Repurposing Synthetic Congeners of a Natural Product Aurone Unveils a Lead Antitumor Agent Inhibiting Folded P-Loop Conformation of MET Receptor Tyrosine Kinase

A library of 24 congeners of the natural product sulfuretin were evaluated against nine panels representing nine cancer diseases. While sulfuretin elicited very weak activities at 10 µM concentration, congener 1t was identified as a potential compound triggering growth inhibition of diverse cell lines. Mechanistic studies in HCT116 colon cancer cells revealed that congener 1t dose-dependently increased levels of cleaved-caspases 8 and 9 and cleaved-PARP, while it concentration-dependently decreased levels of CDK4, CDK6, Cdc25A, and Cyclin D and E resulting in induction of cell cycle arrest and apoptosis in colon cancer HCT116 cells. Mechanistic study also presented MET receptor tyrosine kinase as the molecular target mediating the anticancer activity of compound 1t in HCT116 cells. In silico study predicted folded p-loop conformation as the form of MET receptor tyrosine kinase responsible for binding of compound 1t. Together, the current study presents compound 1t as an interesting anticancer lead for further development.


Introduction
Globally, cancer is the second leading cause of death amongst people in developed and developing countries [1][2][3][4][5].In fact, cancer refers to a group of diseases characterized by common hallmarks including hyperproliferation, stress response survival, angiogenesis, invasive growth, metastasis, and metabolic reprogramming, as well as an altered microenvironment and immune response [6][7][8].Being a complex multifactorial disease, cancer treatments employing multifunctional molecules targeting more than one disease component might be more effective and prone to evolution of resistance [9][10][11][12][13][14].
Inflammation is an axial component in cancer diseases.While acute inflammation response can promote tumor suppression, chronic inflammation was found to create an immunosuppressive microenvironment [15][16][17][18][19][20][21][22].In addition, several inflammatory mediators and signaling pathways such as PGE 2 , cytokines, and NF-κB promote survival, invasion, and metastasis of cancer cells [15].Consequently, targeting inflammation signaling pathways and production of inflammatory mediators has been clinically included in cancer therapy [15].Proliferation of cells follows sequential steps of cell cycle phases that involve duplication of cells' DNA and organelles followed by cell division.Several signaling pathways are involved in initiation and regulation of these phases.Within cancer cells, genetic mutations and/or epigenetic factors usually results in dysregulation of pathways promoting and/or repressing cell cycle and proliferation [23][24][25][26][27][28][29][30][31][32].Therefore, agents interrupting and interfering with the cell cycle are helpful antitumor agents stalling the growth of cancers tissues.In addition, programmed cell death, which is a cell's active self-destruction that might be triggered by apoptosis, necrosis, or autophagy, is a mechanism that several anticancer agents can trigger, inducing death in cancer cells [33][34][35][36].Such programmed cell death occurs in response to several signaling pathways and, thus, agents that impact these pathways could be useful anticancer agents.
Since ancient history, nature has served as a major source for drug discovery and development.Indeed, natural-product-based drug discovery affords higher success rates [37][38][39][40][41].The fact that natural products are privileged structures might be the reason for these higher success rates.In lieu of the above-mentioned literature reports, we have embarked on natural-product-based drug discovery towards the development of multifunctional molecules with anti-inflammatory and antiproliferative activity.Herein, we report our approach and results.

Results and Discussion
2.1.Rational of Profiling a Focused Library of Sulfuretin-Congeners as Anticancer Agents Sulfuretin (Figure 1), an aurone bearing meta and para-dihydroxy substituents at ring-B and a C 6 -hydroxy moiety at ring-A, is a natural product obtained from Rhus verniciflua which exhibits interesting properties [42].Interestingly, sulfuretin was found to induce apoptosis through activation of Fas, Caspase-8, and the mitochondrial death pathway [42].In addition, sulfuretin was reported to elicit anticancer effects, inhibiting cell invasion through inhibition of NF-κB which triggers downregulation of MMP-9 expression [43].Despite the interesting anticancer activity of sulfuretin, these reports are limited, as they incorporated a minimal number of cell lines and, thus, would be far from providing sufficient data on the anticancer spectrum and potency of sulfuretin.
Proliferation of cells follows sequential steps of cell cycle phases that involve duplication of cells' DNA and organelles followed by cell division.Several signaling pathways are involved in initiation and regulation of these phases.Within cancer cells, genetic mutations and/or epigenetic factors usually results in dysregulation of pathways promoting and/or repressing cell cycle and proliferation [23][24][25][26][27][28][29][30][31][32].Therefore, agents interrupting and interfering with the cell cycle are helpful antitumor agents stalling the growth of cancers tissues.In addition, programmed cell death, which is a cell's active self-destruction that might be triggered by apoptosis, necrosis, or autophagy, is a mechanism that several anticancer agents can trigger, inducing death in cancer cells [33][34][35][36].Such programmed cell death occurs in response to several signaling pathways and, thus, agents that impact these pathways could be useful anticancer agents.
Since ancient history, nature has served as a major source for drug discovery and development.Indeed, natural-product-based drug discovery affords higher success rates [37][38][39][40][41].The fact that natural products are privileged structures might be the reason for these higher success rates.In lieu of the above-mentioned literature reports, we have embarked on natural-product-based drug discovery towards the development of multifunctional molecules with anti-inflammatory and antiproliferative activity.Herein, we report our approach and results.

Rational of Profiling a Focused Library of Sulfuretin-Congeners as Anticancer Agents
Sulfuretin (Figure 1), an aurone bearing meta and para-dihydroxy substituents at ring-B and a C 6 -hydroxy moiety at ring-A, is a natural product obtained from Rhus verniciflua which exhibits interesting properties [42].Interestingly, sulfuretin was found to induce apoptosis through activation of Fas, Caspase-8, and the mitochondrial death pathway [42].In addition, sulfuretin was reported to elicit anticancer effects, inhibiting cell invasion through inhibition of NF-κB which triggers downregulation of MMP-9 expression [43].Despite the interesting anticancer activity of sulfuretin, these reports are limited, as they incorporated a minimal number of cell lines and, thus, would be far from providing sufficient data on the anticancer spectrum and potency of sulfuretin.In addition to the antiproliferative activity, sulfuretin was also reported to show potential anti-inflammatory effects through inhibition of the NF-κB signaling pathway, a common pathway for inflammation and cancer [44].However, hispidol (Figure 1), a congener of sulfuretin, was found to be a more potent inhibitor of macrophage production of PGE 2 , an inflammatory mediator that is correlated with both inflammation and cancer [45,46].Nev-ertheless, there is no reported study, to the best of our knowledge, on possible anticancer effects of the sulfuretin congener, hispidol.
Considering the above-mentioned information, the current work was planned to address the profiling of the anticancer activity of sulfuretin against nine panels including diverse cancer cell lines belonging to nine cancer diseases of different origins to explore its anticancer spectrum and potency.Considering that different oxygenation patterns in the form of hydroxy and/or methoxy moieties is a common structural feature in natural products, a library of twenty-four sulfuretin congeners bearing diverse oxygenation patterns on ring-B and a C 6 -hydroxy or methoxy moiety on ring-A (Figure 1), and reported antiinflammatory activity, were selected for profiling their anticancer spectrum in an attempt to obtain insights into SAR against the same panels of diverse cancers used for sulfuretin.The list of the compounds as well as their reported anti-inflammatory effects are presented in Table 1.
Table 1.Sulfuretin and list of its congeners (1a-1x) employed in profiling anticancer activity and their literature reported inhibition of PGE 2 production.
In addition to the antiproliferative activity, sulfuretin was also reported to show potential anti-inflammatory effects through inhibition of the NF-κB signaling pathway, a common pathway for inflammation and cancer [44].However, hispidol (Figure 1), a congener of sulfuretin, was found to be a more potent inhibitor of macrophage production of PGE2, an inflammatory mediator that is correlated with both inflammation and cancer [45,46].Nevertheless, there is no reported study, to the best of our knowledge, on possible anticancer effects of the sulfuretin congener, hispidol.
Considering the above-mentioned information, the current work was planned to address the profiling of the anticancer activity of sulfuretin against nine panels including diverse cancer cell lines belonging to nine cancer diseases of different origins to explore its anticancer spectrum and potency.Considering that different oxygenation patterns in the form of hydroxy and/or methoxy moieties is a common structural feature in natural products, a library of twenty-four sulfuretin congeners bearing diverse oxygenation patterns on ring-B and a C 6 -hydroxy or methoxy moiety on ring-A (Figure 1), and reported anti-inflammatory activity, were selected for profiling their anticancer spectrum in an attempt to obtain insights into SAR against the same panels of diverse cancers used for sulfuretin.The list of the compounds as well as their reported anti-inflammatory effects are presented in Table 1.

