Design, Synthesis, and Antiproliferative Activity of New 5-Chloro-indole-2-carboxylate and Pyrrolo[3,4-b]indol-3-one Derivatives as Potent Inhibitors of EGFRT790M/BRAFV600E Pathways

Mutant EGFR/BRAF pathways are thought to be crucial targets for the development of anticancer drugs since they are over-activated in several malignancies. We present here the development of a novel series of 5-chloro-indole-2-carboxylate 3a–e, 4a–c and pyrrolo[3,4-b]indol-3-ones 5a–c derivatives as potent inhibitors of mutant EGFR/BRAF pathways with antiproliferative activity. The cell viability assay results of 3a–e, 4a–c, and 5a–c revealed that none of the compounds tested were cytotoxic, and that the majority of those tested at 50 µM had cell viability levels greater than 87%. Compounds 3a–e, 4a–c, and 5a–c had significant antiproliferative activity with GI50 values ranging from 29 nM to 78 nM, with 3a–e outperforming 4a–c and 5a–c in their inhibitory actions against the tested cancer cell lines. Compounds 3a–e were tested for EGFR inhibition, with IC50 values ranging from 68 nM to 89 nM. The most potent derivative was found to be the m-piperidinyl derivative 3e (R = m-piperidin-1-yl), with an IC50 value of 68 nM, which was 1.2-fold more potent than erlotinib (IC50 = 80 nM). Interestingly, all the tested compounds 3a–e had higher anti-BRAFV600E activity than the reference erlotinib but were less potent than vemurafenib, with compound 3e having the most potent activity. Moreover, compounds 3b and 3e showed an 8-fold selectivity index toward EGFRT790M protein over wild-type. Additionally, molecular docking of 3a and 3b against BRAFV600E and EGFRT790M enzymes revealed high binding affinity and active site interactions compared to the co-crystalized ligands. The pharmacokinetics properties (ADME) of 3a–e revealed safety and good pharmacokinetic profile.


Introduction
Cancer has been a major public health issue around the world, with an increasing number of patients diagnosed each year [1]. Unfortunately, chemotherapy's effectiveness as a primary mode of cancer treatment is hampered by drug resistance, severe side effects, and poor selectivity [2,3]. Thus, recently, immunotherapy and newly combined, multitargeted therapies have been recommended [4][5][6]. Kinase activation in various cell signaling pathways has been linked to cancer cell survival, invasiveness, and drug resistance [7,8]. As a result, anticancer drugs that target kinases, such as the epidermal growth factor receptor (EGFR) and serine/threonine kinases, such as BRAF, are gaining popularity [9,10]. Motivated by the data presented above, and as part of our ongoing efforts to identify promising lead compounds for dual or multi-targeted anticancer agents [28][29][30], we present herein the design and synthesis of a novel class of indole-2-carboxylates, compounds 3a-e and 4a-c (Scaffold A), as well as 1,2-dihydropyrrolo [3,4-b]indol-3(4H)-ones, compounds 5a-c (Scaffold B) (Figure 1), as dual EGFR/BRAF V600E inhibitors with antiproliferative activity. The new compounds will be evaluated for their safety profile by assessing their effect on the viability of human normal cell lines, while their antiproliferative activity will be evaluated against a panel of four cancer cell lines. The most potent compounds will be evaluated for their ability to inhibit wild-type EGFR (EGFR WT ) and BRAF V600E as a potential mechanistic target for their antiproliferative effects. Furthermore, the most potent EGFR inhibitors will be tested for their inhibitory effect against mutant-type EGFR

