5-Chlorobenzofuran-2-carboxamides: From allosteric CB1 modulators to potential apoptotic antitumor agents

European Journal of Medicinal Chemistry 177 (2019) 1e11

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

5-Chlorobenzofuran-2-carboxamides: From allosteric CB1 modulators to potential apoptotic antitumor agents Bahaa G.M. Youssif a, b, *, Ashraf M. Mohamed c, Essam Eldin A. Osman d, Ola F. Abou-Ghadir b, Dina H. Elnaggar c, Mostafa H. Abdelrahman e, Laurent Treamblu f, Hesham A.M. Gomaa g, h a

Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Aljouf, Sakaka, 2014, Saudi Arabia Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, 71526, Egypt Applied Organic Chemistry Department, National Research Centre, 12622, Dokki, Giza, Egypt d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, 11562, Cairo, Egypt e Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University, Assiut, 71524, Egypt f School of Natural and Computing Sciences, University of Aberdeen, Meston Building, Aberdeen, AB243UE, Ireland g Pharmacology Department, College of Pharmacy, Jouf University, Sakaka, Aljouf, 2014, Saudi Arabia h Biochemistry Department, Faculty of Pharmacy, Nahda University, Beni-Suef, Egypt b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 March 2019 Received in revised form 5 April 2019 Accepted 13 May 2019 Available online 16 May 2019

Cannabinoids as THC and the CB1 allosteric modulator CBD were reported to have antiproliferative activities with no reports for other CB1 allosteric modulators as the 5-chloroindole-2-carboxamide derivatives and their furan congeners. Based on the antiproliferative activity of two 5-chlorobenzofuran-2carboxamide allosteric CB1 modulators, a series of novel derivatives was designed and synthesized. The synthesized compounds were tested in a cell viability assay using human mammary gland epithelial cell line (MCF-10A) where all the compounds exhibited no cytotoxic effects and more than 85% cell viability at a concentration of 50 mM. Some derivatives showed good antiproliferative activities against tumor cells as compounds 8, 15, 21 and 22. The most active compound 15 showed equipotent activity to doxorubicin. Compounds 7, 9, 15, 16, 21 and 22 increased the level of active caspase 3 by 4e8 folds, compared to the control cells in MCF-7 cell line and doxorubicin as a reference drug. Compounds 15 and 21, the most activecaspase-3 inducers, increase the levels of caspase 8 and 9 indicating activation of both intrinsic and extrinsic pathways and showed potent induction of Bax, down-regulation of Bcl-2 protein levels and over-expression of Cytochrome C levels in MCF-7 cell lines. Compound 15 exhibited cell cycle arrest at the Pre-G1 and G2/M phases in the cell cycle analysis of MCF-7 cell line. The drug Likeness profile of the synthesized compounds showed that all the compounds were predicted to have high oral absorption complying with different pharmacokinetics filters. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: Benzofuran Carboxamide CB1 Modulators Anti-proliferative Apoptotic assay Caspases

1. Introduction The endocannabinoid system encompasses three components: (a) G-protein-coupled cannabinoid receptors CB1 and CB2, (b) endocannabinoids (i.e., endogenous CB receptor ligands), and (c) cellular proteins, enzymes, and carriers involved in the formation, function and inactivation of the endocannabinoids [1e3]. Growing

* Corresponding author. Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, 71526, Egypt. E-mail addresses: [email protected] (B.G.M. Youssif), [email protected]. edu.eg (E.E.A. Osman). https://doi.org/10.1016/j.ejmech.2019.05.040 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

evidence suggests the involvement of the endocannabinoid system in a number of physiological processes and pathological conditions as energy balance [4], obesity [5], appetite disorders [6], inflammation [7], pain [8], neurodegeneration [9] and cancer [10e12]. The CB1 receptor is thought to exist in multiple active conformations, each of which may display distinct abilities to regulate individual signaling pathways [13]. While endogenous ligands as anandamide 1 and exogenous ligands as (
)-D9-tetrahydrocannabinols (D9THC) 2 are orthosteric agonists of the cannabinoid receptors, several small molecules were reported to show allosteric modulation of the CB1 receptor. These CB1 allosteric modulators include the non-psychoactive natural cannabinoid cannabidiol (CBD) 3

2

B.G.M. Youssif et al. / European Journal of Medicinal Chemistry 177 (2019) 1e11

[14], the 5-chloro-indole-2-carbxamide ORG compounds, ORG27759 4, ORG27569 5, ORG29647 6 [15,16] and their benzofuran congeners 7 and 8 [17]. While cannabidiol 3 was shown to be a non-competitive negative allosteric modulator of the CB1 receptors [14], ORG compounds affect the CB1 affinity of some orthosteric agonists positively, despite the fact that they inhibit Gprotein coupling induced by these agonists. ORG27569 5 selectively hampers G-protein signaling and promotes b-arrest in 2-mediated internalization of the receptor and b-arrest in 1-mediated activation of kinases independently from the occupancy of CB1 orthosteric site [15,18]. Allosteric ligand-biased signaling via CB1 may lead to useful agents without the CNS side effects associated with direct receptor agonism [19]. Cannabinoids as THC 2 and CBD 3 were shown to affect cancer cells through various mechanisms including induction of apoptosis [20], inhibition of cell growth [21], tumor angiogenesis, invasion and metastasis [22e24]. Moreover, Cannabinoids were claimed to show selective actions against tumor cells and may even protect normal cells from death [25,26]. More specifically, CBD 3 was found to induce programmed cell death, in a concentration-dependent manner, independent of the CB1, CB2, or vanilloid receptors in both estrogen receptorepositive and estrogen receptorenegative breast cancer cell lines while having little effect on nontumorigenic mammary cells [20]. Furthermore, CBD 3 showed anticancer effects in other cells as lung [24] and colon cancers [27]. Other studies demonstrated the antitumor effect of CBD in preclinical models of breast cancer [21,28] (see Fig. 1). No reports, to the best of our knowledge, were describing the screening of other CB1 allosteric modulators as the ORG compounds and their furan congeners, for their antiproliferative effects despite the strong rationale and attractiveness behind this endeavor. The indole-2-carboxamide derivatives 5 and its furan congener 8 were reported to have equilibrium dissociation constant (KB) of 217.3 and 2594 nM, respectively for the CB1 receptor. Despite the fact that 8 was having much higher KB value, it has higher binding cooperativity factor (a) with the orthosteric CB1 agonist compared to 5 [17] and both compounds antagonized the effects of the orthosteric CB1 agonist on the [35S]GTPgS binding [17]. The 5-chlorobenzfuran-2-carboxamide derivatives 7 and 8 were selected as a starting point in this study based on their facile synthesis. Compounds 7 and 8 were synthesized and tested for their antiproliferative activities in four cancer cell lines, namely lung A549, breast MCF-7, pancreatic Panc-1 and colon HT-29 cancer cell lines. The promising anticancer activity of compounds 7 and 8 promoted the synthesis and testing of a small library of twelve novel compounds where the substitution at C3 was varied from ethyl in compounds 14e18 to methyl in compounds 19e25, Fig. 2. The para positions of the phenethyl tails in the newly synthesized derivatives were either unsubstituted as in 14 and 19 or substituted with 4-dimethylamino in 20, morpholin-4-yl in 15 and 21, piperidin-1-yl in 22 or pyrrolidine-1-yl in 16 and 23 to study the effect of substituent variation. The phenethyl amino carbonyl moieties in 7, 8, 14e16 and 19e23 were modified to 4-benzylpiperidin-1-yl carbonyl in compounds 17 and 24, or 4-phenylpiperazin-1-yl carbonyl in compounds 18 and 25 to study the effect of the amidic hydrogen and the nature of the linker on the anticancer activity.

Fig. 1. Structures of some orthosteric and allosteric CB1 modulators.

of 10a-b in the presence of AlCl3 at 150  C gave the corresponding phenols 11a-b. The reaction of 11a-b with ethyl bromoacetate in the presence of K2CO3 afforded 12a-b which were subjected to cyclization using sodium ethoxide to yield 5-chloro-3alkylbenzofuran-2-carboxylic acids 13a-b. The carboxylic acids 13a-b were coupled with amine derivatives using BOP as the coupling reagent in the presence of DIPEA in DCM to obtain the desired target products 7, 8 and 14e25. The 1H NMR spectrum of 15 as a representative example of this series revealed the appearance of a signal at 6.66 ppm assigned to amide NH, two sets of doublets at 7.18 and 6.90 ppm with J ¼ 8.4 Hz each, which is indicative of aromatic para-disubstitution, two signals with two protons integration each at 3.68 (q) and 2.88 (t) ppm attributed to NHCH2CH2 group and ethyl group signals at d 3.19 (q, J ¼ 7.2 Hz, 2H, CH2CH3) and 1.30 (t, J ¼ 8.0 Hz, 3H, CH2CH3) as well as morpholinyl protons. The structure of 15 was also established by HRESI-MS, which gave a molecular ion m/z 413.1625 [M þ H]þ, which is consistent with the molecular formula C23H26ClN2O3 of the desired product. 2.2. Evaluation of biological activities 2.2.1. In vitro anticancer activity 2.2.1.1. Cell viability assay. Cell viability assay was carried out using human mammary gland epithelial cell line (MCF-10A) [28]. 5Chloro-3-alkylbenzofuran-2-carboxamides 7, 8 and 14e25 were incubated with MCF-10A cells for 4 days and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [29] was used to determine the viability of cells. All compounds exhibited no cytotoxic effects and more than 85% cell viability was reported for the majority of the tested compounds at 50 mM.

