Design and synthesis of new energy restriction mimetic agents: Potent anti-tumor activities of hybrid motifs of aminothiazoles and coumarins

The incidence of obesity-related diseases like diabetes, cardiovascular diseases, and different types of cancers shed light on the importance of dietary control as preventive and treatment measures. However, long-term dietary control is challenging to achieve in most individuals. The use of energy restriction mimetic agents (ERMAs) as an alternative approach to affect the energy machinery of cancer cells has emerged as a promising approach for cancer therapy. ERMAs limit the high need for energy in rapidly growing tumor cells, with their survival rate strongly dependent on the robust availability of energy. In this context, initial phenotypic screening of an in-house pilot compound library identified a new class of aminothiazole anchored on coumarin scaffold as potent anticancer lead drug candidates with potential activity as ERMA. The identified chemotypes were able to inhibit glucose uptake and increase ROS content in cancer cells. Compounds 9b, 9c, 9i, 11b, and 11c were highly active against colorectal cancer cell lines, HCT116 and HT-29, with half-maximal inhibitory concertation (IC50) range from 0.25 to 0.38 µM. Further biological evaluations of 9b and 9f using Western blotting, caspase activity, glucose uptake, ROS production, and NADPH/NADP levels revealed the ability of these lead drug candidates to induce cancer cell death via, at least in part, energy restriction. Moreover, the assessment of 9b and 9f synergistic activity with cisplatin showed promising outcomes. The current work highlights the significant potential of the lead compounds, 9b, and 9f as potential anticancer agents via targeting the cellular energy machinery in cancer cells.

Many epidemiological studies indicated that obesity and excess body fat increase the risk of carcinogenesis 1,2 . In the last decade, the observed global rise in the body-mass index has led to an increase in the incidence of cancer-linked obesity 2,3 . The direct link to obesity makes colorectal cancer (CRC), the fourth leading cause of cancer morbidities and the second leading cause of cancer mortality worldwide 4,5 .
The conventional chemotherapeutic protocols such as FOLFIRI (Folinic acid, fluorouracil, and irinotecan) and FOLFOX (Folinic acid, fluorouracil, and oxaliplatin) are the currently available options for the management of advanced CRC 6 . Targeted therapy with epidermal growth factor receptor (EGFR) or vascular endothelial growth factor (VEGF) inhibitors improved the overall survival of CRC patients. However, the heterogeneous nature of CRC and the toxicity of chemotherapeutics continued the urgent need for the development of new agents targeting CRC 7 . On the other hand, caloric restriction (CR) has acquired a great interest as a booster for longevity, cancer prevention, and many therapeutic approaches [8][9][10] . The increased interest in CR is a consequence of the metabolic shift of tumor cells towards the aerobic glycolysis and the upregulation of glucose consumption or what is known as the Warburg effect 11,12 . Therefore, many studies focused on targeting the metabolism in cancer cells by energy restriction mimetic agents (ERMAs) or via reprogramming the energy production in cancer cells 13,14 . ERMAs are compounds that target cellular metabolism and activate the cellular stress response [15][16][17] . In addition to the more straightforward implementation, the use of ERMAs has the advantage of mimicking most of the CR cellular responses without reducing food consumption or causing undesired side effects 8,18 .
In this study, the screening of a pilot compound library against an in-house panel of cancer cell lines identified new anticancer lead drug candidates. These molecules possess a hybrid structure that encompasses an aminothiazole moiety anchored on a coumarin core 19 . Coumarin system, represented by troglitazone analogues 13,19 , were reported as energy restriction mimetic agents. Furthermore, ERMAs such as metformin were also reported to deprive cells of energy 13,19,20 . Inspired by these reports, we envisioned that a hybrid structure encompassing an aminothiazole moiety, a rigid isostere of metformin, and coumarin system might lead to the discovery of potent anticancer lead drug candidates that deprive cellular energy. Henceforth, a pilot library of aminothiazoles anchored on the coumarin ring as potential ERMA was synthesized (Fig. 1). One of our key objectives is to examine the newly designed compounds as potential anticancer lead drug candidates for the treatment of CRC. Besides, the combination of ERMAs with classical chemotherapeutic agents like cisplatin can exploit the maximal benefit through synergistic mechanisms. Targeting the survival signaling in CRC by ERMAs represents a therapeutically relevant approach for treatment, and ultimately might lead to new therapies that improve the treatment and increase the survival of CRC patients.