Blood Cancers
Blood cancers are a group of diverse cancers that may originate from lymphoblastic or myeloid progenitor cells and, furthermore, might be acute or chronic.Towards near inclusive evaluation, five cell lines were employed including: (1) CCRFCEM, a childhood T acute lymphoblastic leukemia (ALL); (2) MOLT4, an acute lymphoblastic leukemia (ALL); (3) HL60(TB), an acute myeloid leukemia (AML); (4) K562, a chronic myelogenous leukemia (CML); and (5) RPMI8226, a multiple myeloma (MM).At the tested concentration, sulfuretin showed very limited activity against CCRFCEM and MOLT4 (Figure 2); Blood cancers are a group of diverse cancers that may originate from lymphoblastic or myeloid progenitor cells and, furthermore, might be acute or chronic.Towards near inclusive evaluation, five cell lines were employed including: (1) CCRFCEM, a childhood T acute lymphoblastic leukemia (ALL); (2) MOLT4, an acute lymphoblastic leukemia (ALL); (3) HL60(TB), an acute myeloid leukemia (AML); (4) K562, a chronic myelogenous leukemia (CML); and (5) RPMI8226, a multiple myeloma (MM).At the tested concentration, sulfuretin showed very limited activity against CCRFCEM and MOLT4 (Figure 2); both are acute lymphoblastic leukemia, ALL.It was almost ineffective against the employed cell lines of myeloid origin (HL60(TB), K562, and RPMI8226).In contrast, the most effective compound was derivative 1t combining polar, hydrogen-bond-donor or acceptor dihydroxy moieties at vicinal orthoand meta-positions of ring-B with the more steric, less polar hydrogenbond-donor-only methoxy moiety at the C 6 -position of ring-A (Figure 2).It was most effective against the ALL cell lines CCRFCEM and MOLT4, triggering growth inhibition near 73% and 58%, respectively.It was less active against cell lines of myeloid origin, especially the CML and K562 cell lines.Shifting the dihydroxy moieties to vicinal metaand para-positions of ring-B of compound 1t to afford derivative 1v resulted in lowered activity (Figure 2), but loss of activity was found for derivative 1u upon placing the dihydroxy moieties at orthoand para-positions of ring-B, i.e., the ring carbons bearing the dihydroxy moieties were separated by one carbon.In comparison, all compounds 1d-1e having dihydroxy moieties at ring-B but possessing a C 6 -hydroxy moiety on ring-A were of low to no activity at the tested concentration regardless of the position of the dihydroxy moieties on ring-B.Compounds incorporating only one hydroxy moiety on ring-B combined with the C 6 -methoxy moiety on ring-A (compounds 1q-1s) or C 6 -hydroxy moiety on ring-A (compounds 1a-1c), derivatives having a methoxy moiety on ring-B combined with C 6 -hydroxy moiety on ring-A (compounds 1h-1j) or C 6 -methoxy moiety on ring-A (compounds 1w and 1x), and trihydroxylated or trimethoxylated ring-B derivatives (compounds 1g and 1o) were found to be almost inactive at the tested concentration.Such an outcome, coupled with the found low to no activity of compounds 1k-1m possessing dimethoxy moieties at ring-B combined with a C 6 -hydroxy moiety on ring-A, might support the conclusion that the vicinal dihydroxy moieties at orthoand meta-positions of ring-B combined with a C 6 -methoxy moiety on ring-A might be optimal for activity.However, compound 1n possessing dimethoxy moieties at both meta-positions of ring-B showed potential activity.Interestingly, its activity was higher on HL60(TB) of myeloid origin; this represents a distinct pattern that is different from compounds 1t and 1v which were more active on cell lines of lymphoblastic origin.
both are acute lymphoblastic leukemia, ALL.It was almost ineffective against the employed cell lines of myeloid origin (HL60(TB), K562, and RPMI8226).In contrast, the most effective compound was derivative 1t combining polar, hydrogen-bond-donor or acceptor dihydroxy moieties at vicinal ortho-and meta-positions of ring-B with the more steric, less polar hydrogen-bond-donor-only methoxy moiety at the C 6 -position of ring-A (Figure 2).It was most effective against the ALL cell lines CCRFCEM and MOLT4, triggering growth inhibition near 73% and 58%, respectively.It was less active against cell lines of myeloid origin, especially the CML and K562 cell lines.Shifting the dihydroxy moieties to vicinal meta-and para-positions of ring-B of compound 1t to afford derivative 1v resulted in lowered activity (Figure 2), but loss of activity was found for derivative 1u upon placing the dihydroxy moieties at ortho-and para-positions of ring-B, i.e., the ring carbons bearing the dihydroxy moieties were separated by one carbon.In comparison, all compounds 1d-1e having dihydroxy moieties at ring-B but possessing a C 6 -hydroxy moiety on ring-A were of low to no activity at the tested concentration regardless of the position of the dihydroxy moieties on ring-B.Compounds incorporating only one hydroxy moiety on ring-B combined with the C 6 -methoxy moiety on ring-A (compounds 1q-1s) or C 6 -hydroxy moiety on ring-A (compounds 1a-1c), derivatives having a methoxy moiety on ring-B combined with C 6 -hydroxy moiety on ring-A (compounds 1h-1j) or C 6 -methoxy moiety on ring-A (compounds 1w and 1x), and trihydroxylated or trimethoxylated ring-B derivatives (compounds 1g and 1o) were found to be almost inactive at the tested concentration.Such an outcome, coupled with the found low to no activity of compounds 1k-1m possessing dimethoxy moieties at ring-B combined with a C 6 -hydroxy moiety on ring-A, might support the conclusion that the vicinal dihydroxy moieties at ortho-and meta-positions of ring-B combined with a C 6 -methoxy moiety on ring-A might be optimal for activity.However, compound 1n possessing dimethoxy moieties at both meta-positions of ring-B showed potential activity.Interestingly, its activity was higher on HL60(TB) of myeloid origin; this represents a distinct pattern that is different from compounds 1t and 1v which were more active on cell lines of lymphoblastic origin.

Non-Small-Cell Lung Cancer
Non-small lung cancer involves multiple subtypes.Accordingly, compounds 1a-1x were profiled against lung adenocarcinoma cells (A549, EKVX, HOP62, and H23), bronchoalveolar carcinoma cells (H322M), and large-cell lung carcinoma cell lines (HOP92 and H460).As shown in Figure 3, an almost null activity was elicited by sulfuretin at the tested concentration.In contrast, two potential compounds were identified (Figure 3), which were compound 1t combining polar, hydrogen-bond-donor or acceptor dihydroxy moieties at vicinal orthoand meta-positions of ring-B with the more steric, less polar hydrogen-bond-donor methoxy moiety at C 6 -position of ring-A and compound 1n having a C 6 -hydroxy moiety ring-A but possessing the less polar dimethoxy moieties at both meta-positions of ring-B.Both compounds elicited almost the same level of activity against the bronchoalveolar carcinoma cell line H322M and the human large-cell lung carcinoma cell line H460, but not HOP92.However, compound 1t was more active against lung adenocarcinoma cell lines A549, EKVX, HOP62, and H23, suggesting different molecular targets impacted by compounds 1t and 1n.Except for compounds 1t and 1n, the activity was limited for all other compounds whether bearing a C 6 -hydroxy moiety at ring-A combined with hydroxy or methoxy moieties on ring-B (compounds 1a-1g and 1h-1p, respectively; Figure 3) or bearing a C 6 -methoxy moiety at ring-A combined with hydroxy or methoxy moieties on ring-B (compounds 1q-1v, 1w, and 1x; Figure 3).

