Development of novel benzofuran-isatin conjugates as potential antiproliferative agents with apoptosis inducing mechanism in Colon cancer

Abstract In the current work, a new set of carbohydrazide linked benzofuran-isatin conjugates (5a–e and 7a–i) was designed and synthesised. The anticancer activity for compounds (5b–d, 7a, 7b, 7d and 7g) was measured against NCI-55 human cancer cell lines. Compound 5d was the most efficient, and thus subjected to the five-dose screen where it showed excellent broad activity against almost all tested cancer subpanels. Furthermore, all conjugates (5a–e and 7a–i) showed a good anti-proliferative activity towards colorectal cancer SW-620 and HT-29 cell lines, with an excellent inhibitory effect for compounds 5a and 5d (IC50 = 8.7 and 9.4 µM (5a), and 6.5 and 9.8 µM for (5d), respectively). Both compounds displayed selective cytotoxicity with good safety profile. In addition, both compounds provoked apoptosis in a dose dependent manner in SW-620 cells. Also, they significantly inhibited the anti-apoptotic Bcl2 protein expression and increased the cleaved PARP level that resulted in SW-620 cells apoptosis.


Introduction
Cancer, a large family of diseases, is characterised by fast and uncontrolled cell division and differentiation mechanisms and has the potential to spread to or invade other body parts 1 . For several decades, cancer is considered one of the major world public health problems, and it remains a serious reason of the death of human beings all over the world 2 . Despite the presence of a variety of cancer treatment strategies, the majority of which induces non-selective cell death by targeting the DNA synthesis [3][4][5][6] and/or the replication machinery [7][8][9][10] . These early strategies are accompanied by severe side effects due to the unspecific cytotoxicity towards the cancer cells in addition to the resistance developed against them 4 . Therefore, the development of safe and effective novel anticancer agents with increased selective treatment strategies towards cancer cells has received more attention and still ongoing active search 11,12 . Recent strategies for anticancer development are to target specific biomarkers required for cancer cells division and/or induction of cell apoptosis such as deregulated, mutated, or over expressed proteins 13 and thus, affect cancer cells selectively with minimum influences on normal cells 14 . Among these targets are the antiapoptotic protein Bcl2 and Poly ADP-ribose polymerase (PARP). In this regard, several reports stated that numerous of cancer cells are characterised by anti-apoptotic proteins (Bcl2) over-expression, which could lead to prevention of cell apoptosis as well as development of drug resistance 15,16 . On the other hand, PARP is a family of proteins involved in numerous cellular functions such as DNA repair and genomic stability [17][18][19] and also, PARP was reported to activate programmed cell death, through cleavage into PAR (Poly ADP-ribose), which motivates mitochondria to produce apoptosis inducing factors 20 . Thus, the development of compounds that inhibit the antiapoptotic Bcl2 proteins and/or potentiate the cleavage of PARP could be a promising approach to identify new anticancer therapies.
Heterocyclic compounds in particular oxygen containing heterocycles represent an important class of compounds possessing interesting pharmacological and biological activities [21][22][23] . Benzofuran nucleus, as a key functional scaffold, represents a basic structure in a diversity of biologically active synthetic and natural products [24][25][26] , with broad range of desirable activities including; anti-Alzheimer's 27 , antibacterial 28 , anti-tubercular 29 , antioxidant 30 , anti-inflammatory 31 , as well as antitumor activities 32 . Benzofuran derivatives exert their antiproliferative activity with diversified mechanisms such as inhibition of tubulin polymerisation 33,34 , HIF-1 35 , Aurora B kinase 36 and VEGFR-2 activity 37 . Furthermore, some benzofurans mediate their antiproliferative activity via apoptosis induction in various human cancer cell lines [38][39][40] . In addition, benzofuran-based conjugates were largely studied and were found to exert significant anticancer activity, such as conjugation of benzofuran with pyrazole 41 , indole 42 and others 43,44 .
On the other hand, isatin is identified as a privileged nucleus that included in many pharmacologically active small molecules, such as antiviral 45 , antimicrobial 46 , anticonvulsant 47 , CNS-acting 48 , as well as anticancer 49,50 agents. Over the last few years, hybridisation of isatin nucleus with different heterocycles has been reported as a successful approach to develop efficient antitumor agents towards different cancer types through diverse enzymatic and cellular mechanisms 49,50 . To name just a few, isatin-phthalazine (compound I) 51 , isatin-thiazolo[3,2-a]benzimidazole (compound II) 52 , isatin-thiazolidinone (compound III) 53 , isatin-indole (compound IV) 54 and isatin-quinazoline (compound V) 55 conjugates were reported to possess promising anticancer activities ( Figure 1).
Encouraged by the aforementioned findings and considering the need to develop safe and effective novel anticancer agents, a new attempt to study the significance of utilisation of heterocycles hybridisation approach to furnish efficient anti-proliferative activity was reported herein. A novel series of benzofuran-isatin conjugates (5a-e and 7a-i, Figure 1) linked by a carbohydrazide group, was designed and synthesised. The new compounds were screened for their potential anticancer activity following NCI, USA protocol against fifty-five different cell lines under nine different cancer panels. In addition, the cytotoxic effect of these conjugates against SW-620 and HT-29 colorectal cancer cell lines was investigated and their ability to induce cell apoptosis was examined. Furthermore, the level of the mitochondrial antiapoptotic protein Bcl2 and the level of cleaved PARP in both SW-620 and HT-29 colorectal cancer cell lines were also determined.
