless solid;[α]D

20 +27.7 (c 0.33, MeOH); 1H NMR (400 MHz, pyridine-d5) δ0.51(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); 13C NMR (125 MHz, pyridine-d5)δ 11.5, 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 forC30H41O11S, 609.2375; found, 609.2365.

4,27-Diacetyl-2,3-dihydrowithaferin A 3β-O-Sulfate (22). Amor-phous colorless solid; [α]D

20 +42.4 (c 0.38, MeOH); 1H NMR (400MHz, pyridine-d5) δ 0.49 (s, 3H), 0.97 (d, J = 6.5 Hz, 3H), 1.50 (s,3H), 1.85 (d, J = 1.0 Hz, 3H), 2.00 (d, J = 1.5 Hz, 3H), 2.02 (s, 3H),2.40 (dd, J = 13.0, 17.5 Hz, 1H), 3.14 (dd, J = 6.5, 17.5 Hz, 1H), 3.19(brs, 1H), 3.65 (dd, J = 10.0, 18.0 Hz, 1H), 4.40 (brd, J = 13.0 Hz,1H), 5.12 (d, J = 11.5 Hz, 1H), 5.22 (d, J = 11.5 Hz, 1H), 5.61 (brt, J =



8.5 Hz, 1H), 5.86 (brs, 1H); 13C NMR (125 MHz, pyridine-d5) δ 11.5,13.5, 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) δ 0.68 (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, 17.6Hz, 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-MS m/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 manganesedioxide (300 mg), and the mixture was stirred at 25 °C. After 16 h, thereaction mixture was filtered, the filtrate was evaporated under reducedpressure, and the residue was purified by preparative TLC (silica gel)using 8% MeOH in CH2Cl2 as eluant to give 27 (18.4 mg, 62%) as awhite powder; mp 273−275 °C (lit.3 272−275 °C); [α]D25 +143 (c 0.8,MeOH) [lit.3 +147 (c 0.83, MeOH)]; APCI-MS (+) m/z: [M + 1]+

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

27-Acetyl-4-dehydrowithaferin A (32). To a solution of 27 (5.0mg) in pyridine (0.1 mL) was added Ac2O (0.05 mL), and the mixturewas stirred at 25 °C for 18 h. The reaction mixture was evaporatedunder reduced pressure and by adding EtOH, and the residue waspurified by preparative TLC (silica gel) using 4% MeOH in CH2Cl2 aseluant to give 32 (5.25 mg, 96%) as a white solid; mp 173−175 °C; 1HNMR (500 MHz, CDCl3) δ 6.85 (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

of 1 (25 mg) in DMF (1.5 mL) were added t-BDMS-Cl (63 mg) and4-PP (78 mg), and the mixture was stirred at 60 °C for 3 h, after whichthe reaction mixture was diluted with EtOAc, washed with brine, driedover anhydrous Na2SO4, and evaporated under reduced pressure, andthe residue was separated on preparative TLC (silica gel) using 3%MeOH in CH2Cl2 as eluant to give 39 as a white solid (28 mg, 90%);mp 178−180 °C; 1H NMR (500 MHz, CDCl3) δ 6.90 (dd, J = 9.9, 5.8Hz, 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 of 39 (20.0 mg) by the usual procedure (Ac2O/pyridine)afforded 40 as 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 reactionmixture was diluted with H2O, evaporated under reduced pressure,and extracted with EtOAc. The EtOAc layer was evaporated underreduced pressure, and the residue was purified by preparative TLC(silica gel) using 5% MeOH in CH2Cl2 as eluant to give 14 as a whitesolid (15 mg, 96%); mp 192−194 °C; 1H NMR data were consistentwith those reported;58 HRMS (ESI): [M + H]+ calcd for C30H41O7,513.2847; 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 wasevaporated, and the residue was separated on preparative TLC (silicagel) using 6% MeOH in CH2Cl2 as eluant to give 28 as a white solid(4.2 mg, 70%); mp 227−228 °C; [α]D

