Development of novel 1,2,4-triazole containing compounds with anticancer and potent anti-CB1 activity

Abstract There is still an unmet need for novel and improved anti-cancer compounds. Nitrogen atoms have heterocyclic ring moieties, which have been shown to have powerful anticancer properties in both natural and synthetic derivatives. Due to their dipole character, hydrogen bonding capacity, rigidity and solubility, 1,2,4-triazoles are particularly effective pharmacophores, interacting with biological receptors with high affinity. Thus, novel 1,2,4-triazole-containing molecular derivatives were synthesized using green chemistry methods, microwave irradiation and ultrasonication, and these methods’ operational simplicity and maximum greener synthetic efficiency with green chemistry metrics calculations will be attractive for academic and industrial research and tested against three distinct human cancer cell lines including PANC1 (pancreatic cancer), DU145 (prostate cancer), MCF7 (breast cancer) and one fibroblast cell line (HDF). Here, we showed that compounds 5e and 5f were similar to CB1 antagonists in structure, binding affinity and poses. In addition, compounds 5e–g decreased the viability of pancreatic and prostate cancer cells, albeit with cytotoxicity to HDF cells. The IC50 values for PANC1 cells were between 5.9 and 7.3 µM for compounds 5e–g. Cell cycle analysis showed that the effect of compounds 5e–g in cancer cell growth was largely due to cell cycle arrest at S-phase. In sum, novel 1,2,4-triazole-containing compounds with anticancer and potent anti-CB1 activity have been developed. Communicated by Ramaswamy H. Sarma


Introduction
Cancer is an extremely deadly disease that causes uncontrolled cell growth and rapid spread, and it is a major public health issue affecting people all over the World (NCBI, n.d.).In 2020, an estimated 19.3 million new cancer cases will be diagnosed worldwide, and more than 10.0 million people will die from cancer (Sung et al., 2021).Chemotherapy, radiation and surgery are the three types of cancer treatment options now accessible.Nitrogen atoms have heterocyclic ring moieties, which are found in both natural and synthetic derivatives and have been shown to have potent anticancer properties against several human cancer cell lines.The structural elucidation of various natural compounds relies heavily on three nitrogen atoms bearing heterocyclic rings, such as 1,2,4-triazoles (Pragathi et al., 2021).The anticancer effects of certain molecular derivatives have been tested against three different human cancer cell lines and one fibroblast cell line, including PANC1 (pancreatic cancer), DU145 (prostate cancer), MCF7 (breast cancer) and HDF (human dermal fibroblast) (Abdelli et al., 2021;Tokala et al., 2018).These derivatives could be used as lead molecules for cancer therapy and may exhibit anticancer properties.
Green chemistry has emerged to tackle health and environmental problems at their source by developing cleaner chemical processes for the chemical industry through the design of innovative and environmentally benign chemical reactions (Kinen et al., 2009).We analyzed the results in terms of the reported green metric parameters and showed that the proposed microwave (MW) and ultrasonication (US) methods for the synthesis of the target compounds fulfill many of the principles of green chemistry, both due to less solvent usage, short reaction time and high reaction yield.In addition, the synthesized triazole derivatives were investigated as anticancer agents, and in silico molecular docking and adsorption, distribution, metabolism and excretion (ADME) calculations were performed.

Chemicals
All chemicals were purchased from Fluka Chemie AG Buchs and Sigma Aldrich, and used without further purification.Melting points of the synthesized compounds were determined in open capillaries on a B€ uchi B-540 melting point apparatus and are uncorrected.Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 aluminum sheets.Fourier transform infrared (FTIR) spectra were recorded using a Perkin Elmer 1600 series FTIR spectrometer. 1 H NMR and 13 C NMR spectra were registered in DMSO-d 6 on a BRUKER AVENE II 400 MHz NMR spectrometer (400.13MHz for 1 H and 100.62 MHz for 13 C).The mass spectra were obtained at a liquid chromatography-tandem mass spectrometry (1200/6210, Agilent) by electrospray ionization.