2. Results and discussion

2.3. Antiproliferative activity

2.1. Chemistry

The antiproliferative activities of 7, 8 and 14e25 against four human cancer cell lines including pancreas cancer cell line (Panc1), breast cancer cell line (MCF-7), colon cancer cell line (HT-29) and epithelial cancer cell line (A-549) using MTT assay and doxorubicin was used as the reference compound. Graph Pad Prism software (Graph Pad Software, San Diego, CA, USA) was used to

The preparation of target compounds 7, 8 and 14e25 having benzofuran nucleus was outlined in Scheme 1 p-chlorophenol 9 was reacted with propionyl or acetyl chloride in toluene to afford pchlorophenyl propionate or acetate 10a-b. The Fries rearrangement

B.G.M. Youssif et al. / European Journal of Medicinal Chemistry 177 (2019) 1e11

3

Fig. 2. Structures of the target compounds 7, 8 and 14e25.

Scheme 1. Synthetic route of 5-chlorobenzofuran-2-carboxamides 7,8 and 14e25. Reagents and conditions: (a) appropriate acyl chloride, toluene, reflux, 5 h; (b) AlCl3, 80  C for 1 h, then 150  C for 2 h; (c) ethylbromoacetate, K2CO3, acetone, reflux, 6 h; (d) NaOEt, toluene, reflux, 12 h, 86%; (e) appropriate amine, BOP, DIPEA, DCM, rt, overnight.

4

B.G.M. Youssif et al. / European Journal of Medicinal Chemistry 177 (2019) 1e11

calculate the median inhibition concentration (IC50) for all compounds. Results were illustrated in Table 1. The previously described CB1 allosteric modulators 7 and 8 showed promising antiproliferative activity but was less potent compared to doxorubicin, Table 1. The 4-piperidin-4-yl derivative 8 was slightly more potent than its 4-dimethylamino derivative 7. Generally, 5-chloro-3-ethylbenzofuran-2-phenethylcarboxamides 7, 8 and 14e16 showed superior antiproliferative activity compared to their methyl counterparts 19e23, Table 1. For example, the unsubstituted derivatives 14 and 19 showed an average GI50 of 7.37 and 12.25 mM, respectively against the tested cell lines and the same trend applies to the 4-dimethylamino substituted derivatives 7 and 20 (mean GI50 ¼ 5.37 and 6.80 mM) or the 4-morpholin-4-yl derivatives 15 and 21 (mean GI50 ¼ 1.35 and 3.45 mM). It worth mentioning that the 3-ethylbenzofuran-2-carboxamides bearing 4piperidin-4-yl 8 or 4-pyrrolidin-1-yl 16 in the pendant phenyl ring were more or less showing the same potency as their methyl congeners 22 and 23 respectively. The para substitution in the phenethyl tail appears to correlate with higher antiproliferative effects and the activity increased with (R’) in the order of morpholinyl > piperidyl  pyrrolidinyl > dimethylamino > H. The 4-morpholin-4-yl phenethyl derivative 15 was the most potent among the synthesized derivatives, with mean GI50 value of 1.35 mM against the four cell lines, being equipotent to the reference doxorubicin (mean GI50 ¼ 1.13 mM) and even more potent than doxorubicin in MCF-7 cell line (GI50 ¼ 0.7 and 0.9 mM, respectively). The unsubstituted derivative 14 was about 6 folds less potent than 15 showing IC50 ranging from 6.0 ± 1.5 in A-549 to 8.3 ± 2.2 in Panc1 cell lines (mean GI50 ¼ 7.37 mM) while the 4-dimethylamino derivative 7 showed mean GI50 ¼ 5.37 mM. Replacement of the 4morpholin-4-yl moiety in compound 15 by 4-piperidin-1-yl or 4pyrrolidin-1-yl in compound 8 and 16 respectively, resulted in at least 3 folds reduction of the mean GI50 values. Moreover, the mean GI50 values of the 4-benzylpiperidin-1-yl carbonyl derivatives 17 and 24, or the 4-phenylpiperazin-1-yl carbonyl derivatives 18 and 25 were the lowest among the tested compounds, suggesting the importance of the N-phenethyl carboxamide architecture for the antiproliferative activity and correlating with the previous SAR studies of the CB1 allosteric modulators [30].

2.2.3. Apoptosis assay Previous reports documented the effect of the CB1 allosteric modulator, CBD (3) as an apoptosis inducer [19]. Therefore, to reveal the proapoptotic potential of our target compounds, the utmost active compounds 7, 8, 15, 16, 21 and 22 were tested for their potential ability to induce the apoptosis cascade in MCF-7 breast cancer cell line. 2.2.3.1. Activation of proteolytic caspases cascade. Activation of caspases plays a key role in the instigation and completing of the apoptotic process [31]. Among the caspases, the executioner caspase-3 is a vital player that cleaves multiple proteins in the cells, leading to apoptotic cell death [32]. The effect of compounds 7, 8, 15, 16, 21 and 22 on caspase 3 was evaluated and compared to doxorubicin as a reference drug. The results revealed that the tested compounds showed an increase in the level of active caspase 3 by 4e8 folds, compared to the control cells, and that 8, 15 and 21 were the most active compounds that possessed remarkable over expression of caspase-3 protein level (375.20 ± 9.27, 532.2 ± 5.13 and 387.40 ± 12.20 pg/mL, respectively) compared to doxorubicin (503.2 ± 4.22 pg/mL). The most active compound 15 showed an

Fig. 3. Caspase-3 level for compounds 7, 8, 15, 16, 21, 22 and doxorubicin in human breast cancer cell line (MCF-7).

Table 1 Antiproliferative activity of compounds 7, 8, 14e25 and Doxorubicin..

Comp.

R

R0

Cell viability %

7 8 14 15 16 17 18 19 20 21 22 23 24 25 Doxorubicin

Et Et Et Et Et Et Et Me Me Me Me Me Me Me –

N(CH3)2 Piperidine H Morpholine Pyrrolidine – – H N(CH3)2 Morpholine Piperidine Pyrrolidine – – –

85 82 89 91 89 90 82 87 90 84 79 89 96 92 e

Antiproliferative activity GI50± SEM (mM) A-549

MCF-7

Panc-1

HT-29

Average

4.8 ± 1.5 4.5 ± 2.6 6.0 ± 1.5 1.8 ± 0.5 4.9 ± 0.7 27.9 ± 3.6 14.7 ± 2.5 11.4 ± 2.5 6.9 ± 0.3 3.5 ± 0.2 4.4 ± 0.4 5.2 ± 0.6 15.4 ± 1.3 29.5 ± 3.8 1.21 ± 0.80

4.7 ± 1.4 3.6 ± 2.2 7.5 ± 1.2 0.7 ± 0.08 4.4 ± 1.0 26.5 ± 2.8 13.6 ± 2.9 12.9 ± 1.8 6.8 ± 1.6 3.1 ± 0.1 4.9 ± 0.6 4.9 ± 0.3 16.4 ± 5.2 35.4 ± 2.7 0.90 ± 0.62

5.5 ± 2.2 4.6 ± 2.9 8.3 ± 2.2 1.3 ± 0.2 5.6 ± 0.5 33.7 ± 3.5 13.8 ± 1.9 12.5 ± 2.3 6.6 ± 1.5 3.3 ± 0.2 4.1 ± 0.2 4.8 ± 0.5 18.6 ± 2.5 36 ± 1.9 1.41 ± 0.58

6.5 ± 2.1 4.8 ± 1.4 7.7 ± 2.4 1.6 ± 0.2 5.4 ± 1.6 27.8 ± 8.2 13.8 ± 1.6 12.2 ± 1.4 6.9 ± 1.1 3.9 ± 0.6 4.9 ± 0.2 4.9 ± 0.8 16.9 ± 3.7 34.7 ± 3.2 1.01 ± 0.82