Results and Discussion
Initial screening. In the search for Energy Restriction Mimetic Agents (ERMAs), we have initially screened an in-house pilot library of more than fifty compounds with diverse scaffolds against a panel of cancer cell lines. Such a phenotypic screen identified an anticancer compound that possesses thiourea derivative anchored on a coumarin scaffold. Thus, a systematic and biased structure-activity relationship (SAR) study was followed, and a small set of compounds was first assembled. Then, screening was performed for their anticancer activities against HCT116 and HT-29 cancer cell lines. The best hits were structurally assessed and subjected to subsequent SAR development. From these experiments and using a predictive physicochemical guideline of small molecules, we concluded that a hybrid structure that connects an aminothiazole to a coumarin moiety is essential for the anticancer activity. Such initiatives led to the development of potent motifs describes in Table 1. Specifically, we intended first to prepare a small library of various aminothiazoles anchored at the 7-position of coumarin through derivatization of the 7-amino coumarin of type 4, as shown in Scheme 1. With the pilot library in hand, screening against cancer cell lines identified several potent anticancer probes, compounds 9b, 9c, 9i, 11b, and 11c, with IC 50 values of less than 0.6 µM ( Table 1). The in vitro antiproliferative activities of the most potent compounds, as indicated by their IC 50 values, are summarized in Fig. 2G.

Synthesis and design.
The synthesis of the designed compounds is contemplated in Schemes 1 and 2. The key starting materials 7-Amino-4-substituted coumarin 4a-d were obtained utilizing modified Pechmann reaction 21 in which m-aminophenol 1 was protected with methoxycarbonyl chloride to afford the urethane 2. The reaction of the latter with the appropriate β-ketoester ester using sulfuric acid produced the coumarins 3a-c. Basic hydrolysis of the carbamates 3a-c delivered the desired 7-amino-4-substituted coumarins 4a-c in good yields 22 .
With the key building blocks 4a-c in hand, it was envisaged that reactions with benzoyl isothiocyanate should deliver N-(4-Substituted-2-oxo-2H-chromen-7-ylcarbamothioyl) benzamide 6a-c. The basic hydrolysis of the latter compounds produced the 7-thiourea coumarin conjugates 7a-c. Construction of the aminothiazole scaffolds 9-11, was achieved by reaction of the thiourea derivatives 7a-c, with various α-bromoacetophenone derivatives 8a-i in refluxing ethanol ( Table 2). The characterization of the new compounds was carried out through 1 H NMR, 13 C NMR, and HRMS.
Initial screening of the developed compounds against HT-29 and HCT116 cancer cell lines (Table 1 and Fig. 2) indicated that compounds 9a, 9b, 9c, 9f, 9i, 11b, and 11c exhibited significant anticancer activities. As a result, compounds possessing electron-withdrawing groups at the phenyl ring of the thiazole moiety are more potent than those with electron-releasing groups. Furthermore, when the coumarin ring carries a methyl group at the   Fig. 2G). Moreover, to understand the structure-activity relationship (SAR) considering the potency and safety of the new compounds, the most potent compound 9b, and the safest compound on normal cells (9f, IC 50 = 7.97 µM), were selected for further biological evaluation and analysis.