Structures of the newly prepared benzofuran-based derivatives 5a-e and 7a-i were verified based on spectral and elemental analyses. 1 H NMR spectra of 5a-e and 7a-i revealed the presence of two singlet peaks for the protons of C-3 CH 3 of benzofuran ring and NH of the hydrazide linker at range d (2.52-2.72) and (11.37-14.10) ppm, respectively. Moreover, structure of compounds 5a-e was confirmed via presence of another singlet D 2 O exchangeable signal attributable to the proton of NH for isatin moieties at d 10.91-11.98 ppm. In addition, 1 H NMR spectra of N-benzyl bearing derivatives 7d-f, 7h and 7i displayed the characteristic singlet signal of the benzylic protons at d 4.98-5.07 ppm, while spectra for hybrids 7a, 7b and 7g revealed the presence of the aliphatic protons corresponding to the N-substituents in these derivatives at d (3.28 ppm), (0.93, 1.66 and 3.76 ppm) and (0.97, 1.69 and 3.80 ppm), respectively.
On the other hand, 13 C NMR spectra for the novel compounds 5a-e and 7a-i showed one signal belonging to the carbon of CH 3 of benzofuran ring at d 8.12-9.49 ppm, also, they showed two signals belonging to the carbon of C ¼ O functionalities for both the hydrazide linker and isatin moiety at range d (161.15-163.62) and (164.08-167.02) ppm, respectively. In addition, the existence of benzylic carbon in N-benzyl bearing derivatives 7d-f, 7h and 7i was confirmed by a signal at d 42.14-46.03 ppm, whereas, the carbons of propyl moiety in compounds 7b and 7g appeared as signals at range d (11.68-13.00), ( and 7g) were screened at a dose of 10 lM for their antiproliferative activity against a panel of fifty-five cancer cell lines. The mean percent growth inhibition values (GI%) for conjugates 5b-d,7a, 7b, 7d and 7g against NCI-55 cancer cell lines were displayed in ( Figure 2, Table 1). The primary assay data analysis revealed that the new benzofuran-isatin hybrids showed weak to moderate inhibitory activity some of the subpanel cancer cell lines except for compound 5d that possessed excellent activity against nearly all the cancer cell lines. Although compound 5b, 7a, 7b, 7d and 7g proved inactive against most of the subpanels cancer cell lines with mean GI% ¼ 0.75%, 0.09%, 5.18%, 6.01%, and À0.8%, respectively, they showed selective moderate anticancer activity against certain cell lines such as Ovarian-IGROV1, Non-small cell lung-EKVX, Renal-UO-31 and Breast/MCF7 cancer cell lines with GI% range 17-53% (Table 1). In particular, compound 5d was the most efficient anti-proliferative agent and exhibited excellent activity against almost all subpanel cancer cell lines with mean growth inhibitory activity of 87.33%. Remarkably, compound 5d exerted excellent growth inhibition properties against Non-small cell lung cancer (NCI-H23), CNS cancer (SF-295, U251), Melanoma (LOX IMVI, SK-MEL- 28    On the other hand, compound 5c showed moderate to good activity against some cell lines with mean GI% ¼ 21.99%. The best results of compound 5c was against cancer cell lines Non-small cell lung-HOP-62 (GI% ¼ 41.41%), Non-small cell lung-NCI-H460 (GI% ¼ 42.60%), Renal-UO-31 (GI% ¼ 43.42%), Ovarian-IGROV1 (GI% ¼ 44.59%), Breast-HS-578T (GI% ¼ 46.27%), CNS-SF-539 (GI% ¼ 57.88%) and Melanoma-MALME-3M (GI% ¼ 64.93%) ( Table 1).
2.2.1.2. In vitro 5 dose full NCI-55 cell panel screening.. The preliminary screening results showed that conjugate 5d (NSC: D-819833/1) was the most potent compound in the present study, and displayed effectiveness towards various cell lines represent numerous tumour subpanels ( Figure 2). Accordingly, 5d was promoted to the five-dose (0.01-100 mM) screening assay. Accordingly, three main response parameters (GI 50 , TGI and LC 50 ) towards each of the examined cancer cell line were calculated for hybrid 5d and displayed in Table 2. Where, GI 50 values represents molar concentration which produces 50% inhibitory effect in the net cell growth; TGI (cytostatic activity) is the molar concentration with total growth inhibition and LC 50 is the cytotoxicity parameter that reflects the molar concentration that results in 50% net cell death. In addition, the mean graph midpoints (MG-MID), representing the GI 50 average for the individual subpanels as well as the full panel cell lines were calculated giving an average potency parameter for the examined compound 5d, (Table 3). Furthermore, by dividing the full panel MID by their individual subpanel MID, the selectivity index of compound 5d was calculated and was used to measure the selectivity of 5d towards different cancer cell subpanels.
Results displayed in Table 2, revealed that conjugate 5d exhibited powerful anti-proliferative activity at a single-digit micromolar level towards all the examined human cancer cell subpanels with GI 50 values range: 1.25 À 6.07 mM, except for Melanoma HL60(TB) cell line (more than100 mM). Moreover, regarding the cytostatic activity, hybrid 5d exhibited excellent cytostatic activity with TGI values range 2.75-7.72 mM against numerous cell lines including NSCLC (HOP-62, NCI-H23 and HOP-92), CNS Cancer (SF-295,   Table 2).
On the other hand, as shown in Table 3, all tested subpanels were sensitive to compound 5d with MG-MID spinning between 1.92 and 3.18 mM and the most susceptible subpanels were Colon Cancer and Renal Cancer that exhibited MG-MID ¼ 1.92 and 1.98 mM, respectively. Furthermore, it is well known that compounds with selectivity index between 3 and 6 are considered to be of a moderate selectivity, ratios more than six indicated high selectivity towards the corresponding cell line, while compounds not meeting either of these values are considered as non-selective 59 . Therefore, as displayed in the Table 3, the calculated selectivity index for compound 5d ranged from 0.78 to 1.30 indicated that conjugate 5d has non-selective, broad spectrum antiproliferative activity against all tested subpanels cancer cells.