Non-Small-Cell Lung Cancer
Non-small lung cancer involves multiple subtypes.Accordingly, compounds 1a-1x were profiled against lung adenocarcinoma cells (A549, EKVX, HOP62, and H23), bronchoalveolar carcinoma cells (H322M), and large-cell lung carcinoma cell lines (HOP92 and H460).As shown in Figure 3, an almost null activity was elicited by sulfuretin at the tested concentration.In contrast, two potential compounds were identified (Figure 3), which were compound 1t combining polar, hydrogen-bond-donor or acceptor dihydroxy moieties at vicinal ortho-and meta-positions of ring-B with the more steric, less polar hydrogenbond-donor methoxy moiety at C 6 -position of ring-A and compound 1n having a C 6 -hydroxy moiety ring-A but possessing the less polar dimethoxy moieties at both meta-positions of ring-B.Both compounds elicited almost the same level of activity against the bronchoalveolar carcinoma cell line H322M and the human large-cell lung carcinoma cell line H460, but not HOP92.However, compound 1t was more active against lung adenocarcinoma cell lines A549, EKVX, HOP62, and H23, suggesting different molecular targets impacted by compounds 1t and 1n.Except for compounds 1t and 1n, the activity was limited for all other compounds whether bearing a C 6 -hydroxy moiety at ring-A combined with hydroxy or methoxy moieties on ring-B (compounds 1a-1g and 1h-1p, respectively; Figure 3) or bearing a C 6 -methoxy moiety at ring-A combined with hydroxy or methoxy moieties on ring-B (compounds 1q-1v, 1w, and 1x; Figure 3).

Colorectal Cancer
Colon adenocarcinoma is the most common type of colon cancers.Consequently, sulfuretin and its congeners were profiled against five colorectal adenocarcinoma cell lines (HCC2998, HCT116, HCT15, KM12, and SW620).In regards to sulfuretin, the growth of only two cell lines (HCT15 and KM12) was weakly inhibited by almost 10% while no inhibition was observed at all against the other three cell lines at the tested concentration (Figure 4).In consensus with the activity pattern revealed from profiling sulfuretin congeners against non-small lung cancers, the results showed that compounds 1t and 1n were the most active.Out of the five cell lines used, the highest growth inhibition activities were measured against HCT116 and HCT15 cell lines (Figure 4).It became clear from profiling results that, in general, all other compounds, whether bearing a C 6 -hydroxy moiety at

Colorectal Cancer
Colon adenocarcinoma is the most common type of colon cancers.Consequently, sulfuretin and its congeners were profiled against five colorectal adenocarcinoma cell lines (HCC2998, HCT116, HCT15, KM12, and SW620).In regards to sulfuretin, the growth of only two cell lines (HCT15 and KM12) was weakly inhibited by almost 10% while no inhibition was observed at all against the other three cell lines at the tested concentration (Figure 4).In consensus with the activity pattern revealed from profiling sulfuretin congeners against non-small lung cancers, the results showed that compounds 1t and 1n were the most active.Out of the five cell lines used, the highest growth inhibition activities were measured against HCT116 and HCT15 cell lines (Figure 4).It became clear from profiling results that, in general, all other compounds, whether bearing a C 6 -hydroxy moiety at ring-A combined with hydroxy or methoxy moieties on ring-B (compounds 1a-1g and 1h-1p, respectively; Figure 4) or bearing a C 6 -methoxy moiety at ring-A combined with hydroxy or methoxy moieties on ring-B (compounds 1q-1v, 1w and 1x; Figure 4), are very weak growth inhibitors for the tested colorectal cancer cell lines.It might be concluded that the structural features of compounds 1t and 1n render them possible hit compounds.
ring-A combined with hydroxy or methoxy moieties on ring-B (compounds 1a-1g and 1h-1p, respectively; Figure 4) or bearing a C 6 -methoxy moiety at ring-A combined with hydroxy or methoxy moieties on ring-B (compounds 1q-1v, 1w and 1x; Figure 4), are very weak growth inhibitors for the tested colorectal cancer cell lines.It might be concluded that the structural features of compounds 1t and 1n render them possible hit compounds.

Brain Cancer
Gliomas are common brain cancers that originate from glial cells.Considering the hematopoietic origin of glial cells [47,48], it might be understandable that profiling results of sulfuretin and its congeners against GBs showed some similarities to the activity pattern against blood cancers rather than against lung and colon cancers.Because of the heterogeneity of gliomas, compounds 1a-1x were profiled against diverse cells including SF268, SNB75, SF295, SF539, SNB19, and U251.As shown in Figure 5, sulfuretin triggered around 20% growth inhibition of astrocytoma cell line SNB75 and glioblastoma cell line SF539 but showed no inhibition at all for glioblastoma multiforme cell line SF295 and only 5% inhibition of anaplastic astrocytoma cell line SF268 and glioblastoma cell lines SNB19 and U251.Meanwhile, the most active amongst the tested compounds was compound 1t, bearing dihydroxy moieties at vicinal ortho-and meta-positions of ring-B coupled with the less polar hydrogen-bond-donor-only methoxy moiety at the C 6 -position of ring-A.It showed almost 10-fold the activity of sulfuretin against the anaplastic astrocytoma SF268 cell line, triggering near 50% growth inhibition.In addition, it induced near 40% growth inhibition of SF539 and U251 glioblastoma cell lines and the SNB75 astrocytoma cell line.Like the activity pattern against blood cancers, compound 1v, incorporating a C 6 -methoxy moiety at ring-A as in compound 1t but with the dihydroxy moieties shifted to vicinal meta-and para-positions of ring-B, showed significant, yet less, activity relative to compound 1t against these four cell lines (Figure 5).Other compounds sharing the C 6 -methoxy moiety at ring-A of compounds 1t and 1v were of much lower activities, regardless of incorporating other hydroxylation or methoxylation patterns on ring-B (compounds 1q-1s, 1u, 1w, and 1x; Figure 5).Out of the compounds having a C 6 -hydroxy moiety on ring-A, compound 1n possessing dimethoxy moieties at both meta-positions of ring-B showed potential activity (Figure 5).It was most active, in order, against the SNB75 astrocytoma cell