Method 3.
Compound 3 (10 mmol) was added to the 3% solution of hydrazine hydrate (25 mmol) in water and the mixture was irradiated in a monomode microwave reactor in a closed vessel under pressure control at 100 � C, 150 W for 5 h.After the completion of the reaction, the solid precipitated by cooling the mixture was filtered off and purified by recrystallization from ethanol (other methods are given in the Supplementary Material).

General methods for the synthesis of compounds 5a-h 2.2.2.1. Method 2.
The solution of compound 4 (10 mmol) and corresponding aldehyde (11 mmol) in ethanol was sonicated in the presence of 1-2 drops of H 2 SO 4 at 80 � C, 40 Hz for 90 min (the reaction progress was monitored by TLC).The solid precipitated was filtered off and purified by recrystallization from ethanol (other methods are given in the Supplementary Material).

MTS assay
PANC1 (pancreatic cancer), DU145 (prostate cancer) and HDF (human dermal fibroblast) were taken out from liquid nitrogen was grown in high glucose Dulbecco's modified eagle medium (Gibco 11965084) containing a nutrient mixture of 10% fetal bovine serum and 1% PSA.7500 cells/200 ml were seeded in 96-well plates and allowed to adhere for 24 h.All of the molecules were dissolved in dimethylsulfoxide (DMSO) as 50 mM master stocks.After 24 h, 1 ml of molecules at the final concentration of 50 mM/well were added to the cells and incubated at 37 � C for 48 h.After 48 h of incubation with the molecules, 20 ml of MTS solution was added to the cells and incubated for 2 h under standard culture conditions.Absorbance was measured at 490 nm using a microplate reader (Multimode Reader Varioskan Lux, ThermoFisher).The absorbance of the blank group was subtracted from the absorbance of the samples The IC50 analysis was done by testing varying doses (0.001-100 mM) of the most effective molecules.DMSO (0.1%) was added to the control wells followed by incubation at 37 � C.After treatment with the molecules, the cells were incubated in the incubator at 37 � C for 48 h.As was done in the MTS assay, absorbance values were measured at 490 nm wavelength.IC50 values were determined using the IC50 Calculator (AAT Bioquest) tool based on a four-parameter logistic regression model as we have done previously (Siyah et al., 2021).Typically, this model resolves as a sigmoid function, or 'S'-shaped curve, which allows the determination of IC50 values.

Apoptosis assay
To determine the effect of molecules on cell apoptosis, PANC-1 and MCF7 cells in the logarithmic growth phase were seeded at a density of 2,000,000 cells/well in six-well plates and HDF cells were seeded at a density of 300,000 cells/well.After 24 h incubation at 37 � C and 5% CO 2 , cells were treated with effective doses (6.25 mM) of small molecules and DMSO (0.1%) in two replicates.After 72 h of the treatment, the cells were washed with ice-cold PBS.Cells were collected from six-well plates and centrifuged at 1500 rpm for 5 min.The pellet was resuspended in 200 ul, 1X binding buffer, then 1 mL Annexin V-FITC was added to the cell suspension.It was mixed and incubated for 10 min at room temperature.After incubation, cells were resuspended in 190 mL of 1X binding buffer.Propidium iodide (PI) was then added to the resuspended cells.Analysis was performed by the flow cytometry (Cytoflex S, Beckman) as we have done previously (Akgol et al., 2021).For the apoptosis assay, we used the 'eBioscience Annexin V-FITC Apoptosis' detection kit.10,000 cells were analyzed per event.Analysis was performed on the FITC-A, PC5.5 and DAPI channels.

Molecular docking
The 3D coordinates of the crystal structure of CB1 with the resolution of 2.6 Å in complex with taranabant (TBM) (PDB code: 5u09) were selected as the receptor model.TBM and water molecules are removed from 5u09 and then converted to PDBQT format using AutoDock Tools (MGL Tools, The Scripps Research Institute).Using the AutoDockTools program, the pocket of TBM was highlighted to determine the grid box locations with 30-30-30 Å search space.3D SDF files of compounds 5a-h, rimonabant and AM6538 were generated using SMILES Translator (Cactus/NIH).Briefly, SMILES codes are provided to the portal at https://cactus.nci.nih.gov/translate/ with SDF, Kekula and 3D output formats.Docking studies were performed using AutoDock Vina 1.1.2and automated using PaDEL-ADV as we have done previously (Turan et al., 2020).To this end, compounds are saved in SDF format in a folder named library.Then, PADEL-ADV was used to initiate docking of the compounds in the ligands folder one by one into the 5u09 receptor according to the grid box coordinates provided.Docking with AutoDock generates docking poses of compounds as well as binding affinities in a csv file.