25 +29.9 (c 1.0, CHCl3);1H

NMR (500 MHz, CDCl3 + CD3OD) δ 6.80 (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 = 6.6Hz, 3H, H3-21), 0.88 (m, 1H), 0.66 (s, 3H, H3-19);

13C NMR (125MHz, CDCl3 + CD3OD) δ 201.4, 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 of 28 (1.0 mg)

by the usual procedure (Ac2O/pyridine) followed by purification onpreparative TLC (silica gel) using 6% MeOH in CH2Cl2 as eluantafforded 35 (1.1 mg, 93%); mp 214−216 °C; [α]D

25 +36.8 (c 1.1,CHCl3);

1H NMR (600 MHz, CDCl3) δ 6.66 (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) δ 201.1, 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 C32H43O8, 555.2952; found,555.2950.

27-Acetyl-4-epi-withaferin A (33). To a stirred solution of 32 (3.0mg) in THF (0.2 mL) and MeOH (0.2 mL) at 0 °C were addedCeCl3·7H2O (65 mg) and NaBH4 (small portion), and the mixturewas stirred at 0 °C. After 10 min, a small ice cube was added to thereaction mixture, the solvent and water were evaporated underreduced pressure, and the residue was partition between H2O andEtOAc. The EtOAc layer was dried over anhydrous Na2SO4 andevaporated under reduced pressure, and the residue was separated onpreparative TLC (silica gel) using 2% MeOH in CH2Cl2 as eluant togive 33 (2.5 mg, 70%) as a white solid; mp 188−190 °C; [α]D25 + 42.4(c 1.0, CHCl3);

1H NMR (500 MHz, CDCl3) δ 6.83 (dd, J = 10.2, 1.4Hz, 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) δ201.2, 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 C30H41O7, 513.2847; found, 513.2854.

4-Dehydrowithaferin A 27-tert-Butyldimethylsilyl Ether (41). To asolution of 27 (11.3 mg) in DMF (0.5 mL) were added t-BDMS-Cl(36.4 mg) and 4-pp (42.9 mg), and the mixture was stirred under anatmosphere of N2 for 1 h at 60 °C. The reaction mixture was thendiluted with EtOAc, washed with brine, and evaporated under reducedpressure, and the residue was separated on preparative TLC usingCH2Cl2 as eluant to give 41 (9.5 mg). APCI-MS (+) m/z: [M + 1]+

583.4-epi-Withaferin A 27-tert-Butyldimethylsilyl Ether (42). To a

solution of 41 (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),and the mixture was stirred at 0 °C. After 10 min, a small ice cube wasadded to the reaction mixture, the solvent and water were evaporated



under reduced pressure, and the residue was partition between H2Oand EtOAc. The EtOAc layer was dried over anhydrous Na2SO4 andevaporated under reduced pressure, and the residue was separated onpreparative TLC (silica gel) using 2% MeOH in CH2Cl2 as eluant togive 42 (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 of 42 (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 evaporatedunder reduced pressure to give 43 (8.0 mg) as a white solid; APCI-MS(+) m/z: [M + 1]+ 627.4-Acetyl-4-epi-withaferin A (34). To a solution of 43 (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 reactionmixture was diluted with H2O, MeOH and THF were evaporatedunder reduced pressure and extracted with EtOAc (3 × 15 mL), thecombined EtOAc layer was washed with H2O, dried over anhydrousNa2SO4, and evaporated under reduced pressure, and the residue wasseparated on preparative TLC (silica gel) using 5% MeOH in CH2Cl2as eluant to give 34 as a white solid (5.3 mg, 70%); mp 236−38 °C;[α]D

25 + 29.7 (c 1.2, CHCl3);1H NMR (600 MHz, CDCl3) δ 6.66 (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 = 12.5Hz, 1H, H-27a), 4.32 (d, J = 12.5 Hz, 1H, H-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);

13CNMR (100 MHz, CDCl3) δ 201.1, 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 C30H41O7, 513.2847; found, 513.2850.Microbial Biotransformation of 1. Small-scale fermentation of