Brain Cancer
Gliomas are common brain cancers that originate from glial cells.Considering the hematopoietic origin of glial cells [47,48], it might be understandable that profiling results of sulfuretin and its congeners against GBs showed some similarities to the activity pattern against blood cancers rather than against lung and colon cancers.Because of the heterogeneity of gliomas, compounds 1a-1x were profiled against diverse cells including SF268, SNB75, SF295, SF539, SNB19, and U251.As shown in Figure 5, sulfuretin triggered around 20% growth inhibition of astrocytoma cell line SNB75 and glioblastoma cell line SF539 but showed no inhibition at all for glioblastoma multiforme cell line SF295 and only 5% inhibition of anaplastic astrocytoma cell line SF268 and glioblastoma cell lines SNB19 and U251.Meanwhile, the most active amongst the tested compounds was compound 1t, bearing dihydroxy moieties at vicinal orthoand meta-positions of ring-B coupled with the less polar hydrogen-bond-donor-only methoxy moiety at the C 6 -position of ring-A.It showed almost 10-fold the activity of sulfuretin against the anaplastic astrocytoma SF268 cell line, triggering near 50% growth inhibition.In addition, it induced near 40% growth inhibition of SF539 and U251 glioblastoma cell lines and the SNB75 astrocytoma cell line.Like the activity pattern against blood cancers, compound 1v, incorporating a C 6 -methoxy moiety at ring-A as in compound 1t but with the dihydroxy moieties shifted to vicinal metaand para-positions of ring-B, showed significant, yet less, activity relative to compound 1t against these four cell lines (Figure 5).Other compounds sharing the C 6 -methoxy moiety at ring-A of compounds 1t and 1v were of much lower activities, regardless of incorporating other hydroxylation or methoxylation patterns on ring-B (compounds 1q-1s, 1u, 1w, and 1x; Figure 5).Out of the compounds having a C 6 -hydroxy moiety on ring-A, compound 1n possessing dimethoxy moieties at both meta-positions of ring-B showed potential activity (Figure 5).It was most active, in order, against the SNB75 astrocytoma cell line, U251 glioblastoma cell line, and SF268 anaplastic astrocytoma cell line.Meanwhile, compounds bearing dimethoxy moieties at the distal orthoand meta-positions or metaand para-positions of ring-B (compounds 1l and 1m, respectively) or bearing a monohydroxy moiety at ring-B (compounds 1a-1c) showed low activity.All other tested derivatives of the C 6 -hydroxy series bearing dihydroxy, polyhydroxy or monomethoxy substituents on ring-B were virtually inactive.Collectively, the results suggest compounds 1t, 1v, and 1n as possible hits possessing significant activities against gliomas.
line, U251 glioblastoma cell line, and SF268 anaplastic astrocytoma cell line.Meanwhile, compounds bearing dimethoxy moieties at the distal ortho-and meta-positions or metaand para-positions of ring-B (compounds 1l and 1m, respectively) or bearing a monohydroxy moiety at ring-B (compounds 1a-1c) showed low activity.All other tested derivatives of the C 6 -hydroxy series bearing dihydroxy, polyhydroxy or monomethoxy substituents on ring-B were virtually inactive.Collectively, the results suggest compounds 1t, 1v, and 1n as possible hits possessing significant activities against gliomas.

Skin Cancer
Melanoma is the most aggressive form of skin cancer.Accordingly, compounds 1a-1x were profiled against diverse melanoma cells, including the following: LOXIMVI, M14, MDAMB435, MALME3M, SKMEL28, SKMEL5, UACC257, and UACC62.As revealed from profiling results, sulfuretin was almost ineffective.It showed no inhibition for five cell lines out of the employed eight cell lines and, furthermore, the growth inhibition found for the other three cell lines was less than 8%.In line with the activity pattern found against lung cancers, compounds 1t and 1n were the most active derivatives (Figure 6).The metastatic amelanotic melanoma LOXIMVI was the cell line most affected by compound 1t, showing a growth inhibition of more than 50%.Compound 1t also triggered significant growth inhibition for all other tested melanoma cell lines except for the UACC257 cell line (Figure 6).However, the UACC257 cell line was significantly inhibited by compound 1n and the metastatic amelanotic melanoma M14 cell line was the most impacted by this compound (Figure 6).Considering the different activity profiles of compounds 1t and 1n coupled with structural feature differences, it might be inferred that different molecular targets might be involved in mediating the antiproliferative activities of each of them.Other than compound 1t, derivatives sharing the C 6 -methoxy moiety on ring-A and bearing hydroxy or methoxy moieties on ring-B (compounds 1q-1v, 1w, and 1x; Figure 6) showed limited activities in general.Other than compound 1n, almost all compounds having the C 6 -hydroxy moiety on ring-A and bearing hydroxy or methoxy moieties on ring-B (compounds 1a-1p) showed almost no significant activity except for compound 1l that showed low activity against most of the employed melanoma cell lines.

Skin Cancer
Melanoma is the most aggressive form of skin cancer.Accordingly, compounds 1a-1x were profiled against diverse melanoma cells, including the following: LOXIMVI, M14, MDAMB435, MALME3M, SKMEL28, SKMEL5, UACC257, and UACC62.As revealed from profiling results, sulfuretin was almost ineffective.It showed no inhibition for five cell lines out of the employed eight cell lines and, furthermore, the growth inhibition found for the other three cell lines was less than 8%.In line with the activity pattern found against lung cancers, compounds 1t and 1n were the most active derivatives (Figure 6).The metastatic amelanotic melanoma LOXIMVI was the cell line most affected by compound 1t, showing a growth inhibition of more than 50%.Compound 1t also triggered significant growth inhibition for all other tested melanoma cell lines except for the UACC257 cell line (Figure 6).However, the UACC257 cell line was significantly inhibited by compound 1n and the metastatic amelanotic melanoma M14 cell line was the most impacted by this compound (Figure 6).Considering the different activity profiles of compounds 1t and 1n coupled with structural feature differences, it might be inferred that different molecular targets might be involved in mediating the antiproliferative activities of each of them.Other than compound 1t, derivatives sharing the C 6 -methoxy moiety on ring-A and bearing hydroxy or methoxy moieties on ring-B (compounds 1q-1v, 1w, and 1x; Figure 6) showed limited activities in general.Other than compound 1n, almost all compounds having the C 6 -hydroxy moiety on ring-A and bearing hydroxy or methoxy moieties on ring-B (compounds 1a-1p) showed almost no significant activity except for compound 1l that showed low activity against most of the employed melanoma cell lines.

Ovarian Cancer
High-grade serous ovarian carcinoma (HGSOC) is the most common ovarian cancer.Meanwhile, endometrioid carcinoma is an invasive ovarian cancer.Accordingly, compounds 1a-1x were profiled against IGROV1 (an endometrioid carcinoma), as well as OVCAR3, OVCAR4, OVCAR5, OVCAR8, and ADRRES (all are HGSOC cells).The results showed that sulfuretin was almost ineffective against all employed cell lines.As shown in Figure 7, potential activity was clear for compound 1t which triggered almost 77% growth inhibition of the high-grade ovarian serous adenocarcinoma OVCAR3 cell lines and near 63% growth inhibition of the endometrioid carcinoma IGROV1 cell line.It also inhibited other high-grade ovarian serous adenocarcinoma cell lines by varying degrees.Other compounds sharing the C 6 -methoxy moiety on ring-A present in compound 1t but with other hydroxy or methoxy substitution patterns on ring-B (compounds 1q-1x) were almost inactive except for minor activity for compound 1v.Except for some activity for compound 1n bearing a dimethoxy substitution pattern at both meta-positions of ring-B and, to a lesser extent, compound 1l possessing a dimethoxy substitution pattern at distal orthoand meta-positions of ring-B, all other compounds with the C 6 -hydroxy moiety on ring-A were almost inactive (compounds 1a-1p, Figure 7).

Ovarian Cancer
High-grade serous ovarian carcinoma (HGSOC) is the most common ovarian cancer.Meanwhile, endometrioid carcinoma is an invasive ovarian cancer.Accordingly, compounds 1a-1x were profiled against IGROV1 (an endometrioid carcinoma), as well as OVCAR3, OVCAR4, OVCAR5, OVCAR8, and ADRRES (all are HGSOC cells).The results showed that sulfuretin was almost ineffective against all employed cell lines.As shown in Figure 7, potential activity was clear for compound 1t which triggered almost 77% growth inhibition of the high-grade ovarian serous adenocarcinoma OVCAR3 cell lines and near 63% growth inhibition of the endometrioid carcinoma IGROV1 cell line.It also inhibited other high-grade ovarian serous adenocarcinoma cell lines by varying degrees.Other compounds sharing the C 6 -methoxy moiety on ring-A present in compound 1t but with other hydroxy or methoxy substitution patterns on ring-B (compounds 1q-1x) were almost inactive except for minor activity for compound 1v.Except for some activity for compound 1n bearing a dimethoxy substitution pattern at both meta-positions of ring-B and, to a lesser extent, compound 1l possessing a dimethoxy substitution pattern at distal orthoand meta-positions of ring-B, all other compounds with the C 6 -hydroxy moiety on ring-A were almost inactive (compounds 1a-1p, Figure 7).

