Utilizing Estra-1,3,5,16-Tetraene Scaffold: Design and Synthesis of Nitric Oxide Donors as Chemotherapeutic Resistance Combating Agents in Liver Cancer

A new series of nitric oxide-releasing estra-1,3,5,16-tetraene analogs (NO-∆-16-CIEAs) was designed and synthesized as dual inhibitors for EGFR and MRP2 based on our previous findings on estra-1,3,5-triene analog NO-CIEA 17 against both HepG2 and HepG2-R cell lines. Among the target compounds, 14a (R-isomer) and 14b (S-isomer) displayed potent anti-proliferative activity against both HepG2 and HepG2-R cell lines in comparison to the reference drug erlotinib. Remarkably, compound 14a resulted in a prominent reduction in EGFR phosphorylation at a concentration of 1.20 µM with slight activity on the phosphorylation of MEK1/2 and ERK1/2. It also inhibits MRP2 expression in a dose-dependent manner with 24% inhibition and arrested the cells in the S phase of the cell cycle. Interestingly, compound 14a (estratetraene core) exhibited a twofold increase in anti-proliferative activity against both HepG2 and HepG2-R in comparison with the lead estratriene analog, demonstrating the significance of the designed ∆-16 unsaturation. The results shed a light on compound 14a and support further investigations to combat multidrug resistance in chemotherapy of hepatocellular carcinoma patients.


