Abstract Melanoma, the most fatal form of skin cancer, often becomes resistant to the current therapeutic approaches in most patients. To explore new treatment options, fused thiazole derivatives were synthesized, and several of these compounds demonstrated potent anti-melanoma activity both in vitro and in vivo. These compounds exhibited significant cytotoxicity against melanoma cell lines at low concentrations. The lead molecules induced apoptosis and caused G2/M phase cell cycle arrest to a lesser extent. These compounds also displayed remarkable antimetastatic activities in several cell-based and molecular assays, significantly inhibiting key processes of metastasis, such as cell migration and adhesion. mRNA sequencing revealed significant downregulation of β-actin (ACTB) and γ-actin (ACTG1) at the transcriptional level, and a similar effect was observed at the protein level by western immunoblotting and proteomics assays. Actin-rich membrane protrusions formation is crucial for facilitating metastasis by promoting cell migration. Fluorescence microscopy demonstrated that compounds E28 and E47 inhibited the formation of these membrane protrusions and impaired actin cytoskeleton dynamics. Docking studies suggested the lead compounds may suppress tumor proliferation and metastasis by targeting the mechanistic target of Rapamycin complex 2 (mTORC2). All these findings unanimously indicated the translational perspective of ethisterone and androstenone fused thiazole derivatives as potent antimetastatic and antimelanoma agents. In a preclinical mouse melanoma model, compounds E2 and E47 significantly reduced tumor growth and greatly improved overall mice survival, while showing a favorable safety profile based on a comprehensive blood plasma metabolite profile. These lead molecules also displayed promising physicochemical properties, making them strong candidates for further drug development studies. __________________________________________________________________ Several fused-thiazole derivatives have been studied for antimelanoma properties. Lead compounds are effective tumor growth inhibitors in mice, which are potent β- and γ-actin cytoskeleton inhibitors with a probable mechanism of β-PARVIN inhibition.[44] graphic file with name d4md00719k-ga.jpg Introduction Malignant melanoma (MM) arising from transformed melanocytes is the most serious type of skin cancers and responsible for over 80% of skin cancer-related deaths.^[45]1 In 2023, an estimated 8000 Americans died of this disease and nearly 100 000 new cases were reported.^[46]2 The cost of treating newly diagnosed MM is expected to increase to $1.6 billion by 2030. Additional efforts are required to prevent the incidence, mortality, and healthcare costs associated with MM.^[47]3 New therapeutic options are urgently needed to treat the increasing incidence of this malignancy.^[48]4 New drugs to treat advanced melanoma have been introduced in recent years, but five-year survival is still less than 20% owing to the development of resistance (e.g., cobimetinib and dabrafenib) or severe adverse effects (e.g., dacarbazine and interleukin-2, IL-2).^[49]4,5 Combination therapy (trametinib and dabrafenib) was approved in 2014 to treat metastatic melanoma patients with BRAF V^600E or V^600K mutation. Complete response rates for these treatments are rare, and progression-free survival is less than 15 months.^[50]6 Due to IL-2-induced vascular leak syndrome and other toxicities, IL-2 therapy is limited to young and otherwise healthy individuals.^[51]7 Recently, immune checkpoint inhibitors (ICIs), particularly anti-CTLA4 and anti-PD-1/L1 antibodies, have shown great success in the management of many cancers, including melanoma.^[52]8 However, despite the enthusiasm generated by the success of ICIs, considerable clinical failures have occurred for checkpoint blockade therapies.^[53]8,9 Therefore, it is important to identify new therapeutic agents for MM to achieve long-term progression-free survival. Thiazole is a five-membered sulfur and nitrogen-containing heterocycle, and it is a privileged scaffold in drug discovery including to develop antimelanoma agents.^[54]10 The thiazole nucleus is found in several natural products, approved drugs (e.g., dabrafenib, [55]Fig. 1), and pharmacologically active synthetic molecules.^[56]11 Similarly, the steroidal skeleton is found in a number of drugs, human hormones, and bioactive natural products.^[57]12 There are several drugs containing heterocycle fused (e.g., Emflaza) and heterocycle attached (e.g., Zytiga) steroidal derivatives to treat different diseases including cancer ([58]Fig. 1).^[59]13 We have developed efficient methodologies to synthesize fused thiazoles with steroidal and decalin skeletons and found several of these as potential therapeutic agents.^[60]14 Ethisterone is an obsolete drug and its related derivatives such as levonorgestrel, danazol, and etonogestrel are among the most widely prescribed drugs.^[61]15 Thus, based on literature precedence and our efficient methodologies, we envisaged that antineoplastic studies of fused thiazole-androstenone and ethisterone derivatives would lead to the discovery of anticancer agents. Fig. 1. Structure of dabrafenib (BRAF inhibitor), androstenedione (endogenous steroidal hormone), ethisterone (progestin medication), and representative example of heterocycle containing steroidal drugs (Emflaza and Zytiga). [62]Fig. 1 [63]Open in a new tab Results and discussion In our effort to find novel azole derivatives as therapeutic agents,^[64]14d,16 we have developed efficient methodologies to synthesize thiazole derivatives.^[65]14a–c,17 In our preliminary cytotoxicity studies, we routinely submit novel compounds to NCI for cytotoxicity screening against 60 cancer cell lines. We found that thiazole-fused with androstenedione ([66]Fig. 2) and ethisterone showed potent activity against the melanoma panel of NCI-60 cell line screening data.^[67]14a–c,18 To further explore the antimelanoma properties of these potent fused thiazole derivatives, we synthesized additional compounds and carried out several in vitro studies to determine their structure activity relationship (SAR). We determined the 50% inhibitory concentration (IC[50]) of our in-house library of fused compounds against a murine melanoma cell line (B16F10) and four human melanoma cell lines (LOX IMVI, SK-MEL-28, SK-MEL- 25, and SK-MEL- 5) using a resazurin cell viability assay. Several in vitro experiments have helped elucidate the mechanism of action, and in vivo studies led to the discovery of non-toxic lead compounds for potential melanoma treatment. Fig. 2. Fused-thiazolo androstenone derivatives. These N-substituted fused thiazole derivatives (A1–A27) have been synthesized by the reaction of thiourea derivatives with 6β-bromo androstenedione (BA). Similar reaction of BA with thioamides afforded other subset of compounds (A28–A53). [68]Fig. 2 [69]Open in a new tab Cytotoxicity studies Several fused thiazolo-androstenedione derivatives ([70]Fig. 2) showed potent inhibition of the tested melanoma cell lines. Compounds showing activity with IC[50] values less than 20 μM are shown in [71]Table 1. Our in-house library of compounds ([72]Fig. 2) has diverse substituent patterns such as aliphatic substituents on the amino group (A1–A7). Aliphatic amine derivatives did not show significant cytotoxicity except the phenylethyl substituted compound (A7), which is active against the tested cell lines with IC[50] values as low as 5.4 μM against the SK-MEL-28 cell line ([73]Table 1). The N-phenyl substituted derivative (A8) showed potent activity with IC[50] values as low as 2.7 μM against human melanoma cell lines and inhibited the growth of the murine melanoma cell line B16F10 with an IC[50] value of 5.8 μM. The tolyl containing compound (A9) effectively inhibited melanoma cell lines with the IC[50] values of ∼5 μM. The 2-methoxyphenyl derivative (A10) did not significantly inhibit the tested melanoma cell lines. Table 1. IC[50] (μM) of potent thiazolo androstenone derivatives (A1–A53) B16 F10 = murine melanoma, LOX IMVI, SK-MEL-28, SK-MEL-25, and SK-MEL-28 = human melanoma cell lines, error bar = SD (standard deviation), # = compound number, and Cisp = cisplatin. # B16 F10 LOX IMVI SK MEL 28 SK MEL 25 SK MEL 5 A7 6.1 ± 0.5 9.0 ± 0.0 5.4 ± 0.6 >20 5.2 ± 0.7 A8 5.8 ± 0.6 2.7 ± 0.1 6.1 ± 0.1 6.8 ± 1.5 10.6 ± 1.1 A9 4.9 ± 0.6 4.2 ± 0.1 4.0 ± 0.5 4.3 ± 1.2 7.3 ± 0.8 A11 6.7 ± 0.2 2.2 ± 0.0 12.6 ± 1.6 4.8 ± 0.8 >20 A12 11.7 ± 1.0 9.3 ± 1.2 >20 12.5 ± 0.5 >20 A13 5.4 ± 0.5 6.7 ± 0.7 6.4 ± 0.6 >20 >20 A14 2.7 ± 0.2 1.6 ± 0.1 4.5 ± 1.4 4.2 ± 1.0 6.7 ± 0.2 A15 4.5 ± 2.7 1.8 ± 0.8 3.4 ± 0.2 1.4 ± 0.1 6.4 ± 0.3 A20 6.1 ± 0.9 3.6 ± 0.5 1.9 ± 0.3 4.4 ± 0.8 6.6 ± 1.8 A21 8.0 ± 1.9 2.5 ± 0.4 2.5 ± 0.7 3.2 ± 0.9 >20 A22 6.4 ± 0.8 6.2 ± 0.1 >20 >20 >20 A23 3.6 ± 0.2 3.0 ± 0.5 4.8 ± 0.7 >20 >20 A24 5.5 ± 0.6 2.8 ± 0.4 5.3 ± 0.1 >20 11.5 ± 1.4 A25 5.9 ± 0.4 2.3 ± 0.2 8.9 ± 1.2 11.8 ± 1.0 7.1 ± 1.1 A26 3.0 ± 0 >20 >20 1.8 ± 0.4 1.3 ± 0.2 Cisp 24.7 ± 0.6 8.4 ± 0.6 24.8 ± 1.7 18.7 ± 3.3 27.4 ± 3.4 [74]Open in a new tab 3-Hydroxy (A11), 4-trifluoromethoxy (A12), and 2-fluoro (A13) substituted derivatives exhibited moderate growth inhibition of melanoma cell lines. A14 was the most potent compound in the series with IC[50] values ranging from 1.6 to 6.7 μM. This 3-fluorophenyl derivative (A14) inhibited the growth of a murine melanoma cell line with an IC[50] value of 2.7 μM. LOX IMVI was the most susceptible cell line to this compound (A14) with an IC[50] value of 1.6 μM. The 4-fluorophenyl derivative (A15) is one of the most active compounds in this series with IC[50] values as low as 1.4 μM. Other mono-substituted derivatives such as 2-chloro (A16), carboxylic acid (A17 and A18), and 2-nitrophenyl (A19) substituted fused compounds did not exhibit any significant cytotoxicity. All the tested disubstituted phenyl derivatives were effective cytotoxic agents against the tested melanoma cell lines ([75]Table 1). Dimethylphenyl derivatives (A20 and A21) were potent against the tested cell lines with IC[50] values ranging from 1.8 to 8.0 μM. The 2,5-dimethoxy derivative (A22) showed moderate activity against B16 and LOX IMVI cell lines. 5-Chloro-2-methoxy (A23) and difluoro (A24) substituted derivatives were moderate cytotoxic agents. Pyridyl (A25), pyrimidinyl (A26), and N-methyl-N-phenyl substituted derivatives (A27) did not show any significant activity against the tested cell lines. We tested compounds obtained by the reaction of thioamide and thiobenzamide derivatives with bromo-androstenone for their activity against melanoma cell lines. Most of these compounds (A28–A53) did not show significant cytotoxicity, likely due to their low solubility in the growth medium and 1% DMSO. Nevertheless, 3-fluorophenyl (A40) and catechol (A48) substituted derivatives showed potent activity against some cell lines, but moderate or weak activity against other cell lines. Overall, these results demonstrated that a subset of derivatives was more potent than cisplatin, which was several-fold less active against these cell lines. We found the IC[50] values of dacarbazine at millimolar concentration in melanoma cell lines (B16F10 – 0.48 mM and SK-MEL-28 – 0.34 mM) which is similar to the literature report.