Skip to main content
NCBI home page
ACS Medicinal Chemistry Letters logoLink to ACS Medicinal Chemistry Letters
. 2020 Nov 20;11(12):2534–2543. doi: 10.1021/acsmedchemlett.0c00534

Synthesis and Biological Evaluations of Electrophilic Steroids Inspired by the Taccalonolides

Nicholas A Clanton †, Shayne D Hastings ‡, Griffin B Foultz †, Julie A Contreras †, Samantha S Yee ‡, Hadi D Arman †, April L Risinger ‡,⊥,*, Doug E Frantz †,*
PMCID: PMC7734803  PMID: 33335677

Abstract

graphic file with name ml0c00534_0013.jpg

Natural products have served as inspirational scaffolds for the design and synthesis of novel antineoplastic agents. Here we present our preliminary efforts on the synthesis and biological evaluation of a new class of electrophilic steroids inspired by the naturally occurring taccalonolides. We demonstrate that these simplified analogs exhibit highly persistent antiproliferative properties similar to the taccalonolides and retain activity against resistant cancer cell lines that warrants further preclinical development.

Keywords: steroids, natural products, triple negative breast cancer, ovarian cancer

Natural products have long served as an inspirational source for the discovery of effective cancer therapeutics.1 In fact, several of Nature’s chemical harvests have directly entered the clinic without structural modification and serve as effective chemotherapies for cancer patients (Figure 1a). Of these, paclitaxel is perhaps the most notable and is recognized on the World Health Organization’s (WHO) list of essential medicines.2 However, the direct preclinical development of most natural products remains a formidable challenge due to significant hurdles that impede the drug discovery process. These hurdles include the lack of early drug supplies due to low natural abundance or synthetic intractability from inherent structural complexity that ultimately inhibits large-scale commercialization.3 Consider the case of halichondrin B (Figure 1b). While displaying excellent antitumor activity, its clinical development was hampered by the synthetic intractability of its complex molecular structure. Subsequent studies from Eisai, however, revealed that truncated analogs that contained the eastern half of halichondrin B retained potent inhibitory cell growth in vitro and antitumor activity in vivo.4,5 This synthetically tractable analog was eventually developed into the drug eribulin. Thus, natural products can also serve as architectural blueprints for validated pharmacophores that inspire and guide medicinal chemistry efforts toward new and effective antineoplastic agents suitable for clinical development.6

Figure 1.

Figure 1

Open in a new tab

(a) Paclitaxel and the vinca alkaloids, vinblastine and vincristine, are examples of natural products with direct clinical applications as antineoplastic agents. (b) Halichondrin B, a complex natural product that provided the architectural blueprint (highlighted in blue) for the drug eribulin.

As a class of natural products, steroids have a proven track record in drug discovery. The core ABCD ring system consisting of 17 carbons has provided a validated pharmacophoric template for designing biologically active small molecules. These terpenoid compounds are active in an unparalleled diversity of biological functions including cell growth and development, immunomodulation, and neurotropism.7−10 Relevant to the current study, a natural metabolite of estradiol with poor estrogenic activity, 2-methoxyestradiol, was evaluated in clinical trials for anticancer efficacy due to its antiangiogenic effects as a microtubule destabilizing agent11,12 but lacked efficacy due to metabolic liabilities.13

Distinct subclasses of steroids have also demonstrated a role in the progression and treatment of hormone-dependent cancers.14−18 For example, estrogen receptor (ER) positive breast cancers are often hormone-dependent and treated with ER antagonists or aromatase inhibitors.19−22 As a result, efforts have led to the identification of small molecules that modulate these pathways including fulvestrant, a semisynthetic steroid that is currently used in the clinic as a selective estrogen receptor degrader for the treatment of ER positive breast cancer.23 Likewise, prostate cancers are often androgen-dependent, making the inhibition of androgen biosynthesis and downstream receptor signaling with drugs such as abiraterone effective against tumor progression.24

A novel class of steroidal natural products produced by the Tacca genus of plants are the taccalonolides (Figure 2a). The C22–C23 epoxidized taccalonolides demonstrate potent microtubule stabilizing and antiproliferative activities by covalently binding to β-tubulin, which allows them to effectively circumvent clinically relevant models of taxane resistance in vitro and in vivo.25−28 Both crystallography and mutational analysis in cells demonstrate that the C22–C23 epoxide of the taccalonolides (highlighted in red in taccalonolide AJ in Figure 2) is critical for covalent modification of the carboxylate of β-tubulin D226, which promotes irreversible microtubule stabilization.29,30 Despite their promising therapeutic potential, total synthesis of the taccalonolides has yet to be achieved due, in part, to their complex molecular architecture that features a highly oxidized steroid core and 20 contiguous stereocenters. This, in turn, has stymied medicinal chemistry efforts to address liabilities such as poor pharmacokinetics and low therapeutic index that have prohibited further preclinical development.31

Figure 2.

Figure 2

Open in a new tab

(a) Naturally occurring taccalonolide A (6) and its potent semisynthetic analog taccalonolide AJ (7) with the steroid core highlighted in blue. (b) Design strategy for the synthesis of simplified analogs of the taccalonolides using the functionalized steroid core of the taccalonolides.