Introduction
Hepatocellular carcinoma (HCC) is one of the most frequent and lethal human cancers, and its incidence-to-mortality ratio is rising [1][2][3][4][5][6]. In 2020, HCC represented 2.4% of all new cancer cases and 5.0% of all cancer deaths [4]. The C subset of the human ATP-binding cassette (ABCC) transporters contains a huge group of membrane transport proteins comprising thirteen subfamily members (1-13) and plays a pivotal role in the recognition and transport of most drugs. Among them, the second member of multidrug resistance protein 2 (MRP2) of the MRP subfamily of the encoded ABCC2 gene located on Chromosome 10q23-q24 with a unique amino acid sequence of the ATP-binding domain, which functions properly in drug resistance mechanisms as it is structurally similar to human MRP1 [7]. MRP2 is overexpressed in many cancers such as the liver, kidney, and small intestine to play a key role in the transport of many drugs such as cisplatin, doxorubicin, epirubicin, etoposide, irinotecan, mitoxantrone, methotrexate, SN-38, and Vinca alkaloids, in addition to endogenous metabolites. Korita and co-workers [8] and our research group [9] recently reported that HCC cells produce drug resistance via MRP2 overexpression, which alkaloids, in addition to endogenous metabolites. Korita and co-workers [8] and our research group [9] recently reported that HCC cells produce drug resistance via MRP2 overexpression, which comprises the major problem associated with the available chemotherapeutic agents in 30-80% of cancer patients [10,11]. Furthermore, the EGFR-MEK1/2-ERK1/2 signaling pathway, as well as MRP2, has been validated as effective targets for liver cancer therapy. So far, the discovered agents have revealed significant activity toward MRP1 and P-gp but rarely against MRP2 [8]. Therefore, it is still important to discover a dual inhibitor drug candidate with good efficacy and low toxicity targeting the EGFR-MEK1/2-ERK1/2 signaling pathway as well as MRP2.
It is worth mentioning that the role of NO in cancer therapy is not only that of enhancing tumor blood flow and oxygen supply [12] but also reversing chemotherapy resistance through the upregulation of efflux pumps [13,14]. Along this line of research, Maksimovic-Ivanic et al. demonstrated that the hybridization of some drugs with NO, such as NO-NSAIDs and NO-HIV-PIs, promoted anticancer activity in a wide range of cancer cell lines and in in vivo models, invariably more potent, less toxic, lower dosing, and fewer side effects than the corresponding des-NO analogs [15]. The mode of action is likely multifactorial as they inhibit tumor cell growth, induce apoptosis, exhibit antiangiogenic and antimetastatic activity, and potentially inhibit P-gp-, MRP-1-, and BCRP1-expressing cells making NO-releasing agents as candidates for the treatment of multidrugresistant tumors [15]. Previously, we detailed the biological studies of NO-releasing cucurbitacin analogs with an estratriene core [16]. Among them, NO-CIEA 17 (Figure 1), exhibits broad-spectrum anti-proliferative activity against both HepG2 and HepG2-R cell lines with IC50 4.69 and 8.21 µ M, respectively [16]. Therefore, overcoming the toxicity and cellular resistance problems encouraged us to look for a more active molecular target anticancer agent with nitric oxide-releasing properties for the treatment of HCC. Enlightened by the aforementioned and the activity profile displayed by NO-CIEA 17 [16], we explored whether further D-ring modification to rigidify the conformations of the estron skeleton would adopt strong interaction inside the target active sites to obtain the highest potency. To this end, a new series of ∆-16 unsaturated analogs NO-∆-16-CIEAs ( Figure 1) was designed, allowing us to establish that the estratetraene core could Enlightened by the aforementioned and the activity profile displayed by NO-CIEA 17 [16], we explored whether further D-ring modification to rigidify the conformations of the estron skeleton would adopt strong interaction inside the target active sites to obtain the highest potency. To this end, a new series of ∆-16 unsaturated analogs NO-∆-16-CIEAs ( Figure 1) was designed, allowing us to establish that the estratetraene core could successfully function as a bioisostere for the estratriene one keeping the NO-releasing  concerning the D-ring and, thus, less constrained by the C-18 methyl group, allowing the approach of the reagent by both sides of the steroid molecule. Consequently, the ZnI2catalysed addition of TMSCN yielded a mixture of epimeric cyanosilylated product 9 (50% yield), as observed by NMR, as well as 16-α-cyano analog 10 (20% yield) [24]. This lack of chemo selectivity in the ZnI2-catalysed TMSCN addition was not surprising. In fact, although the reported ZnI2 catalyzed 1,2-addition to conjugated enones, 1,4-addition was also noticed [24]. 3 KCN (0.1 equiv) 18-crown-6 (0.1 equiv) [27] DCM 0-r.t. 0: 50 30 4 ZnI2 (0.03 equiv) THF r.t. 20:12 31 5 ZnI2 (1 equiv) DCM −20 40:20 3 6 ZnI2 (1 equiv 20 20 Moreover, the mixture of the key intermediate 11a and 11b was isolated from the reaction of epimeric cyanosilylated 9 with methyl lithium in ether at 0 • C. This diastereomeric mixture with ∆R f equal to 0.1 was then separated by column chromatography (5% ethyl acetate in hexane) and, then, identified as (19R)-α-hydroxy methyl ketone (11a, 24.72% yield) and (19S)-α-hydroxy methyl ketone (11b, 36.78% yield). The assignment of the C-19 configuration of the hydroxyl group was made on the basis of 1 H NMR and 13 C NMR chemical shifts in this functional group as well as X-ray crystallographic analysis (Figure 2), and these data were compared to a CUCs side chain containing the function group reported previously by our research group [31]. 1 H NMR showed a singlet signal at 4.07 ppm and 3.90 ppm corresponding to 11a and 11b, respectively. Surprisingly, the protected α,β-enone 8 was also detected in the mixture in 10.20% yield with an unknown mechanism.
Molecules 2023, 28 More specifically, the assignment of the 16-configuration of the cyano group was made based on the 1 H NMR anisotropic shielding effect of this functional group toward the 18-CH3, which appears as a singlet peak at 0.42 ppm, in contrast with 0.73 ppm in the starting compound 8. Such an effect suggests that the methyl group is positioned in the conical area of the cyano substituent. In agreement with this observation, the coupling constant of the doublet due to the 17-H (2.79 ppm) is relatively high, J = 8.7 Hz, pointing to a trans-vicinal coupling with the 16-H (3.41 ppm) [24].
For more study about the SAR, the key intermediates α-hydroxymethylketones 11a,b were then reacted with aromatic or heterocyclic aldehydes through aldol condensation and in situ elimination using NaOH to afford cucurbitacin-inspired estra-1,3,5,16-tetraene analogs 15a-j. Deprotection and finally coupling with furoxan mesylate provided the target compounds 17a-j, Scheme 3. There are some attempts to improve the yield percentages for the target compounds; among them, deprotection of 11b with TBAF and then O-alkylation with furoxan mesylate followed by aldol condensation with different aromatic or heterocyclic aldehydes to afford the desired compounds 20a-d, as described in Scheme 4.