5.375 4.375 7.375 1.350 5.075 28.975 13.975 12.250 6.800 3.450 4.575 4.950 16.825 33.900 1.136

B.G.M. Youssif et al. / European Journal of Medicinal Chemistry 177 (2019) 1e11

5

Table 2 Effects of compounds 7, 8, 15, 16, 21, 22 and doxorubicin on active Caspases 3, 8, 9 and Cytochrome C in MCF-7 breast cancer cell line. Compound Number

Caspase-3

7 8 15 16 21 22 Doxorubicin Control

Caspase-8

Caspase-9

Cytochrome C

Conc (pg/ml)

Fold change

Conc (ng/ml)

Fold change

Conc (ng/ml)

Fold change

Conc (ng/ml)

Fold change

254.70 ± 3.14 375.20 ± 9.27 532.20 ± 5.13 339.80 ± 2.19 387.40 ± 12.2 317.35 ± 3.92 503.20 ± 4.22 65.64

3.87 5.71 8.10 5.17 5.90 4.83 7.66 1

e e 1.64 e 0.89 e 1.75 0.17

e e 9.64 e 5.23 e 10.07 1

e e 14.87 e 12.63 e 16.23 0.93

e e 15.98 e 13.58 e 17.40 1

e e 0.774 e 0.518 e 0.604 0.046

e e 16.83 e 11.26 e 13.13 1

increase in the level of active caspase 3 by 8 folds compared to the control untreated cells, and induced caspase 3 higher than doxorubicin (7.66 folds) (Fig. 3). To outline the contribution of the intrinsic and the extrinsic apoptotic pathway in the antiproliferative effects of compounds 15 and 21, their effect on caspases 8 and caspases 9 was also evaluated. Result revealed that compound 15 increases the levels of caspase 8 and 9 by 9.64 and 15.98 folds respectively, while compound 21 showed 5.23 and 13.58 fold increase in the level of caspase 8 and 9, respectively compared to the control cells which indicates activation of both intrinsic and extrinsic pathways with more prominent effect on the intrinsic pathway since caspase 9 levels were higher [33]. Interestingly, these results were in agreement with the previously described increase in the levels caspase 3 and 9 after CBD (3) treatment [19]. (Table 2). 2.2.3.2. Cytochrome C assay. Cytochrome C concentration within the cell has a significant role for activation of caspases and initiating intrinsic apoptosis pathway [33]. Benzofuran derivatives 15 and 21 were evaluated as Cytochrome C activators in MCF-7 human breast cancer cell line and the results are listed in Table 2. Compounds 15 and 21 resulted in over-expression of Cytochrome C levels in MCF-7 human breast cancer cell line about 17 and 11 folds, respectively higher than untreated control cells. The results add another piece of evidence that apoptosis may be credited to over-expression of Cytochrome C and activation of the intrinsic apoptotic pathway induced by the tested compounds. 2.2.3.3. Bax and Bcl-2 levels assay. The most active caspase activators 15 and 21 were further studied for their effect on Bax and Bacl-2 levels against breast cancer cell line (MCF-7) using doxorubicin as a reference. The results showed that 15 and 21 elicited remarkable increase in Bax level compared to doxorubicin Table 3. Compound 15 showed a comparable induction of Bax (284.70 pg/ mL) compared to doxorubicin (276 pg/mL) with 34 fold higher than control untreated breast cancer cell followed by compound 21 (162.72 pg/mL and 19.7 fold change). Finally, compound 15 caused a down-regulation of Bcl-2 protein level up to 0.885 ng/mL followed by compound 21 (1.978 ng/mL) in MCF-7 cell line compared to doxorubicin (0.98 ng/mL).

2.2.3.4. Flow cytometric cell cycle analysis. Cell cycle analysis was performed in MCF-7 human breast cancer cell line treated with the most active compound 15. The percentages of cells of MCF-7 cell line in G0/G1 phase of the cell cycle in the control was 53.71% which recorded a noteworthy decrease to 24.53% upon treatment with compound 15 while the percentage of cells in the S phase were slightly reduced with compound 15 (31.49%) compared to the control (37.42%) (Fig. 4 and Fig. 5). The percentage of MCF-7 human breast cancer cell line at the G2/M phase was apparently increased to 43.98% upon treatment with 15 compared to the control (8.87%). Furthermore, it is observable that the apoptotic cell percentage in the Pre-G1 phase was increased from 1.93% for control untreated MCF-7 human breast cancer cell to 34.29% and 22.17% in cells treated with 15 and doxorubicin, respectively, (Figs. 5 and 6). According to the above results, it is clear that the compound 15 exhibited mainly cell cycle arrest at the Pre-G1 and G2/M phases. Moreover, it is obvious that the tested compound is not cytotoxic but antiproliferative causing programmed cell death and cell cycle arrest.

2.3. Drug likeness profile Assessment of absorption, distribution, metabolism and excretion (ADME) became an early routine in drug discovery programs where the availability of computer models constitutes valid alternatives to experiments. The drug Likeness profile of the tested compounds 7, 8 and 14e25 were predicted using SwissADME website [35]. The results of the drug likeness profile of these compounds are shown in Table 4. All the compounds were predicted to have high oral absorption and penetrate the blood brain barrier (BBB). All the tested compounds showed no violation to Lipnski (Pfizer) filters except one violation for compounds 17 and 25 and no viloations to Ghose [36], Veber (GSK) [37], Egan (Pharmacia) [38] and Muegge (Bayer) [39] filters. While the compounds were free from Brenk proplematic fragments [40], alerts for Pan

Table 3 Bax and Bcl-2 levels for compounds 15, 21 and Doxorubicin in MCF-7 breast cancer cell line. Compound Number

15 21 Doxorubicin Cont.

Bax

Bcl-2

Conc (pg/ml)

Fold change

Conc (ng/ml)

Fold change

284.70 162.72 276.19 8.26

34.46 19.7 33.42 1

0.885 1.978 0.983 5.086

5.77 2.56 5.10 1.00

Fig. 4. Cell cycle analysis in MCF-7 cell line treated with compound 15.

6

B.G.M. Youssif et al. / European Journal of Medicinal Chemistry 177 (2019) 1e11

(3) was reported to be an inhibitor of CYP enzymes specially CYP2D6 and CYP2C19 in vitro [42,43]. 3. Conclusion

Fig. 5. Percentage of apoptosis and necrosis for compounds 15 in MCF-7 cell line.

Assay Interfering substances (PAINS) [41] were recorded for the tested compounds except the unsubstituted derivatives 14 and 19 and the 3ry amides 17, 18, 24 and 25 since they are lacking the dialkylaniline fragment. The potential of the tested compounds to inhibit CYP enzymes warrants further studying. Surprisingly, CBD

In this work, the antiproliferative effect of 5-chlorobenzofuran2-carboxamide CB1 allosteric modulators was reported for the first time. Based on this, a novel series of derivatives was synthesized and tested in A549 lung, MCF-7 breast, Panc-1 Pancreatic and HT29 colon cancer cell lines. Generally, 5-chloro-3-ethylbenzofuran2-phenethyl carboxamides 7, 8 and 14e16 showed superior antiproliferative activity compared to their methyl counterparts 19e23. This trend was observed when the para position was unsubstituted (14 vs. 19) or substituted with 4-dimethylamino (7 vs. 20) or morpholin-4-yl (15 vs. 21). The 3-ethyl benzofuran-2carboxamides bearing 4-piperidin-4-yl 8 or 4-pyrrolidin-1-yl 16 in the pendant phenyl ring were more or less showing the same potency as their methyl congeners 22 and 23 respectively. The para substitution in the phenethyl tail appears to correlate with higher antiproliferative effects and the activity increased with (R’) in the order of morpholinyl > piperidyl  pyrrolidinyl > dimethylamino > H. The 4-morpholin-4-yl phenethyl derivative 15 was the most

Fig. 6. Cell cycle analysis and Apoptosis induction analysis using Annexin V/PI of compound 15 and control untreated MCF-7 cell line.