^[76]19 The demonstration of the potent activity of fused thiazolo-androstenone derivatives led us to synthesize fused thiazole derivatives with different enone derivatives such as ethisterone and nootkatone. Preliminary studies helped to define nootkatone and ethisterone derivatives as antibacterial and cytotoxic agents respectively.^[77]14b,d In addition to the reported compounds, we synthesized ∼30 new compounds to study the structure–activity relationship (SAR). We found that most of the ethisterone compounds ([78]Fig. 3) were potent cytotoxic agents, functioning at lower doses than thiazolo-androstenone derivatives. Fig. 3. Thiazolo ethisterones: in-house library, novel compounds, and potent cytotoxic agents. The compounds were obtained by the reaction of thiourea and thioamide derivatives with epoxy-ethisterone. [79]Fig. 3 [80]Open in a new tab The cytotoxicity data for fused ethisterone compounds with IC[50] values <20 μM are shown in [81]Table 2. The amino substituted derivative (E1) did not show significant activity against the cell lines, although this may be due to its low solubility in the growth medium. The N-phenyl substituted derivative (E2) was a significant growth inhibitor of the tested cell lines with IC[50] values as low as 1.6 μM. Hydroxy (E3), carboxylic acid (E4 and E5), and nitro (E6) substituted phenyl derivatives did not show any significant activity against the tested cell lines. 3,5-Dimethylphenyl-substituted derivative (E7) showed potent activity with IC[50] values as low as 3.6 μM against the LOX IMVI cell line. Dimethoxy phenyl substituted derivatives (E8 and E9) showed similar activities across the tested cell lines. 2-Chloro-5-methoxy phenyl derivative (E10) was a moderate growth inhibitor of the murine melanoma cell line, but a potent growth inhibitor of human melanoma cell lines with IC[50] values from 3.1 to 4.8 μM. 3-Chloro-4-methyl substituted derivative (E11) was a potent growth inhibitor across the tested cell lines. The last disubstituted compound (E12) was one of the most potent compounds in this series with IC[50] values ∼2 μM across the tested cell lines. This derivative (E12) was most active against the LOX IMVI cell line with an IC[50] value as low as 1.6 μM. Table 2. IC[50] (μM) of potent thiazolo-ethisterone derivatives against melanoma cell lines. # B16 F10 LOX IMVI SK-MEL-28 SK-MEL-25 SK-MEL-5 E2 2.7 ± 0.0 1.6 ± 0.0 1.7 ± 0.2 1.6 ± 0.1 2.4 ± 0.5 E7 5.8 ± 0.1 3.6 ± 0.3 5.0 ± 0.6 3.8 ± 0.2 3.9 ± 0.7 E8 7.7 ± 0.4 3.4 ± 0.2 4.7 ± 0.5 3.3 ± 0.2 3.0 ± 0.1 E9 7.2 ± 1.1 2.4 ± 0.4 5.0 ± 0.9 3.4 ± 0.7 3.5 ± 0.7 E10 9.7 ± 0.7 3.2 ± 0.4 3.4 ± 0.0 3.1 ± 0.0 4.8 ± 0.1 E11 4.9 ± 0.1 3.9 ± 0.1 5.6 ± 0.2 4.6 ± 0.2 4.3 ± 0.6 E12 2.5 ± 0.5 1.6 ± 0.0 1.8 ± 0.1 2.4 ± 0.5 1.9 ± 0.1 E14 7.6 ± 0.4 7.5 ± 0.1 3.4 ± 0.7 4.0 ± 0.7 2.6 ± 0.2 E15 10.7 ± 2.2 2.6 ± 0.2 2.3 ± 0.4 2.1 ± 0.1 3.0 ± 0.1 E16 6.7 ± 0.3 2.5 ± 0.1 4.2 ± 0.8 7.9 ± 1.2 2.9 ± 0.4 E17 2.9 ± 0.0 4.8 ± 0.3 2.4 ± 0.4 >20 5.5 ± 0.4 E18 7.7 ± 0.4 3.6 ± 0.2 4.6 ± 0.7 4.8 ± 0.5 3.7 ± 0.3 E19 6.5 ± 0.4 2.8 ± 0.6 3.0 ± 0.0 5.1 ± 0.2 3.9 ± 0.3 E20 3.1 ± 0.1 5.4 ± 0.9 3.5 ± 0.3 3.2 ± 0.3 2.8 ± 0.4 E21 3.8 ± 0.6 3.0 ± 0.1 2.8 ± 0.1 4.4 ± 0.4 3.6 ± 0.4 E22 3.0 ± 0.1 5.4 ± 0.9 3.7 ± 0.6 8.3 ± 1.2 4.8 ± 0.6 E23 3.8 ± 0.2 5.5 ± 1.0 2.4 ± 0.1 5.7 ± 0.6 2.7 ± 0.2 E24 10.2 ± 0.2 4.1 ± 0.4 5.5 ± 0.7 6.5 ± 0.8 2.5 ± 0.2 E25 13.5 ± 2.4 7.8 ± 1.6 2.8 ± 0.2 5.7 ± 0.7 4.9 ± 0.6 E28 2.8 ± 0.3 7.4 ± 0.2 2.0 ± 0.3 2.8 ± 0.3 2.6 ± 0.2 E38 2.9 ± 0.4 4.1 ± 0.1 6.0 ± 1.1 6.6 ± 0.6 3.5 ± 0.1 E39 4.7 ± 1.1 3.3 ± 0.2 2.5 ± 0.01 3.7 ± 0.6 3.2 ± 0.2 E40 8.5 ± 1.0 15.3 ± 3.3 14.0 ± 0.5 >20 >20 E41 13.9 ± 1.3 15.6 ± 3.0 >20 >20 >20 E46 13.0 ± 0.7 >20 6.1 ± 0.1 4.2 ± 0.6 4.9 ± 0.7 E47 2.6 ± 0.3 4.4 ± 0.2 3.6 ± 0.2 2.5 ± 0.1 3.7 ± 0.5 Cisp 24.7 ± 0.6 8.4 ± 0.6 24.8 ± 1.7 18.7 ± 3.3 27.4 ± 3.4 [82]Open in a new tab N-Methyl-N-phenyl derivative (E13) did not show significant activity against the tested cell lines. Among the pyridyl substituted derivatives (E14, E15, and E16), 3-pyridyl derivative (E15) is the most potent growth inhibitor of the melanoma cell lines with IC[50] values as low as 2.1 μM against the SK-MEL-25 cell line. Methyl substitution on the pyridyl ring (E17, E18, and E19) did not affect potency compared to the unsubstituted pyridyl derivatives. Fluoro (E20) and chloro (E21, E22, and E23) pyridyl derivatives consistently showed potent activities against the tested cell lines. Dimethylpyridyl (E24) and pyrimidinyl (E25) substituted compounds were moderately cytotoxic against most of the tested cell lines. Substituted pyrimidine (E26) and piperidine (E27) derivatives were noncytotoxic against the cell lines up to 20 μM concentrations. The N-methyl piperazine-derived compound (E28) was very potent against all the tested cell lines with significant activity against the B16 cell line with an IC[50] value of 2.8 μM. Thiazoles obtained from thioamides bearing lipophilic substituents (E29–E37) did not show any significant activity. Nevertheless, hydroxy-substituted compounds (E38 and E39) were effective growth inhibitors of melanoma cell lines with IC[50] values as low as 2.5 μM. Fluorophenyl substituted compounds (E40 and E41) showed moderate activity against some of the cell lines, while chloro (E42 and E43), bromo (E44), and trifluoromethyl (E45) derivatives were inactive at the tested concentrations. Benzodioxole-derived fused thiazole (E46) was a moderate growth inhibitor of most of the tested cell lines. The catechol substituted derivative (E47) was one of the most potent compounds in this series with the IC[50] values of 2.6 and 2.5 μM against B16 and SK-MEL-25 cell lines respectively. The dichloro (E48) and pyridinyl derivatives (E49 and E50) showed no significant cytotoxicity against the cell lines. Thus, this series of compounds, chimeric thiazolo-ethisterones, is consistently active against melanoma cell lines. Furthermore, the starting materials, ethisterone, and bromo-androstenedione, did not show any noticeable cytotoxicity up to 40 μM concentrations against the tested melanoma cell lines. Structure–activity relationship (SAR) Based on the antimelanoma potency of more than 100 compounds, we identified distinct SARs ([83]Fig. 4). Thiazole-fused ethisterone derivatives ([84]Fig. 3 and [85]Table 2) were more potent than the thiazole-fused androstenone compounds ([86]Fig. 2 and [87]Table 1). Amino (Z = NH) derivatives were consistently more potent than alkyl and aryl thiazole derivatives (Z = 0). Among the substituted amino thiazole derivatives, alkyl substituted derivatives showed negligible activity except N-methyl piperazine derivative (E28). Among N-aryl substituted compounds, N-phenyl derivatives were good antimelanoma agents. Small lipophilic groups (fluoro and methyl) and hydroxy derivatives showed sufficient overall activity. Disubstitution with these groups increased cytotoxic properties. Ionizable (carboxylic acid) and ionic (nitro) groups almost eliminated the cytotoxic properties of the resultant compounds. Heterocycle-substituted amino derivatives of ethisterone were consistently potent against the cell lines, but androstenone derivatives with similar substitution did not show any significant activity. Due to the potent and consistent activities, we selected E2, E12, E28, and E47 for further in vitro and in vivo studies. Fig. 4. Effect of different groups on the thiazolo-steroidal scaffold on the activity. [88]Fig. 4 [89]Open in a new tab Effect of compounds on cell migration, cell adhesion, and clonogenicity Cell migration and adhesion are key mechanistic phenomena in metastasis.^[90]20 The ability of cells to migrate through the extracellular matrix drives the progression of malignancy, which is mediated by cell membrane protrusions and their adhesion to the extracellular matrix (ECM). Inhibition of the signaling mechanisms that regulate the migration of malignant cells ultimately inhibits the dissemination of cancer cells to secondary organs.^[91]21E12 and E47 at 2 μM significantly inhibited the migration of B16F10 cells relative to the untreated negative control (NC) ([92]Fig. 5A). A similar significant SK-MEL-28 cell migration inhibition was observed for E12 and E47 at 1 μM and 1.2 μM, respectively ([93]Fig. 5B). Additionally, E12 (1.5 μM) and E47 (2.5 μM) inhibited the adhesion of B16F10 ([94]Fig. 6A) and SK-MEL-28 cells ([95]Fig. 6E) significantly relative to the NC. E12 ([96]Fig. 6C) and E47 ([97]Fig. 6D) treatment reduced the number of adherent B16F10 cells compared to those in the NC group ([98]Fig. 6B). Similarly in the SK-MEL-28 cell line, E12 ([99]Fig. 6G) and E47 ([100]Fig. 6H) treatment decreased cell adhesion compared to NC ([101]Fig. 6F). This study further supports our hypothesis that these compounds can inhibit metastasis in vivo. Fig. 5. Wound healing assay of (A) B16F10 cell line and (B) SK-MEL-28 cell line. The bar graph shows the mean wound closure of treatments (E12 and E47) and negative control (NC). Images presented pre (left) and post (right) treated wounds of treatments and NC. NC was less than 0.1% DMSO in complete growth medium. * and **** indicate p values <0.05 and <0.0001, respectively. Error bars show standard deviation (SD). [102]Fig. 5 [103]Open in a new tab Fig. 6. Cell adhesion assay in B16F10 and SK-MEL-28 cell line. Bar graphs A (B16F10) and E (SK-MEL-28) present the mean counts of adherent cells in the negative control (NC), E12, and E47. Fig. 6B–D show the adherent cells treated with NC, E12, and E47 respectively in B16F10 cell line. Fig. 6F–H showed the adherent cells of NC, E12, and E47 respectively in SK-MEL-2 cells. The negative control (NC) contained <0.1% DMSO in complete GM. * and **** show p < 0.05 and <0.0001, respectively. Error bars represent SD. [104]Fig. 6 [105]Open in a new tab After metastasis, unconstrained tumor cells proliferate at organ sites distant from the primary tumor to form metastases. The inhibition of colony proliferation of cells can lead to the inhibition of metastases. Therefore, clonogenic assays enable the measurement of the ability of an adherent cell line to remain viable and multiply over time into a colony of cells. These assays were used to assess the ability of novel compounds to inhibit colony formation in murine and human melanoma cell lines. Compound E12 completely inhibited colony formation at 3 μM ([106]Fig. 7B) and 6 μM ([107]Fig. 7C) in B16F10 cells after six days of colonization. Similarly, complete inhibition of colony formation was observed for E47 at 3 μM ([108]Fig. 7E) and 6 μM ([109]Fig. 7F) concentrations. After eight days of SK-MEL-28 cell colonization, complete inhibition of colony formation was observed in the case of E12 at 3 μM ([110]Fig. 7H) and 6 μM ([111]Fig. 7I), and a few colonies were observed for E47 at 3 μM ([112]Fig. 7K) and 6 μM ([113]Fig. 7L). Cisplatin at 20 μM concentration abolished colony formation in both the cell lines. Fig. 7. Clonogenic assay in B16F10 and SK-MEL-28 cell lines. Fig. 7A, D, B, C, E and F presented NC, cisplatin (20 μM), E12 (3 μM), E12 (6 μM), E47 (3 μM), and E47 (6 μM), respectively in B16F10 cell line. Fig. 7G, J, H, I, K and L illustrated NC, cisplatin (20 μM), E12 (3 