Synthesis and anticancer properties of a hybrid molecule with the testosterone and estradiol head-groups.

This is the first report on a unique hybrid molecule made of estradiol and testosterone (TS). This distinctive hybrid molecule (1) was designed to interact with both the estrogen receptor (ER) and the androgen receptor (AR) found in hormone-dependent female and male cancer cells, and was synthesized using ethynylestradiol (17EE) as the estrogenic component and 7α-(4-azido-but-2-enyl)-4-androsten-17ß-ol-3-one as the androgenic counterpart in a seven-step reaction with âˆ¼ 26 % overall yield. We reasoned that the dual receptor binding ability could allow 1 to act as an antihormone. This was tested on hormone-dependent and hormone-independent breast cancer (BCa) and prostate cancer (PCa) cells. The antiproliferative activity was also assessed on colon and skin cancer cells. We found that 1 was active against MCF7 (ER + ) BCa cells (IC50 of 4.9 µM), had lower inhibitory potency on LNCaP (AR + ) PCa cells (IC50 > 5 µM) and no effect on PC3 and DU145 (AR-) PCa cells. This suggests that the estrogenic component of 1 can interact with the ER on MCF7 cells more effectively than the androgenic component with the AR on LNCaP PCa cells, possibly due to a suboptimal spacer or linkage site(s). Nonetheless, the hybrid 1 was active against colon (HT-29) and melanoma (M21) cancer cells (IC50 of 3.5 µM and 2.3 µM, respectively), and had low cross-reactivity with the drug- and androgen-metabolizing cytochrome P450 3A4 (CYP3A4, IC50 ≫ 5 µM). These findings demonstrate the anticancer potential of 1 and warrant further explorations on this new type of hybrids.

Transcriptional regulation of CYR61 and CTGF by LM98: a synthetic YAP-TEAD inhibitor that targets in-vitro vasculogenic mimicry in glioblastoma cells.

Glioblastoma (GBM) is a highly angiogenic malignancy of the central nervous system that resists standard antiangiogenic therapy, in part because of an alternative process to angiogenesis termed vasculogenic mimicry. Intricately linked to GBM, dysregulation of the Hippo signaling pathway leads to overexpression of YAP/TEAD and several downstream effectors involved in therapy resistance. Little is known about whether vasculogenic mimicry and the Hippo pathway intersect in the GBM chemoresistance phenotype. This study seeks to investigate the expression patterns of Hippo pathway regulators within clinically annotated GBM samples, examining their involvement in vitro regarding vasculogenic mimicry. In addition, it aims to assess the potential for pharmacological targeting of this pathway. In-silico analysis of the Hippo signaling members YAP1, TEAD1, AXL, NF2, CTGF, and CYR61 transcript levels in low-grade GBM and GBM tumor tissues was done by Gene Expression Profiling Interactive Analysis. Gene expression was analyzed by real-time quantitative PCR from human U87, U118, U138, and U251 brain cancer cell lines and in clinically annotated brain tumor cDNA arrays. Transient gene silencing was performed with specific small interfering RNA. Vasculogenic mimicry was assessed using a Cultrex matrix, and three-dimensional capillary-like structures were analyzed with Wimasis. CYR61 and CTGF transcript levels were elevated in GBM tissues and were further induced when in-vitro vasculogenic mimicry was assessed. Silencing of CYR61 and CTGF, or treatment with a small-molecule TEAD inhibitor LM98 derived from flufenamic acid, inhibited vasculogenic mimicry. Silencing of SNAI1 and FOXC2 also altered vasculogenic mimicry and reduced CYR61/CTGF levels. Pharmacological targeting of the Hippo pathway inhibits in-vitro vasculogenic mimicry. Unraveling the connections between the Hippo pathway and vasculogenic mimicry may pave the way for innovative therapeutic strategies.

Total Synthesis of Cyclotripeptidic Natural Products Anacine, Aurantiomide C, Polonimides A and C, and Verrucine F.

The total synthesis of cyclotripeptidic natural products possessing a central piperazino[2,1-b]quinazolin-3,6-dione core is described through an original strategy involving the pivotal cyclocondensation of an electrophilic homoserine lactone intermediate. The alkylidene group was spontaneously installed by autoxidation during the cyclocondensation process, while the propionamide side chain was introduced through the nickel-catalyzed aminocarbonylation of a bromoethyl intermediate. This last reaction is unprecedented on such highly functionalized intermediates. Finally, we explored structural modifications and interconversions of the natural products. Overall, this work led to anacine, aurantiomide C, polonimides A and C, and verrucine F.

Development of HC-258, a Covalent Acrylamide TEAD Inhibitor That Reduces Gene Expression and Cell Migration.

The transcription factor YAP-TEAD is the downstream effector of the Hippo pathway which controls cell proliferation, apoptosis, tissue repair, and organ growth. Dysregulation of the Hippo pathway has been correlated with carcinogenic processes. A co-crystal structure of TEAD with its endogenous ligand palmitic acid (PA) as well as with flufenamic acid (FA) has been disclosed. Here we report the development of HC-258, which derives from FA and possesses an oxopentyl chain that mimics a molecule of PA as well as an acrylamide that reacts covalently with TEAD's cysteine. HC-258 reduces the CTGF, CYR61, AXL, and NF2 transcript levels and inhibits the migration of MDA-MB-231 breast cancer cells. Co-crystallization with hTEAD2 confirmed that HC-258 binds within TEAD's PA pocket, where it forms a covalent bond with its cysteine.