B.G.M. Youssif et al. / European Journal of Medicinal Chemistry 177 (2019) 1e11

7

Table 4 Molecular properties of compounds 7, 8 and 14e25 predicted using Swiss ADME website. Molecule

7

8

14

15

16

17

18

19

20

21

22

23

24

25

MW #Heavy atoms #Aromatic heavy atoms Fraction Csp3 #Rotatable bonds #H-bond acceptors #H-bond donors TPSA Consensus Log P GI absorption BBB permeant Pgp substrate CYP1A2 inhibitor CYP2C19 inhibitor CYP2C9 inhibitor CYP2D6 inhibitor CYP3A4 inhibitor Lipinski #violations Ghose #violations Veber #violations Egan #violations Muegge #violations PAINS #alerts Brenk #alerts

370.87 26 15

410.94 29 15

327.8 23 15

412.91 29 15

396.91 28 15

381.9 27 15

368.86 26 15

313.78 22 15

356.85 25 15

398.88 28 15

396.91 28 15

382.88 27 15

354.83 25 15

367.87 26 15

0.29

0.38

0.21

0.35

0.35

0.35

0.29

0.17

0.25

0.32

0.35

0.32

0.25

0.32

7 2 1 45.48 4.51

7 2 1 45.48 5.17

6 2 1 42.24 4.5

7 3 1 54.71 4.31

7 2 1 45.48 4.9

5 2 0 33.45 5.19

4 2 0 36.69 4.04

5 2 1 42.24 4.2

6 2 1 45.48 4.2

6 3 1 54.71 3.99

6 2 1 45.48 4.87

6 2 1 45.48 4.62

3 2 0 36.69 3.75

4 2 0 33.45 4.9

Yes No

No Yes

Yes Yes

Yes Yes

Yes No

No Yes

No Yes

No Yes

Yes Yes

Yes Yes

Yes Yes

No Yes

Yes Yes

0

0

0

0

1

0

0

0

0

0

0

0

1

1

1

1

1

1

1

1

0

1

0

1

1

0

1

1

1

1

0

1

0

0

0

1

1

1

1

0

0

High Yes No Yes Yes Yes Yes Yes 0 0 0 0

0

potent among the synthesized derivatives, with mean GI50 value of 1.35 mM against the four tested cell lines, being equipotent to the reference standard doxorubicin (mean GI50 ¼ 1.13 mM) and even more potent than doxorubicin in MCF-7 cell with IC50 of 0.7 and 0.9 mM, respectively. The 4-benzylpiperidin-1-yl carbonyl derivatives 17 and 24, or the 4-phenylpiperazin-1-yl carbonyl derivatives 18 and 25 showed the lowest activity among the tested compounds, suggesting the importance of the N-phenethyl carboxamide architecture for the antiproliferative activity and correlating with the previous SAR studies of the CB1 allosteric modulators. Compounds 7, 8, 15, 16, 21 and 22 increased the level of caspase 3 in MCF-7 by 4e8 folds compared to the control cells, and 8, 15 and 21 were the most active compounds. The morpholinyl substituted 3-ethyl derivative 15 increases the levels of caspase 8, 9 and Cytochrome C by 9.64, 15.98 and 17 folds, respectively while its methyl counterpart 21 showed 5.23, 13.58 and 11 folds in MCF-7 breast cancer cell line. 15 and 21 elicited remarkable increase in Bax level and down-regulation of Bcl-2 protein levels in MCF-7 cell line compared to doxorubicin. The drug Likeness profile of the synthesized compounds was predicted and while all the compounds were predicted to have high oral absorption, penetrant the blood brain barrier (BBB) and show no violation to different pharmacokinetics filters, thier potential to inhibit CYP enzymes warrants further studying. 4. Experimental 4.1. Chemistry general details [see Appendix A] 4.1.1. Synthesis of p-chlorophenyl propionate (10a) A mixture of p-chlorophenol 9 (5 g, 38.89 mmol) and propionyl

chloride (3.74 mL, 42.78 mmol) in dry toluene (30 mL) was refluxed for 5 h. The toluene was evaporated in vacuo to give p-chlorophenyl propionate 10a (6.9 g, 96%) as an oil which was used for further step without purification. 4.1.2. Synthesis of 1-(5-chloro-2-hydroxyphenyl)propan-1-one (11a) A mixture of 10a (6.5 g, 35.20 mmol) and AlCl3 (14.07 g, 105.60 mmol) was heated at 80  C for 1 h then at 150  C for 2 h the resulting residue was decomposed with 3 N HCl and extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4, and evaporated under reduced pressure to give crude product which was purified by flash chromatography on silica gel using a mixture of EtOAc, hexanes (1:30) as eluent to yield 11a (5 g, 77%) as white solid; mp 55e57  C. nmax (KBr disc)/cm
1 3296 (OH), 2980, 1781 (C]O), 1610, 1455, 1284, 810, 718, 684. 1H NMR (400 MHz, Chloroform-d) d 12.21 (s, 1H, OH), 7.70 (d, J ¼ 2.6 Hz, 1H, AreH), 7.38 (dd, J ¼ 8.9, 2.6 Hz, 1H, AreH), 6.92 (d, J ¼ 8.9 Hz, 1H, AreH), 3.00 (q, J ¼ 7.2 Hz, 2H, CH2CH3), 1.23 (t, J ¼ 7.2 Hz, 3H, CH2CH3). 13C NMR (100 MHz, CDCl3) d 206.12 (C]O), 160.78, 135.95, 128.99, 123.46, 120.10, 119.76, 31.64, 7.94. HRESI-MS m/z calcd for [MþH]þ C9H10ClO2: 185.0364, found: 185.0367. 4.1.3. Synthesis of ethyl 2-(4-chloro-2-propionylphenoxy)acetate (12a) A mixture of 11a (4.3 g, 23.29 mmol), ethyl bromoacetate (3.10 mL, 27.95 mmol), and K2CO3 (6.44 g, 46.58 mmol) in acetone (80 mL) was heated under reflux for 6 h. After removing of the solvent in vacuo, the residue was extracted with EtOAc, washed with H2O, brine, dried over MgSO4, and evaporated under reduced pressure to give a crude product which was purified by flash

8

B.G.M. Youssif et al. / European Journal of Medicinal Chemistry 177 (2019) 1e11

chromatography on silica gel using a mixture of EtOAc, hexanes (1:4) as eluent to yield 12a (5.2 g, 83%) as white solid; mp 60e62  C. nmax (KBr disc)/cm
1 2981, 1761 (C]O), 1668 (C]O), 1593, 1480, 1287, 822, 756, 703. 1H NMR (400 MHz, Chloroform-d) d 7.65 (d, J ¼ 2.8 Hz, 1H, AreH), 7.34 (dd, J ¼ 8.8, 2.7 Hz, 1H, AreH), 6.75 (d, J ¼ 8.8 Hz, 1H, AreH), 4.68 (s, 2H, OCH2CO), 4.26 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 3.08 (q, J ¼ 7.2 Hz, 2H, CH2CH3), 1.29 (t, J ¼ 7.1 Hz, 3H, OCH2CH3), 1.15 (t, J ¼ 7.2 Hz, 3H, CH2CH3). 13C NMR (100 MHz, CDCl3) d 201.68 (C]O), 167.80 (OCH2CO),155.01, 132.47, 130.23, 130.09, 127.07, 113.69, 65.73, 61.62, 37.09, 14.10, 8.28. HRESI-MS m/z calcd for [MþH]þ C13H16ClO4: 271.0732, found: 271.0734. 4.1.4. Synthesis of 5-chloro-3-ethylbenzofuran-2-carboxylic acid (13a) A mixture of 12a (3.2 g, 11.82 mmol) and sodium ethoxide (20 mL, 21 wt% solution in ethanol) in toluene was heated at reflux under N2 atmosphere. After 12 h, the solvent was removed under reduced pressure and the product was extracted into H2O. The precipitate formed after acidification of with 3 N HCl was filtered and dried to afford 13a (2.3 g, 86%) as an orange solid; mp 205e207  C. nmax (KBr disc)/cm
1 3100, 2976, 1685 (C]O), 1580, 1459, 1310, 1161, 813, 788, 726, 673. 1H NMR (400 MHz, DMSO‑d6) d 7.91 (d, J ¼ 2.2 Hz, 1H, AreH), 7.67 (d, J ¼ 8.8 Hz, 1H, AreH), 7.49 (dd, J ¼ 8.8, 2.2 Hz, 1H, AreH), 3.01 (q, J ¼ 7.5 Hz, 2H, CH2CH3), 1.20 (t, J ¼ 7.6 Hz, 3H, CH2CH3). 13C NMR (100 MHz, DMSO) d 161.08 (COOH), 152.54, 130.43, 129.75, 128.26, 128.16, 121.41, 114.17, 17.21, 14.80. HRESI-MS m/z calcd for [MþH]þ C11H10ClO3: 225.0313, found: 225.0315. 4.1.5. Synthesis of ethyl 2-(2-acetyl-4-chlorophenoxy)acetate (12b) This compound was prepared as described in preparation of ethyl 2-(4-chloro-2-propionylphenoxy)acetate 12a. Yield % 80, mp 61e63  C. 1H NMR (400 MHz, Chloroform-d) d 7.70 (d, J ¼ 2.5 Hz, 1H, AreH), 7.36 (dd, J ¼ 8.8, 2.8 Hz, 1H, AreH), 6.76 (d, J ¼ 8.8 Hz, 1H, AreH), 4.69 (s, 2H, OCH2CO), 4.26 (q, J ¼ 7.1 Hz, 2H, OCH2CH3), 2.68 (s, 3H, CH3), 1.29 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). 13C NMR (100 MHz, CDCl3) d 198.11 (C]O), 167.74 (OCH2CO), 155.45, 132.97, 130.35, 129.82, 127.08, 113.75, 65.72, 61.66, 31.85, 14.10. HRESI-MS m/z calcd for [MþH]þ C12H14ClO4: 257.0575, found: 257.0581. 4.1.6. Synthesis of 5-chloro-3-methylbenzofuran-2-carboxylic acid (13b) This compound was prepared as described in preparation of 5chloro-3-ethylbenzofuran-2-carboxylic acid 13a. Yield % 83, mp 285e287  C. 1H NMR (400 MHz, DMSO‑d6) d 7.81 (d, J ¼ 2.2 Hz, 1H, AreH), 7.63 (d, J ¼ 8.8 Hz, 1H, AreH), 7.45 (dd, J ¼ 8.8, 2.2 Hz, 1H, AreH), 2.48 (s, 3H, CH3). 13C NMR (100 MHz, DMSO) d 161.56, 152.25, 144.08, 130.90, 128.04, 127.77, 123.36, 121.27, 113.90, 9.48. HRESI-MS m/z calcd for [MþH]þ C10H8ClO3: 211.0156, found: 211.0160. 4.1.7. General procedure for coupling of benzofuran carboxylic acids with amines A mixture of benzofuran-2-carboxylic acids 13a-b (1 equiv), BOP (1.5 equiv), and DIPEA (2 equiv) in DCM (0.05 M) was stirred for 10 min at rt before addition of the appropriate amine (1.2 equiv) and the resulting reaction mixture was stirred overnight at rt. After removing of the solvent in vacuo, the residue was extracted with EtOAc, washed with 5% HCl, saturated NaHCO3 solution, brine, dried over MgSO4, and evaporated under reduced pressure to give a crude product which was purified by flash chromatography on silica gel.