Activation of energy restriction cellular responses.
Many ERMAs were reported to induce energy restriction cellular responses, including AMPK phosphorylation, β-TrCP upregulation, downregulation of cyclin D1 and inhibition of Akt phosphorylation 13,23 . Based on this, the energy restriction mediated anticancer activity of the new coumarin tagged aminothiazole derivatives was investigated. The expression levels of PARP, p-Akt, Akt, p-AMPK, AMPK, β-TrCP, LC3A/B-I, LC3A/B-II, caspase-3, cyclin D1, and Bcl-2 proteins were determined by Western blot analysis after 72 h treatment of HT-29 and HCT116 at different concentrations based on the IC 50 values of each compound (Fig. 3A,B). The glucose starvation and apoptosis proteins markers used in this study were found to be differentially expressed upon treatment with the selected compounds 9b and 9f. Moreover, the treatment with 9b and 9f induced caspase 3/7 activation, which was detected by caspase 3/7 activity assay and confirmed by Western blot. The activation of caspases was significant in 9b in both cell lines and significant with 9f in HCT116 cells only and minimal in HT-29 cells (Fig. 3C,D). On the other hand, both 9b and 9f induced dose-dependent proteolytic processing of PARP. 9b induced PARP cleavage, a signature of apoptosis, in a dose-dependent manner in both tested cell lines. However, 9f has shown PARP cleavage only in HT-29 cells at the highest tested dose. Besides, the protein expression level of the anti-apoptotic protein, Bcl-2, was reduced in HCT116 upon treatment with both compounds. These findings suggest the involvement of apoptosis as a mechanism of cell death (Fig. 3A,B).
The treatment with 9b and 9f resulted in the enhancement of AMPK activation through phosphorylation in both HT-29 and HCT116 cells. Besides, the inhibition of Akt phosphorylation was detected upon the treatment Scheme 2. Synthesis of the target compounds 9, 10 and 11. Reagents and conditions: (i) Acetone/reflux 3-4 h; (ii) MeOH/1 N NaOH, reflux, 3-4 h; (iii) α-bromoacetophenone derivative/EtOH, reflux, 4 h.  of HT-29 cells with 9b and 9f. However, Akt phosphorylation was not observed in HCT116 cells (Fig. 3A,B). This finding was in line with many studies in which this mutant PIK3CA cell line exhibited an absence of Akt phosphorylation 24,25 . Surprisingly, 9f induced Akt phosphorylation at high concentrations in the HCT116 cell line only. The induction of Akt phosphorylation was previously demonstrated in different cells treated with the glucose inhibitor, 2-Deoxy-D-glucose (2-DG) and curcumin 26,27 . Additionally, a dose-dependent downregulation of β-TrCP and cyclin D was observed in HT-29 cells on the treatment with both compounds, and the same effect was observed in HCT116 cells treated with 9b (Fig. 3A,B). On the other hand, no significant changes in the expression of cyclin D and β-TrCP were noticed in HCT116 cells on other treatments. These findings are not in parallel to previous studies that showed the ability of ERMAs www.nature.com/scientificreports www.nature.com/scientificreports/ to induce β-TrCP-mediated proteolysis, leading to cell cycle arrest 13,19 . On the other hand, our results confirmed the clinical findings in colorectal cancer tissues, where β-TrCP is overexpressed and could possibly play a role as a tumor oncogene 28 . Also, it has been revealed that β-TrCP is controlled by the mammalian target of rapamycin (mTOR), the cellular metabolism regulator, with consequent downregulation of cyclin E in triple-negative breast cancer cells 29 .