Inspired by the story of halichondrin B, we wondered if simplified analogs of the taccalonolides would be able to retain potent microtubule stabilization by incorporating key structural elements that would mimic the covalent binding to β-tubulin of their naturally occurring cousins (Figure 2b). Furthermore, given the plethora of well-developed synthetic methods capable of functionalizing the ABCD steroid ring system, we envisioned a synthetically tractable approach to rapidly generate truncated analogs of the taccalonolides to accelerate our SAR studies. We therefore sought to pursue a design strategy inspired by the bis-epoxide motif of taccalonolide AJ to access electrophilic steroid-based small molecules that we hypothesized might act as potential microtubule stabilizers through covalent modification of β-tubulin. Inspiration was also garnered from the recent clinical success of several targeted covalent inhibitors with specifically designed electrophilic functional groups for high target specificity.32−34 Herein, we disclose our preliminary results on the synthesis and biological evaluation of a novel class of electrophilic androstanes inspired by the taccalonolides that are readily accessible in a few synthetic steps from commercially available steroid precursors.

Motivated by the synthetic approach to the taccalonolide skeleton by Sorensen et al.,35 we sought to begin our synthetic efforts from readily available steroid precursors that were accessible in large-scale quantities. Both trans-androsterone (8) and trans-dehydroandrosterone (9) are commercially available from multiple vendors and served as an obvious starting point for our synthetic strategy. A detailed description of our synthetic efforts is provided later, while a simple overview of our SAR design strategy starting from 8 and 9 is presented here in Scheme 1. We focused our initial attention on the installation of a bis-epoxide motif at C2–C3 and C16–C17 as a truncated version of taccalonolide AJ (omission of the E and F rings). Variations on this theme were then incorporated in subsequent analogs to study the SARs at various positions on the steroid core while also incorporating epoxide bioisosteres that could serve as electrophilic sites for potential covalent binding to β-tubulin.

Scheme 1. Overview of Analogs Synthesized from trans-Androsterone and trans-Dehydroandrosterone.

Scheme 1

Open in a new tab

The antiproliferative activity of all analogs was initially evaluated against HeLa cells to assess specific SARs around the steroid core. A summary of these results is presented in Table 1 and includes values for concentrations that caused 50% growth inhibition (GI50), total growth inhibition (TGI), and 50% cytotoxicity (LC50) for each analog. Based on these data, some general conclusions can be drawn with respect to important structural features attributed to the antiproliferative activity seen in HeLa cells. For example, most of the analogs that possess an electrophilic site at C15 within the D-ring of the steroid core (either a Michael acceptor or an epoxide) exhibited antiproliferative activity below 1 μM. In contrast, all of the analogs lacking an electrophilic C15 carbon (21, 25, 26, 28, 29, 32, 37, and 38) exhibited little or no antiproliferative activity at concentrations less than 10 μM. Likewise, the δ-lactone analog 13 also demonstrated reduced antiproliferative activity. We were pleased to realize that our original design strategy to incorporate the bis-epoxide motif inspired by the taccalonolides as seen in 12 resulted in an analog with moderate antiproliferative activity in HeLa cells (GI50 = 520 nM). Likewise, the related C2–C3 aziridine analogs 14–16 also demonstrated moderate activity with GI50 values of 450–640 nM. However, the most potent analog with respect to antiproliferative activity in HeLa cells in this study was Michael acceptor 24 with a GI50 of 280 nM. It is interesting to note that analogs where the Michael acceptor is replaced with the C15–C16 epoxide motif and combined with oxidation at C6 (34–37) exhibit reduced antiproliferative and cytotoxic activity as compared to 24. In addition, epoxidation at C2–C3 in tandem with C6 oxidation also appears to reduce antiproliferative activity as can be seen in 22 and 23.

Table 1. Antiproliferative and Cytotoxic SAR on Electrophilic Steroid Analogs in HeLa Cellsa.

graphic file with name ml0c00534_0012.jpg

Open in a new tab
a

Concentrations of each compound that caused 50% growth inhibition (GI50), total growth inhibition (TGI), and 50% cytotoxicity (LC50) as measured in HeLa cells using the SRB assay as average ± SEM (n = 3–5).

In general, cytotoxicity (LC50) profiles parallel antiproliferative activities across all analogs studied. However, while 24 proved to be the most potent with respect to antiproliferation, tosylated aziridine 15 was the most potent cytotoxin in HeLa cells (LC50 = 660 nM) albeit within error of 24 (LC50 = 700 nM). Interestingly, the related Boc-protected aziridine 16 also displayed comparable cytotoxicity (LC50 = 730 nM), while the unprotected aziridine 14 with inverted aziridine stereochemistry demonstrated lower cytotoxicity (LC50 = 1.42 μM) but retained analogous antiproliferative activity.