Synthesis of new 1,2,4-triazole containing compounds
For the synthesis of compound 2, the corresponding 4-chlorophenyl acetonitrile was treated with ethanol in the presence of HCl in an anhydrous cold medium.Compound 3 was obtained by the reaction of synthesized compound 2 with ethyl carbazate in the cold (Figure 1).The current method for the synthesis of compound 4 was improved in terms of both reaction time and yield by applying MW irradiation and US methods in addition to the previously reported procedure (Ikizler & Y€ uksek, 1994;Ikizler et al., 2006).In the reported conventional method, compound 3 was treated with hydrazine hydrate in an aqueous medium at boiling temperature.In the ultrasonication method, the reaction took place with a reaction yield of 72% in 4 h, while in the MW irradiation method, compound 3 was treated with hydrazine hydrate in a closed vessel system in a solvent-free environment with a reaction yield of 95% in 5 h.The corresponding Schiff bases (5a-h) were synthesized by the reaction of aminotriazole compound 4 with various aldehydes such as salicylaldehyde, pyridine-2-carboxaldehyde, pyridine-3-carboxaldehyde, pyridine-4-carboxaldehyde, 3-bromo-4-fluorobenzaldehyde, 2-chloro-6-fluorobenzaldehyde, 4-bromobenzaldehyde, 3-fluorobenzaldehyde.
In order to improve the conventional reaction conditions, MW irradiation and US methods, which are green chemistry techniques that have gained great importance in recent years, have been applied.For this purpose, the synthesizing of compound 5a was selected as a model reaction for both MW and US methods.The MW irradiation method was optimized by varying the power, temperature and time conditions.Accordingly, it was determined that both temperature, power and reaction time were effective on the reaction yield (Table 1).When the optimization conditions were examined, it was observed that the reaction yield increased when the temperature was increased while the applied power remained constant, but when the temperature was increased to 150 � C, the reaction yield decreased.Further, the reaction yields partially decreased when the temperature and power were kept constant and the reaction time was increased.After the temperature and reaction time conditions were determined, the optimization studies were continued by changing the applied power; however, it was noticed that reducing and increasing the power caused a decrease in reaction yield.As a result, the highest reaction yield was obtained at 125 � C, 150 W in 30 min (entry 5).
The second environmentally friendly method, US, was implemented for reaction optimization conditions.Initially, the effect of reaction time on the yield was investigated by keeping the temperature constant (40 � C), and it was determined that the reaction yield increased by raising the time up to 120 min, and thus the highest yield was obtained at 90 min (Table 2).Based on these results, the temperature was increased and the highest yield was achieved at 80 � C (Entry 7).Besides, it was noticed that the reaction yield increased with raising the temperature, the reaction time was again increased to 120 min again, but this condition resulted in a decrease in the reaction yield.
Overall, it was realized that the green chemistry techniques are superior to the conventional method.The reaction, which took place in 3-4 h in the conventional method, was enhanced in terms of both time and yield using green chemistry techniques.Meanwhile, when the reaction yield was examined in these three methods, the highest reaction yield was acquired by the MW irradiation method, except for compounds 5a and 5f.
In the FTIR spectrum of the Schiff base derivatives, it was observed that the NH 2 band of aminotriazole compound (4) disappeared, while NH bands were found in the range of 3170-3200 cm À 1 .Further, new C ¼ N vibrations were detected together with triazole C ¼ O bands appearing in the range of 1701-1722 cm À 1 .The most important evidence for the structural characterization of the target compounds is the disappearance of the NH 2 proton of the aminotriazole compound and the resonance of the singlet protons from the azomethine group in the 1 H NMR spectrum.These single protons resonated about 9.64-10.04ppm in the 1 H NMR spectrum, while they were observed in the range of 147.61-152.69ppm in the 13 C NMR spectrum.Another spectroscopic evidence supporting the structure of Schiff bases were the molecular ion signals noticed in the mass spectra.