In Vitro Anti-Proliferative Activity
The target compounds 14a,b, 17a-j, and 20a-d were examined for their in vitro antiproliferative activity against HepG2 and HepG2-R (erlotinib-resistant [9]) liver cell lines. The cells were treated with serial dilutions of test compounds for 48 h, after which the cell viability was determined by a standard MTT colorimetric assay, using erlotinib as a reference drug.
For more study about the SAR, the key intermediates α-hydroxymethylketones 11a,b were then reacted with aromatic or heterocyclic aldehydes through aldol condensation and in situ elimination using NaOH to afford cucurbitacin-inspired estra-1,3,5,16-tetraene analogs 15a-j. Deprotection and finally coupling with furoxan mesylate provided the target compounds 17a-j, Scheme 3. There are some attempts to improve the yield percentages for the target comp among them, deprotection of 11b with TBAF and then O-alkylation with furoxan m followed by aldol condensation with different aromatic or heterocyclic aldehyde ford the desired compounds 20a-d, as described in Scheme 4. Then, our attention was directed to screen our designed compounds against the HepG2-R cell line. The data revealed that analogs with cucurbitacin side chain 14a and 14b only have a potent cytotoxicity activity (IC 50 3.74 ± 0.66 µM and 6.47 ± 1.41 µM, respectively). Therefore, they may have a good impact on chemotherapeutic resistance.
There are some attempts to improve the yield percentages for the target compounds; among them, deprotection of 11b with TBAF and then O-alkylation with furoxan mesylate followed by aldol condensation with different aromatic or heterocyclic aldehydes to afford the desired compounds 20a-d, as described in Scheme 4.  Furthermore, some important physicochemical parameters were calculated to examine the drug-likeness properties of these compounds [35]. The lipophilicity parameter indicated by CLogP and the polar surface area (PSA) was estimated. As shown in Table 2, compounds 14a and 14b possesses good CLogP (6.39) and PSA (118.25) values among the target final analogs.

Structure-Activity Relationships (SARs) Study
Substitution at CUCs aromatic-like side chain with either electron-withdrawing (EWG) CF 3 and Br (i.e., compounds 17b, 17i,j, and 20c,d) or electron-donating (EDG) CH 3 S and CH 3 O groups, as in compounds 17c-e and 20a, had no significant effect on the anticancer activity. Notably, the EWG group F substitution at the CUCs aromatic-like side chain (i.e., compound 20b) may impact the activity. In contrast, CF 3 substitution caused compound 17a to exhibit significant inhibitory effects in its anticancer activity, indicating that the electron-withdrawing substituents likely led to enhanced potency. On the other hand, replacing both EDG and EWG with a polar t-BuOH group caused compounds 14a and 14b to exhibit significant inhibitory effects in their anticancer activity, indicating that appending the CUCs side chain at C-17 likely led to enhanced potency. Furthermore, the results showed a significant improvement in anticancer activity, especially for compounds 14a and 14b, compared to des-NO-releasing compounds 13a and 13b indicating synergistic efficiency. pounds 14b and 17a possessed higher anti-proliferative activities than the reference drug with IC50 8.10 ± 0.92 and 9.44 ± 0.44 µ M, respectively. In addition, compounds 17g and 17h were almost equipotent to erlotinib with IC50 24.00 ± 3.80 and 25.00 ± 3.12 µ M, respectively. Furthermore, compounds 17b-f, 17i, 17j, and 20a-d did not inhibit the proliferation of HepG2 cells at concentrations as high as 50 µ M. Moreover, compounds linked with an aromatic or heterocyclic side chain at the C-17 position dramatically dropped or totally abolished the anticancer activity unlike compound 14a. Then, our attention was directed to screen our designed compounds against the HepG2-R cell line. The data revealed that analogs with cucurbitacin side chain 14a and HepG2-R  Table 3. Anticancer activity of the ∆-16-CIEA 13a,b and furoxan alcohol in HepG2 and HepG2-R cell lines.
Molecules 2023, 28, x FOR PEER REVIEW 14b only have a potent cytotoxicity activity (IC50 3.74 ± 0.66 µ M and 6.47 ± spectively). Therefore, they may have a good impact on chemotherapeutic r Our consideration was then extended to examine the effect of compilin ring with the estrone core as two pharmacophoric features. Accordingly, furoxan alcohol and cucurbitacin steroidal phenolic derivative 13a and 13 ined ( Table 3). The data revealed that compound 13a showed IC50 18.87 ± 2. 0.72 µ M in HepG2 and HepG2-R cells, respectively, while compounds fu and 13b did not inhibit the proliferation of both cell lines at concentrations µ M. The results indicated that sp 2 -hybridization favored anticancer activi quently improved the anti-proliferative activity of estratetraene derivative by blocking the estrogenic activity [34] of C3 phenolic hydroxy. Furthermore, some important physicochemical parameters were calcul ine the drug-likeness properties of these compounds [35]. The lipophilicity dicated by CLogP and the polar surface area (PSA) was estimated. As show compounds 14a and 14b possesses good CLogP (6.39) and PSA (118.25) valu target final analogs.