Renal Cancer
Renal cell carcinomas (RCC) could be clear-cell renal cell carcinomas (ccRCC; the most common type) or non-clear-cell renal cell carcinomas (nccRCC; heterogeneous and difficult) [49].Consequently, compounds 1a-1x were profiled against a panel composed of the following: (1) 786O, a primary ccRCC cell line; (2) CAKI1, a metastatic ccRCC cell line; (3) ACHN, a metastatic papillary renal cell carcinoma (a subtype of nccRCC); (4) RXF393, an unclassified poorly differentiated renal cell carcinoma; (5) SN12C, an unclassified renal cell carcinoma; and (6) UO31, an unclassified renal cell carcinoma.As illustrated in Figure 8, sulfuretin elicited very weak to no growth inhibition against all types of renal cancer cell lines used.In general, weak to no growth inhibition was the outcome for all derivatives with a C 6 -hydroxy moiety on ring-A coupled with hydroxy functions on ring-B (compounds 1a-1g; Figure 8) or with methoxy functions on ring-B (compounds 1h-1p; Figure 8), except for compound 1n and to a lesser extent compound 1l.Such an activity profile resembles the profiles against colon, melanoma, and ovarian cancers that also showed an increased activity from compound 1n and to a lesser extent compound 1l.The only structural difference between compounds 1n and 1l is that one of the dimethoxy moieties occupying both meta-positions of ring-B in compound 1n is shifted to the ortho-position distant from the other meta-methoxy in compound 1l.The cell lines most inhibited by compound 1n were the metastatic ccRCC CAKI1 cell line, the unclassified renal cell carcinoma UO31 cell line, and, to a lesser extent, the metastatic papillary renal cell carcinoma ACHN cell line.Amongst the compound series sharing the C 6 -methoxy moiety on ring-A, compound 1t elicited potential activity which is in line with the activity profile found against colon, melanoma, and ovarian cancers.Similar to compound 1n, the most affected cell lines for compound 1t were the metastatic ccRCC CAKI1 cell line and the unclassified renal cell carcinoma UO31 cell line.Other compounds with a C 6 -methoxy moiety on ring-A combined with hydroxy functions on ring-B (compounds 1q-1v; Figure 8) or with methoxy functions on ring-B (compounds 1w and 1x; Figure 8) were, in general, much less active, except for a significant inhibition of the unclassified renal cell carcinoma UO31 cell line by compound 1v.Collectively, it might be concluded that the structural features of compounds 1t and 1n suggest them as hit compounds for further development of more potential renal cancer therapeutics.

Renal Cancer
Renal cell carcinomas (RCC) could be clear-cell renal cell carcinomas (ccRCC; the most common type) or non-clear-cell renal cell carcinomas (nccRCC; heterogeneous and difficult) [49].Consequently, compounds 1a-1x were profiled against a panel composed of the following: (1) 786O, a primary ccRCC cell line; (2) CAKI1, a metastatic ccRCC cell line; (3) ACHN, a metastatic papillary renal cell carcinoma (a subtype of nccRCC); (4) RXF393, an unclassified poorly differentiated renal cell carcinoma; (5) SN12C, an unclassified renal cell carcinoma; and (6) UO31, an unclassified renal cell carcinoma.As illustrated in Figure 8, sulfuretin elicited very weak to no growth inhibition against all types of renal cancer cell lines used.In general, weak to no growth inhibition was the outcome for all derivatives with a C 6 -hydroxy moiety on ring-A coupled with hydroxy functions on ring-B (compounds 1a-1g; Figure 8) or with methoxy functions on ring-B (compounds 1h-1p; Figure 8), except for compound 1n and to a lesser extent compound 1l.Such an activity profile resembles the profiles against colon, melanoma, and ovarian cancers that also showed an increased activity from compound 1n and to a lesser extent compound 1l.

Prostate Cancer
Compounds 1a-1x were profiled against two metastatic prostate adenocarcinoma cell lines, PC3 and DU145.As shown in Figure 9, sulfuretin was completely ineffective at the tested concentration against both cell lines.Similarly, all compounds 1a-1g having hydroxy groups on ring-B in conjunction with a C 6 -hydroxy moiety on ring-A had negligible to no effect on both metastatic DU145 and PC3 cell lines.Replacement of the hydroxy substitution patterns on ring-B by methoxy substitution patterns enabled three compounds to trigger growth inhibition of the metastatic DU145 cell line (1i, 1l, and 1n, Figure 9).These three compounds have a common methoxy group in the meta-position on ring-B.The most effective amongst them, compound 1n, has another methoxy moiety at the second meta-position of ring-B; the least active, compound 1i, has no other methoxy moiety on ring-B, while the intermediately active compound 1l possesses a second methoxy group shifted by one carbon relative to that of compound 1n to occupy the ortho-position distant from first methoxy meta-position on ring-B.However, compounds 1k and 1m with a methoxy moiety at meta-position of ring-B and a second methoxy moiety at the vicinal orthoor para-positions on ring-B were inactive.It might be inferred from this that a molecular target in the DU145 cell line, but absent or less expressed in the PC3 cell line, is impacted by compounds with a methoxy moiety at the meta-position on ring-B coupled with a C 6 -hydroxy function on ring-A.It might be also concluded that interactions with such molecular targets are increased by the presence of a second methoxy moiety at the other meta-position and to a lesser degree at the distant ortho-position, but not the paraor the vicinal ortho-position.On the other hand, compounds 1t and 1v possessing dihydroxy moieties on ring-B where one of these hydroxy groups were at the meta-position of ring-B were the active compounds amongst derivatives having a C 6 -methoxy moiety on ring-A.Out of them, compound 1t, having the dihydroxy groups at the vicinal orthoand meta-positions of ring-B, was more active than compound 1v, itself having the dihydroxy groups at the vicinal metaand para-positions (Figure 9).Compounds 1n and 1t might possibly be nominated as hit compounds inhibiting the metastatic DU145 prostate adenocarcinoma cell line.

Prostate Cancer
Compounds 1a-1x were profiled against two metastatic prostate adenocarcinoma cell lines, PC3 and DU145.As shown in Figure 9, sulfuretin was completely ineffective at the tested concentration against both cell lines.Similarly, all compounds 1a-1g having hydroxy groups on ring-B in conjunction with a C 6 -hydroxy moiety on ring-A had negligible to no effect on both metastatic DU145 and PC3 cell lines.Replacement of the hydroxy substitution patterns on ring-B by methoxy substitution patterns enabled three compounds to trigger growth inhibition of the metastatic DU145 cell line (1i, 1l, and 1n, Figure 9).These three compounds have a common methoxy group in the meta-position on ring-B.The most effective amongst them, compound 1n, has another methoxy moiety at the second meta-position of ring-B; the least active, compound 1i, has no other methoxy moiety on ring-B, while the intermediately active compound 1l possesses a second methoxy group shifted by one carbon relative to that of compound 1n to occupy the ortho-position distant from first methoxy meta-position on ring-B.However, compounds 1k and 1m with a methoxy moiety at meta-position of ring-B and a second methoxy moiety at the vicinal ortho-or para-positions on ring-B were inactive.It might be inferred from this that a mo-