Green metric calculations
The descriptions of green chemistry relevant terms, as well as the green metrics are frequently revised in modern literature (Kinen et al., 2009).It is generally agreed that metrics must be clearly identified, basic, measurable and objective rather than subjective (Andraos, 2005;Antenucci et al., 2021;Mohire et al., 2019).Some of the most extensively used metric are the environmental factor based on molecular weight (E mw ), atom economy (AE), mass intensity (MI), reaction mass efficiency (RME), the environmental impact factor based on mass (E m ) and the carbon efficiency (CE) (Antenucci et al., 2021).The applied reaction methods performed in our study provide several of the green chemistry principles with very satisfactory green metrics (Figure 2).The green metric calculations were applied to previously reported method (Sheldon et al., 2022).
Considering the green metric calculation results, the AE% of all three methods are excellent, indicating maximum conversion of raw materials into product and minimum exclusion of waste.Since the reaction yield was found to be higher in both MW and US methods, the CE% value of these methods was found to be better than the conventional method.The same results were also observed for the RME values.Furthermore, it was seen that the MI values in the conventional method were found to be higher due to the use of more solvent compared to other methods.This is significant to point out that the reactions in both MW and US can be achieved with much less solvent.

In silico ADME studies
In silico ADME properties and pharmacokinetic properties of the novel synthesized compounds were examined using an online web-based platform (www.swissadme.ch)(Table 3).The results stated that all compounds obey Lipinski's rule and show favorable pharmacokinetic profiles (Lipinski et al., 1997).Topological polar surface area (Daina et al., 2017), which should not be >140 Å, is another important property associated with drug bioavailability and the obtained results for synthesized final compounds are in the range of 63.04-83.27Å.The prediction of lipophilicity demonstrates that all compounds displayed good lipophilic properties with consensus log P o/w values in the range of 2.46-4.12,while they represented moderate solubility with log S (Ali) values, except for compounds 5b-d, which were found to be water soluble (Ali et al., 2012).Further, the gastrointestinal (GI) absorption and blood-brain barrier (BBB) are two significant pharmacokinetic behaviors for predicting the different levels of the drug discovery processes (Daina & Zoete, 2016).Thus, the compounds exhibited high GI absorption and were estimated to passively permeate through the BBB, except for compound 5a.