4 .1. 7 .1. 5 - C h l o r o - N - ( 4 - ( d i m e t h y l a m i n o ) p h e n e t h y l ) - 3 ethylbenzofuran-2-carboxamide (7). Yield % 83, mp 147-147  C. 1H NMR (400 MHz, Chloroform-d) d 7.33 (s, 1H, AreH), 7.07e7.05 (m, 2H, AreH), 6.86 (d, J ¼ 8.5 Hz, 2H, AreH), 6.46 (d, J ¼ 8.4 Hz, 2H, AreH), 6.40 (t, J ¼ 6.0 Hz, 1H, amide NH), 3.39 (q, J ¼ 7.0 Hz, 2H, NHCH2CH2), 2.86 (q, J ¼ 7.8 Hz, 2H, CH2CH3), 2.66 (s, 6H, N(CH3)2), 2.58 (t, J ¼ 7.1 Hz, 2H, NHCH2CH2), 1.03 (t, J ¼ 7.8 Hz, 3H, CH2CH3). 13 C NMR (100 MHz, CDCl3) d 159.59 (C]O), 151.63, 149.47, 143.54, 130.31, 129.41, 128.69, 127.66, 126.99, 126.49, 120.64, 113.00, 112.68, 40.75, 40.64, 34.80, 17.07, 14.24. HRESI-MS m/z calcd for [MþH]þ C21H24ClN2O2: 371.1521, found: 371.1519. 4.1.7.2. 5-Chloro-3-ethyl-N-(4-(piperidin-1-yl)phenethyl)benzofuran-2-carboxamide (8). Yield % 82, mp 142e144  C. nmax (KBr disc)/cm
1 3344 (NH), 2932, 1648 (C]O), 1610, 1509, 1442, 1290, 1240, 1156, 920, 805, 731, 642. 1H NMR (400 MHz, Chloroform-d) d 7.60 (s, 1H, AreH), 7.34 (d, J ¼ 1.3 Hz, 2H, AreH), 7.14 (d, J ¼ 8.6 Hz, 2H, AreH), 6.91 (d, J ¼ 8.6 Hz, 2H, AreH), 6.66 (t, J ¼ 5.9 Hz, 1H, amide NH), 3.68 (q, J ¼ 7.1 Hz, 2H, NHCH2CH2), 3.19e3.08 (m, 6H, piperidin-H, CH2CH3), 2.86 (t, J ¼ 7.1 Hz, 2H, NHCH2CH2), 1.75e1.68 (m, 4H, piperidin-H), 1.63e1.53 (m, 2H, piperidin-H), 1.30 (t, J ¼ 7.6 Hz, 3H, CH2CH3). 13C NMR (100 MHz, cdcl3) d 159.58 (C]O), 151.63, 150.96, 143.50, 130.30, 129.34, 129.13, 128.70, 127.69, 127.01, 120.64, 116.78, 112.69, 50.78, 40.49, 34.90, 25.88, 24.26, 17.07, 14.24. HRESI-MS m/z calcd for [MþH]þ C24H28ClN2O2: 411.1834, found: 411.1829. 4.1.7.3. 5-Chloro-3-ethyl-N-phenethylbenzofuran-2-carboxamide (14). Yield % 85, mp 85e87  C. 1H NMR (400 MHz, Chloroform-d) d 7.61 (s, 1H, AreH), 7.39e7.21 (m, 7H, AreH), 6.69 (t, J ¼ 6.0 Hz, 1H, amide NH), 3.72 (q, J ¼ 6.7 Hz, 2H, NHCH2CH2), 3.13 (q, J ¼ 7.5 Hz, 2H, CH2CH3), 2.96 (t, J ¼ 7.2 Hz, 2H, NHCH2CH2), 1.31 (t, J ¼ 7.6 Hz, 3H, CH2CH3). 13C NMR (100 MHz, CDCl3) d 159.63 (C]O), 151.63, 143.41, 138.70, 130.27, 128.79, 128.75, 128.69, 127.84, 127.08, 126.59, 120.67, 112.67, 40.36, 35.89, 17.07, 14.23. HRESI-MS m/z calcd for [MþH]þ C19H19ClNO2: 328.1099, found: 328.1101. 4.1.7.4. 5-Chloro-3-ethyl-N-(4-morpholinophenethyl)benzofuran-2carboxamide (15). Yield % 80, mp 110e112  C. 1H NMR (400 MHz, Chloroform-d) d 7.61 (s, 1H, AreH), 7.34 (d, J ¼ 1.5 Hz, 2H, AreH), 7.18 (d, J ¼ 8.3 Hz, 2H, AreH), 6.90 (d, J ¼ 8.4 Hz, 2H, AreH), 6.66 (t, J ¼ 6.1 Hz, 1H, amide NH), 3.89e3.85 (m, 4H, morph-H), 3.68 (q, J ¼ 6.8 Hz, 2H, NHCH2CH2), 3.19e3.08 (m, 6H, morph-H, CH2CH3), 2.88 (t, J ¼ 7.1 Hz, 2H, NHCH2CH2), 1.30 (t, J ¼ 8.0 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 159.60 (C]O), 151.63, 149.90, 143.45, 130.29, 130.18, 129.53, 128.74, 127.79, 127.06, 120.67, 116.02, 112.67, 66.90, 49.53, 40.49, 34.93, 17.07, 14.23. HRESI-MS m/z calcd for [MþH]þ C23H26ClN2O3: 413.1626, found: 413.1625. 4.1.7.5. 5-Chloro-3-ethyl-N-(4-(pyrrolidin-1-yl)phenethyl)benzofuran-2-carboxamide (16). Yield % 79, mp 140-140  C. 1H NMR (400 MHz, Chloroform-d) d 7.61 (s, 1H, AreH), 7.34 (d, J ¼ 1.3 Hz, 2H, AreH), 7.12 (d, J ¼ 8.5 Hz, 2H, AreH), 6.69 (t, J ¼ 5.9 Hz, 1H, amide NH), 6.55 (d, J ¼ 8.5 Hz, 2H, AreH), 3.67 (q, J ¼ 7.0 Hz, 2H, NHCH2CH2), 3.32e3.24 (m, 4H, pyrrolidin-H), 3.14 (q, J ¼ 7.6 Hz, 2H, CH2CH3), 2.85 (t, J ¼ 7.1 Hz, 2H, NHCH2CH2), 2.04e1.96 (m, 4H, pyrrolidin-H), 1.31 (t, J ¼ 7.6 Hz, 3H, CH2CH3). 13C NMR (100 MHz, CDCl3) d 159.59 (C]O), 146.78, 143.58, 130.32, 129.49, 128.68, 127.62, 126.96, 125.08, 120.63, 112.69, 111.87, 47.68, 40.76, 34.87, 25.46, 17.07, 14.25. HRESI-MS m/z calcd for [MþH]þ C23H26ClN2O2: 397.1677, found: 397.1668. 4.1.7.6. (4-Benzylpiperidin-1-yl)(5-chloro-3-ethylbenzofuran-2-yl) methanone (17). Yield % 75, mp 80e82  C. 1H NMR (400 MHz, Chloroform-d) d 7.58 (d, J ¼ 2.1 Hz, 1H, AreH), 7.37 (d, J ¼ 8.8 Hz, 1H,

