Synthesis and Biological Evaluation of Indole-2-Carboxamides with Potent Apoptotic Antiproliferative Activity as EGFR/CDK2 Dual Inhibitors

The apoptotic antiproliferative actions of our previously reported CB1 allosteric modulators 5-chlorobenzofuran-2-carboxamide derivatives VIIa–j prompted us to develop and synthesise a novel series of indole-2-carboxamide derivatives 5a–k, 6a–c, and 7. Different spectroscopic methods of analysis were used to validate the novel compounds. Using the MTT assay method, the novel compounds were examined for antiproliferative activity against four distinct cancer cell lines. Compounds 5a–k, 6a–c, and 7 demonstrated greater antiproliferative activity against the breast cancer cell line (MCF-7) than other tested cancer cell lines, and 5a–k (which contain the phenethyl moiety in their backbone structure) demonstrated greater potency than 6a–c and 7, indicating the importance of the phenethyl moiety for antiproliferative action. Compared to reference doxorubicin (GI50 = 1.10 µM), compounds 5d, 5e, 5h, 5i, 5j, and 5k were the most effective of the synthesised derivatives, with GI50 ranging from 0.95 µM to 1.50 µM. Compounds 5d, 5e, 5h, 5i, 5j, and 5k were tested for their inhibitory impact on EGFR and CDK2, and the results indicated that the compounds tested had strong antiproliferative activity and are effective at suppressing both CDK2 and EGFR. Moreover, the studied compounds induced apoptosis with high potency, as evidenced by their effects on apoptotic markers such as Caspases 3, 8, 9, Cytochrome C, Bax, Bcl2, and p53.


Introduction
In response to increased global morbidity and mortality rates from so-called incurable diseases, medication research and development has never been static but has grown increasingly dynamic. Traditionally, therapeutic drug discovery has relied on the design of highly selective chemical entities that target a single biological entity assumed to play a dominant role in a particular disease [1,2]. By means of this method, researchers hoped to eliminate any unwanted side effects and ensure that drug candidates had more drug-like properties. Highly selective or specific therapeutic medicines focused on single molecular targets, on the other hand, have shown to be ineffective, particularly in the treatment of complicated disorders. Drug resistance has been linked to the use of highly selective therapeutic agents.
However, due to the low efficacy of single target medications against multifactorial disorders whose aetiology is based on a collection of biochemical processes and many bioreceptors acting concurrently, drug design methodologies have to be reconsidered. Over the In the present work, the apoptotic antiproliferative actions of our previously reported 5-chlorobenzofuran-2-carboxamide derivatives VIIa-j ( Figure 1) prompted us to develop and synthesise a novel series of indole-2-carboxamide derivatives 5a-k, 6a-c, and 7 (Figure 2). A small library of fifteen new compounds in which the methyl group was kept as a substituent at C3. To investigate the effect of substituent modification, the para positions of the phenethyl tails in the newly synthesised compounds were left unsubstituted or substituted with 4-dimethylamino, morpholin-4-yl, piperidin-1-yl, or 2-methylpyrrolidine-1yl. To investigate the impact of the linker nature on anticancer activity, the phenethyl amino carbonyl moieties were modified to 4 phenylpiperazin-1-yl carbonyl as in compounds 6a-c or benzyl carbonyl as in compound 7. The position and number of halogen atoms on the indole moiety's phenyl ring were also investigated. The antiproliferative activity of compounds 5a-k, 6a-c, and 7 against a panel of cancer cell lines were investigated. The most active compounds were evaluated for mechanistic activity as multi-targeted kinase inhibitors such as EGFR and CDK2. Furthermore, the compounds were evaluated for apoptotic activity against caspases 3, 8, and 9, as well as Cytochrome C, Bax, Bcl2, and p53. In the present work, the apoptotic antiproliferative actions of our previously reported 5chlorobenzofuran-2-carboxamide derivatives VIIa-j ( Figure 1) prompted us to develop and synthesise a novel series of indole-2-carboxamide derivatives 5a-k, 6a-c, and 7 ( Figure 2). A small library of fifteen new compounds in which the methyl group was kept as a substituent at C3. To investigate the effect of substituent modification, the para positions of the phenethyl tails in the newly synthesised compounds were left unsubstituted or substituted with 4-dimethylamino, morpholin-4-yl, piperidin-1-yl, or 2-methylpyrrolidine-1-yl. To investigate the impact of the linker nature on anticancer activity, the phenethyl amino carbonyl moieties were modified to 4 phenylpiperazin-1-yl carbonyl as in compounds 6a-c or benzyl carbonyl as in compound 7. The position and number of halogen atoms on the indole moiety's phenyl ring were also investigated. The antiproliferative activity of compounds 5a-k, 6a-c, and 7 against a panel of cancer cell lines were investigated. The most active compounds were evaluated for mechanistic activity as multi-targeted kinase inhibitors such as EGFR and CDK2. Furthermore, the compounds were evaluated for apoptotic activity against caspases 3, 8, and 9, as well as Cytochrome C, Bax, Bcl2, and p53.

