STAT3 Inhibitory Activity of Structurally Simplified Withaferin A

Signal transducer and activator of transcription 3 (STAT3) is a component of the JAK/STAT pathway. Therapeutic inhibition of STAT3 has been of high in...
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STAT3 Inhibitory Activity of Structurally Simplified Withaferin A Analogues Teruyuki Tahara,† Ursula Streit, Henry E. Pelish, and Matthew D. Shair* Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, United States S Supporting Information *

ABSTRACT: Signal transducer and activator of transcription 3 (STAT3) is a component of the JAK/STAT pathway. Therapeutic inhibition of STAT3 has been of high interest, as its aberrant activation has been linked to cancer, inflammation, and other human diseases. The withanolide family natural product withaferin A (1) inhibits STAT3 activation. We designed, synthesized, and evaluated simplified withanolide analogues SLW1 (3) and SLW2 (4), and found that SLW1 retained the STAT3 inhibitory activity of withaferin A.

ignal transducer and activator of transcription 3 (STAT3) is a latent cytosolic transcription factor that transmits extracellular signals to the nucleus.1 The binding of several cytokines and growth factors, such as IL-6, IL-10 and epidermal growth factor (EGF), to cell surface receptors activates Janus kinases (JAKs) and tyrosine kinase 2 (Tyk2) to phosphorylate STAT3 at Tyr705.2 Phosphorylated STAT3 dimerizes, translocates to the nucleus and regulates transcription.3 STAT3 has a critical role in various biological activities such as inflammation, embryonic development, angiogenesis, cell proliferation, differentiation, survival, and migration. Since aberrant STAT3 activation can drive human diseases such as psoriasis, cancer, myocardial ischemia/reperfusion injury, rheumatoid arthritis, renal fibrosis, inflammatory bowel disease, and atherosclerosis, it has been considered a promising therapeutic target.4 Withaferin A (1, Figure 1) belongs to the withanolides, a family of over 600 natural products.5 Withanolides have a steroidal backbone bearing a δ-lactone and a highly oxidized A,B-ring system. Withaferin A (1) was isolated from Withania somnifera6 and inhibits STAT3 phosphorylation in MDA-MB231 cells.7 In addition, withaferin A (1) has apparent pleiotropic activity, including antitumor, anti-inflammatory,

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antistress, antifeedant, and antioxidant activities.8 In fact, annexin II,9 vimentin,10 HSP90,11 IKKβ,12 β-tubulin,13 NF-κB essential modulator,14 and AAA+ chaperone p9715 have been identified as cellular targets of withaferin A. Since cysteine residues of these target proteins form covalent bonds between the C3 of withaferin A (1), the 2-en-1-one moiety seems to be the pharmacophore. On the other hand, withaferin A (1) inhibits hedgehog signaling,16 and the pharmacophore is based on the δ-lactone and CD rings, with the A,B-ring system being unnecessary.17 Withacnistin (2, Figure 1), a C18-functionalized congener of withaferin A,18 also inhibits STAT3 phosphorylation.19 Interestingly, an ethanol Michael adduct of withacnistin (2) at C3 does not inhibit JAK/STAT pathway signaling, suggesting that the A-ring enone, common to withaferin A (1) and withacnistin (2), is essential for inhibition of STAT3 phosphorylation.20 The mechanism by which withanolides inhibit JAK/STAT pathway activity has not been determined. Given their activity against the JAK/STAT pathway, withaferin A (1) and withacnistin (2) represent potential starting points for therapeutic discovery. However, only one total synthesis of withaferin A (1) has been reported, in 33 steps from 3β-hydroxy-22,23-bisnorchol-5-enoic acid,21 and no syntheses of withacnistin (2) have been reported. Toward this end, we applied a function-oriented synthesis (FOS) approach22 to withaferin A (1). Namely, we endeavored to prepare simplified withaferin A analogues that maintained or improved on the biological activity of withaferin A and could be efficiently synthesized (Figure 2). Toward this end, we synthesized and evaluated SLW1 (Shair Lab withanolide analogue 1, 3) and SLW2 (4). SLW1 (3) was originally isolated from Physalis viscosa as a decomposition product of withanolide D (5, Figure 1).23 To date, there are no reported

Figure 1. Example withanolides.