Structure-Activity Relationships (SARs) Study
Substitution at CUCs aromatic-like side chain with either electron (EWG) CF3 and Br (i.e., compounds 17b, 17i,j, and 20c,d) or electron-donatin and CH3O groups, as in compounds 17c-e and 20a, had no significant effe cancer activity. Notably, the EWG group F substitution at the CUCs arom HepG2-R It is worth mentioning that the configuration around the C-19 played a crucial role in the biological activity among the most active compounds. Particularly, compound 14a with R-configuration showed higher activity than the corresponding S-isomer 14b in both HepG2 and HepG2-R cell lines. Moreover, compound 17a with R configuration around C-19 showed a potent cytotoxicity activity against the HepG2 cell line while the corresponding S-isomer 17b was inactive up to 50 µM. In addition, the anticancer activity showed a twofold increase against both the HepG2 and HepG2-R cell lines when the estratriene core in NO-CIEA I was replaced with estratetraene core in compound 14a, demonstrating the significance of ∆-16 unsaturation. Appending the furoxan moiety at C3, and utilizing the CUCs side chain at C-17, ring D unsaturation at C-16, and R configuration around C-19 can be suggested as vital elements for the growth inhibitory activity of the hybrids reported here, see Figure 3. In this context, the synthesized analogs are considered promising candidates for the treatment of HCC and overcoming the chemotherapeutic resistance.
C-19 showed a potent cytotoxicity activity against the HepG2 cell line while the corresponding S-isomer 17b was inactive up to 50 µ M. In addition, the anticancer activity showed a twofold increase against both the HepG2 and HepG2-R cell lines when the estratriene core in NO-CIEA I was replaced with estratetraene core in compound 14a, demonstrating the significance of ∆-16 unsaturation. Appending the furoxan moiety at C3, and utilizing the CUCs side chain at C-17, ring D unsaturation at C-16, and R configuration around C-19 can be suggested as vital elements for the growth inhibitory activity of the hybrids reported here, see Figure 3. In this context, the synthesized analogs are considered promising candidates for the treatment of HCC and overcoming the chemotherapeutic resistance.

Cell-Based ELISA
As compound 14a showed a good profile of anti-proliferative activity against both HepG2 and HepG2-R cancer cell lines, the study was extended to explore its mode of action using the In-cell Western assay (ICW). As shown in Figure 4, the Odyssey image shows the detection of total proteins regardless of phosphorylation status as well as the detection of decreasing amounts of phospho-protein as a function of increasing 14a concentrations, while the graphical representations show the quantification of fluorescence. The data reveal the slight activity for compound 14a after incubation for 24 h at a concentration of 4.80 µM to inhibit the phosphorylation of MEK1/2 and ERK1/2 (≈10% and 12% inhibition, respectively) compared to untreated cells (Control), whereas no effects were observed for phosphorylated EGFR. Surprisingly, 14a resulted in a significant reduction in EGFR phosphorylation in treated cells at a concentration of 1.20 µ M with 43% inhibition. Furthermore, as shown in Figure 4, there is a significant reduction in MRP2 expression in the HepG2-R cell line [36] caused by compound 14a in a dose-dependent manner with 24% inhibition compared to control, demonstrating a significant impact on the chemotherapeutic resistance.

Cell-Based ELISA
As compound 14a showed a good profile of anti-proliferative activity against both HepG2 and HepG2-R cancer cell lines, the study was extended to explore its mode of action using the In-cell Western assay (ICW). As shown in Figure 4, the Odyssey image shows the detection of total proteins regardless of phosphorylation status as well as the detection of decreasing amounts of phospho-protein as a function of increasing 14a concentrations, while the graphical representations show the quantification of fluorescence. The data reveal the slight activity for compound 14a after incubation for 24 h at a concentration of 4.80 µM to inhibit the phosphorylation of MEK1/2 and ERK1/2 (≈10% and 12% inhibition, respectively) compared to untreated cells (Control), whereas no effects were observed for phosphorylated EGFR. Surprisingly, 14a resulted in a significant reduction in EGFR phosphorylation in treated cells at a concentration of 1.20 µM with 43% inhibition. Furthermore, as shown in Figure 4, there is a significant reduction in MRP2 expression in the HepG2-R cell line [36] caused by compound 14a in a dose-dependent manner with 24% inhibition compared to control, demonstrating a significant impact on the chemotherapeutic resistance.