Chemistry
Scheme 1 depicts the synthesis of target compounds 5a-k, 6a-c, and 7. Derivatives of phenyl hydrazine Hydrochloride 1a-d under Fisher Indole cyclization were reacted with 2-oxopropanoic acid 2 in the presence of PTSA (p-toluenesulfonic acid) to provide 3methylindole-2-carboxylates 3a-d [24]. The carboxylic acids 4a-d was obtained by alkaline hydrolysis of the esters 3a-d [25]. The appropriate amines were coupled with carboxylic acids 4a-d in the presence of DIPEA in DCM using BOP as a coupling reagent [23], yielding target carboxamides 5a-k, 6a-c, and 7. 1 H NMR, 13 C NMR, and HRESI-MS were used to identify the newly synthesised derivatives. The 1 H NMR spectrum of 5d revealed the appearance of three singlet signals: one at δ 11.34 ppm (1H) consistent with indole NH, one at δ 7.89 ppm (1H) relating to amidic NH, and one at δ 2.41 ppm (3H) corresponding to the methyl group. The spectrum also indicated the existence of signals corresponding to ethylene protons at δ 3.48 (q, J = 7.1 Hz, 2H, NHCH 2 ) and δ 2.77 (t, J = 7.4 Hz, 2H, NHCH 2 ), in addition to the morpholine group's distinctive signals at δ 3.70 (t, J = 4. 8  The MCF-10A (human mammary gland epithelial) cell line was used in the cell viability experiment. Compounds 5a-k, 6a-c, and 7 were incubated with MCF-10A cells for 4 days at 50 µM concentration, and the viability of cells was determined using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test. [26]. All compounds had no cytotoxic effects, and the vitality of the cells was more than 83% for most of the compounds examined.

Antiproliferative Activity
Using the MTT assay with doxorubicin as the reference drug, the antiproliferative activities of 5a-k, 6a-c, and 7 against four human cancer cell lines, including pancreas cancer cell line (Panc-1), breast cancer cell line (MCF-7), colon cancer cell line (HT-29), and epithelial cancer cell line (A-549) were investigated [27]. Table 1 shows the results of calculating the median inhibitory concentration (IC 50 ) for all derivatives. Generally, compounds 5a-k, 6a-c, and 7 demonstrated greater antiproliferative activity against the breast cancer cell line (MCF-7) than other tested cancer cell lines, and 5a-k (which contain the phenethyl moiety in their backbone structure) demonstrated greater potency than 6a-c and 7, indicating the importance of the phenethyl moiety for antiproliferative action.
Compound 5d (R 1 = Cl, R 2 = R 3 = H, R 4 = morpholin-4-yl) rated second in activity with a GI 50 of 1.05 µM against the four cancer cell lines, being somewhat less potent (1.1-fold) than 5e but equipotent to doxorubicin and even more potent than doxorubicin against A-549 and Panc-1 cell lines.
Furthermore, among the tested compounds, the 4-benzyl carbonyl derivative 7 and the 4-phenylpiperazin-1-yl carbonyl derivatives 6a-c had the lowest mean GI 50 values, implying that the N-phenethyl carboxamide architecture is important for antiproliferative action and correlating with previous SAR studies [23].