We selected 20 analogs from Table 1 for additional studies in a panel of molecularly diverse triple negative breast cancer (TNBC) cell lines.36 While we did not observe a dramatic variation in the relative sensitivities between cell lines for most of the analogs (see Supporting Information), we did observe SARs for selective antiproliferative and cytotoxic potency among these lines for at least six of them (Figure 3). In general, the C15–C16 epoxide analogs 12, 18, and 20 demonstrated a noticeable ∼2-fold selectivity for the basal-like BRCA1 mutant cell line HCC1937 that has previously been shown to be resistant to the cytotoxic effects of microtubule targeted agents as well as MEK inhibitors.37,38 Interestingly, when the nature of the electrophile at C15 is changed to a Michael acceptor as in analogs 10 and 31, the selectivity toward HCC1937 cells is lost and replaced by selectivity toward the mesenchymal BT-549 cell line, which is also resistant to the cytotoxic effects of microtubule targeted agents.37 Enigmatically, aziridine analog 15 also demonstrates slight cytotoxic selectivity toward the BT-549 cell line despite possessing the C15–C16 epoxide. The relative shift in cell line selectivity between these analogs suggests they may have distinct mechanisms of action. Most importantly, each analog demonstrated full cytotoxic efficacy against each of the TNBC cell lines while demonstrating measurable specificity against cancer cells. For example, an LC50 value could not be achieved for 12 in the nontumorigenic breast cell line MCF 10A at concentrations up to 50 μM.

Figure 3.

Figure 3

Open in a new tab

Concentration–response curves and average LC50 values of selected analogs in four molecularly diverse TNBC cell lines and HeLa cells.

We further evaluated the antiproliferative and cytotoxic efficacy of the C15–C16 epoxide 12 in four ovarian cancer cell lines as compared to paclitaxel and taccalonolide AJ (Figure 4). Although less potent than paclitaxel or taccalonolide AJ, compound 12 demonstrated superior cytotoxic efficacy against each of these ovarian cancer cell lines as evidenced by a more complete inhibition of growth (more negative values on the y-axis). Additionally, the ability of 12 to circumvent drug resistance due to P-glycoprotein expression was evaluated by directly comparing the relative potencies in the parental SK-OV-3 line and the isogenic cell line transduced with MDR1, SK-OV-3/MDR-1-M6/6 (SK-OV-3 M6/6).39 We found that 12 was only 5-fold less sensitive in the P-glycoprotein expressing cell line than the parental line at the TGI, demonstrating a similarity to the taccalonolides in that it has less vulnerability to this drug resistance mechanism than paclitaxel, which suffers over a 25-fold decrease in potency when this drug efflux pump is expressed.

Figure 4.

Figure 4

Open in a new tab

Concentration–response of (a) 12, (b) paclitaxel (1), and (c) taccalonolide AJ (7) in ovarian cancer cell lines.

Intrigued by the high degree of cytotoxic efficacy of 12, particularly in drug resistant cancer cell lines, we examined the timing of these cytotoxic effects to gain some initial insight into the biological mechanism. We found that concentrations of 12 that caused 50–100% cytotoxicity in the 48 h SRB assay (Table 1) did not show evidence of activity within the first 8 h of treatment as evidenced by trypan blue exclusion, demonstrating that this compound has a delayed mechanism of action similar to that of the microtubule targeted agents. We further examined whether 8 h of acute drug treatment was sufficient to produce cytotoxicity in long-term clonogenic assays even after the drug was removed as a measure of the relative cellular persistence of the antiproliferative and cytotoxic effects of the compound. Indeed, an 8 h treatment of HeLa cells with a LC50 concentration of 1.4 μM 12, which was not cytotoxic within the treatment period, was sufficient to completely inhibit the formation of colonies 11 days after removal from the medium (Figure 5). This is similar to the long-term cellular persistence of the irreversible microtubule stabilizing taccalonolides and distinct from paclitaxel, which binds reversibly to microtubules. Together, these data indicate that although 12 has a delayed mechanism of cytotoxicity, even short treatments have irreversible cellular effects, further suggesting that they may have inherited the irreversible target engagement of their inspiration, the taccalonolides.

Figure 5.

Figure 5

Open in a new tab

(a) Colony formation of HeLa cells 11 days after treatment for 8 h with vehicle, 10 nM paclitaxel (1), 10 nM taccalonolide AJ (7), or 1.4 μM 12 prior to drug washout. ***p < 0.001 for 1-way ANOVA with Dunnett’s post hoc test compared to vehicle treated cells. (b) Representation of colonies formed 11 days after 8 h treatment of HeLa cells with EtOH vehicle (V), 10 nM paclitaxel (1), 10 nM taccalonolide AJ (7), or 1.4 μM 12.

Encouraged by these results, we pursued studies to compare the interactions of these simplified analogs with microtubules to those of the more complex taccalonolides. We first examined the impact of 10, 12, 15, 22, 23, and 31 on the polymerization of purified tubulin (Figure 6a). All analogs with the Michael acceptor in the D-ring (10, 22, 23, and 31) demonstrated measurable efficacy in inhibiting both the rate and extent of tubulin polymerization. In contrast, analogs with the C15–C16 epoxide (12 and 15) did not show any appreciable inhibition of tubulin polymerization suggesting a possible role of the α,β-unsaturated ketone in microtubule destabilization. This is in contrast to the taccalonolides, which are potent irreversible microtubule stabilizers that increase both the rate and extent of tubulin polymerization.28 Of the analogs studied, 10 proved to be the most potent, demonstrating concentration dependent microtubule depolymerizing activity at concentrations as low as 5 μM (Figure 6b). To examine the possibility of cellular microtubule destabilization as a mechanism of action of the antiproliferative and cytotoxic efficacy of this compound, we treated BT-549 cells with 1 μM 10, a concentration that is sufficient to cause complete cytotoxicity in this most sensitive cell line after a 48 h incubation. However, in contrast to the dramatic microtubule loss or bundling elicited by the microtubule destabilizer colchicine or the microtubule stabilizer paclitaxel, respectively, 10 does not impart any noticeable effects on intracellular microtubules within this time period. Therefore, in spite of the inspiration of these compounds by the taccalonolide skeleton, their potent antiproliferative and cytotoxic effects, and the ability of a subset to directly inhibit microtubule polymerization in biochemical preparations, their cellular mechanism of action appears to be independent of any gross alterations to microtubule structure.40

Figure 6.