Flow Cytometric Study
After the 24 h incubation of compounds 14a (2.4 µM) and 14b (8.1 µM) and vehicle DMSO as the control, quantitation of the results showed that 14a exhibited a decrease in the fraction of cells in the G0/G1 phase (34.66% compared to 44.11% for the control) and an increase in the proportion of cells in the S phase (59.05 % compared to 48.82%) and the G2/M phase (6.29% compared to 7.06%) ( Figure 5A,B). Moreover, compound 14b also exhibited an increase in the fraction of cells in the G2/M phase (17.08% compared to 7.06% for the control) and a decrease in the proportion of cells in the S phase (39.99% compared to 48.82%) and the G0/G1 phase (42.93% compared to 44.11%) ( Figure 5A

Flow Cytometric Study
After the 24 h incubation of compounds 14a (2.4 µM) and 14b (8.1 µM) and vehicle DMSO as the control, quantitation of the results showed that 14a exhibited a decrease in the fraction of cells in the G0/G1 phase (34.66% compared to 44.11% for the control) and an increase in the proportion of cells in the S phase (59.05 % compared to 48.82%) and the G2/M phase (6.29% compared to 7.06%) ( Figure 5A,B). Moreover, compound 14b also exhibited an increase in the fraction of cells in the G2/M phase (17.08% compared to 7.06% for the control) and a decrease in the proportion of cells in the S phase (39.99% compared to 48.82%) and the G0/G1 phase (42.93% compared to 44.11%) ( Figure 5A,C). Therefore, compound 14a arrested the cells in the S phase while 14b arrested the cells in the G2/M phase of the cell cycle.

Flow Cytometric Study
After the 24 h incubation of compounds 14a (2.4 µM) and 14b (8.1 µM) and vehicle DMSO as the control, quantitation of the results showed that 14a exhibited a decrease in the fraction of cells in the G0/G1 phase (34.66% compared to 44.11% for the control) and an increase in the proportion of cells in the S phase (59.05 % compared to 48.82%) and the G2/M phase (6.29% compared to 7.06%) ( Figure 5A,B). Moreover, compound 14b also exhibited an increase in the fraction of cells in the G2/M phase (17.08% compared to 7.06% for the control) and a decrease in the proportion of cells in the S phase (39.99% compared to 48.82%) and the G0/G1 phase (42.93% compared to 44.11%) ( Figure 5A,C). Therefore, compound 14a arrested the cells in the S phase while 14b arrested the cells in the G2/M phase of the cell cycle.