In vitro anti-cancer activity against SW-620 and HT-29colorectal cancer cell lines
In the present investigation a new set of benzofuran-isatin hybrids (5a-e and 7a-i) was synthesised to be evaluated for their potential anticancer activity towards two human colorectal cancer cell lines, SW-620 and HT-29. The anticancer activity of the new conjugates was assessed using MTT assay 60 , and the results were shown in Figure 3. The most active compound in the NCI assay (5d), in addition to another one from untested compounds by NCI (5a), were selected to explore their activity. Both, SW-620 and HT-29 cells were treated with 10 mM of each compound for 24 h and the percent cell viability was calculated using MTT assay. Regarding impact of the target conjugates towards SW-620 cancer cells viability, compound 5d exhibited about 52% inhibition, whereas, compound 5a showed 46% inhibition. On the other hand, the results showed that seven compounds (5a, 5d, 7b, 7c, 7e, 7h and 7i) showed >50% inhibition of HT-29 cancer cells viability ( Figure 3).
The results revealed that compounds 5a and 5d exhibited promising cytotoxic activity for both cell lines. For this reason, compounds 5a and 5d were pursued for further studies. Starting with determination of IC 50s and cytotoxic selectivity studies. Serial concentrations of compounds 5a and 5d were used to examine their impact on cell viability using MTT protocol. Results of concentration vs percent viability were charted, and the IC 50 was calculated for SW-620 and HT-29 cell lines using Graph Pad prism 8 ( Figure 4). Compound 5a was found to have IC 50 ¼ 9.4 mM and 8.7 mM against SW-620 and HT-29 cell lines, respectively. In addition, the IC 50 for compound 5d equals 9.8 mM and 6.5 mM against SW-620 and HT-29 cell lines, respectively, compared to IC 50 of Irinotecan, a reference drug, which was found to be 1.0 mM against SW-620 cell line and 6.18 mM against HT-29 cell line ( Figure 4).
Furthermore, selective cytotoxicity of compounds 5a and 5d was studied on human skin fibroblast (HFF-1) normal cells. Both conjugates were found to possess a little effect on fibroblast normal cell viability ( Figure 5). These results revealed that compounds 5a and 5d possessed a selective cytotoxicity against SW-620 and HT-29 cancer cell lines with non-significant effect on normal fibroblast cells.