Figure 6

Open in a new tab

Effect of selected analogs on tubulin polymerization. (a) Purified porcine brain tubulin (20 μM) was incubated with 20 μM of each compound or DMSO vehicle, and microtubule polymerization was monitored turbidimetrically after shift to 37 °C. (b) Concentration dependent effect of 10 on purified tubulin polymerization. (c) Microtubules were visualized by immunofluorescence using a β-tubulin antibody after treatment of BT-549 cells for 6 h with DMSO vehicle (Veh), 100 nM colchicine (Col), 1 μM paclitaxel (1), or 1 μM 10.

The synthetic routes toward the analogs tested in this study are highlighted in Schemes 2–5. To begin with, analogs 10–16, as well as 30, are highlighted in Scheme 2. trans-Androsterone (8) served as the starting point for this series of analogs with 10 serving as the linchpin intermediate for the remaining set of compounds. Briefly, 8 was dehydrated using trifluoromethanesulfonic anhydride (Tf2O)/pyridine to yield the C2–C3 olefin 39 in 81% yield. Saegusa–Ito oxidation of the D-ring in 39 afforded 10 in 83% overall yield. Divergent epoxidation at this stage with either meta-chloroperoxybenzoic acid (mCPBA) or NaOCl yielded the C2–C3 epoxide 31 or the C15–C16 epoxide 11, respectively. Interestingly, attempted subsequent C2–C3 epoxidation of 11 yielded the Bayer–Villiger product 13 as the major product instead of the desired bis-epoxide. Conversely, using dimethyldioxirane (DMDO) yielded the bis-epoxide 12 in near quantitative yield. Finally, aziridine analogs 14–16 were obtained using the recent Rh-catalyzed approaches developed by Kürti et al.41,42 Using O-(2,4-dinitrophenyl)hydroxylamine (DPH) as the aminating agent provided aziridine 40, which was not isolated but protected directly to provide either the tosylaziridine 15 or Boc-aziridine 16. Curiously, when the aminating agent was switched to NH3SO4, the only aziridine product we were able to isolate was 14 with inverted stereochemistry.

Scheme 2. Synthetic Routes Towards Analogs 10–16 and 31.

Scheme 2

Open in a new tab

Reagents and conditions: (a) Tf2O, pyridine, DCM, 0 °C; (b) LDA, TMSCl, THF, −78 °C to rt; (c) 10% Pd(OAc)2, DMSO, DCE, O2 (balloon); (d) mCPBA, DCM, 0 °C; (e) NaClO, pyridine, ethanol; (f) DPH, 1% Rh2(esp)2, TFE; (g) TsCl, NEt3, DCM; (h) Boc2O, NEt3,DCM; (i) NH3SO4, Rh2(esp)2, pyridine, HFIP; (j) DMDO, acetone, −10 °C; (k) 2 equiv of mCPBA, DCM, 0 °C.

Scheme 5. Synthetic Routes towards 25–27, 32, 37, and 38.

Scheme 5

Open in a new tab

Reagents and conditions: (a) TMSOTf, NEt3, DCM; (b) 10% Pd(OAc)2, DMSO, DCE, O2 (balloon); (c) K2CO3, MeOH; (d) TPAP, NMO, DCM; (e) DMDO, acetone, −10 °C; (f) oxone, NaHCO3, water, acetone; (g) TPAP, NMO, DCM.

Scheme 3 outlines the routes to 17–21 and 28–30. Protection of the free alcohol in 8 with tert-butyldimethylchlorosilane (TBSCl) followed by silyl enol ether formation and subsequent Saegusa–Ito oxidation provided intermediate enone 41 in 66% over three steps. Deprotection of the alcohol provided 17 in 78% isolated yield. Successive epoxidation of 17 with NaOCl gives direct access to 18 in 44% yield. Alternatively, oxidation of 17 yields the C3 ketone 19, which can then undergo epoxidation to give epoxide 20 in 25% isolated yield. Access to 21 began with 39, which was subjected to a Shapiro reaction to provide the D-ring olefin 42 in 26% yield. Both the A-ring and D-ring olefins in 42 were then epoxidized with mCPBA to give the bis-epoxide 21. The remaining analogs 28–30 were derived from 11 by first undergoing a Wharton reaction to give allylic alcohol 43 in 34% yield. Intermediate 44 was obtained through Ley oxidation and was then carried on to 28 via mCPBA A-ring epoxidation in 36% overall yield. Diverting allylic alcohol 43 to global olefin epoxidation provided 29, which was used to access 30 via Ley oxidation.

Scheme 3. Synthetic Routes towards 17–21 and 28–30.