B.G.M. Youssif et al. / European Journal of Medicinal Chemistry 177 (2019) 1e11

AreH), 7.34e7.23 (m, 3H, AreH), 7.25e7.17 (m, 1H, AreH), 7.18e7.11 (m, 2H, AreH), 4.68e4.63 (m, 1H, piperidin-H), 4.01e3.98 (m, 1H, piperidin-H), 3.04e2.70 (m, 4H, piperidin-H, CH2CH3), 2.58 (d, J ¼ 7.1 Hz, 2H, PhCH2), 1.86e1.74 (m, 4H, piperidin-H), 1.33e1.28 (m, 4H, piperidin-H, CH2CH3). 13C NMR (100 MHz, CDCl3) d 160.35 (C]O), 151.99, 145.49, 129.68, 129.08, 128.54, 128.33, 126.09, 126.06, 125.01, 120.16, 112.78, 42.93, 38.32, 17.21, 14.23. HRESI-MS m/z calcd for [MþH]þ C23H25ClNO2: 382.1568, found: 382.1569. 4.1.7.7. (5-Chloro-3-ethylbenzofuran-2-yl)(4-phenylpiperazin-1-yl) methanone (18). Yield % 76, mp 108e110  C. 1H NMR (400 MHz, Chloroform-d) d 7.76 (s, 1H, AreH), 7.60e7.39 (m, 4H, AreH), 7.13e7.06 (m, 3H, AreH), 4.07e4.02 (m, 4H, piperazin-H), 3.43e3.39 (m, 4H, piperazin-H), 3.06 (q, J ¼ 7.5 Hz, 2H), 1.47 (t, J ¼ 7.6 Hz, 3H). 13C NMR (100 MHz, CDCl3) d 160.39 (C]O), 151.98, 150.89, 144.74, 129.61, 129.27, 128.73, 126.55, 126.47, 120.64, 120.32, 116.70, 112.82, 49.81, 48.00, 46.69, 42.61, 17.30, 14.24. HRESI-MS m/z calcd for [MþH]þ C21H22ClN2O2: 369.1364, found: 369.1366. 4.1.7.8. 5-Chloro-3-methyl-N-phenethylbenzofuran-2-carboxamide (19). Yield % 84, mp 80e82  C. 1H NMR (400 MHz, Chloroform-d) d 7.54 (d, J ¼ 2.0 Hz, 1H, AreH), 7.38e7.22 (m, 7H, AreH), 6.71 (t, J ¼ 5.9 Hz, 1H, amide NH), 3.73 (q, J ¼ 7.2 Hz, 2H, NHCH2CH2), 2.95 (t, J ¼ 7.2 Hz, 2H, NHCH2CH2), 2.59 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) d 159.87 (C]O), 151.46, 143.91, 138.70, 131.11, 128.80, 128.78, 128.69, 127.17, 126.59, 121.73, 120.52, 112.55, 40.39, 35.89, 8.82. HRESI-MS m/z calcd for [MþH]þ C18H17ClNO2: 314.0942, found: 314.0945. 4 .1. 7 . 9 . 5 - C h l o r o - N - ( 4 - ( d i m e t h y l a m i n o ) p h e n e t h y l ) - 3 methylbenzofuran-2-carboxamide (20). Yield % 79, mp 125e127  C. 1 H NMR (400 MHz, Chloroform-d) d 7.26 (s, 1H, AreH), 7.04 (d, J ¼ 2.0 Hz, 2H, AreH), 6.83 (d, J ¼ 8.4 Hz, 2H, AreH), 6.46e6.33 (m, 3H, AreH, amide NH), 3.37 (q, J ¼ 6.8 Hz, 2H, NHCH2CH2), 2.64 (s, 6H, N(CH3)2), 2.55 (t, J ¼ 7.0 Hz, 2H, NHCH2CH2), 2.29 (s, 3H, CH3). 13 C NMR (100 MHz, CDCl3) d 159.82 (C]O), 151.47, 149.47, 144.05, 131.18, 129.41, 128.76, 127.09, 126.48, 121.55, 120.52, 113.00, 112.58, 40.74, 40.64, 34.81, 8.82. HRESI-MS m/z calcd for [MþH]þ C20H22ClN2O2: 357.1364, found: 357.1362. 4.1.7.10. 5-Chloro-3-methyl-N-(4-morpholinophenethyl)benzofuran2-carboxamide (21). Yield % 82, mp 180e182  C. 1H NMR (400 MHz, Chloroform-d) d 7.57 (d, J ¼ 1.8 Hz, 1H, AreH), 7.35e7. 33 (m, 2H, AreH), 7.17 (d, J ¼ 8.4, 2H, AreH), 6.89 (d, J ¼ 8.4 Hz, 2H, AreH), 6.66 (t, J ¼ 5.9 Hz, 1H, amide NH), 3.90e3.83 (m, 4H, morph-H), 3.68 (q, J ¼ 6.7 Hz, 2H, NHCH2CH2), 3.18e3.11 (m, 4H, morph-H), 2.88 (t, J ¼ 7.1 Hz, 2H, NHCH2CH2), 2.59 (s, 3H). 13C NMR (100 MHz, CDCl3) d 159.83 (C]O), 151.47, 149.97, 143.97, 131.16, 130.09, 129.52, 128.80, 127.16, 121.67, 120.55, 115.98, 112.56, 66.92, 49.48, 40.49, 34.93, 8.82. HRESI-MS m/z calcd for [MþH]þ C22H24ClN2O3: 399.1470, found: 399.1470. 4.1.7.11. 5-Chloro-3-methyl-N-(4-(piperidin-1-yl)phenethyl)benzofuran-2-carboxamide (22). Yield % 80, mp 155e157  C. 1H NMR (400 MHz, Chloroform-d) d 7.56 (d, J ¼ 1.8 Hz, 1H, AreH), 7.37e7.31 (m, 2H, AreH), 7.13 (d, J ¼ 8.6 Hz, 2H, AreH), 6.91 (d, J ¼ 8.6 Hz, 2H, AreH), 6.65 (t, J ¼ 5.8 Hz, 1H, amide NH), 3.68 (q, J ¼ 7.1 Hz, 2H, NHCH2CH2), 3.18e3.10 (m, 4H, piperidin-H), 2.86 (t, J ¼ 7.1 Hz, 2H, NHCH2CH2), 2.59 (s, 3H, CH3), 1.77e1.66 (m, 4H, piperidin-H), 1.63e1.53 (m, 2H, piperidin-H). 13C NMR (100 MHz, CDCl3) d 159.81 (C]O), 151.47, 150.98, 144.01, 131.17, 129.34, 129.10, 128.77, 127.11, 121.59, 120.52, 116.77, 112.58, 50.77, 40.49, 34.90, 25.88, 24.27, 8.82. HRESI-MS m/z calcd for [MþH]þ C23H26ClN2O2: 397.1677, found: 397.1673.