Code
Another anticancer mechanism of energy restriction is the induction of autophagy 30 . To explore the ability of the new compounds to induce autophagy, the expression levels of LC3-II (LC3A-II and LC3B-II), which is a central component of the autophagosome membrane, were assessed using Western blot 31 . The results indicated that treatment with compounds 9b and 9f induced the expression of LC3A-II and LC3B-II in CRC cell lines (Fig. 3A,B). These results provided further evidence that these new compounds could possibly activate autophagy via the AMPK-TSC1/2-mTOR signaling pathway. Therefore, we have analyzed the protein expression levels of p-mTOR (Ser2448), mTOR and p-p70S6K (Thr389) (Fig. 3E, F). Interestingly, we observed an initial increase in p-mTOR and p-p70S6K at 0.5 µM of 9b in HT-29 and HCT116 cell lines. This increase could possibly serve as a protective mechanism to maintain cellular homeostasis. Earlier studies have also found an initial increase in mTOR due to the exposure to cellular stress such as radiation or treatment with hydrogen peroxide 32,33 . On the other hand, we have observed a decrease in p-mTOR and p-p70S6K levels at higher concentrations of 9b and 9f in both cell lines except at doses 1 and 2 µM in HCT116 cells, where induced p-mTOR was observed. In addition, decreased p-mTOR and p-p70S6K was parallel to an increase in p-AMPK level and a decrease in p-Akt in HT-29 cells. Furthermore, in HCT116 cells, which have mutant PIK3CA 24,25 , the observed increase in mTOR expression was not correlated to the increase in p-p70S6K and the upregulation of p-p70S6K was independent of mTOR, which requires further investigation. However, these results confirmed that the mechanism of anticancer activity of the new compounds was mediated, at least in part, through the activation of AMPK, which promotes the inhibition of mTOR 34 .

Induction of cell cycle arrest at G1 phase in HT-29 cells upon treatment with 9b and 9f.
To further investigate the effect of the synthesized compounds on cell cycle progression, we examined cell cycle profile of cells upon 24 h treatment in comparison to metformin, a known AMPK activator that inhibits cell proliferation in CRC cells through p53-independent manner 35 . In HT-29 cells, we have observed the accumulation of cells in G1 and a decrease in the G2/M phase upon the treatment with 9b, 9f, and metformin (Fig. 4A, C). These changes in cell cycle were in correlation with the observed reduction in cyclin D1 expression in this p53-mutant cell line (Fig. 3A). These results could suggest that the induced cell cycle arrest by the candidate compounds is p53 independent. Whereas, in HCT116, a p53 wild type expressing cell line, 9b treatment increased the number of cells in the sub-G1 cell population, which represents the dead cells. Besides, 9f caused a slight increase in the G1 cell population (Fig. 4B,C). However, in Western blot, the expression of cyclin D1 was not reduced in HCT116 cells (Fig. 3B).
Impact of 9b and 9f on glucose uptake and ROS production in CRC. According to previous studies, ERMAs inhibited glucose utilization in cancer cells 13,19 . Therefore, we investigated glucose uptake (Fig. 5A) upon treatment with 9b and 9f in HT-29 and HCT116 cells at different concentrations based on the IC 50 of each compound. Glucose uptake was inhibited significantly in both cell lines in a dose-dependent manner in 9b and 9f after 16 h treatment. Further investigations of 9b and 9f activities have shown a significant decrease in NADPH/ NADP + ratio (Fig. 5B). This effect is known to result in the accumulation of reactive oxygen species (ROS) and could play a role in glucose deprivation-induced cytotoxicity and oxidative stress in cancer cells 36 . Interestingly, 9b and 9f significantly increased the generation of hydrogen peroxide (H 2 O 2 ) and ROS in HCT116, while only 9b caused the same effect in HT-29 cells (Fig. 5C). The observed increase in ROS levels could lead to oxidative stress and extensive cell death in the absence of NADPH detoxification 36 .