Scheme 3

Open in a new tab

Reagents and conditions: (a) TBSCl, imidazole, DCM; (b) LDA, TMSCl, THF, −78 °C to rt; (c) 10% Pd(OAc)2, DMSO, DCE, O2 (balloon); (d) BF3–OEt2, DCM, 0 °C; (e) TPAP, NMO, DCM; (f) NaClO, pyridine, ethanol, −10 °C; (g) NaClO, pyridine, ethanol, −10 °C; (h) p-toluenesulfonyl hydrazide, EtOH, reflux; (i) nBuLi, diethyl ether; (j) mCPBA, DCM, −10 °C; (k) hydrazine hydrate, HOAc, MeOH; (l) TPAP, NMO, DCM; (m) mCPBA, DCM, −10 °C; (n) 2 equiv of mCPBA, DCM, −10 °C; (o) TPAP, NMO, DCM.

Our strategy to access C6-oxygenated analogs utilized trans-dehydroandrosterone (9) and exploited the intrinsic C5–C6 olefin to explore SAR at the C6-position in tandem with C15 electrophilicity (Scheme 4). Efforts began with protection of the C3-alcohol and D-ring ketone through silylation and ketal formation, respectively, to give 45 in 94% yield over two steps. Hydroboration of the C5–C6 olefin followed by acetate protection provided 47 in 93% yield. Mild acid treatment of 47 resulted in deprotection of both the silyl alcohol and ketal to give 48, which was subjected to dehydration using Tf2O/pyridine to give 49. Base-mediated deprotection of the C6-alcohol was achieved using K2CO3 in MeOH followed by Saegusa–Ito oxidation to provide 24 in 12% overall yield from 48. Leveraging 24 as another key linchpin intermediate, 22, 23, and 33–36 were made available through standard epoxidation or oxidation manipulations.

Scheme 4. Synthetic Routes towards C6-Oxygenated 22–24 and 33–36.

Scheme 4

Open in a new tab

Reagents and conditions: (a) ethylene glycol, TsOH–H2O, toluene, Dean–Stark; (b) TBSCl, imidazole, DCM; (c) BH3–SMe2, THF, then 10 M NaOH, 30% H2O2; (d) Ac2O, pyridine, DCM; (e) 1 M HCl, acetone; (f) Tf2O, pyridine, DCM, 0 °C; (g) K2CO3, MeOH, (h) TMSOTf, NEt3, DCM; (i) Pd(OAc)2, DMSO, DCE, O2 (balloon), then 1 M HCl, acetone; (j) NaClO, pyridine, EtOH, −10 °C; (k) TPAP, NMO, DCM; (l) DMDO, acetone, −10 °C.

Highlighted in Scheme 5 are the routes used to synthesize 25–27, 32, 37, and 38. Subjecting 49 to a Saegusa–Ito oxidation provided enone 51 in 46% yield. Base-promoted conjugate addition of MeOH occurred simultaneously with acetate deprotection to directly provide 37 in good overall yield. Epoxidation of 37 using DMDO allowed access to 25 in excellent yield, while Ley oxidation provided 38 in moderate isolated yield. Conversion of 38 via DMDO epoxidation provided 26, again in excellent yield. Finally, we were able to isolate 32 by epoxidizing intermediate 39 via in situ generation of DMDO, whereas oxidation of 24 provided C6-ketone 27 in good isolated yield.

Together, these data demonstrate that this new class of C15 electrophilic steroids have promising efficacy against drug-resistant TNBC and ovarian cancer cell lines. Experiments are ongoing in our laboratories to determine the specific cellular target(s) of these compounds.

Experimental Procedures

For methods, see the Supporting Information.

Acknowledgments

D.E.F. thanks the Max and Minnie Tomerlin Voelcker Fund for providing unrestricted funds in support of this work. A.L.R. also thanks the Max and Minnie Tomerlin Voelcker Fund for supporting this work through its Young Investigator Award. The UTSA NMR and X-ray facilities are supported by the NSF (CHE-1625963 and CHE-1920057).

Glossary

Abbreviations

Boc

tert-butyloxycarbonyl

BRCA

breast cancer gene

DCE

dichloroethane

DMDO

dimethyldioxirane

DPH

O-(2,4-dinitrophenyl)hydroxylamine

GI50

50% growth inhibition

HeLa

immortal cell line derived from cervical cancer cells

HFIP

hexafluoroisopropanol

LC50

50% lethal concentration

LDA

lithium diisopropylamide

mCPBA

meta-chloroperoxybenzoic acid

MEK

mitogen-activated extracellular signal-regulated kinase

NMO

4-methylmorpholine N-oxide

SRB

sulforhodamine B

TBSCl

tert-butyldimethylsilylchlorosilane

Tf2O

trifluoromethanesulfonic anhydride

TFE

2,2,2-trifluoroethanol

TGI

total growth inhibition

TMSCl

trimethylsilyl chloride

TMSOTf

trimethylsilyl triflate

TNBC

triple-negative breast cancer

TPAP

tetrapropylammonium perruthenate

TsCl

tosyl chloride

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00534.