9

4.1.7.12. 5-Chloro-3-methyl-N-(4-(pyrrolidin-1-yl)phenethyl)benzofuran-2-carboxamide (23). Yield % 80, mp 140e142  C. 1H NMR (400 MHz, Chloroform-d) d 7.56 (d, J ¼ 1.4 Hz, 1H, AreH), 7.35e7.32 (m, 2H, AreH), 7.12 (d, J ¼ 8.4 Hz, 2H, AreH), 6.68 (t, J ¼ 5.7 Hz, 1H, amide NH), 6.55 (d, J ¼ 8.5 Hz, 2H, AreH), 3.67 (q, J ¼ 7.1 Hz, 2H, NHCH2CH2), 3.32e3.24 (m, 4H, pyrrolidin-H), 2.85 (t, J ¼ 7.0 Hz, 2H, NHCH2CH2), 2.59 (s, 3H, CH3), 2.04e1.96 (m, 4H, pyrrolidin-H). 13 C NMR (100 MHz, CDCl3) d 159.81 (C]O), 151.47, 146.79, 144.09, 131.19, 129.49, 128.74, 127.06, 125.08, 121.50, 120.50, 112.58, 111.87, 47.68, 40.76, 34.87, 25.45, 8.82. HRESI-MS m/z calcd for [MþH]þ C22H24ClN2O2: 383.1521, found: 383.1517. 4.1.7.13. (4-Benzylpiperidin-1-yl)(5-chloro-3-methylbenzofuran-2yl)methanone (24). Yield % 75, mp 100e102  C. 1H NMR (400 MHz, Chloroform-d) d 6.93 (d, J ¼ 2.0 Hz, 1H, AreH), 6.81e6.52 (m, 7H, AreH), 4.11e4.02 (m, 1H, piperidin-H), 3.46e3.42 (m, 1H, piperidin-H), 2.46e2.42 (m, 1H, piperidin-H), 2.16e2.10 (m, 1H, piperidin-H), 1.99 (d, J ¼ 7.1 Hz, 2H, PhCH2), 1.77 (s, 3H, CH3), 1.29e1.12 (m, 4H, piperidin-H), 0.79e0.64 (m, 1H, piperidin-H). 13C NMR (100 MHz, CDCl3) d 160.38 (C]O), 151.82, 145.83, 139.87, 130.52, 129.07, 128.61, 128.32, 126.26, 126.09, 119.98, 112.65, 42.94, 38.36, 8.75. HRESI-MS m/z calcd for [MþH]þ C22H23ClNO2: 368.1412, found: 368.1413. 4.1.7.14. (5-Chloro-3-methylbenzofuran-2-yl)(4-phenylpiperazin-1yl)methanone (25). Yield % 76, mp 140e142  C. 1H NMR (400 MHz, Chloroform-d) d 7.57 (d, J ¼ 2.1 Hz, 1H, AreH), 7.44e7.24 (m, 4H, AreH), 7.00e6.88 (m, 3H, AreH), 3.92e3.87 (m, 4H, piperazin-H), 3.30e3.27 (m, 4H, piperazin-H), 2.44 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) d 160.40 (C]O), 151.81, 150.89, 145.17, 130.47, 129.27, 128.80, 126.67, 120.85, 120.63, 120.16, 116.70, 112.71, 49.83, 48.00, 46.73, 42.62, 8.92. HRESI-MS m/z calcd for [MþH]þ C20H20ClN2O2: 355.1208, found: 355.1209. 4.2. Biological evaluation 4.2.1. Cytotoxic activity using MTT assay and evaluation of IC50 4.2.1.1. MTT assay. MTT assay was performed to investigate the effect of the synthesized compounds on the viability of mammary epithelial cells (MCF-10A) [44,45]. See Appendix A. 4.2.1.2. Assay for antiproliferative effect. To explore the antiproliferative potential of compounds MTT assay was performed according to previously reported procedure [46,47] using different cell lines. See Appendix A. 4.2.1.3. Caspase-3, 8 and 9 activation assay. Cell line cells of human breast MCF-7 cell line were obtained from ATCC. RPMI 1640 containing 10% FBS was used to allow cells to grow at 37  C, stimulated with the compounds to be tested for caspase-3, 8 or caspase-9 [48]. See Appendix A. 4.2.1.4. Cytochrome C assay. Cells were obtained from American Type Culture Collection, cells were grown in RPMI 1640 containing 10% fetal bovine serum at 37  C, stimulated with the compounds to be tested for Cytochrome C [48]. See Appendix A. 4.2.1.5. Evaluation of bax and Bcl-2 expressions. m RNA isolation was carried out using RNeasy extraction kit, up to 1  107 cells. They were disrupted in Buffer RLT and homogenized according to a previously reported procedure [49]. See Appendix A. 4.2.1.6. Cell apoptosis assay. Apoptosis was determined by flow cytometry based on the Annexin-V-fluoresce in isothiocyanate (FITC) and propidium iodide (PI) stainingkit (BD Pharmingen, San

10

B.G.M. Youssif et al. / European Journal of Medicinal Chemistry 177 (2019) 1e11

Diego, USA) [50,51]. See Appendix A. Conflicts of interest The authors declare no conflict of interest. Acknowledgment The authors thank The Deanship of Scientific Research, Jouf University, Saudi Arabia for financially supporting this work through the Grant Number (39/550). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.05.040. References [1] V. Di Marzo, M. Bifulco, L. De Petrocellis, The endocannabinoid system and its therapeutic exploitation, Nat. Rev. Drug Discov. 3 (2004) 771e784. [2] D. Piomelli, The endocannabinoid system: a drug discovery perspective, Curr. Opin. Investig. Drugs 6 (2005) 672e679. [3] V. Di Marzo, Targeting the endocannabinoid system: to enhance or reduce? Nat. Rev. Drug Discov. 7 (2008) 438e455. [4] I. Matias, V. Di Marzo, Endocannabinoids and the control of energy balance, Trends Endocrinol. Metabol. 18 (2007) 27e37. [5] L. Bellocchio, G. Mancini, V. Vicennati, R. Pasquali, U. Pagotto, Cannabinoid receptors as therapeutic targets for obesity and metabolic diseases, Curr. Opin. Pharmacol. 6 (2006) 586e591. [6] C. Li, P.M. Jones, S.J. Persaud, Role of the endocannabinoid system in food intake, energy homeostasis and regulation of the endocrine pancreas, Pharmacol. Ther. 129 (2011) 307e320. [7] P. Nagarkatti, R. Pandey, S.A. Rieder, V.L. Hegde, M. Nagarkatti, Cannabinoids as novel anti-inflammatory drugs, Future Med. Chem. 1 (2009) 1333e1349. [8] J. Guindon, A.G. Hohmann, The endocannabinoid system and pain, CNS Neurol. Disord. - Drug Targets 8 (2009) 403e421. [9] T. Bisogno, V. Di Marzo, The role of the endocannabinoid system in Alzheimer's disease: facts and hypotheses, Curr. Pharmaceut. Des. 14 (2008) 2299e3305. [10] A.M. Malfitano, E. Ciaglia, G. Gangemi, P. Gazzerro, C. Laezza, M. Bifulco, Update on the endocannabinoid system as an anticancer target, Expert Opin. Ther. Targets 15 (2011) 297e308. [11] D.J. Hermanson, L.J. Marnett, Cannabinoids, endocannabinoids, and cancer, Cancer Metastasis Rev. 30 (2011) 599e612. [12] G. Velasco, C. Sanchez, M. Guzman, Endocannabinoids and cancer, Handb. Exp. Pharmacol. 231 (2015) 449e472. [13] B.D. Hudson, T.E. Hebert, M.E. Kelly, Ligand- and heterodimer-directed signaling of the CB(1) cannabinoid receptor, Mol. Pharmacol. 77 (2010) 1e9. [14] R.B. Laprairie, A.M. Bagher, M.E. Kelly, E.M. Denovan-Wright, Cannabidiol is a negative allosteric modulator of the cannabinoid CB1 receptor, Br. J. Pharmacol. 172 (2015) 4790e4805. [15] K.H. Ahn, M.M. Mahmoud, D.A. Kendall, Allosteric modulator ORG27569 induces CB1 cannabinoid receptor high affinity agonist binding state, receptor internalization, and Gi protein-independent ERK1/2 kinase activation, J. Biol. Chem. 287 (2012) 12070e12082. [16] E.E. Cawston, W.J. Redmond, C.M. Breen, N.L. Grimsey, M. Connor, M. Glass, Real-time characterization of cannabinoid receptor 1 (CB1 ) allosteric modulators reveals novel mechanism of action, Br. J. Pharmacol. 170 (2013) 893e907. [17] M.M. Mahmoud, H.I. Ali, K.H. Ahn, A. Damaraju, S. Samala, V.K. Pulipati, S. Kolluru, D.A. Kendall, D. Lu, Structure-activity relationship study of indole2-carboxamides identifies a potent allosteric modulator for the cannabinoid receptor 1 (CB1), J. Med. Chem. 56 (2013) 7965e7975. [18] K.H. Ahn, M.M. Mahmoud, J.Y. Shim, D.A. Kendall, Distinct roles of betaarrestin 1 and beta-arrestin 2 in ORG27569-induced biased signaling and internalization of the cannabinoid receptor 1 (CB1), J. Biol. Chem. 288 (2013) 9790e9800. [19] L. Khurana, K. Mackie, D. Piomelli, D.A. Kendall, Modulation of CB1 cannabinoid receptor by allosteric ligands: pharmacology and therapeutic opportunities, Neuropharmacology 124 (2017) 3e12. [20] A. Shrivastava, P.M. Kuzontkoski, J.E. Groopman, A. Prasad, Cannabidiol induces programmed cell death in breast cancer cells by coordinating the crosstalk between apoptosis and autophagy, Mol. Cancer Ther. 10 (2011) 1161e1172. [21] S.D. McAllister, R. Murase, R.T. Christian, D. Lau, A.J. Zielinski, J. Allison, C. Almanza, A. Pakdel, J. Lee, C. Limbad, Y. Liu, R.J. Debs, D.H. Moore, P.Y. Desprez, Pathways mediating the effects of cannabidiol on the reduction of breast cancer cell proliferation, invasion, and metastasis, Breast Canc. Res.