Anticancer activity of compounds
Compounds 5a-h were subjected to cancer cell viability analysis after treatment in a pancreatic adenocarcinoma cell line PANC1.PANC-1 is a human pancreatic cancer cell line derived from ductal cell carcinoma in the pancreas.The cells have an epithelial morphology and the ability to metastasize but do not to differentiate.PANC1 cells have been utilized to assess the cytotoxic effect of various drug candidates, as well as to investigate cancer cell migration in response to physiological changes.Here we assessed the effect of compounds 5a-h initially at 50 mM to determine if they have any antiproliferative effect in PANC1 cells.After 2 days of treatment with the compounds, the MTS assay was carried out to determine cell viability.Since compounds were dissolved in DMSO, the control group was treated with 1 mL of DMSO/well.We have found that compounds 5e-g exhibited remarkable inhibition of PANC1 growth (approximately 50% reduction) (Figure 3(A)).Compounds 5b and 5c also  demonstrated mild growth inhibition (about 15-25% reduction compared to control treatment) (Figure 3(A)).Next, we repeated these studies with varying doses of selected compounds, namely compounds 5g (Figure 3(B)), 5e (Figure 3(C)) and 5f (Figure 3(D)) to determine inhibitory concentration %50 (IC50).We found compounds with IC50 values lower than 10 mM, indicating a significant effect in the inhibition of pancreatic cancer growth (Figure 3(E)).Compounds 5g, 5e and 5f demonstrated IC50 values of 5.9, 7.3 and 6.4 mM, respectively.Next, compounds 5e-g were tested in DU145 prostate cancer (Figure 3(F)) and HDF cells (Figure 3(G)).Compounds 5g and 5f decreased the cell viability of both of DU145 and HDF cell lines to 40% and 10%, respectively.This suggests that the effect of compounds 5g and 5f on cancer cell viability is similar in two pathologically distinct cancers, prostate and pancreatic cancer, albeit with cytotoxicity to fibroblasts.
We next assessed the effect of selected compounds on apoptosis to determine whether the effect of cancer growth inhibition was due to cytotoxicity.We treated PANC1 cells with DMSO (control) and compounds 5e-g (Figure 4(A) and (B)) at a concentration of 6.25 mM, which is approximately their IC50 values.We then evaluated the apoptosis and necrosis post three days with a flow cytometer.PANC1 cells were stained with Annexin V and PI and were analyzed for the percentage of early-apoptotic (Annexin V þ PIÀ ), lateapoptotic (Annexin V þ PIþ) and necrotic cells (Annexin V À PIþ).Quantification of early apoptotic cells in PANC1 cells treated with compounds 5g exhibited increased cell death compared to the DMSO treatment.This suggests that the effect of compounds 5e and 5f (and probably for compound 5g as well) is a cytostatic effect rather than cytotoxicity.This is also evident when compounds 5e-g were tested in MCF7 (Supplementary Figure S3(A) and (B)) and HDF cells (Supplementary Figure S3(C) and (D)).Therefore, we investigated the cell cycle of PANC1 cells after treatment with compounds 5e-g.Cell cycle analysis showed that compounds 5e-g increased the percentage of cells in S phase, suggesting a potential S-phase cell cycle arrest in PANC1 cells (Figure 4(C) and (D)).These findings showed that the anticancer effects of compounds 5e-g were associated with cancer cell cycle arrest and were largely cytostatic.Therefore, we next looked at the pathways and similar compounds with cytostatic and anticancer properties.

Novel anticancer compounds with potent CB1 targeting
We have performed structural analysis of similar compounds in the PubChem database.For this purpose, we provided SMILES of compounds to the PubChem portal at https://pubchem.ncbi.nlm.nih.gov/ and analyzed the presence of identical or similar compounds in PubChem.The search did not yield any compounds identical to compounds 5e and 5f.However, there were 20 similar compounds that were further analyses in terms of their known patents and previously reported bioactivities.We have found that PubChem CID 11164161 has similar ring and structural features to compound 5f.PubChem CID 11164161 has been reported in patents as a member of cannabinoid receptor modulators, including rimonabant (PubChem CID: 104850), which is similar to both compound 5f and PubChem CID: 11164161.Rimonabant is the first selective cannabinoid receptor CB1 receptor blocker to be marketed in 38 countries.A recent study reported that a new derivative of rimonabant inhibits breast cancer cell growth and metastasis more effectively in vitro and in vivo (Li et al., 2021).Apart from studies in patients with brain tumors, the use of cannabis in cancer treatment is currently limited to chemotherapy-and radiotherapy-induced nausea and cancer-associated pain (Brown et al., 2013).
Here, we performed molecular docking studies to determine how compounds 5e or 5f can interact with the cannabinoid receptor CB1 in comparison to known synthetic antagonists or inverse agonists such as rimonabant and AM6538 (Shahbazi et al., 2020).The CB1 (PDB:5u09) structure was prepared for docking by AutoDockTools.The docking and calculation of binding affinity of compounds 5a-h, rimonabant and AM6538 were performed using PaDelAdv as we have done previously (Kocabas ¸ & Ergin, 2016;Siyah et al., 2021;Turan et al., 2020).We have found that rimonabant and AM6538 had À 10.0 ± 0.3 and À 7.8 kcal/mol binding affinity to CB1, respectively (Figure 5(A)).Compounds 5e and 5f showed the highest binding with À 9.7 ± 0.3 and À 9.8 ± 0.3 kcal/mol calculated affinity to CB1, similar to rimonabant (Figure 5