  • Experimental procedures, characterization data, and dose–response curves for all reported analogs in Table 1, 1H and 13C NMR spectra for all compounds (PDF)

  • Crystallographic information file for 15 (CIF)

  • Crystallographic information file for 31 (CIF)

  • Crystallographic information file for 11 (CIF)

This work was supported by the Cancer Prevention & Research Institute of Texas (CPRIT no. RP170714).

The authors declare no competing financial interest.

Supplementary Material

ml0c00534_si_001.pdf (8.9MB, pdf)
ml0c00534_si_002.cif (30.3KB, cif)
ml0c00534_si_003.cif (991.2KB, cif)
ml0c00534_si_004.cif (19.1KB, cif)

References

  1. Newman D. J.; Cragg G. M. Natural Products as Sources of New Drugs over the Nearly Four Decades from 01/1981 to 09/2019. J. Nat. Prod. 2020, 83, 770–803. 10.1021/acs.jnatprod.9b01285. [DOI] [PubMed] [Google Scholar]
  2. Wani M. C.; Horwitz S. B. Nature as a remarkable chemist: a personal story of the discovery and development of Taxol. Anti-Cancer Drugs 2014, 25, 482–7. 10.1097/CAD.0000000000000063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beutler J. A. Natural Products as a Foundation for Drug Discovery. Curr. Protoc. in Pharmacol. 2009, 46, 9.11.1–9.11.21. 10.1002/0471141755.ph0911s46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Seletsky B. M.; Wang Y.; Hawkins L. D.; Palme M. H.; Habgood G. J.; DiPietro L. V.; Towle M. J.; Salvato K. A.; Wels B. F.; Aalfs K. K.; Kishi Y.; Littlefield B. A.; Yu M. J. Structurally simplified macrolactone analogues of halichondrin B. Bioorg. Med. Chem. Lett. 2004, 14, 5547–5550. 10.1016/j.bmcl.2004.08.068. [DOI] [PubMed] [Google Scholar]
  5. Zheng W.; Seletsky B. M.; Palme M. H.; Lydon P. J.; Singer L. A.; Chase C. E.; Lemelin C. A.; Shen Y.; Davis H.; Tremblay L.; Towle M. J.; Salvato K. A.; Wels B. F.; Aalfs K. K.; Kishi Y.; Littlefield B. A.; Yu M. J. Macrocyclic ketone analogs of halichondrin B. Bioorg. Med. Chem. Lett. 2004, 14, 5551–5554. 10.1016/j.bmcl.2004.08.069. [DOI] [PubMed] [Google Scholar]
  6. Chen G.; Jiang Z.; Zhang Q.; Wang G.; Chen Q.-H. New Zampanolide Mimics: Design, Synthesis, and Antiproliferative Evaluation. Molecules 2020, 25, 362. 10.3390/molecules25020362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dembitsky V. M.; Savidov N.; Poroikov V. V.; Gloriozova T. A.; Imbs A. B. Naturally occurring aromatic steroids and their biological activities. Appl. Microbiol. Biotechnol. 2018, 102, 4663–4674. 10.1007/s00253-018-8968-7. [DOI] [PubMed] [Google Scholar]
  8. Bansal R.; Singh R. Exploring the potential of natural and synthetic neuroprotective steroids against neurodegenerative disorders: A literature review. Med. Res. Rev. 2018, 38, 1126–1158. 10.1002/med.21458. [DOI] [PubMed] [Google Scholar]
  9. Drucker W. D. Biologic activity and metabolism of androgenic hormones: the role of the adrenal androgens. Bull. NY Acad. Med. 1977, 53, 347–358. [PMC free article] [PubMed] [Google Scholar]
  10. Shahidi N. A review of the chemistry, biological action, and clinical applications of anabolic-androgenic steroids. Clin. Ther. 2001, 23, 1355–1390. 10.1016/S0149-2918(01)80114-4. [DOI] [PubMed] [Google Scholar]
  11. Fotsis T.; Zhang Y.; Pepper M. S.; Adlercreutz H.; Montesano R.; Nawroth P. P.; Schweigerer L. The endogenous oestrogen metabolite 2-methoxyoestradiol inhibits angiogenesis and suppresses tumour growth. Nature 1994, 368, 237–239. 10.1038/368237a0. [DOI] [PubMed] [Google Scholar]
  12. Cushman M.; He H. M.; Katzenellenbogen J. A.; Lin C. M.; Hamel E. Synthesis, antitubulin and antimitotic activity, and cytotoxicity of analogs of 2-methoxyestradiol, an endogenous mammalian metabolite of estradiol that inhibits tubulin polymerization by binding to the colchicine binding site. J. Med. Chem. 1995, 38, 2041–9. 10.1021/jm00012a003. [DOI] [PubMed] [Google Scholar]
  13. Verenich S.; Gerk P. M. Therapeutic promises of 2-methoxyestradiol and its drug disposition challenges. Mol. Pharmaceutics 2010, 7, 2030–9. 10.1021/mp100190f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kattan S. W.; Nafie M. S.; Elmgeed G. A.; Alelwani W.; Badar M.; Tantawy M. A. Molecular docking, anti-proliferative activity and induction of apoptosis in human liver cancer cells treated with androstane derivatives: Implication of PI3K/AKT/mTOR pathway. J. Steroid Biochem. Mol. Biol. 2020, 198, 105604. 10.1016/j.jsbmb.2020.105604. [DOI] [PubMed] [Google Scholar]