Anticancer activity of 9b and 9f in combination with cisplatin in CRC cells. Cisplatin, a
platinum-based anticancer agent, has been used in different combination regimes in CRC therapy to combat single drug cell resistance 37 . Therefore, we have explored cisplatin and compounds 9b and 9f combination as a potential therapeutic protocol. HT-29 and HCT116 cells were treated with different doses of cisplatin (1.5 and 3.0 µg/ml), 9b (0.2 and 0.4 µM) and 9f (2.0 and 4.0 µM) alone or in combinations. As a single compound, the IC 50 of cisplatin was in the range of 1.81-1.93 µg/ml in HT-29 and HCT116 cells, respectively. Whereas the IC 50 values of 9b were 0.38 and 0.53 µM in HCT116 and HT-29 cells, respectively. While in 9f, the IC 50 values were 2.56 and 3.96 µM in HT-29 and HCT116 cells, respectively. Furthermore, the combination treatment of cisplatin with 9b was weakly synergistic at 1.5 µg/ml concentration of cisplatin and 0.2 µM of 9b in HT-29 cells. An additive effect was detected in HT-29 at 0.4 µM of 9b with 1.5 and 3 µg/ml of cisplatin. While no synergistic effect was observed in HCT116 cells in any of the tested doses (Fig. 6), Interestingly, the combination treatment of cisplatin and 9f was synergistic in all the tested concentrations (Fig. 7). These promising results need further investigations to illustrate the exact mechanism of synergy.

conclusions
In summary, we have identified a new series of low molecular weight compounds that possess potent anticancer activities with potential use as ERMA leads. The synthetic methodologies followed were simple, efficient, and economical. Several of the developed low molecular weight scaffolds possess high potency with IC 50 values ranging from 0.25 µM to 3.96 µM in CRC cell lines. Several molecular techniques used in this study revealed the anticancer role of the developed compounds through the activation of energy restriction cellular responses and induction of cell apoptosis. Moreover, the tested compounds mediated glucose uptake inhibition, decreased NADPH/ NADP+ ratio, and facilitated ROS generation, ultimately promoted cell death. The promising anticancer activity www.nature.com/scientificreports www.nature.com/scientificreports/ of the synthesized compounds and their ability to synergize cisplatin in targeting CRC cell lines supported the possibility for their combination with conventional chemotherapeutic agents specifically with compound 9f. Finally, the current work highlights the potential role of the newly developed ERMAs, individually or in combination, in targeting CRC. However, further investigations are required to explore the potential benefits of these novel compounds in different types of cancers and their compatibility with other chemotherapeutic agents.

Methods
General chemistry. All used reagents were purchased from commercial suppliers without further purification. The reactions were carried out in oven-dried or flamed graduated vessels. Solvents were dried and purified by conventional methods before use. All reactions were monitored using Merck aluminum plated pre-coated with silica gel PF254 and detected by visualization of the plate under UV lamp (λ = 254 or 365). Column chromatography was performed using silica gel 60, 0.040-0.063 mm (230-400 mesh). 1 H and 13 C NMR spectra were recorded on a Bruker Avance DPX-300 MHz and DPX-500 MHz instruments. Splitting patterns are designated as s, singlet; d, doublet; dd, doublet of doublet; t, triplet; q, quartet; m, multiplet; br, broad. Chemical shifts (δ) are given in ppm with reference to TMS as an internal standard. High-resolution mass spectra (HRMS) were measured by Electrospray Ionization (ESI) on a Bruker APEX-IV instrument. The samples were dissolved in acetonitrile and infused using a syringe pump with a flow rate of 120 μL/min. External calibration was conducted using Arginine cluster in a mass range m/z 175-871. For all HRMS data, mass error: 0.00-0.50 mDa.

General procedure for the preparing 7-Methoxycarbonylamino-4-substituted Coumarins 3 a-c.
A mixture of 10 mmol (1 eq) of m-(N-methoxycarbonylamino) phenol 2 was added in portions to 15 ml of concentrated H 2 SO 4 followed by dropwise addition of 11 mmol (1.1 eq) of appropriate β-Ketoester ester derivatives with starring and cooling (10-15 °C). The mixture was further stirred for 3 h, upon completion 40 ml of ice-water www.nature.com/scientificreports www.nature.com/scientificreports/ was added and stirred until crystals formed. The precipitate was filtrated, washed with water and methanol and dried to give the desired product, which was used in the next steps without further purification.