NO Detection
The levels of NO produced intracellularly from compounds 14a,b and 17a along with JS-K (a known NO donor prodrug) were examined using NO-sensitive fluorophore DAF-FM DA, (Figure 6A-D). It was observed that compound 14a showed a significant increase in fluorescence (2.9-fold compared to untreated cells) after 6 h of incubation in a dose-dependent manner and produced amounts of NO in tumor cells comparable to JS-K [16], which also showed a significant increase in fluorescence (2.9-fold compared to untreated cells) after 1 h of incubation in a dose-dependent manner. It was also found that compounds 14b and 17a showed a relative increase in fluorescence by about 1.38 and 1.13-fold compared to untreated cells (Control) after 1 h and 6 h of incubation in a dose-dependent manner, respectively. Furthermore, these results strongly suggest that compound 14a is a far superior source of controlled NO release in comparison with the reference prodrug JS-K.
which also showed a significant increase in fluorescence (2.9-fold compared to untreated cells) after 1 h of incubation in a dose-dependent manner. It was also found that compounds 14b and 17a showed a relative increase in fluorescence by about 1.38 and 1.13-fold compared to untreated cells (Control) after 1 h and 6 h of incubation in a dose-dependent manner, respectively. Furthermore, these results strongly suggest that compound 14a is a far superior source of controlled NO release in comparison with the reference prodrug JS-K. Additionally, compound 14a was tested again for its NO-releasing activity in the HepG2-R cell line and the data revealed a marked increase in fluorescence intensity after incubation for 6 h with concentrations lower than the IC50. Moreover, treatment with higher concentrations than IC50 or a prolonged incubation showed a marked decrease in fluorescence intensity in a dose-dependent manner compared to the DMSO control (Figure 7). Therefore, the potent inhibitory effects of compound 14a might be related to the obvious release of NO, estratetraene skeleton, and the absolute configuration of C-19 in the cucurbitacin side chain.
It is worth mentioning that our previously reported lead furoxan hybrid NO-CIEA 17 with IC50 values of 4.69 and 8.21 µ M against both HepG2 and HepG2-R cell lines, respectively, showed an increase in fluorescence after 1 h of incubation in a dose-dependent manner and produced a significant concentration of NO in tumor cells compared to JS-K (reference drug). In addition, NO-CIEA 17 released a maximum level of NO at a concentration of 32.92 and 25 µ M compared to the reference prodrug JS-K, which produced the maximum release at 50 µ M. Additionally, compound 14a was tested again for its NO-releasing activity in the HepG2-R cell line and the data revealed a marked increase in fluorescence intensity after incubation for 6 h with concentrations lower than the IC 50 . Moreover, treatment with higher concentrations than IC 50 or a prolonged incubation showed a marked decrease in fluorescence intensity in a dose-dependent manner compared to the DMSO control ( Figure 7). Therefore, the potent inhibitory effects of compound 14a might be related to the obvious release of NO, estratetraene skeleton, and the absolute configuration of C-19 in the cucurbitacin side chain. In addition, in a dose-dependent manner, the tested compounds showed a marked decrease in NO level compared to the DMSO control after incubation for 24 h. The incubation of cells with the target compounds decreased the overall metabolic activity of the cells, as determined by MTT assay within the concentration range and incubation periods tested; thus, the low NO intracellular content at high concentrations of NO donors was caused by their cytotoxicity activity. Moreover, the DAF-FM DA probe was sufficient for detecting the increased fluorescence within a wide range of NO concentrations, as shown by the measurements after 1 h of incubation. These findings document that any potential quenching of the specific DAF-FM DA fluorescence after incubation for long periods by In addition, in a dose-dependent manner, the tested compounds showed a marked decrease in NO level compared to the DMSO control after incubation for 24 h. The incubation of cells with the target compounds decreased the overall metabolic activity of the cells, as determined by MTT assay within the concentration range and incubation periods tested; thus, the low NO intracellular content at high concentrations of NO donors was caused by their cytotoxicity activity. Moreover, the DAF-FM DA probe was sufficient for detecting the increased fluorescence within a wide range of NO concentrations, as shown by the measurements after 1 h of incubation. These findings document that any potential quenching of the specific DAF-FM DA fluorescence after incubation for long periods by increased NO production at higher concentrations of the tested compound did not happen.

Lead-Optimization and Library Design
The lead compound NO-CIEA 17, in our previous work [16], was used for the efficient design of potent molecules using OpenEye's EON software in lead optimization and library design. EON calculates the electrostatic similarity between the designed compounds and the existing lead compound, NO-CIEA 17, in the form of an Electrostatic Tanimoto (ET) score [37]. The lower EON-ranked compounds have the highest shape and electrostatic similarity to the reference, as shown in Table 4. * The value of electrostatic Tanimoto (ET) using only the coulombic part of Poisson-Boltzmann (PB) electrostatics. ** The value of ET using full PB electrostatics. *** The shape Tanimoto between the given molecule and the query. **** Sum of ET-pb and EON-shape-tani.

Structure-Based Design
A molecular docking study was carried out to examine the binding modes of the designed compounds in the active sites of EGFR (PDB:ID 1M17), ERK2 (PDB:ID 5NHJ), MEK1 (PDB:ID 1S9J), and MRP1 (PDB:ID 2CBZ) using the OpenEye Scientific software [38], as shown in Table 5. Among the docked compounds, 14a scored the best Chemgauss4 scores within the target active sites [39]. As shown in Figure 8A, the FRED view of compound 14a inside EGFR active site showed an overlay with the co-crystallized ligand erlotinib in addition to the inverted binding mode to the lead NO-CIEA I with three hydrogen bonding formations between (1) C-19-OH and a hydroxyl group of THR:830:A with bond length of 1.54 Å, (2) C23-OH and a hydroxyl group of THR:766:A with bond length of 2.22 Å, and (3) C23-OH and a carbonyl group of LEU:764:A with bond length of 1.59 Å. Inside the active site of both ERK2 and MEK1, compound 14a scored the highest Chemgauss4 overlaying with the co-crystallized ligands with hydrophobic interactions as shown in Figure 8B,C, respectively. In the active site of MRP1, the compound 14a showed an overlay with the adenosine moiety of the ATP ligand, forming a hydrogen bond coming from C23-OH and the hydroxyl group of THR:666:A with bond length of 1.91 Å ( Figure 8D).