Discussion
Novel and better anticancer chemicals are currently in short supply.Nitrogen atoms have heterocyclic ring moieties, which have been found to have potent anticancer effects in both natural and synthetic derivatives.1,2,4-Triazoles are excellent pharmacophores, interacting with biological receptors with high affinity due to their dipole character, hydrogen bonding ability, rigidity and solubility.As a result, new 1,2,4-triazole-containing molecular derivatives were synthesized and evaluated against three unique human cancer cell lines.Compounds 5e and 5f were found to be structurally and functionally comparable to CB1 antagonists.Furthermore, compounds 5e-g reduced pancreatic and prostate cancer cell viability while causing cytotoxicity.Compounds 5e-g also reduced pancreatic and prostate cancer cell viability while being cytotoxic to HDF cells.Compounds 5eg had values from 5.9 to 7.3 M in PANC1 cells.Cell cycle studies revealed that the effect of compounds 5e-g on cancer cell growth was mainly due to S-phase cell cycle arrest.In conclusion, new 1,2,4-triazole-containing compounds with anticancer and anti-CB1 activity were discovered.
Cannabinoids are a class that includes various chemical compounds that act on cannabinoid receptors in cells that alter neurotransmitter release in the brain.Natural cannabis (D9-THC, tetrahydrocannabinol) is obtained from the cannabis plant (Cannabis Sativa) and its therapeutic effects have been known since ancient times (Deiana, 2013).Today, cannabis is used for cancer, MS, AIDS, anti-inflammatory and in Crohn's disease.There are drugs containing natural, synthetic cannabinoids or cannabinoid analogs (Dronabinol, Nabilone, Rimonabant).The main psychoactive component in natural cannabis that affects the central nervous system is D9-THC.CB1 (Central receptor) and CB2 (Peripheral receptor) have been identified as two important cannabinoid receptors.It has been shown that D9-THC exerts its effects on the brain through the CB1 receptor (Tomiyama & Funada, 2011).CB1 receptors are located on central and peripheral nerve endings, and these receptors are found in cortical and subcortical regions, spinal cord and peripheral nerve regions.Activation of CB1 receptors causes mood elevation, euphoria, decreased nausea and increased appetite.CB2 receptors are mostly found in cells belonging to the immune system, and some of them are located in the central nervous system.These receptors are predominantly found in macrophages, B and T lymphocytes, monocytes, leukocytes with polymorphous nuclei and astrocytes (Grotenhermen, 2003).
Recently, the role of cannabinoids in the field of cancer research has been investigated in detail and many studies have been conducted.The use of cannabinoids in cancer therapy depends on their ability to target and kill tumors.Several preclinical studies show that natural and synthetic cannabinoids have anticancer effects against lung carcinoma, thyroid epithelioma, lymphoma, skin carcinoma, uterine carcinoma, breast cancer, prostate carcinoma and pancreas cancer in vitro (Sarfaraz et al., 2008).In vitro studies have also been supported by in vivo studies, and most of the effects of cannabinoids appear to be mediated by CB1 and CB2 receptors.The widespread distribution of cannabinoid receptors (CB1/2, TRPV1) regulates various central and peripheral physiological functions in the body.Importantly, these CB1/2 receptors are responsible for proliferation, motility, invasion, adhesion and apoptosis of cancer cells both in vitro and in vivo.CB1/2 receptor activation leads to a variety of events, including the modulation of Ca 2þ and K þ channels in many tissues and models, the modulation of adenylyl cyclase and cyclic AMP (c-AMP) levels, and the regulation of members of the mitogen-activated protein kinase family (Bosier et al., 2010;Howlett, 2005).One of the important aspects of an effective antitumor drug is its ability to inhibit the proliferation of cancer cells.Cannabinoids have proven to be antiproliferative and apoptotic drugs (Chakravarti et al., 2014).
In conclusion, a series of 1,2,4-triazole-Schiff base derivatives were synthesized via conventional and green chemistry approaches, MW irradiation and US.Considering the reaction yields, it was determined that both environmentally friendly methods had a clear advantage over the conventional method.It resulted from the green metric calculations that the reactions using MW and US methods have better CE than the conventional method, which in this case is equal to the yield.Along the same lines, lower solvent usage and higher yields produce very good RME values in these methods.Moreover, since not too much solvent was used in these green chemistry methods, the MI values were also calculated at an acceptable level.The synthesized compounds were investigated against PANC-1 cell line, and among them, compounds 5g, 5e and 5f exhibited remarkable inhibitory activity with IC50 values of 5.9 mM, 7.3 mM and 6.4 mM, respectively.Furthermore, it resulted from structure-activity relationship that the nature and number of the substituents in the phenyl ring had an effect on activity.The presence of a pyridine linked to the triazole ring caused a decrease in activity, whereas the compounds containing di-substituted electron-withdrawing group were found to be potent against the PANC-1 cell line.In addition, among the compounds including mono-substituent in phenyl, compound 5g with a bulky electron-withdrawing group, bromine, was found to be more effective than the corresponding ones.Compared with the literature studies containing 1,2,4-triazole cores and examining their activities against the PANC-1 cell line, it was determined that while a superior inhibition advantage was observed over some compounds, they were also comparable with others (El-Sherief et al., 2018;Gomaa et al., 2020;Mentes¸e et al., 2021;Mohassab et al., 2021;Yang et al., 2021).In addition, while pancreatic tumor cells express cannabinoid receptors (McKallip et al., 2005(McKallip et al., , 2006)), MCF7 cells have low levels of CB1 and CB2 expression (Mangal et al., 2021).This is consistent with our findings regarding why there is no reduction in cell growth following 5e-g treatments as potent CB inhibitors.