Development of LM-41 and AF-2112, two flufenamic acid-derived TEAD inhibitors obtained through the replacement of the trifluoromethyl group by aryl rings.

The Hippo pathway regulates organ size and tissue homeostasis by controlling cell proliferation and apoptosis. The YAP-TEAD transcription factor, the downstream effector of the Hippo pathway, regulates the expression of genes such as CTGF, Cyr61, Axl and NF2. Aberrant Hippo activity has been identified in multiple types of cancers. Flufenamic acid (FA) was reported to bind in a liphophilic TEAD palmitic acid (PA) pocket, leading to reduction of the expression of Axl and NF2. Here, we show that the replacement of the trifluoromethyl moiety in FA by aromatic groups, directly connected to the scaffold or separated by a linker, leads to compounds with better affinity to TEAD. Co-crystallization studies show that these compounds bind similarly to FA, but deeper within the PA pocket. Our studies identified LM-41 and AF-2112 as two TEAD binders that strongly reduce the expression of CTGF, Cyr61, Axl and NF2. LM-41 gave the strongest reduction of migration of human MDA-MB-231 breast cancer cells.

Investigating a new C2-symmetric testosterone dimer and its dihydrotestosterone analog: Synthesis, antiproliferative activity on prostate cancer cell lines and interaction with CYP3A4.

The synthesis of a 17α-linked C2-symmetric testosterone dimer and its dihydrotestosterone analog is reported. The dimers were synthesized using a short five-step reaction sequence with 28% and 38% overall yield for the testosterone and dihydrotestosterone dimer, respectively. The dimerization reaction was achieved by an olefin metathesis reaction with 2nd generation Hoveyda-Grubbs catalyst. The dimers and their corresponding 17α-allyl precursors were tested for the antiproliferative activity on androgen-dependent (LNCaP) and androgen-independent (PC3) prostate cancer cell lines. The effects on cells were compared with that of the antiandrogen cyproterone acetate (CPA). The results showed that the dimers were active on both cell lines, with an increased activity towards androgen-dependent LNCaP cells. However, the testosterone dimer (11) was fivefold more active than the dihydrotestosterone dimer (15), with an IC50 of 11.7 µM vs. 60.9 µM against LNCaP cells, respectively, and more than threefold more active than the reference drug CPA (IC50 of 40.7 µM). Likewise, studies on the interaction of new compounds with drug-metabolizing cytochrome P450 3A4 (CYP3A4) showed that 11 was a fourfold stronger inhibitor than 15 (IC50 of 3 µM and 12 µM, respectively). This suggests that changes in the chemical structure of sterol moieties and the manner of their linkage could largely affect both the antiproliferative activity of androgen dimers and their crossreactivity with CYP3A4.

Innovative C2-symmetric testosterone and androstenedione dimers: Design, synthesis, biological evaluation on prostate cancer cell lines and binding study to recombinant CYP3A4.

The synthesis of two isomeric testosterone dimers and an androstenedione dimer is reported. The design takes advantage of an efficient transformation of testosterone leading to the synthesis of the key diene, 7α-(buta-1,3-dienyl)-4-androsten-17ß-ol-3-one, through an elimination reaction. It was found that in some instances the same reaction led to partial epimerization of the 17ß-hydroxyl group into the 17α-hydroxyl group. The specific orientation of the hydroxyl function was confirmed by NMR spectroscopy. Capitalizing on this unforeseen side reaction, several dimers were assembled using an olefin metathesis reaction with Hoveyda-Grubbs catalyst. This led to the formation of two isomeric testosterone dimers with 17α-OH or 17ß-OH (14α and 14ß) as well as an androstenedione dimer (14). The new dimers and their respective precursors were tested on androgen-dependent (LNCaP) and androgen independent (PC3 and DU145) prostate cancer cells. It was discovered that the most active dimer was made of the natural hormone testosterone (14ß) with an average IC50 of 13.3 µM. In LNCaP cells, 14ß was ∼5 times more active than the antiandrogen drug cyproterone acetate (IC50 of 12.0 µM vs. 59.6 µM, respectively). At low concentrations (0.25-0.5 µM), 14α and 14ß were able to completely inhibit LNCaP cell growth induced by testosterone or dihydrotestosterone. Furthermore, cross-reactivity of androgen-based dimers with sterol-metabolizing cytochrome P450 3A4 was explored and the results are disclosed herein.

Recent Advances in the Use of the Dimerization Strategy as a Means to Increase the Biological Potential of Natural or Synthetic Molecules.

The design of C2-symmetric biologically active molecules is a subject of interest to the scientific community. It provides the possibility of discovering medicine with higher biological potential than the parent drugs. Such molecules are generally produced by classic chemistry, considering the shortness of reaction sequence and the efficacy for each step. This review describes and analyzes recent advances in the field and emphasizes selected C2-symmetric molecules (or axial symmetric molecules) made during the last 10 years. However, the description of the dimers is contextualized by prior work allowing its development, and they are categorized by their structure and/or by their properties. Hence, this review presents dimers composed of steroids, sugars, and nucleosides; known and synthetic anticancer agents; polyphenol compounds; terpenes, known and synthetic antibacterial agents; and natural products. A special focus on the anticancer potential of the dimers transpires throughout the review, notwithstanding their structure and/or primary biological properties.