General Chemical Techniques
1 H and 13 C NMR spectra were acquired on a Bruker AVANCE-400 or 600 MHz NMR spectrometer, in DMSO-d6, CDCl3, or (CD3)2CO using the solvent residual peak as the in- As a result, the rigidification of the D-ring through the installation of a double bond at C-16, particularly in compound 14a with R-configuration around C-19, improved the binding affinity toward the tested protein active sites, which might serve as a good candidate for EGFR-ERK signal transduction inhibitors.

General Chemical Techniques
1 H and 13 C NMR spectra were acquired on a Bruker AVANCE-400 or 600 MHz NMR spectrometer, in DMSO-d 6 , CDCl 3, or (CD 3 ) 2 CO using the solvent residual peak as the internal standard, with the reporting of coupling constants in Hz and the signal multiplicities are reported as singlet (s), doublet (d), triplet (t), quartet (q), doublet of doublets (dd), doublet of triplets (dt), multiplet (m), or broad (br). HRMS data were obtained using EI ionization on a ThermoFinnigan MAT 95 XL mass spectrometer at Buffalo mass spectroscopy facility, Buffalo, NY, USA. TLC analysis was performed using pre-coated silica gel PE sheets. Products were purified via column chromatography using silica gel 40-63 um (230-400 mesh) and prep-plates (Alltech preparative column, Econosil C18 10u, length 250 mm, ID 22 mm). All reagents and solvents were obtained from commercial suppliers and used as received. All chemical reactions requiring anhydrous conditions were performed with oven-dried glassware under an atmosphere of nitrogen.

General Procedure A for C3 Phenolic OH Protection
To a stirred solution of crude mixture of phenolic enones 5 and 6 (0.51 mmol) in DMF (1.5 mL), imidazole (0.092 g, 1.35 mmol) and TBSCl (0.113 g, 0.75 mmol) were added. The reaction mixture was stirred for 12 h at room temperature then extracted with ethyl acetate (3 × 10 mL) and evaporated to give a brown oil. Column chromatography (10-30% ethyl acetate in hexane) was used to purify the crude products, which gives the target enone tert-butyldimethylsilyl ether 7 (62.20 %) as a white powder in addition to the side product 8 (24.15 %) as a white powder [23,44,45].

General Procedure C for Preparation of the Key Intermediated α-Hydroxy Ketones
To a stirred solution of diastereomers 9 (0.54 g, 1.06 mmol) in a dry diethyl ether (3.10 mL), methyl lithium (2.00 mL, 3.19 mmol) was added drop wise at 0 • C. The reaction mixture was stirred for 2 h at 0 • C, and then quenched by adding glacial acetic acid (0.40 mL) in one portion at 0 • C and allowed to stir for 30 min at 0 • C. Saturated sodium bicarbonate solution was added to neutralize the acidic mixture, and the aqueous layer was extracted by ethyl acetate (3 × 50 mL), dried under anhydrous sodium sulfate, and then concentrated under vacuo. The resulting crude products were separated by silica gel column chromatography (5% ethyl acetate in hexane) to give the hydroxyl methyl ketones 11a (25%) and 11b (37%) and the side product 8 (10.20%) [26].

General Procedure D for Preparation of C-17 CUCs Side Chain Precursors
To a stirred solution of α-hydroxy ketone 11a,b (0.500 g, 0.79 mmol) in THF (1.6 mL), LDA (1.42 mL, 2.83 mmol, 2 M in THF) was added at −78 • C and stirred for 1 h. A solution of 2-((tert-butyldimethylsilyl)oxy)-2-methylpropanal (0.3997 g, 1.98 mmol) in THF (5.3 mL) was then added at −78 • C, and the reaction mixture was allowed to slowly warm to room temperature and stirred for 20 h. The reaction mixture was then quenched by the addition of saturated NH 4 Cl (20 mL), followed by extraction with EtOAc (3 × 50 mL), dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by silica gel column chromatography with hexanes/EtOAc (9.5:0.5) to yield the desired products 12a,b.