μM), E12 (6 μM), E47 (3 μM), and E47 (6 μM), respectively in SK-MEL-28 cell line. Negative control (NC) included <0.1% DMSO. [114]Fig. 7 [115]Open in a new tab Flow cytometry assays Cell cycle analysis Cell cycle arrest at any phase of mitosis can prevent cancer cells from proliferation. Propidium iodide (PI) staining and flow cytometry analysis were used to determine the effects of compounds on the cell cycle. The percentage of cells in each phase of the cell cycle for B16F10 and SK-MEL-28 cell lines are shown in [116]Fig. 8A and B respectively. Treatment with 3 μM of E12 ([117]Fig. 8G) and E47 ([118]Fig. 8H) significantly arrested the cell cycle at the G2/M phase in SK-MEL-28 cells compared to NC ([119]Fig. 8F). Still, in the B16F10 cell line, only E47 ([120]Fig. 8E) at 3 μM concentration arrested the cell cycle significantly in the G2/M phase relative to NC ([121]Fig. 8C). Fig. 8. Cell cycle analysis. Percentage of cells in each phase of cell cycle for B16F10 (Fig. 8A) and SK-MEL-28 (Fig. 8B) cell lines. Fig. 8C–E respectively showed the cell cycle of NC, E12 and E47 at 3 μM in B16F10 cell line. Fig. 8F–H depicted the cell cycle of NC, E12, and E47 at 3 μM in SK-MEL-28 cell line, respectively. Negative control (NC) contained <0.1% DMSO. *, ** and **** correspond to p < 0.05, 0.01 and 0.0001, respectively. (iii) Error bars depict SD. [122]Fig. 8 [123]Open in a new tab Apoptosis study Annexin V-fluorescein (FITC) and propidium iodide (PI) flow cytometry assays were used to measure the extent of apoptosis induced by the fused thiazole derivatives. This assay utilizes annexin V conjugated with FITC and PI. PI enables the differentiation of live cells (PI-negative cells, cells in Q3 and Q4 quadrants) and dead cells (PI-positive cells, cells in Q1 and Q2 quadrants). In the early apoptotic stage, cells are alive (PI negative) but have flipped phosphatidylserine out (annexin V positive, detected via FITC) which indicates the Q4 cell population. Cells in the late stage of apoptosis are dead (PI positive) and have externalized phosphatidylserine (annexin V positive), which are denoted as the Q2 cell population. At 8 μM treatment, E12 ([124]Fig. 9B), and E47 ([125]Fig. 9C) induced statistically significant apoptosis relative to the NC ([126]Fig. 9A) in B16F10 cells after 24 h of treatment. Fig. 9. Annexin V - PI flow cytometry apoptosis assay. The bar graph negative control (NC) (Fig. 9A), 8 μM treatment of B16F10 cell with E12 (Fig. 9B) and E47 (Fig. 9C). Fig. 9D indicated the percentage of apoptotic cells (Q2 + Q4) in treatments and NC. Negative control (NC) contained <0.1% DMSO. * and ** indicate p < 0.05 and <0.01, respectively. [127]Fig. 9 [128]Open in a new tab Mode of action studies Genomics Total mRNA sequencing of the human melanoma cell line SK-MEL-28 treated with E28 at 2 μM for 24 h revealed 148 differentially expressed genes (DEGs). Among the identified DEGs, 117 were upregulated and 31 were downregulated relative to those in the control groups ([129]Fig. 10A). In contrast, treatment with E47 at 2 μM for 24 h resulted in 1549 DEGs. Of the 1549 DEGs, 806 were overexpressed and 743 were downregulated ([130]Fig. 10B). E28 and E47 treatments shared 86 DEGs, of which 65 were upregulated, and 21 were downregulated. Among the downregulated genes, E47 significantly reduced the expression of β-actin (ACTB) and γ-actin (ACTG1) in SK-MEL-28 cells after 2 μM treatment 24 h ([131]Tables 3 and [132]4), indicating a possible mechanism for the inhibition of cell migration ([133]Fig. 5). ACTB and ACTG1 are essential components of actin cytoskeleton dynamics and homeostasis. Under normal conditions, the actin cytoskeleton performs a range of cellular functions, including control of cell shape, cell adhesion, cell migration, cell division, regulation of muscle contraction, cellular junctions, transcriptional regulation, and vesicle trafficking in various cell types.^[134]20 Fig. 10. Differentially expressed genes (DEGs) by E28 (A) and E47 (B) in comparison to NC <0.1% DMSO. (C) KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment analysis for DEGs by E47. (D) Gene ontology (biological function) enrichment analysis of DEGs by E47. The absolute value of log[2] fold change (log[2]FC) was >0.5 with a significant adjusted p < 0.05. [135]Fig. 10 [136]Open in a new tab Table 3. mRNA expression (mRNA sequencing) of ACTB and ACTG1 in E47 treatment. Gene Log[2]FC P value ACTB −0.95 7.2 × 10^−41 ACTG1 −0.70 1.3 × 10^−25 [137]Open in a new tab Table 4. Subset of DEPs following treatment with E47 (2 μM) for 24 h. Genes Log[2]FC P value ACTB −0.4 0.001 ACTG1 −0.4 0.001 PARVB 2.4 0.100 [138]Open in a new tab ACTB primarily regulates cellular contraction and adhesion, whereas ACTG1 regulates cell motility and flexibility.^[139]22 Although actin regulates crucial physiological cellular functions during normal homeostasis, it has also been exploited during cancer progression to drive metastasis in diverse cancer phenotypes. In human colon cancer cells, ACTB and ACTG1 overexpression are associated with constant remodeling of the actin cytoskeleton, and an increase in invasiveness and migration of tumor cells, both of which are known to drive metastasis.^[140]20 Cell migration is facilitated by forming membrane protrusions (lamellipodia, filopodia, and invadopodia), a process mediated by the polymerization of actin filaments in contact with the plasma membrane.^[141]23ACTB is upregulated in several other cancers including sarcoma, colon adenocarcinoma, hepatoma, melanoma, gastric cancer, esophageal cancer, lung cancer, breast cancer, lymphoma, and cervical cancer. Similarly, ACTG1 is upregulated in skin cancer, hepatoma, non-clear cell renal cell carcinoma, colorectal cancer, lung cancer, cervical cancer, leukemia, neuroblastoma, breast cancer, prostate cancer, pancreatic cancer, and testicular cancer.^[142]24 In addition to the actin cytoskeleton, E47 also affected microtubule organization, protein and macromolecule trafficking, terpenoid (sterols) biosynthesis, and the cell cycle ([143]Fig. 10C and D). Our total mRNA sequencing results indicated that E28 and E47 treatment induced significant shifts in transcriptome composition, including the downregulation of factors necessary for cytoskeleton dynamics, implicating a possible mechanism for the reduced invasive and metastatic potential of melanoma cells following treatment with these compounds. Determination of differential protein expressions using proteomics We performed quantitative proteomics to identify differentially expressed proteins (DEPs) resulting from the treatment of a melanoma cell line with different thiazolo-ethisterone derivatives to better understand the effects of these small molecules on melanoma biology. Treatment of SK-MEL-25 cells with E12 at 1.5 μM concentration for 24 h resulted in the differential expression of 88 proteins, out of which 47 were downregulated and 41 were upregulated ([144]Fig. 11A). Treatment with E47 at a 2 μM concentration for 24 h resulted in the differential expression of 136 proteins, of which 28 proteins were downregulated and 108 proteins were upregulated ([145]Fig. 11B). Among the 224 DEPs resulting from the two treatments, 43 were common to both thiazolo ethisterone derivatives (E12 and E47), 15 of which were downregulated and 28 were upregulated. Among the DEPs common to both treatments, the expression of ACTB and ACTG1 was significantly downregulated with a log[2]FC value of 0.4 ([146]Table 4). Together with our mRNA sequencing results, this indicated that exposure to E12 and E47 resulted in the downregulation of ACTB and ACTG1 at both transcriptional and translational levels. Interestingly, β-parvin was significantly upregulated in the proteomics dataset ([147]Table 5), although it was not identified among the DEGs using mRNA sequencing. The integrin-linked kinase (ILK)-cysteine histidine-rich protein (PINCH)-parvin complex acts as a cell signaling pathway bridge between the cytoplasmic actin cytoskeleton and extracellular integrin, thereby communicating extracellular signals.^[148]25 Overexpression of PARVB prevents lamellipodium and ILK-α parvin complex formation which is essential for promoting cell growth, migration, and inhibiting apoptosis.^[149]24 Coupled with the downregulation of ACTB and ACTG1, the identification of PARVB upregulation by thiazolo ethisterone derivative (E47) treatment begins to define the mechanism of action of these putative drugs. According to previous reports, upregulation of PARVB is sufficient to inhibit cancer cell growth and migration in a model of upper urinary tract urothelial carcinoma (UUT-UC), and PARVB expression is downregulated in breast cancer.^[150]26,27 Fig. 11. Volcano plot of differentially expressed proteins (proteomics) by E12 (A) and E47 (B) in comparison to NC <0.1% DMSO. The absolute value of log[2] fold change (log[2]FC) was >1 with a significant p < 0.05. [151]Fig. 11 [152]Open in a new tab Table 5. β-Parvin (PARVB) upregulation by E47 (2 μM) for 24 h by qPCR. Log[2]FC p value Cell line 1.90 0.000025 LOX IMVI 2.16 0.010837 SK MEL 25 [153]Open in a new tab Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western immunoblot We measured the expression of ACTB protein in the SK-MEL-28 cell line and found that E28 and E47 downregulated this protein compared to the NC (<0.1% DMSO) ([154]Fig. 12) which supported our genomics and proteomics findings. We observed upregulation of PARVB mRNA by E47 (2 μM for 24 h) in the SK-MEL-25 cell line using qRT-PCR, which was similar to proteomics findings ([155]Table 5). Fig. 12. β-Actin (ACTB) expression in SK-MEL-28 cell line in a Western immunoblot assay in the presence of lead compounds E28 and E47. [156]Fig. 12 [157]Open in a new tab Fluorescence imaging of actin cytoskeleton In SK-MEL-28 cells, we observed actin dynamics after treatment with E28 and E47 using fluorescence microscopy. Cellular actin filaments were stained with Phalloidin (Invitrogen™ Alexa Fluor™ 488, Catalog No. A12379) after 24 h of treatment with 2 μM E28 and E47. At 2 μM, E28 ([158]Fig. 13B) and E47 ([159]Fig. 13C) inhibited the protrusions of cell membranes (lamellipodia, filopodia, and invadopodia) and cell spreading compared to NC (<0.1% DMSO) ([160]Fig. 13A) that indicated cells lack filamentous actin to retain the shape and protrude the membranes for lamellipodia, filopodia, and invadopodia formation. It is possible that E28 and E47 inhibited actin polymerization via downregulation of ACTB and ACTG1. The outcome of actin polymerization inhibition is supposed to culminate as the inhibition of cell adhesion and motility, which aligns with the findings of our in vitro cell adhesion ([161]Fig. 6) and cell migrations ([162]Fig. 5) assays. Therefore, the results of cell adhesion, cell migration, fluorescence microscopy, qRT-PCR, western immunoblotting, proteomics, and mRNA sequencing assays unanimously validated the potent antimetastatic properties of the thiazolo-ethisterone derivatives. Fig. 13. Actin filament (F actin) staining. (A)–(C) showed the F actin (stained green), cell spreading and membrane protrusions in negative control (NC), E28 (2 μM) and E47 (2 μM) treated SK-MEL-28 cells, respectively. [163]Fig. 13 [164]Open in a new