Cunninghamella echinulata (ATCC 10028B) was performed in anErlenmeyer flask (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 inErlenmeyer flasks (3 × 250 mL) holding 50 mL of the medium in eachflask, which was inoculated with 15% of the 1 day old inoculum. Asolution of 1 (5 mg in 0.5 mL of DMF) was added to each flaskcontaining 24 h old second cultivation. After 72 h, fermentation brothswere combined, and mycelium was filtered off and washed with H2O(100 mL), which was combined with filtrate 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 giveEtOAc extract (42 mg). Gel-permeation chromatography of thisextract over a column of Sephadex LH-20 (3.0 g) followed bypreparative TLC (silica gel) gave 12β-hydroxywithaferin A (29, 3.8mg, 24%) and 15β-hydroxywithaferin A (30, 4.9 mg, 32%).12β-Hydroxywithaferin A (29). mp 120−121 °C (lit.38 119−120

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

15β-Hydroxywithaferin A (30). mp 271−273 °C (lit.38 270−274°C); 1H 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 themixture was stirred at 25 °C for 18 h. The reaction mixture wasevaporated under reduced pressure and by adding EtOH, and theresidue was purified on preparative TLC (silica gel) using 4% MeOHin DCM as eluant to give 36 (1.2 mg, 95%) as a white amorphoussolid. 1H 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 themixture was stirred at 25 °C for 18 h. The reaction mixture wasevaporated under reduced pressure and by adding EtOH, and theresidue was purified on preparative TLC (silica gel) using 4% MeOHin DCM as eluant to give 37 (1.2 mg, 95%) as a white amorphoussolid. 1H NMR data were consistent with those reported.38

Withaferin A N-Acetylcysteine Adduct (38). To a solution of 1(40.0 mg) in MeOH (4.0 mL) was added N-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 separatedby HPLC [Phenomenex Luna C18 (5 μ) 10 × 250 mm, gradientsolvent system from 60 to 80% in 20 min, 3 mL/min flow rate,detection at 230 nm] to afford 38 (22.0 mg; 66% based on recovered1) (tR = 13.0 min) and 1 (9.4 mg) (tR = 14.8 min). 38: off-whiteamorphous solid; 1H NMR (400 MHz, CDCl3) δ 4.36 (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 and2.40 (1H each, m, H2-2), 2.00 (6H, s, Cys-CH3 and H3-28), 1.23 (3H,s, H3-19), 0.93 (3H, d, J = 6.4 Hz, H3-21), 0.61 (3H, s, H3-18);

13CNMR (100 MHz, pyridine-d5) δ 209.8 (C-1), 174.3 (Cys-CON),170.6 (Cys-CO2H), 166.7 (C-26), 154.2 (C-3), 128.9 (C-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 andmyeloma cell line H929 were cultured at 37 °C under 6% CO2 inRPMI 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 Mycoplasmacontamination. Cultures were passaged twice weekly, and cells inexponential growth were used for experiments. Stock solutions ofcompounds were formulated in DMSO and maintained at −20 °Cprotected from light. To measure acute toxicity, CHP-100 cells wereseeded in flat-bottom 96-well plates (7500 cells/well) and allowed toadhere overnight. Serial dilutions of compounds or DMSO vehiclecontrol (not exceeding 0.2%) were added, and the relative viable cellnumber was determined 24 h later by dye-reduction assay using thesubstrate [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide] (MTT). Cytotoxicity over 3 days against H929 and 293Tcells was measured in 384-well format (3000 cells/well), and therelative viable cell number was determined by reduction of the dyeresazurin (Alamar Blue) as previously described.40