General Procedure E for Aldol Condensation
In an open 20 mL glass vial, aromatic aldehyde derivatives (1.5 mmol) and NaOH (2.9 mmol) were added to a solution of hydroxyl methyl ketone 11a,b and 19 (1 mmol) in 1 mL dry THF. The reaction mixture was heated at 110 • C for 10-45 min and THF was continuously added at a rate of~0.5 mL per 1 min till the completion of the reaction (monitored by TLC). The reaction mixture was quenched with water and then the aqueous layer was extracted using ethyl acetate (3 × 10 mL), dried over anhydrous sodium sulfate, and concentrated under vacuo. The crude product was then purified using silica gel column chromatography (5% ethyl acetate in hexane) to yield enone products 15a-j and NO-CIETA 20a-d in good yields [46,47].

General Procedure F for Deprotection
To a stirred solution of protected enone 12a,b, 15a-j, and 11b (1 mmol) in THF (3 mL), TBAF (3.1 mmol) was added and stirred for 12 h. The reaction mixture was quenched with saturated ammonium chloride solution and then extracted with ethyl acetate (3 × 20 mL), dried over sodium sulfate anhydrous, filtrated, and concentrated in vacuo. Silica gel column chromatography was used to purify the crude material (15 % ethyl acetate in hexane) to afford NO-CIETA 13a,b, 16a-j, and 18 [18].

DAF-FM DA Assay for Intracellular Measurement of NO
The NO-sensitive fluorophore DAF-FM DA was used for quantification of NO intracellular levels. Briefly, HepG2 cells were seeded in a six-well plate (7.5 × 10 5 cells/well) and allowed to grow overnight. Cells were incubated for 30 min with 2 mL per well of 2.5 µM DAF-FM DA at 37 • C and 5% CO 2 , rinsed twice, and then loaded with different concentrations of target compounds. After incubation time (1, 6, 12, and 24 h), cells were washed twice with HPSS, trypsinized, and pipetted into flow cytometer test tubes. The fluorescence formed intracellularly was analyzed by BD Acuuri C6 flow cytometer (Becton-Dickinson, Mountain View, CA, USA) with the excitation source at 495 nm and emission at 515 nm. Data are expressed as mean values of at least two runs ± SD [33,49,57,58].

Computational Studies
OpenEye ® software (version 2022.2.1) was used for molecular Modeling studies. This software package generates chemguas4 scoring, which is a filtering process to obtain virtual binding affinity, the lower score, the better the binding affinity of the ligands toward the receptor. Briefly, a virtual library of NO-∆-16-CIEAs was energy minimized using MMFF94 force field, followed by generation of multi-conformers using OMEGA ® application. The whole library was then subjected to shape and electrostatic similarity with the lead compound NO-CIEA I using EON application. The designed library was then docked along with the prepared EGFR (PDB:ID 1M17), ERK2 (PDB:ID 5NHJ), MEK1 (PDB:ID 1S9J), and MRP1 (PDB:ID 2CBZ) using FRED ® application. Finally, Vida application was used to show the potential binding interactions of the ligands to the receptor of interest [37][38][39][59][60][61][62].

Conclusions
A new series of NO-∆-16-CIEAs was synthesized and evaluated for their anti-prolifera tive activity against hepatocellular carcinoma. Among them, 14a,b showed broad-spectrum potent anti-proliferative activity against both HepG2 and HepG2-R cell lines in comparison to erlotinib. Moreover, compound 17a showed remarkably high inhibitory activity against the HepG2 cell line comparable to erlotinib, while 17b did not show any activity at a concentration as high as 50 µM. Furthermore, compounds 17g and 17h were almost equipotent to Erlotinib. Compound 14a showed a significant increase in fluorescence after 6 h of incubation in a dose-dependent manner and produced amounts of NO in tumor cells comparable to the reference prodrug JS-K. Compound 14a arrested the cells in the S phase while 14b arrested the cells in the G2/M phase of the cell cycle. Compound 14a has a slight inhibitory activity toward the EGFR/MEK/MAPK pathway as shown by In-Cell-Based ELISA screening. It also showed a significant reduction in MRP2 expression in the HepG2-R cell line in a dose-dependent manner, demonstrating a significant impact on the chemotherapeutic resistance. The molecular docking studies were found to be well correlated with cell study. The findings indicated that ∆-16 unsaturated analog 14a with R configuration around C-19 and furoxan moiety at C-3 improved the anticancer activity twofold more than the corresponding estratriene analog NO-CIEA 17. The data support further investigation of 14a as a promising anti-liver cancer agent.