  15. Papadopoulou N.; Papakonstanti E. A.; Kallergi G.; Alevizopoulos K.; Stournaras C. Membrane androgen receptor activation in prostate and breast tumor cells: Molecular signaling and clinical impact. IUBMB Life 2009, 61, 56–61. 10.1002/iub.150. [DOI] [PubMed] [Google Scholar]
  16. Sasano H.; Suzuki T.; Miki Y.; Moriya T. Intracrinology of estrogens and androgens in breast carcinoma. J. Steroid Biochem. Mol. Biol. 2008, 108, 181–185. 10.1016/j.jsbmb.2007.09.012. [DOI] [PubMed] [Google Scholar]
  17. Vihko P.; Härkönen P.; Oduwole O.; Törn S.; Kurkela R.; Porvari K.; Pulkka A.; Isomaa V. 17β-Hydroxysteroid dehydrogenases and cancers. J. Steroid Biochem. Mol. Biol. 2002, 83, 119–122. 10.1016/S0960-0760(02)00278-9. [DOI] [PubMed] [Google Scholar]
  18. Salvador J. A. R.; Carvalho J. F. S.; Neves M. A. C.; Silvestre S. M.; Leitão A. J.; Silva M. M. C.; Sáe Melo M. L. Anticancer steroids: linking natural and semi-synthetic compounds. Nat. Prod. Rep. 2013, 30, 324–374. 10.1039/C2NP20082A. [DOI] [PubMed] [Google Scholar]
  19. Amaral C.; Varela C. L.; Mauricio J.; Sobral A. F.; Costa S. C.; Roleira F. M.F.; Tavares-da-Silva E. J.; Correia-da-Silva G.; Teixeira N. Anti-tumor efficacy of new 7α-substituted androstanes as aromatase inhibitors in hormone-sensitive and resistant breast cancer cell. J. Steroid Biochem. Mol. Biol. 2017, 171, 218–228. 10.1016/j.jsbmb.2017.04.002. [DOI] [PubMed] [Google Scholar]
  20. Ghosh D.; Lo J.; Morton D.; Valette D.; Xi J.; Griswold J.; Hubbell S.; Egbuta C.; Jiang W.; An J.; Davies H. M. L. Novel Aromatase Inhibitors by Structure-Guided Design. J. Med. Chem. 2012, 55, 8464–8476. 10.1021/jm300930n. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ghosh D.; Lo J.; Egbuta C. Recent Progress in the Discovery of Next Generation Inhibitors of Aromatase from the Structure-Function Perspective. J. Med. Chem. 2016, 59, 5131–5148. 10.1021/acs.jmedchem.5b01281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Roleira F. M. F.; Varela C.; Amaral C.; Costa S. C.; Correia-da-Silva G.; Moraca F.; Costa G.; Alcaro S.; Teixeira N. A. A.; Tavares da Silva E. J. C-6α- vs C-7α-Substituted Steroidal Aromatase Inhibitors: Which Is Better? Synthesis, Biochemical Evaluation, Docking Studies, and Structure-Activity Relationships. J. Med. Chem. 2019, 62, 3636–3657. 10.1021/acs.jmedchem.9b00157. [DOI] [PubMed] [Google Scholar]
  23. Li J.; Shao Z.; Wang Z. Fulvestrant in the treatment of hormone receptor-positive/human epidermal growth factor recepter 2-negative advanced breast cancer: A review. Cancer Med. 2019, 8, 1943–1957. 10.1002/cam4.2095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Garrido M.; Peng H.-M.; Yoshimoto F. K.; Upadhyay S. K.; Bratoeff E.; Auchus R. J. A-ring modified steroidal azoles retaining similar potent and slowly reversible CYP17A1 inhibition as abiraterone. J. Steroid Biochem. Mol. Biol. 2014, 143, 1–10. 10.1016/j.jsbmb.2014.01.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Balaguer F. A.; Muhlethaler T.; Estevez-Gallego J.; Calvo E.; Gimenez-Abian J. F.; Risinger A. L.; Sorensen E. J.; Vanderwal C. D.; Altmann K. H.; Mooberry S. L.; Steinmetz M. O.; Oliva M. A.; Prota A. E.; Diaz J. F. Crystal Structure of the Cyclostreptin-Tubulin Adduct: Implications for Tubulin Activation by Taxane-Site Ligands. Int. J. Mol. Sci. 2019, 20 (6), 1392. 10.3390/ijms20061392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li J.; Risinger A. L.; Peng J.; Chen Z.; Hu L.; Mooberry S. L. Potent taccalonolides, AF and AJ, inform significant structure-activity relationships and tubulin as the binding site of these microtubule stabilizers. J. Am. Chem. Soc. 2011, 133, 19064–7. 10.1021/ja209045k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ola A. R. B.; Risinger A. L.; Du L.; Zammiello C. L.; Peng J.; Cichewicz R. H.; Mooberry S. L. Taccalonolide Microtubule Stabilizers Generated Using Semisynthesis Define the Effects of Mono Acyloxy Moieties at C-7 or C-15 and Disubstitutions at C-7 and C-25. J. Nat. Prod. 2018, 81, 579–593. 10.1021/acs.jnatprod.7b00967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Risinger A. L.; Li J.; Bennett M. J.; Rohena C. C.; Peng J.; Schriemer D. C.; Mooberry S. L. Taccalonolide binding to tubulin imparts microtubule stability and potent in vivo activity. Cancer Res. 2013, 73, 6780–92. 