Annexin V-FITC/propidium iodide apoptosis assay
Further investigation for compounds 5a and 5d concerning their potential role of apoptosis induction, using Annexin V-FITC/PI double staining assay 61 , was performed to evaluate their impact on both early and late apoptosis percentages in SW-620 cancer cell lines ( Figure 6). The assay findings showed that compounds 5a and 5d resulted in a dose dependent induction of apoptosis for SW-620 cancer cells. As shown, compound 5a induced approximately 1.7-folds and 3.8-folds total increase in apoptosis at concentration of 5 mM and 10 mM, respectively, in comparison to the control untreated SW-620 cell line (Figure 6(A)).
Similarly, compound 5d, at concentration of 5 mM and 10 mM approximately induced 2.9-folds and 3.8-folds total increase in apoptosis, respectively, when incubated with SW-620 cell line, compared to the untreated cells ( Figure 6(B)). Encouraged by these results compounds 5a and 5d were further investigated for their effect on the anti-apoptotic mitochondrial protein Bcl2 and their effect on the level of cleaved PARP in SW-620 colorectal cancer cell line.

Effect of compounds 5a and 5d on the anti-apoptotic markers Bcl2 and the level of cleaved PARP
To further examine the possible mechanism of apoptosis, the effect of compounds 5a and 5d on certain apoptosis-related proteins was studied. Bcl2 protein as a critical component of the mitochondrial apoptotic pathway is reported to be overexpressed in numerous tumours causing survival of cancer cell 62 . In addition, it was reported that caspase activation during apoptosis leads to proteolytic cleavage of several cellular substrates participating in DNA reparation including [poly (ADP-ribose) polymerase] 63 . Therefore, the impact of compounds 5a and 5d on the anti-apoptotic protein Bcl2 and the level of cleaved PARP was examined (Figure 7). The results showed that, Western blot analysis of the extracts prepared from SW-620 cells incubated with compound 5a (5 lM and 10 lM) for 24 h, resulted in a dose dependent inhibition of Bcl2 protein expression and significant increase in the level of cleaved PARP (Figure 7(A)).
Similarly, compound 5d was found to follow the same pattern with significant inhibition of the anti-apoptotic Bcl2 protein expression and significant increase in the level of cleaved PARP in SW-620 cancer cells (Figure 7(B)). These findings indicated that both compounds 5a and 5d inhibited SW-620 cells viability by deregulating apoptosis-related proteins (anti-apoptotic Bcl2 and cleaved PARP) resulting in the induction of apoptosis.