Received: February 1, 2017

© XXXX American Chemical Society

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Organic Letters Scheme 2. Synthesis of SLW1 (3)

Figure 2. Structures of SLW1 (3) and SLW2 (4).

total syntheses of 3, and it has not been evaluated for biological activity. A compound related to SLW2 (4), but lacking a hydroxyl group at C7, was synthesized in 10 steps and in 2.6% overall yield from the Wieland−Miescher ketone by Ikekawa and co-workers24 and its antibiotic activity was reported.25 Although all withanolides possess a ketone or hydroxyl group at C1, installing an oxygen functionality at this position proved challenging. To this end, the Birch reduction of a 1,4,6-trien-3one was exploited to form a 1β-hydroxyl group toward the total synthesis of withaferin A (1).21 Toward this end, we developed a Shapiro reaction/Riley oxidation sequence for the elaboration of SLW1 (3). We also implemented a modified version of Ikekawa’s synthetic strategy for the synthesis of SLW2 (4) by utilizing a hydroxyl group at C4 as a directing group and stereoselectively constructed the 5,6-β-epoxy group. Retrosynthetically, we envisioned to access SLW1 (3) from ketone 6 via a previously reported three-step sequence of (1) methanesulfonylation, (2) dihydroxylation and (3) intramolecular SN2 reaction26 (Scheme 1). Ketone 6 was to be Scheme 1. Retrosynthesis of SLW1 (3)

provided 2,4-dien-1-one 12 in 53% yield. Trimethylsilyl substitution at C3 was critical to the success of the Riley allylic oxidation.31 Oxidation of substrates lacking TMS substitution led to the formation of a 1,4-dien-3-one derivative of 6. Efforts to desilylate compound 12 were not fruitful. Even 10 equiv of TBAF under refluxing conditions only resulted in removal of the TBS group at C20, with the C3 TMS group remaining intact. To heighten the reactivity of the silane functionality, enone 12 was reduced using DIBAL-H to give allylic alcohol 13. Treatment of 13 with TBAF readily provided the desilylated product. Allylic oxidation with activated manganese dioxide subsequently afforded enone 14 in 42% yield over two steps.32 Further oxidation using Dess−Martin periodinane (DMP) and cleavage of the MOM group under acidic conditions yielded diketo alcohol 6. Finally, compound 6 was converted to SLW1 (3) by methanesulfonylation, dihydroxylation, and intramolecular SN2 reaction in 35% yield. We next turned our attention to the synthesis of SLW2 (4). We envisioned constructing the enone functionality from the corresponding ketone (15) by treatment with LDA followed by Mukaiyama’s reagent (Scheme 3). The C4 alcohol in 16 would be exploited as a directing group for an epoxidation to

prepared from hydrazone 8 using the aforementioned Shapiro reaction/Riley oxidation sequence. Finally, hydrazone 8 would be derived from known alcohol 9, readily accessible from commercially available pregnenolone in two steps.27,28 Our synthesis of SLW1 (3) commenced with the preparation of hydrazone 8 (Scheme 2). Silyl protection of alcohol 9 and subsequent saponification afforded alcohol 10 in 93% yield, which was then converted to enone 11 in 73% yield over three steps: (1) epoxidation of the trisubstituted olefin with m-CPBA, (2) oxidation of the C3 alcohol using PDC, and (3) epoxide opening under basic conditions. After MOM protection of alcohol 11, the resulting enone was treated with 2,4,6triisopropylbenzenesulfonylhydrazide in the presence of sodium sulfate at room temperature to obtain an inseparable mixture of E and Z isomers of hydrazone 8 in quantitative yield.29 Treatment of hydrazone 8 with excess n-BuLi followed by exposure to trimethylsilyl chloride afforded silyldiene 7 in 65% yield.30 Subsequent selenium dioxide treatment of silyldiene 7