tab In vivo efficacy studies After encouraging findings in human and murine cell lines, in vivo tumor growth inhibition was measured in a subcutaneous mouse melanoma model to study the efficacy of the most potent fused thiazole derivatives, E2 and E47. In C57BL/6 mice, E2 inhibited tumor growth at 40 mg kg^−1 body weight (bwt) once daily (SID) via intraperitoneal injection from day 6th to day 9th of tumor cell injection and then every alternate day till day 18th E2 (4D + Alt) ([165]Fig. 14B). Similarly, E47 inhibited tumor growth at 40 mg kg^−1 bwt SID from day 6th to day 9th of tumor cell injection and then every alternate day for up to day 18th E47 (4D + Alt) ([166]Fig. 14B). On the day 19th, the tumor weights of the E2 and E47 treated mice were remarkably lower than those of the vehicle treated mice ([167]Fig. 14C). In another treatment setup, administration of E2 at 40 mg kg^−1 bwt every alternate day from day 6th to day 18th of tumor cell injection E2 (Alt) and E47 at 40 mg kg^−1 from day 6th to day 12th followed by every alternate day up to day 18th E47 (7D + Alt) significantly inhibited mouse tumor growth compared to the vehicle ([168]Fig. 15B). However, tumor growth inhibition was more prominent with the E47 (7D + Alt) treatment, whereas the E2 (Alt) treatment was less effective ([169]Fig. 15B). In addition to tumor volume and weight, [E2 (4D + Alt)] and [E47 (4D + Alt)] treated mice had 19 days overall survival of around 80% while 19 days survival of vehicle treated mice was 0% ([170]Fig. 14A). Similarly, [E2 (Alt)] and [E47 (7D + Alt)] treated mice showed 19 days overall survival around 10% and 80%, respectively while the 19th-day overall survival chance of vehicle treated mice dropped to 0% ([171]Fig. 15A). On day 19th, the tumor weight of [E47 (7D + Alt)] treated mice was significantly lower than that of the vehicle, whereas [E2 (Alt)] did not yield a significant difference ([172]Fig. 15C). This finding indicates that diluting the treatment dose mitigated the drug efficacy while increasing the treatment dose and frequency making the treatment more effective. Fig. 14. In vivo efficacy study. (A) Kaplan–Meier survival curve presented percentage of survival in [E2 (4D + Alt)] and [E47 (4D + Alt)] treated mice (B) tumor volume of mice after treating with [E2 (4D + Alt)] and [E47 (4D + Alt)]. (C) Mean tumor weight (gm) of mice on 19th day of treatment where error bars show SD and * indicates p value <0.05. (D) Images of excised mice tumors. [173]Fig. 14 [174]Open in a new tab Fig. 15. In vivo efficacy study. (A) Kaplan–Meier curve showed the percentage of survival in [E2 (Alt)], [E47 (7D + Alt)] and vehicle treated mice. (B) Tumor volume of mice after treating with [E2 (Alt)] and [E47 (7D + Alt)]. (C) Mean tumor weight (gm) of mice at 19th day of treatment where error bars show SD and *** presents p-value <0.001. (D) Images of excised tumors. [175]Fig. 15 [176]Open in a new tab In vivo safety and toxicology studies Deleterious and life-threatening adverse effects of new antitumor agents are major challenges in anticancer drug development.^[177]28 To overcome this problem with our potent compounds, we performed comprehensive toxicology studies of treated mice by analyzing 14 key plasma markers that were indicative of organ function. E2 (4D + Alt) and E47 (4D + Alt) treatments appeared to be safe for mice and the treatments did not affect the body weight (bwt) of mice during the treatment ([178]Fig. 16). The chronic in vivo effect of 14 days of treatment with compounds (E2 and E47) was assessed using 14 key parameters of organ function as previously described by our group.^[179]16a,29 In the plasma metabolite profile, a normal concentration of albumin in the blood plasma showed that these lead compounds were unlikely to cause toxicity to the kidneys or liver ([180]Fig. 16). The unaffected kidneys of treated mice were further confirmed by the normal concentration of blood urea nitrogen (BUN), creatine, potassium, and sodium in the blood plasma. E2 treatment showed similar amylase concentrations and E47 caused insignificant elevation of this metabolite indicating normal and healthy function of the pancreas. Similar concentrations of calcium and phosphorus to the untreated control confirmed the presence of several healthy organs, such as the bones, liver, and kidneys of the treated mice. Furthermore, the unaffected total protein and globulin showed healthy kidneys as well as the liver and did not elicit an immune response or suppress the immune system in mice. Finally, normal glucose levels also confirm the proper functioning of several organs such as the kidneys, pancreas, liver, and muscles.^[181]30 The absence of a significant elevation in alkaline phosphatase or total bilirubin in E2 (4D + Alt) indicated the absence of hepatotoxicity ([182]Fig. 16). Thus, these potent compounds are safe even after multiple exposures in mice. Fig. 16. E47 (4D + Alt) and E2 (4D + Alt) plasma metabolite profiles. The individual panel of bar graphs shows plasma levels of physiologically important metabolites. Blue, red, and green bars indicate E47 (4D + Alt), E2 (4D + Alt) and vehicle treatments, respectively, and error bars represent SD. [183]Fig. 16 [184]Open in a new tab Molecular docking analysis Molecular docking studies are widely used to find ligand interactions with target proteins in modern drug discovery. In silico docking analyses of potent compounds (E2 and E47) with the target protein, the actin filament uncapping complex^[185]31 (PDB: 7CCC) were conducted using the AutoDock Vina ([186]https://vina.scripps.edu/) program. The target protein was selected based on genomics, proteomics, and other in vitro studies as discussed above. The 3D-PDB structure was downloaded from Protein Data Bank ([187]https://www.rcsb.org/) and the ligands were prepared using PyMol and Discovery Studios. All three potent compounds showed strong interaction with the protein with the binding energy (BE) values in the range of 9.1 to 9.6 kcal mol^−1 ([188]Table 6). As shown in [189]Fig. 17A, the potent compound (E2) interacted with the amino acids of the protein via different connections. Amino and hydroxy groups of E2 made hydrogen bonding with aspartic acid (ASP) residues of proteins. The phenyl substituent showed Pi-anion interaction with the glutamic acid (GLU) of the protein. In addition, several van der Waals, carbon–hydrogen, and alkyl interactions were observed with the different amino acids and the hydrophobic parts of E2. The lead compound E12 showed ([190]Fig. 17C) a similar interaction in addition to the interaction of fluorine atoms with arginine (ARG) and aspartic acid (ASP). Another lead compound (E47) showed different interactions with the target protein ([191]Fig. 17C). Hydroxy groups of catechol and propargyl alcohol made a hydrogen bonding with histidine and glutamine residues respectively. The thiazole ring interacted with the protein via π-anion and π-alkyl interactions. Furthermore, several van der Waals interactions were observed among the lipophilic residues of the molecule with different amino acids of the target proteins. Table 6. Docking studies of potent compounds (comp: E2, E12, and E47) with the target proteins: 7CCC = actin filament protein and 7PE8 = human mTOR complex 2. Comp Protein (PDB) BE (kcal mol^−1) E2 7CCC −9.6 E12 7CCC −9.1 E47 7CCC −9.4 E2 7PE8 −10 E12 7PE8 −9.8 E47 7PE8 −10 [192]Open in a new tab Fig. 17. 2D Representation of the interaction of compounds (E2, E12, and E47) with the actin filament protein (PDB: 7CCC) and human mTOR complex 2 (7PE8). [193]Fig. 17 [194]Open in a new tab The mammalian target of rapamycin (mTOR) is a key regulator of tumor cell growth, proliferation, migration, and invasion.^[195]32 Previous studies reported that inhibitors of the mTOR signaling pathway inhibit cell migration and cell invasion in multiple malignancies by affecting actin cytoskeleton organization.^[196]33 We found our lead compounds interacted with mTOR and actin with high binding energy, which suggests that our lead compounds inhibited cell migration, cell adhesion, and tumor growth through interfering mTOR signaling. Therefore, we studied the docking of human mTOR complex 2 (7PE8) with compounds ([197]Fig. 17D–F). These potent compounds showed slightly more binding energy than the actin filament protein ([198]Table 6). The docking of compounds with the proteins is shown in 3D representations in [199]Fig. 18. Thus, genomics, proteomics, spectroscopic, Western blot, and docking studies helped to identify the possible protein target of our potent compounds. Nevertheless, further validation studies will be required to confirm the targets. Fig. 18. 3D Representation of docking of compounds (E2, E12, and E47) with the actin filament protein (PDB: 7CCC) and human mTOR complex 2 (7PE8). [200]Fig. 18 [201]Open in a new tab Calculated physicochemical properties We calculated different physicochemical properties of the lead compounds (E2, E12, E28, and E47) using [202]http://www.swissadme.ch/index.php ([203]Table 7). Topological total surface area (TPSA) is an important parameter that indicates the possibility of drug molecules for passive transport through the cell membrane.^[204]34 The TPSA values of these compounds are within the range of 60 to 90 Å indicating the possible complete absorption by passive transport through the cell membrane. The n-octanol/water partition coefficient (ilog P) is one of the key parameters in drug development, and it indicates the possibility of aqueous solubility of drug candidates.^[205]35 Our lead compounds showed ilog P values of 3.66 to 4.63 which is within the range of most approved drugs. Based on the physicochemical calculations, our lead compounds will not interact with P-glycoproteins and did not show any PAINS alerts except for E47 due to its catechol moiety. Catechol moiety is ascribed as a PAINS. Nevertheless, catechol-containing compounds such as l-dopa and cefiderocol are widely used drugs, and several compounds are in different stages of drug development.^[206]36 Based on these facts, we studied the catechol-derived compound (E47) after finding its potent activity. These results are promising to further develop these lead compounds as anticancer agents. To study the selectivity of our compounds, we investigated the possible inhibition of five isoforms of cytochrome P450 (CYP) enzymes. Our lead compounds did not inhibit these enzymes except CYP2C9. These results are very significant as CYP inhibition can lead to drug accumulation leading to drug–drug interaction and possible clinical toxicity.