Breast Cancer
Breast cancer could be hormone-sensitive or resistant and, in addition, primary or metastatic.Accordingly, compounds 1a-1x were profiled against four breast cancer cells including the following: (1) MCF7, a metastatic invasive hormone-responsive breast adenocarcinoma; (2) BT549, a primary triple-negative invasive breast adenocarcinoma; (3) MDAMB231, a metastatic triple-negative breast adenocarcinoma; and (4) MDAMB468, a metastatic triple-negative breast adenocarcinoma.As shown in Figure 10, sulfuretin had almost negligible to no activity against the cell lines employed.As revealed from the results, the profile of sulfuretin congeners' activity against the primary triple-negative BT549 cell line was distinct from the activity profile against the metastatic hormone-responsive MCF7 cell line and metastatic MDAMB231 and MDAMB468 triple-negative cell lines.In general, all C 6 -hydroxy derived compounds having hydroxy substituents on ring-B did not inhibit the growth of any primary or metastatic cell lines (compounds 1a-1g, Figure 10).Meanwhile, out of the C 6 -hydroxy derived compounds 1h-1p having methoxy substituents at ring-B, compounds 1i, 1l, and 1n with at least one methoxy substituent at meta-position of ring-B or compound 1j with one methoxy substituent at the para-position of ring-B triggered growth inhibition of the primary triple-negative BT549 cell line (Figure 10).Amongst them, the most effective was compound 1n, possessing two methoxy moieties at both meta-positions of ring-B, while the equally active compounds 1i and 1l have either only one methoxy moiety at meta-position of ring-B or the second methoxy moiety shifted by one carbon relative to that of compound 1n to occupy the ortho-position distant from first methoxy meta-position on ring-B.This indicates that a methoxy moiety at ortho-position of ring-B has no role in mediating the activity, which might be supported by the found no activity of compound 1h with one methoxy moiety at ortho-position of ring-B.In the case of the least active compound 1j, it has only one methoxy moiety but shifted to the para-position of ring-B.Accordingly, it might be inferred that the methoxy moiety at meta-position is more optimal for activity against the primary triple-negative BT549 cell line.In the case of metastatic cell lines, all C 6 -hydroxy derived compounds 1h-1p with methoxy substituents at ring-B did not trigger significant growth inhibition except for compound 1n, possessing two methoxy moieties at both meta-positions of ring-B, that showed some inhibition of the metastatic hormone-responsive MCF7 cell line.However, some members of C 6 -methoxy derived compounds 1q-1v, having hydroxy substituents at ring-B, showed potential inhibition of the metastatic hormone-responsive MCF7 cell line (Figure 10).As revealed from the results, this antiproliferative activity against the metastatic hormone-responsive MCF7 cells might be associated with hydroxy moieties at the orthoand meta-positions of ring-B (compounds 1q and 1r, respectively).When both vicinal orthoand meta-positions were substituted simultaneously by dihydroxy moieties, the activity was more enhanced (compound 1t).In addition, compound 1t, amongst the C 6 -methoxy derived compounds 1q-1x, showed the best inhibition of the growth of the primary triple-negative BT549 cell line and metastatic triple-negative MDAMB231 cell line.However, the growth of the triple-negative MDAMB468 cell was more inhibited by compound 1w, possessing a ortho-methoxy substituent on ring-B relative to compound 1t possessing vicinal orthoand meta-methoxy substituents on ring-B.Such a difference in structural feature requirements suggests the involvement of different molecular targets.Collectively, the results might nominate compound 1t as a hit candidate for the development of antiproliferative compounds against metastatic hormone responsive MCF7 or triple-negative primary and metastatic triple-negative BT549 and MDAMB231 breast cancers.In addition, compound 1n might be nominated as a hit compound against primary triple-negative BT549 cells.

Compound 1t Induces Cell Cycle Arrest in HCT116 Colon Cancer Cells
To assess the mechanism mediating the potential antiproliferative activity of compound 1t, the potency of compound 1t was first assessed against three cell lines that were available to us and included in the panels used to profile the activity that involved lung cancer A549, colon cancer HCT116, and breast cancer MDAMB231 cell lines.As shown in Table 2, compound 1t showed potent inhibitory activity for HCT116 cell growth with a low micromolar IC 50 value in the range of 8.68 µM.Consequently, the mechanism of action of compound 1t was studied employing colon cancer HCT116 cells.
Because sulfuretin was reported to induce apoptosis through activation of Fas, Caspase-8, and the mitochondrial death pathway in HL60(TB) blood cancer [42], a flow cytometry assay was addressed for compound 1t in HCT116 cells at different doses to explore possible apoptotic and/or necrotic activity.The results showed that compound 1t could significantly induce the apoptosis in HCT116 cells (Figure 11A).Accordingly, Western blotting was addressed to assess proteins involved in apoptosis.The results showed that compound 1t dose-dependently increased the levels of cleaved-caspase 8 and 9 and cleaved-PARP and significantly decreased levels of caspase 8, and 9 as well as PARP in a dose-dependent manner in HCT116 cells (Figure 11B).These results further confirmed that compound 1t can induce apoptotic death of HCT116 cells.Furthermore, assessment of cycle distribution was addressed to explore possible correlation of the antiproliferative activities of compound 1t with cell cycle arrest.

Breast Cancer
Breast cancer could be hormone-sensitive or resistant and, in addition, primary or metastatic.Accordingly, compounds 1a-1x were profiled against four breast cancer cells including the following: (1) MCF7, a metastatic invasive hormone-responsive breast adenocarcinoma; (2) BT549, a primary triple-negative invasive breast adenocarcinoma; (3) MDAMB231, a metastatic triple-negative breast adenocarcinoma; and (4) MDAMB468, a metastatic triple-negative breast adenocarcinoma.As shown in Figure 10, sulfuretin had almost negligible to no activity against the cell lines employed.As revealed from the results, the profile of sulfuretin congeners' activity against the primary triple-negative BT549 cell line was distinct from the activity profile against the metastatic hormone-responsive MCF7 cell line and metastatic MDAMB231 and MDAMB468 triple-negative cell lines.In general, all C 6 -hydroxy derived compounds having hydroxy substituents on ring-B did not inhibit the growth of any primary or metastatic cell lines (compounds 1a-1g, Figure 10).Meanwhile, out of the C 6 -hydroxy derived compounds 1h-1p having methoxy substituents at ring-B, compounds 1i, 1l, and 1n with at least one methoxy substituent at meta-position of ring-B or compound 1j with one methoxy substituent at the para-position of ring-B triggered growth inhibition of the primary triple-negative BT549 cell line (Figure 10).Amongst them, the most effective was compound 1n, possessing two methoxy moieties at both meta-positions of ring-B, while the equally active compounds 1i and 1l have either only one methoxy moiety at meta-position of ring-B or the second methoxy moiety shifted by one carbon relative to that of compound 1n to occupy the ortho-position distant from first methoxy meta-position on ring-B.This indicates that a methoxy moiety at orthoposition of ring-B has no role in mediating the activity, which might be supported by the found no activity of compound 1h with one methoxy moiety at ortho-position of ring-B.In the case of the least active compound 1j, it has only one methoxy moiety but shifted to the para-position of ring-B.Accordingly, it might be inferred that the methoxy moiety at meta-position is more optimal for activity against the primary triple-negative BT549 cell line.In the case of metastatic cell lines, all C 6 -hydroxy derived compounds 1h-1p with methoxy substituents at ring-B did not trigger significant growth inhibition except for compound 1n, possessing two methoxy moieties at both meta-positions of ring-B, that some members of C 6 -methoxy derived compounds 1q-1v, having hydroxy substituents at ring-B, showed potential inhibition of the metastatic hormone-responsive MCF7 cell line (Figure 10).As revealed from the results, this antiproliferative activity against the metastatic hormone-responsive MCF7 cells might be associated with hydroxy moieties at the ortho-and meta-positions of ring-B (compounds 1q and 1r, respectively).When both vicinal ortho-and meta-positions were substituted simultaneously by dihydroxy moieties, the activity was more enhanced (compound 1t).In addition, compound 1t, amongst the C 6methoxy derived compounds 1q-1x, showed the best inhibition of the growth of the primary triple-negative BT549 cell line and metastatic triple-negative MDAMB231 cell line.However, the growth of the triple-negative MDAMB468 cell was more inhibited by compound 1w, possessing a ortho-methoxy substituent on ring-B relative to compound 1t possessing vicinal ortho-and meta-methoxy substituents on ring-B.Such a difference in structural feature requirements suggests the involvement of different molecular targets.Collectively, the results might nominate compound 1t as a hit candidate for the development of antiproliferative compounds against metastatic hormone responsive MCF7 or triplenegative primary and metastatic triple-negative BT549 and MDAMB231 breast cancers.In addition, compound 1n might be nominated as a hit compound against primary triplenegative BT549 cells.