Treat. 129 (2011) 37e47. [22] C. Blazquez, M.L. Casanova, A. Planas, T. Gomez Del Pulgar, C. Villanueva, M.J. Fernandez-Acenero, J. Aragones, J.W. Huffman, J.L. Jorcano, M. Guzman, Inhibition of tumor angiogenesis by cannabinoids, FASEB J. 17 (2003) 529e531. [23] A. Vaccani, P. Massi, A. Colombo, T. Rubino, D. Parolaro, Cannabidiol inhibits human glioma cell migration through a cannabinoid receptor-independent mechanism, Br. J. Pharmacol. 144 (2005) 1032e1036. [24] R. Ramer, K. Bublitz, N. Freimuth, J. Merkord, H. Rohde, M. Haustein, P. Borchert, E. Schmuhl, M. Linnebacher, B. Hinz, Cannabidiol inhibits lung cancer cell invasion and metastasis via intercellular adhesion molecule-1, FASEB J. 26 (2012) 1535e1548. [25] G. Velasco, C. Sanchez, M. Guzman, Towards the use of cannabinoids as antitumour agents, Nat. Rev. Canc. 12 (2012) 436e444. [26] B.J. Cridge, R.J. Rosengren, Critical appraisal of the potential use of cannabinoids in cancer management, Cancer Manag. Res. 5 (2013) 301e313. [27] B. Romano, F. Borrelli, E. Pagano, M.G. Cascio, R.G. Pertwee, A.A. Izzo, Inhibition of colon carcinogenesis by a standardized Cannabis sativa extract with high content of cannabidiol, Phytomedicine 21 (2014) 631e639. [28] M.M. Caffarel, C. Andradas, E. Mira, E. Perez-Gomez, C. Cerutti, G. MorenoBueno, J.M. Flores, I. Garcia-Real, J. Palacios, S. Manes, M. Guzman, C. Sanchez, Cannabinoids reduce ErbB2-driven breast cancer progression through Akt inhibition, Mol. Cancer 9 (2010) 196. [29] H.D. Soule, T.M. Maloney, S.R. Wolman, W.D. Peterson Jr., R. Brenz, C.M. McGrath, J. Russo, R.J. Pauley, R.F. Jones, S.C. Brooks, Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10, Cancer Res 50 (1990) 6075e6086. [30] D.A. Scudiero, R.H. Shoemaker, K.D. Paull, A. Monks, S. Tierney, T.H. Nofziger, M.J. Currens, D. Seniff, M.R. Boyd, Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines, Cancer Res. 48 (1988) 4827e4833. [31] T. Nguyen, J.X. Li, B.F. Thomas, J.L. Wiley, T.P. Kenakin, Y. Zhang, Allosteric modulation: an alternate approach targeting the cannabinoid CB1 receptor, Med. Res. Rev. 37 (2017) 441e474. [32] G.M. Cohen, Caspases: the executioners of apoptosis, Biochem. J. 326 (Pt 1) (1997) 1e16. [33] E.A. Slee, C. Adrain, S.J. Martin, Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis, J. Biol. Chem. 276 (2001) 7320e7326. [35] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules, Sci. Rep. 7 (2017) 42717. [36] A.K. Ghose, V.N. Viswanadhan, J.J. Wendoloski, A knowledge-based approach in designing combinatorial or medicinal chemistry libraries for drug discovery. 1. A qualitative and quantitative characterization of known drug databases, J. Comb. Chem. 1 (1999) 55e68. [37] D.F. Veber, S.R. Johnson, H.Y. Cheng, B.R. Smith, K.W. Ward, K.D. Kopple, Molecular properties that influence the oral bioavailability of drug candidates, J. Med. Chem. 45 (2002) 2615e2623. [38] W.J. Egan, K.M. Merz Jr., J.J. Baldwin, Prediction of drug absorption using multivariate statistics, J. Med. Chem. 43 (2000) 3867e3877. [39] I. Muegge, S.L. Heald, D. Brittelli, Simple selection criteria for drug-like chemical matter, J. Med. Chem. 44 (2001) 1841e1846. [40] R. Brenk, A. Schipani, D. James, A. Krasowski, I.H. Gilbert, J. Frearson, P.G. Wyatt, Lessons learnt from assembling screening libraries for drug discovery for neglected diseases, ChemMedChem 3 (2008) 435e444. [41] J.B. Baell, G.A. Holloway, New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays, J. Med. Chem. 53 (2010) 2719e2740. [42] S. Yamaori, Y. Okamoto, I. Yamamoto, K. Watanabe, Cannabidiol, a major phytocannabinoid, as a potent atypical inhibitor for CYP2D6, Drug Metab. Dispos. 39 (2011) 2049e2056. [43] R. Jiang, S. Yamaori, Y. Okamoto, I. Yamamoto, K. Watanabe, Cannabidiol is a potent inhibitor of the catalytic activity of cytochrome P450 2C19, Drug Metab. Pharmacokinet. 28 (2013) 332e338. [44] H.L. Qin, J. Leng, B.G.M. Youssif, M.W. Amjad, M.A.G. Raja, M.A. Hussain, Z. Hussain, S.N. Kazmi, S.N.A. Bukhari, Synthesis and mechanistic studies of curcumin analog-based oximes as potential anticancer agents, Chem. Biol. Drug Des. 90 (2017) 443e449. [45] H.A.M. El-Sherief, B.G.M. Youssif, S.N. Abbas Bukhari, A.H. Abdelazeem, M. Abdel-Aziz, H.M. Abdel-Rahman, Synthesis, anticancer activity and molecular modeling studies of 1,2,4-triazole derivatives as EGFR inhibitors, Eur. J. Med. Chem. 156 (2018) 774e789. [46] B.G.M. Youssif, M.H. Abdelrahman, A.H. Abdelazeem, M.A. Abdelgawad, H.M. Ibrahim, O.I.A. Salem, M.F.A. Mohamed, L. Treambleau, S.N.A. Bukhari, Design, synthesis, mechanistic and histopathological studies of smallmolecules of novel indole-2-carboxamides and pyrazino[1,2-a]indol-1(2H)ones as potential anticancer agents effecting the reactive oxygen species production, Eur. J. Med. Chem. 146 (2018) 260e273. [47] M.S. Abdelbaset, G.E.A. Abuo-Rahma, M.H. Abdelrahman, M. Ramadan, B.G.M. Youssif, S.N.A. Bukhari, M.F.A. Mohamed, M. Abdel-Aziz, Novel pyrrol2(3H)-ones and pyridazin-3(2H)-ones carrying quinoline scaffold as antiproliferative tubulin polymerization inhibitors, Bioorg. Chem. 80 (2018) 151e163. [48] M.S. Abdelbaset, M. Abdel-Aziz, G.E.A. Abuo-Rahma, M.H. Abdelrahman,

B.G.M. Youssif et al. / European Journal of Medicinal Chemistry 177 (2019) 1e11 M. Ramadan, B.G.M. Youssif, Novel quinoline derivatives carrying nitrones/ oximes nitric oxide donors: design, synthesis, antiproliferative and caspase-3 activation activities, Arch. Pharm. (Weinheim) 352 (2019), e1800270. [49] T. Mitupatum, K. Aree, S. Kittisenachai, S. Roytrakul, S. Puthong, S. Kangsadalampai, P. Rojpibulstit, mRNA expression of Bax, bcl-2, p53, cathepsin B, caspase-3 and caspase-9 in the HepG2 cell line following induction by a novel monoclonal ab Hep88 mAb: cross-talk for paraptosis and apoptosis, Asian Pac. J. Cancer Prev. APJCP 17 (2016) 703e712.

11

[50] M.H. Abdelrahman, A.S. Aboraia, B.G.M. Youssif, B.E.M. Elsadek, Design, synthesis and pharmacophoric model building of new 3-alkoxymethyl/3-phenyl indole-2-carboxamides with potential antiproliferative activity, Chem. Biol. Drug Des. 90 (2017) 64e82. [51] H.A.M. El-Sherief, B.G.M. Youssif, S.N.A. Bukhari, M. Abdel-Aziz, H.M. AbdelRahman, Novel 1,2,4-triazole derivatives as potential anticancer agents: design, synthesis, molecular docking and mechanistic studies, Bioorg. Chem. 76 (2018) 314e325.