^[207]37 Table 7. Calculated physicochemical properties to determine important descriptors for the lead compounds (E2, E12, E28, and E47). # and parameters E2 E12 E28 E47 TPSA (Å) 73.39 73.39 67.84 101.82 iLog P 4.03 4.41 4.63 3.66 P-gp substrate No No No No PAINS (alert) 0 0 0 1 CYP1A2 inhibitor Yes No No No CYP2C19 inhibitor No No No No CYP2C9 inhibitor Yes Yes Yes Yes CYP2D6 inhibitor No No No No CYP3A4 inhibitor No No No No [208]Open in a new tab Materials and methods Chemistry All the reactions were carried out in a normal air atmosphere in round-bottom flasks. Reaction materials, and solvents were purchased from Fischer Scientific (Hanover Park, IL, USA) and Oakwood Chemical (Estill, SC, USA). No chemicals were further purified. Proton (^1H) and carbon (^13C) spectra were recorded on a Variance NMR Spectrometer at 300 MHz for ^1H and 75 MHz for ^13C in CDCl[3] or DMSO-d[6] solvents. The spectra of some compounds were recorded on a JEOL spectrometer at 400 MHz for ^1H, 101 MHz for ^13C in CDCl[3] or DMSO-d[6] solvents. ^1H NMR spectra were described as chemical shifts (δ, ppm), and multiplicities were designated as follows: s = singlet, d = doublet, dd = doublet of doublets, ddd = doublet of doublets of doublets, t = triplet, and m = multiplet. Bruker Apex II-FTMS was used to obtain high-resolution mass spectrometry (HRMS) data. The following novel compounds were synthesized using our previously reported procedures.^[209]14a,c,d Synthesis of aminothiazole-derived androstenones, A1–A27 (Scheme S1[210]†) Androstenedione (0.5 mmol), thiourea derivatives (0.55 mmol), sodium acetate (0.55 mmol), and 15 mL anhydrous ethanol were taken in a 50 mL round bottom flask. The reaction mixture was refluxed for 8 h. After completion of the reaction, distilled water was added to the reaction mixture to precipitate the formed. Solid precipitates were thoroughly washed with distilled water, filtered, and vacuum drying provided pure fused-thiazolo androstenedione product. Synthesis of aminothiazole-derived androstenones, A28–A53 (Scheme S2[211]†) Androstenedione (0.5 mmol), thiobenzamide derivatives (0.55 mmol), sodium acetate (0.50 mmol), and 10 mL hexafluoroisopropanol (HFIP) were taken in a 50 mL round bottom flask. The reaction mixture was refluxed for 12 h. After completion of the reaction, HFIP was distilled out and methanol (10 mL) was added. The solid precipitate was filtered and washed with ∼10 mL methanol and ∼20 mL water under vacuum to afford the pure products. Synthesis of thiazole-fused ethisterone (E1–E50) Synthesis of epoxy ethisterone (Scheme S3[212]†) Ethisterone (1 mmol), dichloromethane (3 mL), methanol (4 mL), 10% NaOH (0.28 mL), 30% H[2]O[2] (0.56 mL) were taken in a 50 mL round bottom flask. The reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, liquid–liquid extraction of the reaction mixture was conducted with dichloromethane and water. The dichloromethane portion was collected, washed with brine, and dried by passing through anhydrous sodium sulfate. Evaporation of dichloromethane by rotatory evaporator provided a solid product. The solid precipitates were solubilized in ethyl acetate and insoluble impurities were filtered out. Drying ethyl acetate provided pure white solid ethisterone-epoxyketone. Synthesis of thiazolo-ethisterone (Scheme S4[213]†) Ethisterone-epoxyketone (0.5 mmol), thiourea/thioamide derivatives (0.55 mmol), and 10 mL acetic acid were taken in a 25 mL round bottom flask. The reaction was conducted at 100 °C for 8 h. After completion of the reaction, sodium bicarbonate was added slowly to neutralize some of the acetic acid. Distilled water was added to form precipitates. The precipitates were washed thoroughly with distilled water, filtered, and vacuum drying provided pure fused thiazolo-ethisterone products. In case of impurities, compounds were recrystallized from methanol to get the pure products. Experimental data of new compounds (1S,2R,13R,14S,18S)-7-(4-Fluoroanilino)-2,18-dimethyl-8-thia-6-azapentacyclo[ 11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-one (A15) [214]graphic file with name d4md00719k-u1.jpg Light yellowish powder (361 mg, 83%); ^1H-NMR (400 MHz, CDCl[3]) δ 7.88–7.84 (m, 2H), 7.10–7.05 (m, 2H), 5.80 (s, 1H), 3.00–2.92 (m, 1H), 2.87–2.79 (m, 1H), 2.51–2.33 (m, 2H), 2.14–1.77 (m, 7H), 1.60–1.29 (m, 5H), 1.21–1.16 (m, 1H), 1.04 (s, 3H), 0.91 (s, 3H); ^13C-NMR (101 MHz, CDCl[3]) δ 220.9, 163.7 (^1J[C–F] = 250.5 Hz), 163.1, 150.6, 136.6, 131.8, 130.3 (^4J[C–F]= 3.4 Hz), 128.3 (^3J[C–F] = 8.2 Hz), 121.7, 116.0 (^2J[C–F] = 22.2 Hz), 51.8, 48.2, 47.7, 36.8, 35.9, 34.4, 31.5, 31.2, 30.9, 24.2, 21.9, 20.8, 18.8, 13.7; HRMS (ESI-FTMS) mass (m/z): calcd for C[26]H[29]FN[2]OS [M + H]^+ = 437.2057, found 437.2054. (1S,2R,13R,14S,18S)-7-(3,5-Dimethylanilino)-2,18-dimethyl-8-thia-6-azapentacy clo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-one (A21) [215]graphic file with name d4md00719k-u2.jpg Yellowish solid (347 mg, 78%); ^1H-NMR (400 MHz, CDCl[3]) δ 6.91 (s, 2H), 6.70 (s, 1H), 5.47–5.46 (m, 1H), 2.69–2.61 (m, 2H), 2.50–2.43 (m, 1H), 2.33–2.29 (m, 7H), 2.14–2.05 (m, 1H), 1.99–1.75 (m, 6H), 1.60–1.28 (m, 5H), 1.20–1.14 (m, 1H), 1.04 (s, 3H), 0.91 (s, 3H); ^13C-NMR (101 MHz, CDCl[3]) δ 221.2, 162.8, 145.2, 140.2, 139.3, 136.8, 125.2, 120.1, 117.4, 116.6, 51.9, 48.2, 47.7, 36.8, 35.9, 34.4, 31.5, 31.3, 30.7, 24.1, 21.9, 21.5, 20.8, 18.8, 13.8; HRMS (ESI-FTMS) mass (m/z): calcd for C[28]H[34]N[2]OS [M + H]^+ = 447.2465, found 447.2472. 3-[[(1S,2R,13R,14S,17R,18S)-17-Ethynyl-17-hydroxy-2,18-dimethyl-8-thia-6-azap entacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-7-yl]amino]benzoi c acid (E4) [216]graphic file with name d4md00719k-u3.jpg Grey solid (390 mg, 80%); ^1H-NMR (300 MHz, DMSO-d[6]) δ 10.40 (s, 1H), 8.15 (s, 1H), 7.93 (d, J = 8.0 Hz, 1H), 7.51–7.38 (m, 2H), 5.38–5.33 (m, 2H), 2.66 (s, 2H), 2.21–2.03 (m, 4H), 1.99–1.81 (m, 2H), 1.75–1.62 (m, 6H), 1.50–1.25 (m, 5H), 1.13–1.07-0.98 (m, 4H), 0.77 (s, 3H); ^13C-NMR (75 MHz, DMSO-d[6]) δ 167.9, 160.1, 146.1, 141.6, 136.6, 132.1, 129.6, 122.3, 121.3, 120.4, 118.6, 118.0, 89.3, 78.5, 75.6, 50.9, 48.0, 46.7, 36.5, 34.3, 32.8, 32.2, 31.4, 24.4, 23.3, 21.6, 21.1, 19.0, 13.2; HRMS (ESI-FTMS) mass (m/z): calcd for C[29]H[32]N[2]O[3]S [M + H]^+ = 489.2206, found 489.2204. 4-[[(1S,2R,13R,14S,17R,18S)-17-Ethynyl-17-hydroxy-2,18-dimethyl-8-thia-6-azap entacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-7-yl]amino]benzoi c acid (E5) [217]graphic file with name d4md00719k-u4.jpg Grey solid (376 mg, 77%); ^1H NMR (300 MHz, DMSO-d[6]) δ 7.87–7.85 (m, 2H), 7.69–7.67 (m, 2H), 5.41 (s, 1H), 3.32 (s, 2H), 2.67–2.65 (m, 2H), 2.20–1.95 (m, 3H), 1.89–1.81 (m, 4H), 1.74–1.61 (m, 5H), 1.49–1.19 (m, 4H), 0.97 (s, 3H), 0.76 (s, 3H); ^13C-NMR (75 MHz, DMSO-d[6]) δ 172.7, 167.7, 159.6, 146.1, 144.9, 136.5, 131.1, 124.1, 121.1, 119.0, 116.4, 89.3, 78.5, 75.5, 50.8, 48.0, 46.7, 36.5, 34.3, 32.8, 32.2, 31.4, 24.4, 23.2, 21.7, 19.0, 13.2; HRMS (ESI-FTMS) mass (m/z): calcd for C[29]H[32]N[2]O[3]S [M + H]^+ = 489.2206, found 489.2202. (1S,2R,13R,14S,17R,18S)-7-(3,5-Dimethylanilino)-17-ethynyl-2,18-dimethyl-8-th ia-6-azapentacyclo[11.7.0.02,10.05,9.014,18]icosa-5(9),6,10-trien-17-ol (E7) [218]graphic file with name d4md00719k-u5.jpg Brick red solid (401 mg, 85%); ^1H-NMR (300 MHz, CDCl[3]) δ 6.92 (s, 2H), 6.73 (s, 1H), 5.47 (s, 1H), 2.69–2.60 (m, 3H), 2.32–1.91 (m, 13H), 1.74–1.19 (m, 10H), 1.05 (s, 3H), 0.91 (s, 3H); ^13C-NMR (75 MHz, CDCl[3]) δ 163.1, 144.0, 140.0, 139.2, 136.4, 125.2, 119.7, 118.0, 116.5, 87.6, 29.8, 74.0, 50.8, 47.7, 46.7, 38.9, 36.6, 34.2, 32.5, 32.2, 31.2, 23.6, 23.2, 21.4, 21.1, 18.7, 12.8; HRMS (ESI-FTMS) mass (m/z): calcd for C[30]H[36]N[2]OS [M + H]^+ = 473.2621, found 473.2629. (1S,2R,13R,14S,17R,18S)-7-(3-Chloro-4-methyl-anilino)-17-ethynyl-2,18-dimethy l-8-thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17 -ol (E11) [219]graphic file with name d4md00719k-u6.jpg Off white solid (409 mg, 83%); ^1H-NMR (400 MHz, DMSO-d[6]) δ 7.80 (d, J = 3.7 Hz, 1H), 7.34–7.31 (m, 1H), 7.21–7.18 (m, 1H), 5.33–5.28 (m, 2H), 3.30 (s, 1H), 2.60 (s, 2H), 2.21–2.01 (m, 4H), 1.94–1.78 (m, 2H), 1.70–1.15 (m, 11H), 1.03–1.00 (m, 1H), 0.94 (s, 3H), 0.73 (s, 3H); ^13C-NMR (101 MHz, DMSO-d[6]) δ 160.1, 146.1, 140.7, 136.6, 133.7, 131.8, 127.9, 120.4, 118.7, 117.2, 116.2, 89.4, 78.6, 75.6, 50.9, 48.0, 46.8, 39.3, 36.6, 34.4, 32.8, 32.3, 31.4, 24.5, 23.3, 21.6, 19.4, 19.1, 13.3; HRMS (ESI-FTMS) mass (m/z): calcd for C[29]H[33]ClN[2]OS [M + H]^+ = 493.2075, found 493.2070. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-2,18-dimethyl-7-(N-methylanilino)-8-thia-6 -azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E13) [220]graphic file with name d4md00719k-u7.jpg Grey solid (403 mg, 88%); ^1H-NMR (400 MHz, DMSO-d[6]) δ 7.41–7.39 (m, 4H), 7.25–7.21 (m, 1H), 5.28 (d, J = 3.7 Hz, 1H), 5.17 (s, 1H), 3.38 (s, 3H), 3.28 (s, 1H), 2.54–2.46 (m, 3H), 2.08–1.77 (m, 4H), 1.61–1.20 (m, 10H), 0.99–0.91 (m, 4H), 0.72 (s, 3H); ^13C-NMR (101 MHz, DMSO-d[6]) δ 166.2, 146.5, 146.3, 136.8, 130.2, 126.8, 125.4, 120.1, 117.5, 89.4, 78.6, 75.6, 50.9, 48.0, 46.7, 40.3, 39.3, 36.5, 34.4, 32.8, 32.2, 31.4, 24.4, 23.3, 21.1, 19.1, 13.3; HRMS (ESI-FTMS) mass (m/z): calcd for C[29]H[34]N[2]OS [M + H]^+ = 459.2464, found 459.2462. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-2,18-dimethyl-7-(3-pyridylamino)-8-thia-6- azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E15) [221]graphic file with name d4md00719k-u8.jpg Light red solid (356 mg, 80%); ^1H-NMR (400 MHz, DMSO-d[6]) δ 10.33 (s, 1H), 8.69 (d, J = 2.5 Hz, 1H), 8.14–8.09 (m, 2H), 7.28 (dd, J = 8.4, 4.7 Hz, 1H), 5.36–5.35 (m, 1H), 5.29 (s, 1H), 3.31 (s, 1H), 2.66–2.55 (m, 2H), 2.18–1.78 (m, 5H), 1.71–1.19 (m, 9H), 1.08–0.98 (m, 1H), 0.94 (s, 3H), 0.73 (s, 3H); ^13C-NMR (101 MHz, DMSO-d[6]) δ 160.0, 146.1, 142.5, 139.4, 138.2, 136.6, 124.2, 123.8, 120.9, 118.9, 89.4, 78.6, 75.7, 50.9, 48.0, 46.8, 39.3, 36.6, 34.3, 32.8, 32.3, 31.4, 24.4, 23.3, 21.1, 19.1, 13.3; HRMS (ESI-FTMS) mass (m/z): calcd for C[27]H[31]N[3]OS [M + H]^+ = 446.2261, found 446.2258. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-2,18-dimethyl-7-(4-pyridylamino)-8-thia-6- azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E16) [222]graphic file with name d4md00719k-u9.jpg Light yellowish solid (373 mg, 84%); ^1H NMR (300 MHz, DMSO-d[6], CDCl[3]) δ 8.22–8.09 (m, 4H), 5.5 (s, 1H), 2.81–2.50 (m, 4H), 2.27–2.12 (m, 2H), 2.00–1.84 (m, 3H), 1.74–1.22 (m, 9H), 1.18–1.04 (m, 2H), 0.95 (s, 3H), 0.77 (s, 2H); ^13C NMR (75 MHz, DMSO-d[6], CDCl[3]) δ 156.5, 153.5, 146.1, 140.0, 135.9, 125.8, 121.0, 112.3, 88.7, 78.7, 73.4, 50.6, 47.6, 46.6, 39.0, 36.5, 34.1, 32.5, 32.1, 31.4, 24.2, 23.2, 21.0, 18.9, 12.9; HRMS (ESI-FTMS) mass (m/z): calcd for C[27]H[31]N[3]OS [M + H]^+ = 446.2261, found 446.2268. (1S,2R,13R,14S,17R,18E17S)-17-Ethynyl-2,18-dimethyl-7-[(3-methyl-2-pyridyl)am ino]-8-thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien -17-ol (E17) [223]graphic file with name d4md00719k-u10.jpg Light yellowish solid (394 mg, 86%); ^1H NMR (300 MHz, CDCl[3]) δ 7.52 (t, J = 7.8 Hz, 1H), 6.84–6.76 (m, 2H), 5.73 (d, J = 2.0 Hz, 1H), 2.72–2.60 (m, 3H), 2.54 (s, 3H), 2.38–2.20 (m, 2H), 2.13 (s, 3H), 2.07–1.99 (m, 2H), 1.89–1.66 (m, 4H), 1.62–1.36 (m, 4H), 1.24–1.16 (m, 1H), 1.03 (s, 3H), 0.92 (s, 3H); ^13C NMR (75 MHz, CDCl[3]) δ 177.6, 159.6, 156.1, 149.7, 138.5, 138.2, 135.9, 121.8, 118.9, 116.4, 108.4, 87.4, 79.8, 74.1, 50.7, 47.6, 46.7, 38.9, 36.5, 33.9, 32.5, 32.2, 31.2, 23.7, 23.2, 21.9, 18.6, 12.7; HRMS (ESI-FTMS) mass (m/z): calcd for C[28]H[33]N[3]OS [M + H]^+ = 460.2417, found 460.2412. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-2,18-dimethyl-7-[(5-methyl-2-pyridyl)amino ]-8-thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17 -ol (E18) [224]graphic file with name d4md00719k-u11.jpg Light red solid (390 mg, 85%); ^1H NMR (300 MHz, CDCl[3]) δ 8.20 (d, J = 5.1 Hz, 1H), 6.71 (m, 2H), 5.69 (s, 1H), 2.74–2.72 (m, 2H), 2.60 (s, 1H), 2.39–2.01 (m, 10H), 1.87–1.17 (m, 10H), 1.05 (s, 3H), 0.92 (s, 3H); ^13C NMR (75 MHz, CDCl[3]) δ 178.4, 158.7, 151.4, 149.0, 146.5, 142.1, 136.5, 122.6, 118.1, 111.1, 87.6, 79.8, 74.0, 50.8, 47.7, 46.7, 39.0, 36.4, 34.3, 32.6, 32.2, 31.3, 23.4, 23.2, 22.3, 21.1, 18.7, 12.8; HRMS (ESI-FTMS) mass (m/z): calcd for C[28]H[33]N[3]OS [M + H]^+ = 460.2417, found 460.2423. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-2,18-dimethyl-7-[(6-methyl-2-pyridyl)amino ]-8-thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17 -ol (E19) [225]graphic file with name d4md00719k-u12.jpg Light yellowish solid (403 mg, 88%); ^1H-NMR (400 MHz, DMSO-d[6]) δ 7.53–7.49 (m, 1H), 6.78 (d, J = 8.1 Hz, 1H), 6.71 (d, J = 7.2 Hz, 1H), 5.46 (s, 1H), 3.42–3.37 (m, 2H), 3.29 (s, 1H), 2.58–2.57 (m, 2H), 2.39 (s, 3H), 2.18–1.81 (m, 4H), 1.72–1.23 (m, 10H), 1.04–0.99 (m, 1H), 0.93 (s, 3H), 0.74 (s, 3H); ^13C-NMR (101 MHz, DMSO-d[6]) δ 157.1, 155.8, 151.4, 144.2, 138.6, 137.3, 121.9, 117.3, 115.4, 108.0, 89.4, 78.6, 75.6, 51.0, 48.2, 46.8, 39.2, 36.5, 34.5, 32.9, 32.3, 31.4, 24.1, 24.0, 23.4, 22.3, 19.1, 13.3; HRMS (ESI-FTMS) mass (m/z): calcd for C[28]H[33]N[3]OS [M + H]^+ = 460.2417, found 460.2417. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-7-[(5-fluoro-2-pyridyl)amino]-2,18-dimethy l-8-thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17 -ol (E20) [226]graphic file with name d4md00719k-u13.jpg Off white solid (407 mg, 88%); ^1H NMR (300 MHz, CDCl[3]) δ 8.19 (s, 1H), 7.38 (s, 1H), 6.95–6.93 (m, 1H), 5.67 (s, 1H), 2.69–2.60 (m, 3H), 2.29–2.04 (m, 7H), 1.83–1.19 (m, 10H), 1.04 (s, 3H), 0.92 (s, 3H); ^13C NMR (75 MHz, CDCl[3]) δ 177.9, 155.1 (^1J[C–F] = 246.2 Hz), 147.5, 140.2, 136.1, 133.6 (^2J[C–F] = 25.4 Hz), 125.8 (^2J[C–F] = 20.8 Hz), 118.6, 112.1 (^3J[C–F] = 4.1 Hz), 87.5, 79.8, 74.1, 50.8, 47.6, 46.7, 38.9, 36.4, 34.1, 32.5, 32.2, 31.2, 29.7, 23.1, 22.4, 21.9, 18.6, 12.7; HRMS (ESI-FTMS) mass (m/z): calcd for C[28]H[33]N[3]OS [M + H]^+ = 464.2166, found 464.2159. (1S,2R,13R,14S,17R,18S)-7-[(4-Chloro-2-pyridyl)amino]-17-ethynyl-2,18-dimethy l-8-thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17 -ol (E21) [227]graphic file with name d4md00719k-u14.jpg Off white powder (408 mg, 85%); ^1H-NMR (300 MHz, CDCl[3]) δ 8.28 (d, J = 5.5 Hz, 1H), 7.26 (s, 1H), 7.05 (dd, J = 1.5, 1.6 Hz, 1H), 5.80 (s, 1H), 2.84–2.72 (m, 2H), 2.60 (s, 1H), 2.39–2.24 (m, 2H), 2.13–2.00 (m, 5H), 1.90–1.17 (m, 10H), 1.04 (s, 3H), 0.93 (s, 3H); ^13C NMR (75 MHz, CDCl[3]) δ 176.6, 159.8, 150.1, 147.4, 146.0, 134.6, 122.2, 121.4, 119.2, 112.6, 87.3, 79.8, 74.2, 50.6, 47.4, 46.7, 38.9, 36.5, 33.4, 32.4, 32.1, 31.2, 23.1, 21.1, 20.9, 18.5, 12.7; HRMS (ESI-FTMS) mass (m/z): calcd for C[27]H[30]ClN[3]OS [M + H]^+ = 480.1871, found 480.1869. (1S,2R,13R,14S,17R,18S)-7-[(5-Chloro-2-pyridyl)amino]-17-ethynyl-2,18-dimethy l-8-thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17 -ol (E22) [228]graphic file with name d4md00719k-u15.jpg White powder (398 mg, 83%); ^1H-NMR (400 MHz, DMSO-d[6]) δ 8.27–8.25 (m, 1H), 7.75–7.71 (m, 1H), 7.05–7.01 (m, 1H), 5.47 (s, 1H), 5.29 (s, 1H), 3.30–3.28 (m, 2H), 2.58 (s, 2H), 2.16–2.03 (m, 2H), 1.95–1.82 (m, 4H), 1.67–1.23 (m, 8H), 1.01 (s, 1H), 0.92 (s, 3H), 0.73 (s, 3H); ^13C-NMR (101 MHz, DMSO-d[6]) δ 156.7, 150.9, 145.3, 144.4, 138.3, 137.0, 122.5, 122.3, 117.9, 112.8, 89.4, 78.6, 75.6, 50.9, 48.1, 46.8, 39.3, 36.5, 34.4, 32.9, 32.2, 31.5, 24.0, 23.3, 21.6, 19.1, 13.3; HRMS (ESI-FTMS) mass (m/z): calcd for C[27]H[30]ClN[3]OS [M + H]^+ = 480.1871, found 480.1874. (1S,2R,13R,14S,17R,18S)-7-[(6-Chloro-2-pyridyl)amino]-17-ethynyl-2,18-dimethy l-8-thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17 -ol (E23) [229]graphic file with name d4md00719k-u16.jpg Off white solid (384 mg, 80%); ^1H-NMR (300 MHz, CDCl[3]) δ 4.48 (t, J = 8.2 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H), 6.79 (d, J = 7.6 Hz, 1H), 5.58 (s, 1H), 2.70–2.50 (m, 3H), 2.20–2.11 (m, 2H), 1.97–1.84 (m, 3H), 1.76–1.64 (m, 6H), 1.53–1.01 (m, 5H), 0.94 (s, 3H), 0.72 (s, 3H); ^13C-NMR (75 MHz, DMSO-d[6], CDCl[3]) δ 157.5, 151.5, 148.1, 141.5, 140.0, 136.2, 122.7, 118.8, 115.8, 109.7, 88.7, 78.8, 78.2, 73.4, 50.6, 47.7, 46.6, 39.0, 36.4, 34.1, 32.6, 32.1, 31.3, 23.2, 21.2, 18.6, 12.9; HRMS (ESI-FTMS) mass (m/z): calcd for C[27]H[30]ClN[3]OS [M + H]^+ = 480.1871, found 480.1868. (1S,2R,13R,14S,17R,18S)-7-[(4,6-Dimethyl-2-pyridyl)amino]-17-ethynyl-2,18-dim ethyl-8-thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trie n-17-ol (E24) [230]graphic file with name d4md00719k-u17.jpg Light yellowish solid (416 mg, 88%); ^1H-NMR (300 MHz, CDCl[3]) δ 6.77 (s, 1H), 6.65 (s, 1H), 5.72 (s, 1H), 2.74–2.59 (m, 4H), 2.51 (s, 1H), 2.44–2.24 (m, 3H), 2.13–2.99 (m, 6H), 1.91–1.32 (m, 10H), 1.25–1.04 (m, 5H), 0.92 (s, 3H); ^13C-NMR (75 MHz, CDCl[3]) δ 177.1, 161.9, 159.9, 149.4, 137.0, 135.6, 121.7, 119.6, 118.4, 109.2, 87.4, 79.8, 77.2, 74.1, 50.7, 47.6, 46.7, 38.9, 36.5, 33.8, 32.5, 32.2, 31.2, 23.1, 21.8, 21.5, 21.0, 18.6, 12.7; HRMS (ESI-FTMS) mass (m/z): calcd for C[29]H[35]N[3]OS [M + H]^+ = 474.2574, found 474.2572. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-7-[(4-methoxy-6-methyl-pyrimidin-2-yl)amin o]-2,18-dimethyl-8-thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9 ),6,10-trien-17-ol (E26) [231]graphic file with name d4md00719k-u18.jpg Light red solid (391 mg, 80%); ^1H-NMR (300 MHz, CDCl[3]) δ 6.21 (s, 1H), 5.69 (s, 1H), 4.08 (s, 1H), 2.84–2.71 (m, 2H), 2.60 (s, 1H), 2.53–2.20 (m, 6H), 2.13–2.01 (m, 6H), 1.88–1.17 (m, 10H), 1.04 (s, 3H), 0.92 (s, 3H); ^13C-NMR (75 MHz, CDCl[3]) δ 176.5, 171.1, 166.9, 159.8, 155.5, 135.7, 123.1, 119.8, 110.2, 87.4, 79.8, 74.1, 55.0, 50.7, 47.6, 46.7, 38.9, 36.5, 33.8, 32.5, 32.2, 31.2, 23.1, 22.9, 22.0, 21.0, 18.6, 12.7; HRMS (ESI-FTMS) mass (m/z): calcd for C[28]H[34]N[4]O[2]S [M + H]^+ = 491.2475, found 491.2474. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-2,18-dimethyl-7-(1-piperidyl)-8-thia-6-aza pentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E27) [232]graphic file with name d4md00719k-u19.jpg Grey solid (327 mg, 75%); ^1H-NMR (400 MHz, DMSO-d[6]) δ 5.22 (s, 1H), 3.37–3.28 (m, 3H), 2.15–2.00 (m, 2H), 1.92–1.78 (m, 2H), 1.71–1.52 (m, 18H), 1.44–1.11 (m, 3H), 1.03–0.91 (m, 4H), 0.72 (s, 3H); ^13C-NMR (101 MHz, DMSO-d[6]) δ 167.8, 146.9, 137.0, 119.3, 116.7, 89.4, 78.6, 75.6, 50.9, 49.4, 48.1, 46.7, 39.2, 36.5, 34.4, 32.8, 32.3, 31.4, 25.2, 25.0, 24.5, 24.1, 23.3, 19.1, 13.3; HRMS (ESI-FTMS) mass (m/z): calcd for C[27]H[36]N[2]OS [M + H]^+ = 437.2621, found 437.2630. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-2,18-dimethyl-7-(4-methylpiperazin-1-yl)-8 -thia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E28) [233]graphic file with name d4md00719k-u20.jpg Off white solid (333 mg, 74%); ^1H-NMR (400 MHz, DMSO-d[6]) δ 5.29 (s, 1H), 3.99–3.93 (m, 2H), 3.60 (s, 2H), 3.40–3.37 (m, 2H), 3.11 (s, 2H), 2.76 (s, 3H), 2.64 (s, 1H), 2.50 (s, 2H), 2.13–1.79 (m, 4H), 1.68–0.98 (m, 12H), 0.90 (s, 3H), 0.72 (s, 3H); ^13C-NMR (101 MHz, DMSO-d[6]) δ 166.8, 146.7, 136.8, 121.4, 117.9, 89.4, 78.6, 75.7, 51.8, 50.9, 48.0, 46.7, 45.5, 42.7, 39.2, 36.6, 34.3, 32.8, 32.2, 31.4, 24.4, 23.3, 21.1, 19.1, 13.3; HRMS (ESI-FTMS) mass (m/z): calcd for C[27]H[37]N[3]OS [M + H]^+ = 452.273, found 452.2728. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-2,7,18-trimethyl-8-thia-6-azapentacyclo[11 .7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E29) graphic file with name d4md00719k-u21.jpg White powder (256 mg, 70%); ^1H-NMR (300 MHz, CDCl[3]) δ 5.68 (s, 1H), 2.94–2.72 (m, 2H), 2.67–2.58 (4H), 2.45–1.89 (m, 6H), 1.85–1.10 (m, 10H), 1.03 (s, 3H), 0.92 (s, 3H); ^13C-NMR (75 MHz, CDCl[3]) δ 162.8, 148.3, 136.3, 131.2, 121.3, 87.4, 79.8, 74.1, 50.7, 47.7, 46.7, 38.9, 36.7, 34.4, 32.5, 32.1, 31.3, 23.9, 23.1, 21.1, 19.3, 18.6, 12.7; HRMS (ESI-FTMS) mass (m/z): calcd for C[23]H[29]NOS [M + H]^+ = 368.2043, found 368.2042. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-2,18-dimethyl-7-(m-tolyl)-8-thia-6-azapent acyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E34) [234]graphic file with name d4md00719k-u22.jpg Light yellowish powder (341 mg, 77%); ^1H-NMR (300 MHz, CDCl[3]) δ 7.77–7.67 (m, 2H), 7.34–7.20 (m, 2H), 5.83–5.81 (m, 1H), 3.06–2.83 (m, 2H), 2.61 (s, 1H), 2.41–1.99 (s, 9H), 1.84–1.13 (m, 10H), 1.07 (s, 3H), 0.92 (s, 3H); ^13C-NMR (75 MHz, CDCl[3]) δ 164.5, 150.0, 138.7, 136.4, 133.5, 131.7, 130.7, 128.8, 126.9, 123.7, 122.1, 87.4, 79.8, 74.1, 51.0, 47.7, 46.7, 38.9, 36.6, 34.4, 32.5, 32.1, 31.5, 24.1, 23.2, 21.4, 21.1, 18.7, 12.8; HRMS (ESI-FTMS) mass (m/z): calcd for C[29]H[33]NOS [M + H]^+ = 444.2356, found 444.2360. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-7-(3-methoxyphenyl)-2,18-dimethyl-8-thia-6 -azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E36) [235]graphic file with name d4md00719k-u23.jpg Grey solid (330 mg, 72%); ^1H-NMR (400 MHz, DMSO-d[6]) δ 7.38–7.35 (m, 3H), 6.99 (d, J = 6.3 Hz, 1H), 5.76 (s, 1H), 3.78 (s, 3H), 2.87–2.74 (m, 2H), 2.22–2.03 (m, 3H), 1.85–1.24 (m, 11H), 1.06–1.00 (m, 2H), 0.95 (s, 3H), 0.74 (s, 3H); ^13C-NMR (101 MHz, DMSO-d[6]) δ 163.1, 160.2, 150.8, 136.4, 135.0, 131.9, 130.9, 123.0, 119.1, 116.6, 110.9, 89.4, 78.6, 75.7, 55.8, 50.9, 48.0, 46.8, 36.7, 34.3, 32.8, 32.2, 31.7, 24.3, 23.3, 21.2, 19.1, 19.0, 13.3; HRMS (ESI-FTMS) mass (m/z): calcd for C[29]H[33]NO[2]S [M + H]^+ = 460.2305, found 460.2310. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-7-(4-fluorophenyl)-2,18-dimethyl-8-thia-6- azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E41) [236]graphic file with name d4md00719k-u24.jpg White powder (335 mg, 75%); ^1H-NMR (300 MHz, CDCl[3]) δ 7.96–7.92 (m, 2H), 7.16–7.10 (m, 2H), 5.84 (s, 1H), 3.02–2.89 (m, 2H), 2.61 (s, 1H), 2.34–2.00 (m, 6H), 1.90–1.23 (m, 10H), 1.08 (s, 3H), 0.93 (s, 3H); ^13C-NMR (75 MHz, CDCl[3]) δ 163.7 (^1J[C–F] = 248.5 Hz), 163.0, 150.0, 136.3, 132.0, 129.9, 128.3 (^3J[C–F] = 7.9 Hz), 122.3, 116.0 (^2J[C–F] = 21.5 Hz), 87.5, 79.7, 74.0, 50.7, 47.7, 46.7, 38.9, 36.7, 34.3, 32.5, 32.1, 31.5, 24.0, 23.1, 21.1, 18.7, 12.7; HRMS (ESI-FTMS) mass (m/z): calcd for C[28]H[30]FNOS [M + H]^+ = 448.2105, found 448.2104. (1S,2R,13R,14S,17R,18S)-7-(3-Chlorophenyl)-17-ethynyl-2,18-dimethyl-8-thia-6- azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E43) [237]graphic file with name d4md00719k-u25.jpg Off white powder (370 mg, 73%); ^1H-NMR (400 MHz, CDCl[3]) δ 7.91–7.89 (m, 1H), 7.74 (s, 1H), 7.33–7.32 (m, 2H), 5.82 (s, 1H), 2.99–2.81 (m, 2H), 2.59–2.57 (m, 1H), 2.34–2.23 (m, 2H), 2.10–1.32 (m, 13H), 1.20–1.15 (m, 1H), 1.03 (s, 3H), 0.89 (s, 3H); ^13C-NMR (101 MHz, CDCl[3]) δ 162.3, 150.7, 136.4, 135.6, 135.0, 132.7, 130.2, 129.6, 126.3, 124.5, 122.6, 87.5, 79.9, 74.2, 50.8, 47.8, 46.8, 39.0, 36.7, 34.4, 32.6, 32.2, 31.6, 24.3, 23.2, 21.2, 18.8, 12.8. (1S,2R,13R,14S,17R,18S)-7-(4-Bromophenyl)-17-ethynyl-2,18-dimethyl-8-thia-6-a zapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E44) [238]graphic file with name d4md00719k-u26.jpg White powder (381 mg, 75%); ^1H-NMR, (300 MHz, CDCl[3]) δ 7.79–7.76 (m, 2H), 7.55–7.52 (m, 2H), 5.82 (s, 2H), 3.01–2.88 (m, 2H), 2.59 (s, 1H), 2.30–2.04 (m, 6H), 1.79–1.15 (m, 9H), 1.05 (s, 3H), 0.91 (s, 3H); ^13C-NMR (75 MHz, CDCl[3]) δ 162.8, 150.3, 136.3, 132.5, 132.4, 132.1, 127.8, 124.0, 122.6, 87.4, 79.8, 74.1, 50.7, 47.7, 46.7, 38.9, 36.7, 34.3, 32.5, 32.1, 31.5, 24.1, 23.2, 21.1, 18.7, 12.8; HRMS (ESI-FTMS) mass (m/z): calcd for C[28]H[30]BrNOS [M + H]^+ = 508.1304, found 474.2572. (1S,2R,13R,14S,17R,18S)-7-(1,3-Benzodioxol-5-yl)-17-ethynyl-2,18-dimethyl-8-t hia-6-azapentacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E46) [239]graphic file with name d4md00719k-u27.jpg Light yellowish solid (321 mg, 68%); ^1H-NMR (300 MHz, CDCl[3]) δ 7.44–7.41 (m, 2H), 6.86–6.83 (m, 1H), 6.03 (s, 2H), 5.79 (dd, J = 2.16, 2,85 Hz, 1H), 3.01–2.79 (m, 2H), 2.61 (s, 1H), 2.39–1.99 (m, 6H), 1.88–1.13 (m, 10H), 1.07 (s, 3H), 0.92 (s, 3H); ^13C-NMR (300 MHz, CDCl[3]) δ 148.2, 150.1, 149.3, 148.2, 136.4, 131.2, 128.3, 121.8, 120.9, 108.6, 106.6, 101.5, 87.4, 79.8, 74.1, 50.7, 47.7, 46.7, 38.9, 36.6, 34.4, 32.5, 32.2, 31.5, 24.2, 23.2, 21.1, 18.7, 12.7; HRMS (ESI-FTMS) mass (m/z): calcd for C[29]H[31]NO[3]S [M + H]^+ = 474.2097, found 474.2103. 