CDK2 Inhibitory Assay
Compounds 5d, 5e, and 5h-k were further examines for their ability to inhibit the CDK2 enzyme [29]. The IC 50 values are shown in Table 2. In comparison to the reference dinaciclib (IC 50 = 20 nM), all investigated derivatives inhibited CDK2 effectively, with IC 50 values ranging from 11 nM to 34 nM. Three derivatives, 5e, 5h, and 5k, were shown to be superior to the standard dinaciclib as CDK2 inhibitors, with IC 50 values of 13, 11, and 19 nM, respectively. Compound 5e, the most potent antiproliferative derivative, displayed significant anti-CDK2 activity with an IC 50 value of 13 nM, which is 1.5-fold more active than the reference dinaciclib. On the other hand, compounds 5d, 5i, 5j, and 5k exhibited significant activity against CDK2 (IC 50 = 23, 27, 34, and 19 µM) comparable to dinaciclib. The findings of the EGFR and CDK2 tests revealed that the examined compounds exhibit significant antiproliferative activity and are efficient at suppressing both CDK2 and EGFR.

Apoptosis Assay
A previous report has shown that CBD (2), a CB1 allosteric modulator, can trigger apoptosis [30]. Therefore, to assess the proapoptotic potential of our target compounds, we evaluated the most active compounds 5d, 5e, and 5h for their capacity to initiate the apoptosis cascade in the breast cancer (MCF-7) cell line.