Figure 3 .
Figure 3. Analysis of cancer cell viability.(A) PANC1 pancreatic adenocarcinoma cell line is treated with 50 mM of compounds (5a-h) and analyzed for cell viability post 2 days.Note that compounds numbered 5g, 5e and 5f reduced pancreas cancer growth.Selected compounds (B) 5g, (C) 5e and (D) were further tested for their varying doses (0.001-100 mM) to determine their IC50 values against PANC1.(E) Quantification of IC50 values of 5g, 5e and 5f for PANC 1. (F) DU145 prostate cancer cell line and (G) HDFs were treated with 25 mM of compounds 5g, 5e and 5f and analyzed for cell viability post 2 days.Note that compounds numbered 5g and 5f reduced prostate cancer and HDF growth.UT: Untreated.
(B)).Rimonabant (Figure 5(C)), compound 5e (Figure 5(D)) and compound 5f (Figure 5(E)) docked into the same pocket and interacted with similar residues in CB1.Rimonabant, compounds 5e and 5f were also overpositioned in CB1 (Figure 5(F)).Overall, we have found that compounds 5e and 5f could be potent CB1 antagonists with similar mechanisms of action to the synthetic antagonist or inverse agonist rimonabant and AM6538.However, further validations are required involving pathway analysis and animal studies.

Figure 4 .
Figure 4. Apoptosis and cell cycle analysis by flow cytometer.(A) Representative flow plots and (B) quantification of Annexin V/PI staining of PANC1 cells.(C) Representative flow plots and (D) quantification of Pyronin Y & Hoechst staining of PANC1 cells post treatment with DMSO (control), 5g, 5e and 5f compounds.

Figure 5 .
Figure 5. Molecular docking of selected compounds into CB1.(A) AM6538, rimonabant and compounds were docked into CB1 to determine their binding affinity by AutoDock Vina & PaDel-ADV.(B) Compounds 5e and 5f that had the highest binding affinity to CB1 were similar to rimonabant by structure and binding affinity value.Docking pose of (C) rimonabant, (D) compound 5e and (E) compound 5f with the most optimum pose and binding affinity to CB1. (F) Superpositioning of rimonabant (gray) compound 5e (purple) and compound 5f (red) in the CB1.

Table 1 .
Optimization conditions of the reaction in microwave irradiation.

Table 2 .
Optimization conditions of the reaction in ultrasound sonication.

Table 3 .
In silico ADME results of the synthesized compounds 5a-h.
a Number of hydrogen bond acceptors.b Number of hydrogen bond donors.c Number of rotatable bonds.d Logarithm of partition coefficient between n-octanol and water.e Aqua solubility parameter.f Gastrointestinal absorption.g Blood-brain barrier permeation.h Lipinski's rule of five.TPSA: topological polar surface area.