Conclusions
In summary, a novel series of benzofuran-isatin conjugates linked by a carbohydrazide group, (5a-e and 7a-i) was designed and synthesised. Seven compounds (5b-d and 7a,b,d,g) were selected according to NCI's DTP selection guidelines for the assessment of their antitumor activity against NCI-55 human cancer cell lines. All compounds proved effective against diverse cell lines among which compound 5d was promoted to the five-dose screen and  showed good to excellent growth inhibitory activity against almost all subpanel cancer cell lines. In addition, the novel conjugates (5a-e and 7a-i) showed good anti-proliferative activity against two human colorectal cancer cell lines, SW-620 and HT-29, with excellent inhibitory activity for compounds 5a and 5d that showed IC 50 ¼ 8.7 mM and 9.4 mM for 5a and IC 50 ¼ 6.5 mM and Figure 6. (A) AnnexinV/PI apoptosis assay for compound 5a. Tow concentrations (5 and 10 mM) of compound 5a, in addition untreated plate as a control were used to test the apoptotic effect by using Annexin V/PI in SW-620 cell line. Cells were treated with the compound 5a for 24 h. (B) AnnexinV/PI apoptosis assay for compound 5d. Tow concentrations (5 and 10 mM) of compound 5d, in addition untreated plate as a control were used to test the apoptotic effect by using Annexin V/PI in SW-620 cell line. Cells were treated with the compound 5d for 24 h. 9.8 mM for 5d against SW-620 and HT-29 cell lines, respectively, and proved to have selective cytotoxicity with increased safety profile to fibroblast (HFF-1) normal cells. Further mechanistic studies revealed that both compounds 5a and 5d were able to induce apoptosis in a dose dependent manner with an approximately 1.7-3.8 folds and 2.9-3.8 folds total increase in apoptosis for compounds 5a and 5d, respectively, compared to the control untreated SW-620 cell line. Furthermore, both conjugates significantly inhibited the expression of the anti-apoptotic Bcl2 protein and increased the level of the cleaved PARP and resulted in SW-620 cells apoptosis. Collectively, the significant potency and high selective cytotoxicity of this series specially compounds 5a and 5d suggested that these conjugates might serve as starting point for additional optimisation to develop potential anticancer agents and apoptotic inducers.

General
Solvents of HPLC grade have been used and purchased from Thermo Fisher. Follow up of reactions has been performed utilising precoated TLC F 254 Merck plates. Schimadzu FT-IR spectrometer has been used for functional groups analysis for the synthesised derivatives. NMR spectrometric analyses have been conducted using Bruker-Avance 400 NMR spectrometer (100 MHz for 13 CNMR and 400 MHz for 1 H NMR). Chemical shifts have been recorded in ppm. Multiplicities have been reported with their 1st order J coupling constants (Hz) for doublets (d); Stuart apparatus has been used to determine the melting points. FLASH 2000 CHNS/O analyser has been adopted to perform the elemental analysis.

Synthesis of target derivatives 5a-e and 7a-i
To stirred hot solution of 3-methylbenzofuran-2-carbohydrazide 3 (0.25 g, 1.3 mmol) in 13 ml of absolute EtOH with catalytic drops of ethanoic acid, equivalent amount of appropriate indoline-2,3dione compounds 4a-e or 6a-i has been added. The reaction mixture has been then refluxed for (3)(4)(5)(6) h. The produced precipitate, after cooling, was collected by filtration, washed with water then recrystallized from glacial acetic acid to produce target derivatives 5a-e and 7a-i, respectively in a good yield (70-87%).
Full characterisation (NMR, IR, and elemental analysis) data for target compounds (5a-e and 7a-i) have been presented in the Supporting Materials.

Biological evaluation
All in vitro biological assays in this study; NCI anticancer screening 67,68 , MTT cell viability assay 54 , Annexin V-FITC/PI assay 54 and Western blot analysis 54 were performed as reported earlier. All experimental procedures were provided in the Supporting materials.

Disclosure statement
No potential conflict of interest was reported by the author(s).