Activation of Proteolytic Caspases Cascade
Caspases play a crucial role in the initiation and completion of the apoptotic process [31]. Caspase-3 is a crucial caspase that cleaves a variety of proteins in cells, causing apoptosis [32]. The effects of compounds 5d, 5e, and 5h on caspase 3 were assessed and compared to doxorubicin, which was used as a control [33]. The results showed that when compared to control cells, the tested compounds increased the level of active caspase 3 by 8-10 folds and that 5d, 5e, and 5h induce outstanding overexpression of caspase-3 protein level (570.00 ± 5.00, 635.50 ± 5.50 and 537.50 ± 5.00 pg/mL, respectively) compared to doxorubicin (503.50 ± 4.50 pg/mL). In comparison to the control untreated cells, the most active antiproliferative derivative 5e increases caspase 3 levels by 9.70 times.  Compounds 5d, 5e, and 5h-k were further examines for their ability to inhibit the CDK2 enzyme [29]. The IC50 values are shown in Table 2. In comparison to the reference dinaciclib (IC50 = 20 nM), all investigated derivatives inhibited CDK2 effectively, with IC50 values ranging from 11 nM to 34 nM. Three derivatives, 5e, 5h, and 5k, were shown to be superior to the standard dinaciclib as CDK2 inhibitors, with IC50 values of 13, 11, and 19 nM, respectively. Compound 5e, the most potent antiproliferative derivative, displayed significant anti-CDK2 activity with an IC50 value of 13 nM, which is 1.5-fold more active than the reference dinaciclib. On the other hand, compounds 5d, 5i, 5j, and 5k exhibited significant activity against CDK2 (IC50 = 23, 27, 34, and 19 µ M) comparable to dinaciclib. The findings of the EGFR and CDK2 tests revealed that the examined compounds exhibit significant antiproliferative activity and are efficient at suppressing both CDK2 and EGFR.  Compounds 5d, 5e, and 5h-k were further examines for their ability to inhibit the CDK2 enzyme [29]. The IC50 values are shown in Table 2. In comparison to the reference dinaciclib (IC50 = 20 nM), all investigated derivatives inhibited CDK2 effectively, with IC50 values ranging from 11 nM to 34 nM. Three derivatives, 5e, 5h, and 5k, were shown to be superior to the standard dinaciclib as CDK2 inhibitors, with IC50 values of 13, 11, and 19 nM, respectively. Compound 5e, the most potent antiproliferative derivative, displayed significant anti-CDK2 activity with an IC50 value of 13 nM, which is 1.5-fold more active than the reference dinaciclib. On the other hand, compounds 5d, 5i, 5j, and 5k exhibited significant activity against CDK2 (IC50 = 23, 27, 34, and 19 µ M) comparable to dinaciclib. The findings of the EGFR and CDK2 tests revealed that the examined compounds exhibit significant antiproliferative activity and are efficient at suppressing both CDK2 and EGFR.  Compounds 5d, 5e, and 5h-k were further examines for their ability to inhibit the CDK2 enzyme [29]. The IC50 values are shown in Table 2. In comparison to the reference dinaciclib (IC50 = 20 nM), all investigated derivatives inhibited CDK2 effectively, with IC50 values ranging from 11 nM to 34 nM. Three derivatives, 5e, 5h, and 5k, were shown to be superior to the standard dinaciclib as CDK2 inhibitors, with IC50 values of 13, 11, and 19 nM, respectively. Compound 5e, the most potent antiproliferative derivative, displayed significant anti-CDK2 activity with an IC50 value of 13 nM, which is 1.5-fold more active than the reference dinaciclib. On the other hand, compounds 5d, 5i, 5j, and 5k exhibited significant activity against CDK2 (IC50 = 23, 27, 34, and 19 µ M) comparable to dinaciclib. The findings of the EGFR and CDK2 tests revealed that the examined compounds exhibit significant antiproliferative activity and are efficient at suppressing both CDK2 and EGFR.  Compounds 5d, 5e, and 5h-k were further examines for their ability to inhibit the CDK2 enzyme [29]. The IC50 values are shown in Table 2. In comparison to the reference dinaciclib (IC50 = 20 nM), all investigated derivatives inhibited CDK2 effectively, with IC50 values ranging from 11 nM to 34 nM. Three derivatives, 5e, 5h, and 5k, were shown to be superior to the standard dinaciclib as CDK2 inhibitors, with IC50 values of 13, 11, and 19 nM, respectively. Compound 5e, the most potent antiproliferative derivative, displayed significant anti-CDK2 activity with an IC50 value of 13 nM, which is 1.5-fold more active than the reference dinaciclib. On the other hand, compounds 5d, 5i, 5j, and 5k exhibited significant activity against CDK2 (IC50 = 23, 27, 34, and 19 µ M) comparable to dinaciclib. The findings of the EGFR and CDK2 tests revealed that the examined compounds exhibit significant antiproliferative activity and are efficient at suppressing both CDK2 and EGFR.  Compounds 5d, 5e, and 5h-k were further examines for their ability to inhibit the CDK2 enzyme [29]. The IC50 values are shown in Table 2. In comparison to the reference dinaciclib (IC50 = 20 nM), all investigated derivatives inhibited CDK2 effectively, with IC50 values ranging from 11 nM to 34 nM. Three derivatives, 5e, 5h, and 5k, were shown to be superior to the standard dinaciclib as CDK2 inhibitors, with IC50 values of 13, 11, and 19 nM, respectively. Compound 5e, the most potent antiproliferative derivative, displayed significant anti-CDK2 activity with an IC50 value of 13 nM, which is 1.5-fold more active than the reference dinaciclib. On the other hand, compounds 5d, 5i, 5j, and 5k exhibited significant activity against CDK2 (IC50 = 23, 27, 34, and 19 µ M) comparable to dinaciclib. The findings of the EGFR and CDK2 tests revealed that the examined compounds exhibit significant antiproliferative activity and are efficient at suppressing both CDK2 and EGFR.  Compounds 5d, 5e, and 5h-k were further examines for their ability to inhibit the CDK2 enzyme [29]. The IC50 values are shown in Table 2. In comparison to the reference dinaciclib (IC50 = 20 nM), all investigated derivatives inhibited CDK2 effectively, with IC50 values ranging from 11 nM to 34 nM. Three derivatives, 5e, 5h, and 5k, were shown to be superior to the standard dinaciclib as CDK2 inhibitors, with IC50 values of 13, 11, and 19 nM, respectively. Compound 5e, the most potent antiproliferative derivative, displayed significant anti-CDK2 activity with an IC50 value of 13 nM, which is 1.5-fold more active than the reference dinaciclib. On the other hand, compounds 5d, 5i, 5j, and 5k exhibited significant activity against CDK2 (IC50 = 23, 27, 34, and 19 µ M) comparable to dinaciclib. The findings of the EGFR and CDK2 tests revealed that the examined compounds exhibit significant antiproliferative activity and are efficient at suppressing both CDK2 and EGFR.
The impact of compounds 5d, 5e, and 5h on caspases 8 and 9 was also investigated to highlight the involvement of the intrinsic and extrinsic apoptotic pathways in the antiproliferative actions of these compounds, Table 3. When compared to control cells, compound 5e increased caspase 8 and 9 levels by 10.90 and 18.15 folds, respectively, while compound 5d increased caspase 8 and 9 levels by 9.70 and 17.80 folds, respectively, indicating activation of both intrinsic and extrinsic pathways with a stronger effect on the intrinsic pathway because caspase 9 levels were higher [34]. Cytochrome C Assay The quantity of cytochrome C within the cell is important for activating caspases and commencing the intrinsic apoptosis process [35]. Table 3 shows the findings of testing indole-2-carboxamide derivatives 5d, 5e, and 5h as Cytochrome C activators in the MCF-7 human breast cancer cell line. Compounds 5d, 5e, and 5h increased Cytochrome C levels in the MCF-7 human breast cancer cell line by 14, 16, and 13 times, respectively, compared to untreated control cells. The findings add to the evidence that apoptosis can be attributed to Cytochrome C overexpression and activation of the intrinsic apoptotic pathway triggered by the investigated compounds.