Heat-Shock Reporter Assays. Reporter cells were generated byinfecting 293T cells (American Type Culture Collection) with apreviously reported lentiviral vector encoding a fusion proteinconsisting of enhanced GFP fused to firefly luciferase under controlof HSP70B′ promoter elements.54 The plasmid encoding the fusionprotein was generously provided by Khalid Shah (MassachusettsGeneral Hospital, Boston, MA, USA). To isolate a homogeneouspopulation of high responding cells, a transduced culture was heat-shocked at 42 °C for 1 h and then processed 8 h later by fluorescence-activated cell sorting (FACS). Prior to use, cells were reverse-selectedby FACS to eliminate a minority population of cells constitutivelyexpressing the reporter in the absence of induction. To evaluatecompounds, cells were seeded in white 384-well plates (20 000 cells/well). The following day, serial compound dilutions were added toquadruplicate wells, and incubation was continued overnight.Measurement of relative luciferase activity was achieved using anEnvision plate luminometer (PerkinElmer) and Steady-Glo reagent(Promega) per the manufacturer’s recommendations. As a con-firmatory assay for some compounds, 3T3-Y9-B12 reporter cells wereseeded in black flat-bottom 96-well plates (20 000/well) and allowedto adhere overnight as previously reported.23 Cells were thenincubated for 24 h in the presence of WA or analogues (1, 2, or 4μM). After washing with PBS, fluorescence was quantified using anAnalyst AD (LJL Biosystems) plate reader with excitation andemission filters set at 485 and 530 nm, respectively.

Pharmacodynamic Studies in Mice. All experimentationinvolving mice was performed in accordance with a protocol approvedby the MIT Committee on Animal Care (CAC). Mice were randomlyassigned to receive either control vehicle (hydroxpropyl β-cyclodextrin30% w/v; DMSO 10% v/v) or test compound in an equal volume ofthe same vehicle. Treatment was administered via subcutaneousinjection, and mice were euthanized by CO2 inhalation followed by



tissue harvesting approximately 18 h postdosing. Organs were snap-frozen in liquid nitrogen and pulverized, and lysates were prepared innonionic detergent buffer. Samples (15 μg of total protein/lane) werefractionated by SDS-PAGE, transferred to nitrocellulose, and blottedwith antibodies to HSP72 (clone C92F3A-5, Stressmarq Biosciences,1:3000) and β-actin (mABGEa, ThermoFisher, 1:1000). Chemilumi-nescent detection was performed using a ChemiDoc MP imagingsystem (Bio-Rad), and integrated band intensity was determined withImage Lab software (version 4.1, Bio-Rad). Plotting of the data andstatistical analysis were performed using GraphPad Prism 6 software.The statistical significance cutoff for all comparisons was p < 0.05.

■ ASSOCIATED CONTENT*S Supporting InformationChemical characterization of new withanolide analogues 15,17−19, 21−26, 28, 31, 33−35, and 38; screen for acutecytotoxic activity of 1 and analogues 2−37 using CHP-100cells; HPLC evidence for time-dependent conversion of 31 to 1in cell culture medium; and relative reactivity of 1 and 35 withN-acetylcysteine. This material is available free of charge via theInternet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Authors*(L.W.) Phone: +617-452-3542; E-mail: [email protected].*(A.A.L.G.) Phone: +520-621-9932; E-mail: [email protected] Contributions#E.M.K.W. and Y.-M.X. contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSFinancial support for this work from the Arizona BiomedicalResearch Commission, the United States Department ofAgriculture, and the University of Arizona College ofAgriculture and Life Sciences is gratefully acknowledged.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior(CAPES), Brazil, is thanked for the award of a graduatefellowship (to D.D.R.). S.L. is an Investigator of the HowardHughes Medical Institute.

■ ABBREVIATIONS USEDCHP-100, human Ewing’s sarcoma cell line; DMEM,Dulbecco’s modified Eagle’s medium; DMF, N,N-dimethylfor-mamide; DMSO, dimethyl sulfoxide; FACS, fluorescence-activated cell sorting; H929, plasmacytoma myeloma; HMBC,heteronuclear multibond correlation; HSA, heat-shock-inducingactivity; HSE, heat-shock element; HSF1, heat-shock factor 1;HSP, heat-shock protein; HSR, heat-shock response; NOE,nuclear Overhauser effect; PBS, phosphate-buffered saline;SAR, structure−activity relationship; t-BDMS-Cl, tert-butyldi-methylsilyl chloride; TLC, thin-layer chromatography; WA,withaferin A; 293T, transformed human embryonic kidney cells

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