10.1158/0008-5472.CAN-13-1346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Du L.; Yee S. S.; Ramachandran K.; Risinger A. L. Elucidating target specificity of the taccalonolide covalent microtubule stabilizers employing a combinatorial chemical approach. Nat. Commun. 2020, 11, 654. 10.1038/s41467-019-14277-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wang Y.; Yu Y.; Li G. B.; Li S. A.; Wu C.; Gigant B.; Qin W.; Chen H.; Wu Y.; Chen Q.; Yang J. Mechanism of microtubule stabilization by taccalonolide AJ. Nat. Commun. 2017, 8, 15787. 10.1038/ncomms15787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Risinger A. L.; Li J.; Du L.; Benavides R.; Robles A. J.; Cichewicz R. H.; Kuhn J. G.; Mooberry S. L. Pharmacokinetic Analysis and in Vivo Antitumor Efficacy of Taccalonolides AF and AJ. J. Nat. Prod. 2017, 80, 409–414. 10.1021/acs.jnatprod.6b00944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Baillie T. A. Targeted covalent inhibitors for drug design. Angew. Chem., Int. Ed. 2016, 55, 13408–13421. 10.1002/anie.201601091. [DOI] [PubMed] [Google Scholar]
  33. Lonsdale R.; Ward R. A. Structure-based design of targeted covalent inhibitors. Chem. Soc. Rev. 2018, 47, 3816–3830. 10.1039/C7CS00220C. [DOI] [PubMed] [Google Scholar]
  34. Singh J.; Petter R. C.; Baillie T. A.; Whitty A. The resurgence of covalent drugs. Nat. Rev. Drug Discovery 2011, 10, 307–317. 10.1038/nrd3410. [DOI] [PubMed] [Google Scholar]
  35. Danielsson J.; Sun D. X.; Chen X.-Y.; Risinger A. L.; Mooberry S. L.; Sorensen E. J. A stereocontrolled annulation of the taccalonolide epoxy lactone onto the molecular framework of trans-androsterone. Org. Lett. 2017, 19, 4892–4895. 10.1021/acs.orglett.7b02349. [DOI] [PubMed] [Google Scholar]
  36. Lehmann B. D.; Bauer J. A.; Chen X.; Sanders M. E.; Chakravarthy A. B.; Shyr Y.; Pietenpol J. A. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J. Clin. Invest. 2011, 121, 2750–67. 10.1172/JCI45014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Risinger A. L.; Dybdal-Hargreaves N. F.; Mooberry S. L. Breast Cancer Cell Lines Exhibit Differential Sensitivities to Microtubule-targeting Drugs Independent of Doubling Time. Anticancer Res. 2015, 35 (11), 5845–50. [PMC free article] [PubMed] [Google Scholar]
  38. Gu Y.; Helenius M.; Vaananen K.; Bulanova D.; Saarela J.; Sokolenko A.; Martens J.; Imyanitov E.; Kuznetsov S. BRCA1-deficient breast cancer cell lines are resistant to MEK inhibitors and show distinct sensitivities to 6-thioguanine. Sci. Rep. 2016, 6, 28217. 10.1038/srep28217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Risinger A. L.; Jackson E. M.; Polin L. A.; Helms G. L.; LeBoeuf D. A.; Joe P. A.; Hopper-Borge E.; Luduena R. F.; Kruh G. D.; Mooberry S. L. The taccalonolides: microtubule stabilizers that circumvent clinically relevant taxane resistance mechanisms. Cancer Res. 2008, 68, 8881–8. 10.1158/0008-5472.CAN-08-2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. We would like to thank a reviewer who suggested we examine the activity of the D-ring saturated version of analog 10 (lacking a Michael acceptor) to support the covalent engagement of a cellular target by our active compounds. In HeLa cells, this analog demonstrated >10-fold less potency in antiproliferative activity supporting the hypothesis that an electrophilic site at C15 within the D-ring is critical to the engagement of an intracellular target(s). See Supporting Information for details.
  41. Jat J. L.; Paudyal M. P.; Gao H.; Xu Q.-L.; Yousufuddin M.; Devarajan D.; Ess D. H.; Kürti L.; Falck J. R. Direct Stereospecific Synthesis of Unprotected N-H and N-Me Aziridines from Olefins. Science 2014, 343, 61–65. 10.1126/science.1245727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Ma Z.; Zhou Z.; Kürti L. Direct and Stereospecific Synthesis of N-H and N-Alkyl Aziridines from Unactivated Olefins Using Hydroxylamine-O-Sulfonic Acids. Angew. Chem., Int. Ed. 2017, 56, 9886–9890. 10.1002/anie.201705530. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ml0c00534_si_001.pdf (8.9MB, pdf)
ml0c00534_si_002.cif (30.3KB, cif)
ml0c00534_si_003.cif (991.2KB, cif)
ml0c00534_si_004.cif (19.1KB, cif)

Articles from ACS Medicinal Chemistry Letters are provided here courtesy of American Chemical Society

RESOURCES