Bax and Bcl-2 Levels Assay
The most potent caspase activators, 5d and 5e, were investigated further for their influence on Bax and Bacl-2 levels in a breast cancer cell line (MCF-7) using doxorubicin as a control [36]. Table 4 shows that 5d and 5e caused a significant increase in Bax levels when compared to doxorubicin. Compound 5e demonstrated a comparable induction of Bax (296.50 pg/mL) compared to doxorubicin (276 pg/mL) with a 36-fold increase over control untreated breast cancer cells, followed by compound 5d (290 pg/mL and 35-fold rise). Finally, compound 5e reduced the anti-apoptotic Bcl-2 protein levels to 0.87 ng/mL in MCF-7 cells, followed by compound 5d (0.89 ng/mL) in comparison to doxorubicin (0.98 ng/mL). Effect of Compounds 5d and 5e on p53 Transcription in MCF-7 p53 is a unique protein that participates in several physiological processes such as cell metabolism [37], stem cell maintenance [38], and cell adhesion [39]. Because p53 is frequently inactivated in cancer cells, the cells are unable to undergo apoptosis [40,41]. Similarly, activating, or stabilizing p53 aids cancer cells in normalizing p53-controlled physiological processes and increasing apoptotic activity [42]. The effects of 5d and 5e on p53 transcription were evaluated and compared to doxorubicin as a control [43], Table 5. The results revealed an increase of at least 27-folds in p53 level compared to the test cells and that the p53 protein level of 5d and 5e was significantly inductive (1375 and 1435 pg/mL, respectively) in relation to doxorubicin (1265 pg/mL).