Compound 1t Induces Cell Cycle Arrest in HCT116 Colon Cancer Cells
To assess the mechanism mediating the potential antiproliferative activity of compound 1t, the potency of compound 1t was first assessed against three cell lines that were available to us and included in the panels used to profile the activity that involved lung cancer A549, colon cancer HCT116, and breast cancer MDAMB231 cell lines.As shown in Table 2, compound 1t showed potent inhibitory activity for HCT116 cell growth with a  Because sulfuretin was reported to induce apoptosis through activation of Fas, Caspase-8, and the mitochondrial death pathway in HL60(TB) blood cancer [42], a flow cytometry assay was addressed for compound 1t in HCT116 cells at different doses to explore possible apoptotic and/or necrotic activity.The results showed that compound 1t could significantly induce the apoptosis in HCT116 cells (Figure 11A).Accordingly, Western blotting was addressed to assess proteins involved in apoptosis.The results showed that compound 1t dose-dependently increased the levels of cleaved-caspase 8 and 9 and cleaved-PARP and significantly decreased levels of caspase 8, and 9 as well as PARP in a dose-dependent manner in HCT116 cells (Figure 11B).These results further confirmed that compound 1t can induce apoptotic death of HCT116 cells.Furthermore, assessment of cycle distribution was addressed to explore possible correlation of the antiproliferative activities of compound 1t with cell cycle arrest.Using variable concentrations of compound 1t, analysis of HCT116 cell distribution in the different phases of cell cycle progression revealed that compound 1t dose-dependently increased the G 0 /G 1 cell population (Figure 12A).Therefore, Western blotting was addressed to assess changes in levels of proteins involved in cell cycle regulation.In line with cell cycle analysis results, compound 1t was found to suppress CDK4, CDK6, Cdc25A, and Cyclin D and E protein levels in a concentration-dependent manner (Figure 12B).Together, these results indicate that the antiproliferative effects of the compound 1t are associated with G 0 /G 1 cell cycle arrest.
in the different phases of cell cycle progression revealed that compound 1t dose-dependently increased the G0/G1 cell population (Figure 12A).Therefore, Western blotting was addressed to assess changes in levels of proteins involved in cell cycle regulation.In line with cell cycle analysis results, compound 1t was found to suppress CDK4, CDK6, Cdc25A, and Cyclin D and E protein levels in a concentration-dependent manner (Figure 12B).Together, these results indicate that the antiproliferative effects of the compound 1t are associated with G0/G1 cell cycle arrest.

Compound 1t Inhibits Activation of MET Receptor Tyrosine Kinase and Downregulates Its Downstream in HCT116 Colon Cancer Cells
Activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways is involved in cancer cells' increased motility, proliferation, migration, invasion, survival, and angiogenesis [50][51][52].It was reported that the PI3K/AKT/mTOR intracellular pathway plays a key role in the regulation of the cell cycle in colon cancer [53].Their biochemical function is diametrically related to cellular quiescence, metastasis, tumor, and lifespan [54,55].PI3K/Akt/mTOR pathways have been elucidated for their biofunctional involvement in colorectal tumorigenesis.As clinical pathological biological features, the abnormal regulation of these signals has frequently been detected with concomitant overexpression of related proteins.Interestingly, these cellular signaling pathways are downstream components of the MET signaling pathway.These observations and the elucidated role of MET signaling pathways in the invasive growth of cancer have enhanced enthusiasm for the development of MET inhibitors as anticancer cancer agents.

Compound 1t Inhibits Activation of MET Receptor Tyrosine Kinase and Downregulates Its Downstream in HCT116 Colon Cancer Cells
Activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathways is involved in cancer cells' increased motility, proliferation, migration, invasion, survival, and angiogenesis [50][51][52].It was reported that the PI3K/AKT/mTOR intracellular pathway plays a key role in the regulation of the cell cycle in colon cancer [53].Their biochemical function is diametrically related to cellular quiescence, metastasis, tumor, and lifespan [54,55].PI3K/Akt/mTOR pathways have been elucidated for their biofunctional involvement in colorectal tumorigenesis.As clinical pathological biological features, the abnormal regulation of these signals has frequently been detected with concomitant overexpression of related proteins.Interestingly, these cellular signaling pathways are downstream components of the MET signaling pathway.These observations and the elucidated role of MET signaling pathways in the invasive growth of cancer have enhanced enthusiasm for the development of MET inhibitors as anticancer cancer agents.
Western blotting was used to verify whether mTOR is involved in mediating the cell cycle arrest and apoptotic effects of compound 1t in HCT116 cells.As evident in Figure 13, phosphorylated to unphosphorylated forms (p-mTOR/mTOR) were concentration-dependently reduced.As mTOR is downstream of AKT, Western blotting was addressed for phosphorylated to unphosphorylated forms of AKT and results unveiled a concentration-dependent decrease in p-AKT/AKT.Subsequently, checking PI3K as upstream of AKT also showed a concentration-dependent decrease in p-PI3K/PI3K.Finally, dose-dependent inhibition of the activation of the upstream MET receptor tyrosine kinase (AKA HGFR; hepatocyte growth factor receptor) without affecting the total protein expression was confirmed, as shown in Figure 13.Together, these findings suggest that the antiproliferative effects of compound 1t are mediated by suppression of MET receptor tyrosine kinase activation, and invariably lead to down-regulation of the PI3K/AKT/mTOR signaling pathways.However, it is possible the suppression of activation of MET found might arise from other factors and signaling pathways rather than direct interaction with MET, and this might be explored in the future.
cycle arrest and apoptotic effects of compound 1t in HCT116 cells.As evident in Figure 13, phosphorylated to unphosphorylated forms (p-mTOR/mTOR) were concentration-dependently reduced.As mTOR is downstream of AKT, Western blotting was addressed for phosphorylated to unphosphorylated forms of AKT and results unveiled a concentrationdependent decrease in p-AKT/AKT.Subsequently, checking PI3K as upstream of AKT also showed a concentration-dependent decrease in p-PI3K/PI3K.Finally, dose-dependent inhibition of the activation of the upstream MET receptor tyrosine kinase (AKA HGFR; hepatocyte growth factor receptor) without affecting the total protein expression was confirmed, as shown in Figure 13.Together, these findings suggest that the antiproliferative effects of compound 1t are mediated by suppression of MET receptor tyrosine kinase activation, and invariably lead to down-regulation of the PI3K/AKT/mTOR signaling pathways.However, it is possible the suppression of activation of MET found might arise from other factors and signaling pathways rather than direct interaction with MET, and this might be explored in the future.