4-[(1S,2R,13R,14S,17R,18S)-17-Ethynyl-17-hydroxy-2,18-dimethyl-8-thia-6-azape ntacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-7-yl]benzene-1,2-d iol (E47) [240]graphic file with name d4md00719k-u28.jpg Grey solid (300 mg, 65%); ^1H-NMR (400 MHz, DMSO-d[6]) δ 9.43 (s, 1H), 9.28 (s, 1H), 7.25 (d, J = 1.4 Hz, 1H), 7.13–7.11 (m, 1H), 6.74 (d, J = 8.4 Hz, 1H), 5.67 (s, 1H), 5.29 (s, 1H), 2.77–2.69 (m, 2H), 2.20–1.98 (m, 3H), 1.82–1.03 (m, 12H), 0.94 (s, 3H), 0.74 (s, 3H); ^13C-NMR (101 MHz, DMSO-d[6]) δ 164.0, 150.3, 148.3, 146.2, 136.5, 130.1, 125.4, 122.0, 118.5, 116.5, 113.5, 89.4, 78.6, 75.7, 56.6, 50.9, 48.0, 46.8, 36.7, 34.3, 32.8, 32.2, 31.6, 24.3, 23.3, 21.2, 19.1, 13.3; HRMS (ESI-FTMS) mass (m/z): calcd for C[28]H[31]NO[3]S [M + H]^+ = 462.2097, found 462.2094. (1S,2R,13R,14S,17R,18S)-17-Ethynyl-2,18-dimethyl-7-(4-pyridyl)-8-thia-6-azape ntacyclo[11.7.0.0.^2,100.^5,90^14,18]icosa-5(9),6,10-trien-17-ol (E50) [241]graphic file with name d4md00719k-u29.jpg Light red solid (309 mg, 72%); ^1H NMR (300 MHz, CDCl[3]) δ 7.83 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 2H), 5.82 (s, 1H), 2.99–2.89 (m, 2H), 2.61 (s, 1H), 2.40–2.26 (m, 5H), 2.13–2.00 (m, 3H), 1.90–1.21 (m, 8H), 1.08 (s, 3H), 0.93 (s, 3H); ^13C NMR (75 MHz, CDCl[3]) δ 163.5, 149.7, 140.3, 136.3, 131.4, 129.6, 126.4, 122.1, 87.4, 79.8, 74.1, 50.7, 47.7, 46.7, 38.9, 36.7, 34.3, 32.5, 32.2, 31.5, 24.0, 23.2, 21.5, 21.1, 12.7; HRMS (ESI-FTMS) mass (m/z): calcd for C[27]H[30]N[2]OS [M + H]^+ = 431.2152, found 431.2155. In vitro cytotoxicity studies The cytotoxic effects of various compounds were assessed in multiple melanoma cell lines using the resazurin cell viability assay.^[242]16a,38 Cells were seeded into a 96-well plate at a density of 6000 cells per well and incubated for 24 h followed by the treatment with potential drugs for 24 h. After the treatment, resazurin dye was added to each well, and the cells were incubated for another 4 h. Fluorescence was then measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm using Cytation™ 5 plate reader (BioTek, Winooski, VT, USA). The half-maximal inhibitory concentration (IC[50]) was determined using GraphPad Prism 9.4.1 (Dotmatics, San Diego, CA, USA) by nonlinear regression analysis of drug concentrations against the percentage of viable cells. Wound healing assay The cell migration inhibitory effects of fused thiazole compounds were evaluated using a wound-healing assay on confluent monolayers of cancer cells.^[243]39 B16-F10 (CRL-6475™, ATCC) and SK-MEL-28 (HTB-72™, ATCC) cells were seeded at a density of 10^6 cells per well in 6-well tissue culture plates and incubated for 24 h to establish confluent monolayers. Wounds were manually created by dragging a P200 pipette tip across the cell monolayers, followed by gentle washing with phosphate-buffered saline (PBS) to remove cellular debris. Afterward, the compounds and controls were added, and the cells were incubated. Wound closure was monitored by capturing images at different time points using Cytation 5 (BioTek, Winooski, VT, USA) imaging system. Cell adhesion assay Fibronectin serves as the foundation for cellular adhesion to the matrix, and plates coated with fibronectin can simulate the physiology of the extracellular matrix and allow us to determine the effect of compounds on cell adhesion.^[244]40 Individual wells of six-well tissue culture plates were coated with 1 mL of a 10 μg mL^−1 fibronectin solution for 45 min. A total of ∼50 000 B16-F10 (CRL-6475™, ATCC) and SK-MEL-28 (HTB-72™, ATCC) cells from the treatment and control groups were seeded into each well. After allowing the cells to adhere for 1.5 h, the plates were carefully rinsed with PBS without disrupting the adherent cells. Citation 5 (BioTek, Winooski, VT, USA) was used to capture images of the wells at 4× magnification. Clonogenic assay After exposure to different treatments, the ability of a single cell to grow into colonies was determined using a clonogenic assay in vitro.^[245]41 In six-well plates, a thousand B16F10 (CRL-6475™, ATCC) and SK-MEL-28 (HTB-72™, ATCC) cells were cultured for 24 h. The cells were then incubated with drugs and controls for 24 h. Cells were left to colonize with a complete growth medium for several days, and the medium was changed every two days. The growth medium was taken out, and the cells were fixed in a 10% formalin solution for 30 min. Following fixation, the cells were stained with 0.1% crystal violet for 15 min. After staining, the cells were thoroughly washed with phosphate-buffered saline (PBS) to remove excess dye. Images of the visible colonies were then captured for comparison. Cell cycle analysis To assess whether the compounds disrupted the cell cycle and identify the specific stage affected, a flow cytometry propidium iodide (PI) assay was performed, as cell size and DNA content vary at different phases of the cell cycle.^[246]42 B16F10 (CRL-6475™, ATCC) and SK-MEL-28 (HTB-72™, ATCC) cells were seeded at a density of 1 × 10^6 per well on six-well tissue culture plates and incubated for 24 h. Following 24 h of compound treatment, the cells were fixed in 70% ethanol. PI staining was carried out by incubating the cells at room temperature for 15 min, and the samples were analyzed using a BD FACS Aria™ cell sorter (BD Biosciences, Franklin Lakes, NJ, USA). Cell cycle analysis was performed using histograms generated by FCS Express 7 software (Dotmatics, San Diego, California, USA). Apoptosis study To assess early and late apoptosis, B16F10 (CRL-6475™, ATCC) cells were seeded at a density of 1 × 10^6 cells per well in six-well plates and cultured for 24 h before treatment. After treatment, the cells were harvested and washed twice with 1× phosphate-buffered saline (PBS). The cells were then resuspended in 500 μL 1× binding buffer, and a 100 μL aliquot of the cell suspension was transferred to a 5 mL culture tube. The cell suspension was incubated with 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) in the dark for 15 min. Following incubation, the cells were analyzed using a flow cytometer with appropriate gating and wavelength settings. The percentage of apoptosis was determined by measuring the fluorescence intensities of Annexin V-FITC and PI using scatterplots generated with BD FACSDiva Software v6.1.3. mRNA sequencing In a 6-well plate, SK-MEL-28 cells (1 × 10^6 cells per well) were cultured for 24 h followed by the treatment with compounds for 24 h. RNA sequencing was outsourced to the Genomics Core Facility, University of Arkansas Medical Science (UAMS), Little Rock, AR, USA. Briefly, total RNA was extracted from treated and control cells using the Quick-DNA/RNA Miniprep Plus Kit (Zymo Research, Irvine, CA, USA) and the yields were quantified using the Qubit RNA BR Assay (Thermo Fisher, Waltham, MA, USA). To prepare for sequencing, 0.1–1.0 μg of total RNA was used as input to generate sequencing libraries using the TrueSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA), following the manufacturer's protocol. The concentration of the newly generated libraries was measured with the Qubit 1× dsDNA HS Assay (Thermo Fisher, Waltham, MA, USA), and the libraries were checked for quality using a Fragment Analyzer System (Agilent Tech, Santa Clara, CA, USA) and the HS NGS Fragment kit (1–6000 bp) from Agilent Technologies. Libraries were then sequenced either on a NextSeq2000 or NovaSeq 6000 (Illumina, San Diego, CA, USA) in paired-end mode (read 1: 101 cycles, read 2: 101 cycles, i7: 8 cycles, i5: 8 cycles). We used the ShinyGO 0.77 bioinformatics tool to perform gene enrichment and pathway analyses.^[247]43 Protein extraction Cells were seeded at a density of 1 × 10^6 cells per well in a six-well plate and cultured for 24 h before being treated with experimental compounds. After treatment, the growth medium was removed, and the cells were washed with 1× phosphate-buffered saline (PBS). Lysis buffer (CelLytic MT, Sigma-Aldrich) was then added to each well, and the plate was shaken for 15 min to lyse the cells. The resulting lysates were transferred to 1.5 mL microcentrifuge tubes and centrifuged at 12 000 × g for 15 min to separate the cell debris from the supernatant, which contained the extracted proteins. The protein samples were stored at −70 °C until further analysis. Protein concentrations were measured using the Pierce Coomassie Plus (Bradford) assay kit (Bradford, USA). Proteomics Extracted protein samples were sent to the IDeA National Resource for Quantitative Proteomics, University of Arkansas for Medical Science (UAMS), Little Rock, AR, USA for proteomics and data analysis. Briefly, total protein extracts from each sample were reduced, alkylated, and purified using chloroform/methanol extraction before digestion with sequencing-grade, modified porcine trypsin (Promega, Madison, WI, USA). Tryptic peptides were then separated using reverse-phase XSelect CSH C18 2.5 μm resin (Waters, Milford, MA, USA) on an in-line 150 × 0.075 mm column using an UltiMate 3000 RSLCnano system (Thermo Fisher, Waltham, MA, USA). Peptides were eluted using a 60 min gradient from 98 : 2 to 65 : 35 buffer A : B ratio, and the eluted peptides were ionized by electrospray ionization (2.2 kV) followed by mass spectrometric analysis on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher, Waltham, MA, USA). To assemble the chromatogram library, six gas-phase fractions were acquired on the Orbitrap Exploris with four m/z DIA spectra (4 m/z precursor isolation windows at 30 000 resolution, normalized AGC target 100%, maximum injection time 66 ms) using a staggered window pattern from narrow mass ranges and optimized window placements. Precursor spectra were acquired after each DIA duty cycle, spanning the gas-phase fraction's m/z range, i.e., 496–602 m/z, 60 000 resolution, normalized AGC target 100%, maximum injection time 50 ms. For wide-window acquisitions, Orbitrap Exploris was configured to acquire a precursor scan (385–1015 m/z, 60 000 resolution, normalized AGC target 100%, maximum injection time 50 ms) followed by 50 × 12 m/z DIA spectra (12 m/z precursor isolation windows at 15 000 resolution, normalized AGC target 100%, maximum injection time 33 ms) using a staggered window pattern with optimized window placements. Precursor spectra were acquired after each DIA duty cycle. Proteomics data were further analyzed for pathway analysis. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) qRT-PCR protocol was followed as described previously.^[248]44 Primers for beta Parvin (PARVB) were designed using the National Center for Biotechnology Information (NCBI) database and the PrimerQuest tool (Integrated DNA Technologies, CA, USA). The forward primer sequence was 5′-CCCTGTCTCCAAATACCATCAC-3′, and the reverse primer sequence was 5′-CTCCCGGTACAAGGCAAATTA-3′, producing an amplicon of 92 base pairs. We used glyceraldehyde-3-phosphate dehydrogenase (forward primer 5′- TTGGCTACAGCAACAGGGTG-3′ and reverse primer 5′-GGGGAGATTCAGTGTGGTGG-3′)^[249]45 and peptidylprolyl isomerase A (forward primer 5′ TCCTTTCTCTCCAGTGCTCAG 3′ and reverse primer 5′ CACCGTGTTCTTCGACATTG3′)^[250]46 as reference genes. Cells were seeded at a density of 1 × 10^6 cells per well in six-well plates, incubated for 24 h, and then treated with the experimental compounds and vehicle controls for another 24 h. Total RNA was extracted and purified using the RNeasy Mini Kit (Qiagen, Maryland, USA) with DNase treatment using RNase-Free DNase Set (Qiagen, Maryland, USA). The concentration of the extracted RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, CA, USA). Complementary DNA (cDNA) was synthesized from the RNA using the RT2 First Strand Kit (Qiagen, Maryland, USA) and stored at −80 °C for subsequent qPCR analysis. For qPCR amplification, a reaction volume of 12.5 μL was prepared, containing 6.25 μL of RT2 SYBR Green Mastermix (Qiagen, Maryland, USA), 1 μL each of forward and reverse primers at a final concentration of 400 nM, 1 μL of cDNA (equivalent to 9 ng of RNA), and 3.25 μL of nuclease-free water. Real-time amplification was carried out using the CFX384 Real-Time System with a C1000 Thermal Cycler (Bio-Rad, USA). The amplification protocol consisted of an initial denaturation at 95 °C for 3 min, followed by 39 cycles of denaturation at 95 °C for 15 s, and annealing/extension at 55 °C for 1 min. A melting curve analysis was performed at the end to verify the purity of the amplified products. Gene expression levels were analyzed using Bio-Rad CFX Maestro software (Bio-Rad, USA). Western immunoblot Standard immunoblotting, combined with enhanced chemiluminescence (ECL), generates signals that provide an indirect measure of the relative abundance of the target protein.^[251]47 Extracted protein samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and the proteins were transferred from the polyacrylamide gel to the nitrocellulose membrane. The membrane was incubated with a primary antibody (β-actin (13E5) rabbit mAb # 4970, cell signaling) and an enzyme-linked secondary antibody (anti-rabbit IgG, HRP-linked antibody #7074, cell signaling) to form an antigen-antibody–antibody complex. The exposure of substrate emits luminescence through enzyme activity and luminescence was used to estimate protein expression using Odyssey FC imaging (LI-COR Biosciences, USA). Actin cytoskeleton imaging SK-MEL-28 cells (6 × 10^6 cells per well) were seeded in a 96-well plate for 24 h and treated with the compounds for 24 h. The cells were stained with Invitrogen Alexa Fluor™ 488 Phalloidin (catalog no. A12379) and imaged using Citation 5 (BioTek, Winooski, VT, USA) following the manufacturer's protocol. In vivo antitumor studies The Institutional Animal Care and Use Committee (IACUC) at Arkansas State University, Jonesboro, AR accredited the in vivo experimental protocol (FY21-22-130). B16F10 subcutaneous melanoma was induced in C57BL/6 mice (Strain: 000664, The Jackson Laboratory, Bar Harbor, ME) according to a previously reported protocol.^[252]48 Tumors were induced in male mice by subcutaneous injection of 5 × 10^5 B16F10 cells per mouse. On day six after tumor cell injection, the mice were randomized (nine mice per group) and treated with E2 and E47 intraperitoneally at different doses using a vehicle containing 5% DMSO and 5% Tween 80 in PBS. E2 was injected at 40 mg kg^−1 body weight once daily via intraperitoneal injection from day 6th to day 9th of tumor cell injection and then every alternate day till day 18th E2 (4D + Alt). Similarly, E47 was injected at 40 mg kg^−1 from day 6th to day 9th of tumor cell injection and then every alternate day until day 18th E47 (4D + Alt). In another treatment setup, E2 was administered at 40 mg kg^−1 for every alternate day from day 6th to day 18th of tumor cell injection E2 (Alt) and E47 at 40 mg kg^−1 from day 6th to day 12th followed by every alternate day till day 18th E47 (7D + Alt). Tumor diameters were measured using slide calipers and tumor volumes were calculated using the following formula: volume = 0.5 × length × width.^[253]2 We measured the weight of mice every alternate day and compared the treatments with the vehicle. We considered a standard tumor volume of 1000 mm^3 as an endpoint of the survival study. At the end of the experiment, sufficient blood was obtained under anesthesia (gaseous 1% isoflurane) to harvest plasma, and samples were profiled for important metabolites. After blood collection, anesthetized mice were euthanized in a CO[2] chamber. Toxicology testing in vivo At the end of the experiment, plasma was harvested, and plasma metabolite profiling was outsourced to the DNA Damage and Toxicology Core at UAMS, Little Rock, AR. Toxicity was assessed by measuring 14 blood markers in the plasma as previously described.^[254]16a,29 Blood markers of organ function were available in the Comprehensive Diagnosis Kit (Union City, CA, USA) and VetScan VS2 instrument (Abaxis) to analyze alanine, albumin, alkaline phosphatase, transaminase, amylase, blood urea nitrogen, calcium, creatinine, globulin, glucose, phosphorus, potassium, sodium, total bilirubin, and total protein. Our criteria for toxicity were the combination of statistically significant differences from the vehicle control and consistency between several functional markers of the same organ. Computational studies The proteins were downloaded from the Protein Data Bank (PDB) ([255]https://www.rcsb.org/) as .pdb files. ChemSketch ([256]https://www.acdlabs.com/resources/free-chemistry-software-apps/ch emsketch-freeware/) was used to draw the compound structures and saved as .mol files and then converted into .pdb files by using the Online SMILES Translator ([257]https://cactus.nci.nih.gov/translate/). Before starting the docking, Python programming was installed to run the docking software. AutoDock Tools and AutoDock Vina ([258]https://autodock.scripps.edu/download-autodock4/) were used to study the compound interaction with the protein. For docking, the protein was opened as a .pdb file into the AutoDock Tools to clean proteins by removing the preexisting ligand, water, hetatm, and other unnecessary residues. Hydrogen and the Kollman charges were added and saved as .pdb file followed by turning into an Ad4 type. The clean protein was opened through the Grid option, into AutoDock, and saved in the same location as a .pdbqt file. The compound was opened as a normal file from the Ligand option and turned into a .pdbqt file. A Grid Box was set up at the same spot from where the preexisting ligand was removed or other binding sites. Then the protein as .gpf, and the ligand as .dpf files were saved. Before running the program in the same location, the configuration file was written as a .txt file. Afterward, the program was run into the Command Prompt to get the binding scores. Furthermore, the Discovery Studio ([259]https://www.3ds.com/products/biovia/discovery-studio/visualizatio n) was used to look into the protein and ligand interaction in 3D and 2D modes to collect the pictures. Statistical analysis Statistical analysis was performed in GraphPad Prism 9 software (GraphPad, Boston, MA). Kruskal Wallis test (non-parametric one-way analysis of variance) was used to measure the significance of treatment and the Kaplan–Meier survival curve was used to represent the overall mice survival. *, **, ***, and **** depicted significant p values <0.05, 0.01, 0.001, and 0.0001, respectively. Conclusions This study identified fused-thiazole derivatives with potent antimelanoma activities in vitro and in vivo. We found that the lead thiazolo-ethisterone derivatives had a minimum IC[50] value of 1.6 μM in melanoma cell lines and significantly inhibited the cell migration and colony formation. Flow cytometry studies showed that these compounds induced mild arrest in the G2/M phase of the mitotic cell cycle. The potent compounds also induced moderate apoptosis as determined by annexin V/PI flow cytometry. Using mRNA sequencing, we found β-actin (ACTB) and γ-actin (ACTG1) expression were downregulated by E47 which was further validated by proteomics and Western blotting studies. Downregulation of ACTB and ACTG1 ultimately culminated in the inhibition of lamellipodia, filopodia, and invadopodia formation owing to the lack of actin filaments as determined by fluorescence microscopy. In line with our mechanism of action studies, E12 and E47 significantly inhibited cell migration and adhesion to the extracellular matrix (ECM) in in vitro assays. Proteomics and qRT-PCR data in the treated cell lines showed upregulation of β-parvin (PARVB), an integrin-linked kinase (ILK) adapter protein that is known to inhibit ILK signaling when upregulated indicating a possible mechanism for the inhibition of migration and adhesion. Finally, E2 and E47 effectively inhibited tumor growth in a subcutaneous mouse melanoma model, consistent with our in vitro findings that these compounds effectively inhibited melanoma cell proliferation through cell cycle arrest. Toxicological studies after multiple doses were found to be safe for administration of these compounds in mice. These lead compounds are the first reported potential drugs that downregulate β-actin and γ-actin and upregulate β-parvin in melanoma cell lines to confer an antimelanoma effect both in vitro and in vivo. Associated content The data underlying this study are available in published article and online ESI.[260]† Ethical statement All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Arkansas State University and approved by the Animal Ethics Committee of Arkansas State University (IRB# FY21-22-130). Data availability Data provided in the ESI[261]† and on request to the corresponding author. Author contributions Conceptualization: M. A. A., S. A., N. A.; validation: M. A. A., S. A., data curation: M. A. A., S. A., S. R., S. C., A. S., A. T., S. B., N. A., A. B., M. M.; writing – original draft: S. A., S. R., M. A. A.; writing – review and editing: M. A. A., S. A., S. R., S. C., A. S., A. T., S. B., N. A., A. B., M. M.; supervision: M. A. A., A. T., N. A.; funding acquisition: M. A. A. Conflicts of interest Sanjay Adhikary was a graduate student in the biology department at Arkansas State University, Jonesboro, Arkansas, 72467, United States. Part of this manuscript was prepared as Sanjay's thesis. Supplementary Material MD-OLF-D4MD00719K-s001 [262]MD-OLF-D4MD00719K-s001.pdf^ (5.6MB, pdf) MD-OLF-D4MD00719K-s002 [263]MD-OLF-D4MD00719K-s002.xlsx^ (99.3KB, xlsx) Acknowledgments