Docking Study
Interestingly, running docking simulations of compounds 5d and 5e within EGFR active site revealed docking scores (S; −6.90 and −6.79 kcal/mol; respectively), so much close to that of co-crystallized ligand, erlotinib (−7.30 kcal/mol), which co-insides with what obtained in-vitro against EGFR enzyme (as shown in Table 2). Moreover, visual inspection of the best docking poses of compounds 5d and 5e showed their close distance to key amino acids lining EGFR active site. Additionally, compounds 5d and 5e showed a number of H-bonding and pi-H interactions with LEU 694 and THR 766 amino acid residues (as listed in Table 6 and shown in Figure 3).  Both compounds 5d and 5e showed a common settling profile within EGFR active represented by U-shaped bending of the whole molecule, so its indole ring interacts with LEU 694 (as shown in Figure 3). On the other hand, compound 5d showed additional Hacceptor bonding with THR 766 that resulted in its better docking score over its congener, compound 5e. Additionally, and as shown in Table 6, MDs of compound 5e within the CDK2 active site revealed its better docking score (S = −6.99 kcal/mol) over its congener 5d (S = −6.03 kcal/mol), although its inability to have strong H-bonding with amino acid residues lining active site, its close proximity to key amino acid residues (revealed by its proximity contour as shown in Figure 4) could explain its better scoring over 5d. Both compounds 5d and 5e showed a common settling profile within EGFR active represented by U-shaped bending of the whole molecule, so its indole ring interacts with LEU 694 (as shown in Figure 3). On the other hand, compound 5d showed additional H-acceptor bonding with THR 766 that resulted in its better docking score over its congener, compound 5e.
Additionally, and as shown in Table 6, MDs of compound 5e within the CDK2 active site revealed its better docking score (S = −6.99 kcal/mol) over its congener 5d (S = −6.03 kcal/mol), although its inability to have strong H-bonding with amino acid residues lining active site, its close proximity to key amino acid residues (revealed by its proximity contour as shown in Figure 4) could explain its better scoring over 5d.   The MCF-10A (human mammary gland epithelial) cell line was used in the cell viability experiment. Compounds 5a-k, 6a-c, and 7 were incubated with MCF-10A cells for 4 days at 50 µM concentration, and the viability of cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test [26].

Antiproliferative Activity
Using the MTT assay with doxorubicin as the reference drug, the antiproliferative activities of 5a-k, 6a-c, and 7 against four human cancer cell lines, including pancreas cancer cell line (Panc-1), breast cancer cell line (MCF-7), colon cancer cell line (HT-29), and epithelial cancer cell line (A-549) were investigated [27].

EGFR Inhibitory Activity
The inhibitory efficacy of 5d, 5e, and 5h-k against EGFR was evaluated using the EGFR-TK assay [28].

Apoptosis Assay Activation of Proteolytic Caspases Cascade
The effects of compounds 5d, 5e, and 5h on caspases 3, 8, and 9 were assessed and compared to doxorubicin, which was used as a control [33].

Bax and Bcl-2 Levels Assay
The most potent caspase activators, 5d and 5e, were investigated for their influence on Bax and Bacl-2 levels in a breast cancer cell line (MCF-7) using doxorubicin as a control [36].
Effect of Compounds 5d and 5e on p53 Transcription in MCF-7 The effects of 5d and 5e on p53 transcription were evaluated and compared to doxorubicin as a control [43].

Conclusions
A new series of EGFR/CDK2 dual inhibitors containing indole-2-carboxamides has been reported. A total of fifteen target compounds were synthesized and evaluated in vitro against four cancer cell lines as well as these two kinases. The majority of the compounds examined had promising antiproliferative activity. The most effective of these compounds were 5d, 5e, 5h, 5i, 5j, and 5k. The novel compounds induced apoptosis and increased Caspase 3,8,9, and Cytochrome C levels. Furthermore, the investigated compounds increased Bax and p53 levels while decreasing anti-apoptotic Bcl2 protein levels. Following optimization, these compounds form a novel class of compounds capable of acting as potent apoptotic anticancer agents for both EGFR and CDK2.