In Silico Docking Simulation
The molecular target identified, MET receptor tyrosine kinase, is amongst few kinases that were recently found to possess distinct rare loop conformations [56][57][58].These conformations are important to achieve selective inhibition.In addition to the regularly extended phosphate-binding loop (p-loop) conformation of MET receptor tyrosine kinase, researchers from the pharmaceutical industry company AstraZeneca have recently reported a rare, folded p-loop conformation of MET receptor tyrosine kinase which is collapsed or arranged into the ATP binding site that might provide a structural basis for selective targeting of MET receptor tyrosine kinase [57].Moreover, another rearranged αC helix conformation was also reported recently by researchers from AstraZeneca to form two helices and occupy the DFG-pocket, which provides an alternative structural basis for selective targeting of MET receptor tyrosine kinase [58].To explore which conformation might be involved in the binding of compound 1t with MET receptor tyrosine kinase, an in silico study was conducted employing crystal structures of folded p-loop, extended ploop, and rearranged αC helix conformations of MET receptor tyrosine kinase (protein data bank accession codes: 7b41, 7b3w, and 8an8, respectively).The results showed that compound 1t was able to dock perfectly into the p-folded MET receptor tyrosine kinase conformation (PDB: 7b41), showing a predicted binding mode that overlayed the reported

In Silico Docking Simulation
The molecular target identified, MET receptor tyrosine kinase, is amongst few kinases that were recently found to possess distinct rare loop conformations [56][57][58].These conformations are important to achieve selective inhibition.In addition to the regularly extended phosphate-binding loop (p-loop) conformation of MET receptor tyrosine kinase, researchers from the pharmaceutical industry company AstraZeneca have recently reported a rare, folded p-loop conformation of MET receptor tyrosine kinase which is collapsed or arranged into the ATP binding site that might provide a structural basis for selective targeting of MET receptor tyrosine kinase [57].Moreover, another rearranged αC helix conformation was also reported recently by researchers from AstraZeneca to form two helices and occupy the DFG-pocket, which provides an alternative structural basis for selective targeting of MET receptor tyrosine kinase [58].To explore which conformation might be involved in the binding of compound 1t with MET receptor tyrosine kinase, an in silico study was conducted employing crystal structures of folded p-loop, extended p-loop, and rearranged αC helix conformations of MET receptor tyrosine kinase (protein data bank accession codes: 7b41, 7b3w, and 8an8, respectively).The results showed that compound 1t was able to dock perfectly into the p-folded MET receptor tyrosine kinase conformation (PDB: 7b41), showing a predicted binding mode that overlayed the reported X-ray co-crystallized ligand (Figure 14A).It elicited a favorable score of −6.98019 and established a network of favorable interactions.Importantly, amongst these interactions was a crucial hydrophobic interaction with the conserved aromatic residue of the c-MET P-loop, Phe1089.In addition, two hydrogen-bonding interactions were established between the 2 ,3 -dihydroxy groups of compound 1t with Ala1226 and Arg1227 residues in the β-turn motif formed from the A-loop residues 1223-1227.Moreover, a hydrophobic interaction was formed with the catalytic residue Lys1110.On the other side, the prediction of the binding mode of compound 1t to the extended p-loop conformation (PDB: 7b3w) revealed that the predicted binding has lower score (−6.27824) relative to the folded p-conformation and is not aligned over the co-crystalized ligand (Figure 14B).More importantly, it was unable to establish crucial interactions with Pro1158 and Met1160 residues of hinge region residues that were indispensable for binding of the co-crystallized ligand with the extended p-loop conformation.While the co-crystallized ligand in the rearranged αC helix conformation of MET receptor tyrosine kinase (PDB: 8an8) inserts between the two helices resulting from rearrangement, the prediction of the binding mode of compound 1t unveiled its inability to insert between these two helices (Figure 14C) and, thus, it was unable to establish the crucial hydrogen bonding with the backbone of Ser1122 in the hinge connecting these helices.Together, these results might nullify the possible binding of compound 1t to any of the extended p-loop or the rearranged αC helix conformations but suggests its binding to the folded p-loop conformations of MET receptor tyrosine kinase.In fact, compounds that inhibit the activation loop can function as a switch control and such phenomena was reported for ripretinib with KIT and PDGFRA kinases [59].loop, Phe1089.In addition, two hydrogen-bonding interactions were established between the 2′,3′-dihydroxy groups of compound 1t with Ala1226 and Arg1227 residues in the βturn motif formed from the A-loop residues 1223-1227.Moreover, a hydrophobic interaction was formed with the catalytic residue Lys1110.On the other side, the prediction of the binding mode of compound 1t to the extended p-loop conformation (PDB: 7b3w) revealed that the predicted binding has lower score (−6.27824) relative to the folded p-conformation and is not aligned over the co-crystalized ligand (Figure 14B).More importantly, it was unable to establish crucial interactions with Pro1158 and Met1160 residues of hinge region residues that were indispensable for binding of the co-crystallized ligand with the extended p-loop conformation.While the co-crystallized ligand in the rearranged αC helix conformation of MET receptor tyrosine kinase (PDB: 8an8) inserts between the two helices resulting from rearrangement, the prediction of the binding mode of compound 1t unveiled its inability to insert between these two helices (Figure 14C) and, thus, it was unable to establish the crucial hydrogen bonding with the backbone of Ser1122 in the hinge connecting these helices.Together, these results might nullify the possible binding of compound 1t to any of the extended p-loop or the rearranged αC helix conformations but suggests its binding to the folded p-loop conformations of MET receptor tyrosine kinase.In fact, compounds that inhibit the activation loop can function as a switch control and such phenomena was reported for ripretinib with KIT and PDGFRA kinases [59].

Chemistry
Synthesis and structural elucidation of compounds was reported earlier [45,60,61] and indicated in the supporting information.

In Vitro Profiling against Human Cancer Cells Panels
Screening against the cancer cell lines was carried out according to the known standard NCI protocol [9].

In Vitro Evaluation of Antiproliferative Mechanisms
Evaluation of cytotoxic mechanisms was conducted following standard protocols as described in Supplementary Materials [62,63].

Figure 1 .
Figure 1.Design of sulfuretin's congeners focused library for profiling anticancer activities.Figure 1. Design of sulfuretin's congeners focused library for profiling anticancer activities.

Figure 1 .
Figure 1.Design of sulfuretin's congeners focused library for profiling anticancer activities.Figure 1. Design of sulfuretin's congeners focused library for profiling anticancer activities.

Figure 4 .
Figure 4. % growth inhibition of growth of diverse colorectal cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 4 .
Figure 4. % growth inhibition of growth of diverse colorectal cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 5 .
Figure 5. % growth inhibition of growth of diverse CNS cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 5 .
Figure 5. % growth inhibition of growth of diverse CNS cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 6 .
Figure 6.% growth inhibition of growth of diverse melanoma cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 6 .
Figure 6.% growth inhibition of growth of diverse melanoma cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 7 .
Figure 7. % growth inhibition of growth of ovarian cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 7 .
Figure 7. % growth inhibition of growth of ovarian cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 8 .
Figure 8. % growth inhibition of growth of diverse renal cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 9 .
Figure 9. % growth inhibition of growth of prostate cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 10 .
Figure 10.% Growth inhibition of growth of diverse breast cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 10 .
Figure 10.% Growth inhibition of growth of diverse breast cancer cell lines triggered by 10 µM concentrations of sulfuretin and its congeners (1a-1x).

Figure 12 .
Figure 12.Impact of compound 1t on the cell cycle distribution in HCT116 cells.(A) HCT116 cell cycle distributions after treatment with different concentrations of compound 1t.The graphs show the quantified results.(B) CDK4, CDK6, Cyclin D, Cyclin E, and Cdc25A protein expressions after treatment with different concentrations of compound 1t.

Figure 12 .
Figure 12.Impact of compound 1t on the cell cycle distribution in HCT116 cells.(A) HCT116 cell cycle distributions after treatment with different concentrations of compound 1t.The graphs show the quantified results.(B) CDK4, CDK6, Cyclin D, Cyclin E, and Cdc25A protein expressions after treatment with different concentrations of compound 1t.

.
Sulfuretin and list of its congeners (1a-1x) employed in profiling anticancer activity and their literature reported inhibition of PGE2 production.

Table 2 .
Inhibitory effects of compound 1t on the proliferation of three human cancer cell lines.