Scheme 3. Retrosynthesis of SLW2 (4)

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Organic Letters construct the 5,6-β-epoxide in 15. This alcohol would be installed at an early stage of the synthesis via the key Rubottom oxidation of a vinylogous enol ether,33 which could in turn be obtained from known compound 17.34 Optically pure, monoprotected Wieland−Miescher ketone (17) was employed as a starting material, which was synthesized following a literature protocol35 (Scheme 4). Scheme 4. Synthesis of SLW2 (4)

Figure 3. Immunoblot showing the effect of withaferin and withaferin analogues on levels of phospho-STAT3 Tyr705 in MDA-MB-231. (%) is vehicle-normalized % intensity of pSTAT3 Tyr705 to total STAT3.

In conclusion, we synthesized and evaluated SLW1 (3) and SLW2 (4). SLW1 (3) was obtained in 17 steps and in 1.7% overall yield from known compound 6 and retained STAT3 phosphorylation inhibitory activity. Our Shapiro reaction/Riley oxidation sequence provided facile construction of the 4βhydroxy-5,6-β-epoxy-2-en-1-one moiety, which is the common substructure for several withanolides including withaferin A (1), withacnistin (2), withanolide D (5), witharistatin, withangulatin B, and phyperunolide A.5 Given its facile synthesis and STAT3 inhibtory activity, SLW1 (3) represents a promising probe to investigate the JAK/STAT pathway. Understanding its mechanism of action may also facilitate the development of a new therapeutic strategy for human diseases that are dependent on aberrant STAT3 activation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b00332. Experimental procedures, supporting results, and proton and carbon NMR for new compounds (PDF)

Treatment of 17 with TMSCl and triethylamine afforded the corresponding thermodynamic vinylogous silyl enol ether, which was found to be unstable under acidic conditions and was therefore directly used in the next step without further purification.36 The key Rubbotom oxidation was carried out under CH2Cl2/ water biphasic conditions using m-CPBA in the presence of sodium bicarbonate to give alcohol 16 in 51% yield. The minor diastereomer 18 was also obtained in 7% yield. Installation of the C4 alcohol was key to our synthetic route because it was subsequently employed to impart stereoselection on the remainder of our route. Reduction of enone 16 under Luche conditions37 afforded allylic alcohol 19 with high diastereoselectivity (dr = 9:1). Diastereoselective epoxidation of olefin 19 was achieved with m-CPBA followed by cleavage of the ketal moiety to give ketone 20. For the next step, the two hydroxyl groups in 20 were protected as TES ethers. Treatment of 15 with LDA afforded the corresponding lithium enolate, which was then treated with Mukaiyama’s reagent in situ to give enone 21 in 60% yield.38 Finally, removal of the silyl protecting groups provided SLW2 (4). To evaluate SLW1 (3) and SLW2 (4), immunoblot analysis was conducted with the breast cancer cell line MDA-MB-231, which has constitutive JAK/STAT pathway activation (Figure 3). Withaferin A (1) and SLW1 (3) dose-dependently inhibited the phosphorylation of STAT3 (Tyr705), with more than 50% inhibition at 1 μM SLW1 (3). SLW2 (4) showed only weak STAT3 phosphorylation inhibitory activity at 80 μM (∼50%). Withaferin A (1), SLW1 (3), and SLW2 (4) did not inhibit phosphorylation of pJAK1 (Tyr1022/1023), pJAk2 (Tyr1007/ 1008), and pSTAT3 (Ser727) (see the Supporting Information).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Teruyuki Tahara: 0000-0001-6608-8752 Present Address † (T.T.) Maruho Co., Ltd., Kyoto R&D Center, 93-Awata-cho, Chudoji, Simogyo-ku, Kyoto, 600-8815, Japan.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Chemiderm, Inc. for financial support and Dr. Ludovic Decultot (Harvard University) for reviewing this manuscript.

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REFERENCES

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