General procedure for preparing 7-amino-4-substituted Coumarins 4a-c. A suspension of
15 mmol of (3a-c) in 15 ml of 45% of KOH was stirred at 80-90 °C for 3 h until completion. The mixture was cooled and diluted with 50 ml water; the solution was acidified with concentrated HCl to 7-8 pH with stirring and cooling until crystallization occurred. The precipitate was filtrated, washed with water and ether then air-dried.   13

7-(4-(4-Ethoxyphenyl)thiazol-2-ylamino)-4-methyl-2H-chromen-2-one (9d). The title com-
pound was obtained starting from the reaction of 7a and 8d (brown solid, 86% yield, mp 222-224 °C). 1     www.nature.com/scientificreports www.nature.com/scientificreports/ Cell cycle analysis. The cell cycle was assessed by flow cytometry, in which cells were treated at the indicated concentrations with the tested compounds for 24h 39 . Then cells were harvested, fixed with 70% ethanol, and stained with propidium iodide (Sigma-Aldrich, St. Louis, MO, USA), a DNA fluorescent binding dye. BD FACS Aria (Becton-Dickinson, Ann Arbor, MI, USA) was used to acquire the results and data were analyzed using FlowJo V.10 software (Ashland, OR, USA). DMSO was used as vehicle control and metformin (Sigma-Aldrich-St. Louis, MO, USA) as a positive control Glucose uptake assay. CRC cells (1 × 10 4 ) were seeded into a 96-well plate overnight. Cells were treated with test compounds or vehicle (negative control) glucose-free RPMI-1640 medium for 16 h; then, the cells were incubated with 2 mM 2-DG for 10 minutes. Eventually, glucose uptake of cells was measured using Glucose Uptake-Glo Assay (Promega, Madison, WI, USA) according to the manufacturer's instructions. Luminescence was measured post 2 h of incubation at room temperature using Varioskan Flash multimode reader (Thermo Scientific). Glucose uptake was normalized to the total protein content in the samples using DC protein assay kit (Bio-Rad, Hercules, CA, USA).

Measurement of NADPH/NADP+ and ROS Levels.
NADPH/NADP+ and ROS concentrations were measured using the NADP/NADPH-Glo Assay Kit (Promega, Madison, WI, USA) and ROS-Glo H2O2 Assay (Promega, Madison, WI, USA), respectively, based on the manufacturer instructions. In NADPH/NADP+ assay, CRC cells were seeded into a 96-well flat-bottomed plate and incubated overnight. On the following day, cells were treated with the testing compound or vehicle for 48 h. At the endpoint of treatment, the media were aspirated and replaced with 60 µl PBS/well, then cells were lysed in 60 µl base solution with 1% dodecyl (trimethyl) ammonium bromide (Sigma-Aldrich-St. Louis, MO, USA). Afterward, cell lysate was transferred into a white opaque 96-well plate to measure NADP+ and NADPH individually. Luminescence was measured after 30 minutes using Varioskan Flash multimode reader (Thermo Scientific). Whereas in ROS assay cells were plated in 96-well flat-bottomed plates in a total volume of 70 µl overnight. Later, cells were treated with different concentrations of compounds or the vehicle, followed by 20 µl of the H 2 O 2 substrate solution for a total volume of 100 µl. After the 6 h incubation at 37 °C in a 5% CO 2 incubator, 50 µl of each reaction mixture was transferred to a white opaque 96-well plate and mixed with 50 µl of the ROS-Glo detection solution. Luminescence was measured after 20 min incubation at room temperature.
Statistical analysis. The data are expressed as mean ± SD, and the statistical analysis was performed using unpaired student's t-test using Graphpad Prism V. 6.01 software. Differences were considered significant at p < 0.05.

Data availability
The data generated and analyzed that support the scientific findings and claims of this study are presented in this published article.