Chemistry
The synthesis of target compounds 3a-e, 4a-c, and 5a-c is depicted in Scheme 1. 5-chloro-3-formyl indole-2-carboxylate 1 [31] was reacted with amines 2a-e [32] through reflux in ethanol followed by reduction of the intermediate imine with NaBH 4 under reductive-amination conditions to yield secondary amines 3a-e which subjected to saponification with LiOH to afford a carboxylic acids 4a-c. The structures of compounds 3a-e and 4a-c were confirmed using 1 H NMR, 13 C NMR, and HRESI-MS spectroscopy (Varian Inova, University of Aberdeen, Meston Building, Aberdeen AB24 3UE, UK). 1 H NMR spectrum of compound 3c revealed the presence of a singlet signal δ 9.12 ppm of indole NH, the characteristic signals of ethyl group in the form of quartet at δ 4.33 ppm (2H) and triplet at δ 1.35 ppm (3H), a singlet signal at δ 4.18 ppm (2H) of CH 2 NH-group, and two triplets (each of 2H) at δ 2.88 and 2.74 ppm of NHCH 2 CH 2 group. Moreover, the spectrum revealed the presence of the characteristic signals of both piperidine and aromatic protons. HRESI-MS m/z of 3c calcd for [M + H] + C 25 H 31 ClN 3 O 2 : 440.2099, found: 440.2100. The disappearance of the characteristic signals of the ethyl group in the form quartet at δ 4.33 ppm (2H) and triplet at δ 1.35 ppm (3H) and the appearance of a singlet signal at δ 3.43 ppm (2H) corresponding to COOH and NHCH 2 characterize the 1 H NMR spectrum of 4c.
The synthesis of target compounds 3a-e, 4a-c, and 5a-c is depicted in Scheme 1. 5chloro-3-formyl indole-2-carboxylate 1 [31] was reacted with amines 2a-e [32] through reflux in ethanol followed by reduction of the intermediate imine with NaBH4 under reductive-amination conditions to yield secondary amines 3a-e which subjected to saponification with LiOH to afford a carboxylic acids 4a-c. The structures of compounds 3a-e and 4a-c were confirmed using 1 H NMR, 13 C NMR, and HRESI-MS spectroscopy (Varian Inova, University of Aberdeen, Meston Building, Aberdeen AB24 3UE, UK). 1 H NMR spectrum of compound 3c revealed the presence of a singlet signal δ 9.12 ppm of indole NH, the characteristic signals of ethyl group in the form of quartet at δ 4.33 ppm (2H) and triplet at δ 1.35 ppm (3H), a singlet signal at δ 4.18 ppm (2H) of CH2NH-group, and two triplets (each of 2H) at δ 2.88 and 2.74 ppm of NHCH2CH2 group. Moreover, the spectrum revealed the presence of the characteristic signals of both piperidine and aromatic protons. HRESI-MS m/z of 3c calcd for [M + H] + C25H31ClN3O2: 440.2099, found: 440.2100. The disappearance of the characteristic signals of the ethyl group in the form quartet at δ 4.33 ppm (2H) and triplet at δ 1.35 ppm (3H) and the appearance of a singlet signal at δ 3.43 ppm (2H) corresponding to COOH and NHCH2 characterize the 1 H NMR spectrum of 4c. Scheme 1. Synthesis of compounds 3a-e, 4a-c, and 5a-c. Reagents and conditions: (a) NaBH 4 , EtOH, reflux, 12 h to rt, 1 h, 78%; (b) LiOH, THF/ H 2 O, 40 • C, 90%; (c) BOP, DIPEA, DMF, rt, overnight, 70%. Intramolecular coupling of the carboxylic acids 4a-c using BOP as the coupling reagent in the presence of DIPEA in DMF provided target compounds 5a-c. 1 H NMR spectrum of compound 5c revealed the presence of a singlet signal δ 12.01 ppm of indole NH, a singlet siganl at δ 4.34 ppm (2H) of CH 2 N-group, and two triplets (each of 2H) at δ 3.66 and 2.79 ppm of NHCH 2 CH 2 group. Furthermore, the disappearance of the singlet signal at 3.43 ppm (2H) corresponding to COOH and NHCH 2 confirms cyclization.

Cell Viability Assay
The viability test was performed on a normal human mammary gland epithelial cell line (MCF-10A). The MTT test was used to assess the viability of compounds 3a-e, 4a-c, and 5a-c [33,34]. After four days of incubation with MCF-10A cells, the results showed that none of the substances tested were cytotoxic, and that the majority of those tested at 50 µM had cell viability levels greater than 87%.

Antiproliferative Assay
Using the MTT assay [35,36] and erlotinib as the reference drug, compounds 3a-e, 4a-c, and 5a-c were tested for antiproliferative efficacy against four human cancer cell lines: Panc-1 (pancreatic cancer cell line), MCF-7 (breast cancer cell line), HT-29 (colon cancer cell line), and A-549 (epithelial cancer cell line). The median inhibitory concentration (IC 50 ) calculated (Graph Pad Software, San Diego, CA, USA) is shown in Table 1. For ease of manipulation, the average (GI 50 ) versus the four cancer cell lines was used. When compared to the reference drug erlotinib, which had a GI 50 of 33 nM, compounds 3a-e, 4a-c, and 5a-c all had substantial antiproliferative activity with GI 50 values ranging from 29 nM to 78 nM. According to Table 1's findings, 3a-e were superior to 5a-c and 4a-c in their inhibitory actions against the tested cancer cell lines.
Compared to erlotinib (GI 50 = 33 nM), the indole-2-carboxylate 3a-e had the most antiproliferative effects, with GI 50 values between 29 nM and 42 nM. Compound 3e (R = m-piperidin-1-yl) was the most potent derivative, with a GI 50 of 29 nM, outperforming the reference erlotinib, which had a GI 50 of 33 nM. Compound 3e was found to be more effective than erlotinib against Panc-1 (pancreatic cancer cell line), MCF-7 (breast cancer cell line), and A-549 (epithelial cancer cell line), Table 1. The substitution of the m-piperidine moiety in compound 3e with the p-piperidine moiety in compound 3c (R = p-piperidin-1-yl) resulted in a significant decrease in the antiproliferative activity of compound 3c, which has a GI 50 of 42 nM and is 1.5-fold less potent than 3e, indicating the importance of the substitution position on the antiproliferative activity, where the meta position is better tolerated than the para one. Compound 3b (R = p-pyrrolidin-1-yl) is the second most active antiproliferative compound, with a GI 50 value of 31 nM, which is also higher than the reference compound erlotinib (GI 50 = 33). Compound 3b is more effective than erlotinib against the MCF-7 cancer cell line, with an IC 50 value of 32 nM compared to 40 nM for erlotinib. With a mean GI 50 value of 35 nM, the unsubstituted derivative 3a (R = H) ranks third in activity against the four cancer cell lines and is even more potent than erlotinib against the MCF-7 cancer cell line, Table 1. The antiproliferative activity of the 2-methylpyrrolidine derivative 3d (R = p-2-methylpyrrolidin-1-yl) was promising, with a GI 50 of 38 nM, which is 1.3-fold less potent than 3e. These findings demonstrated the importance of the substitution pattern on the phenyl group of the phenethyl moiety, with activity increasing in the order m-piperidine > p-pyrrolidine > H > p-2-methylpyrrolidine > p-piperidine.
Compounds 5a-c had lower antiproliferative activity than compounds 3a-e, with GI 50 values of 48 nM, 62 nM, and 54 nM, respectively, compared to their congeners 3a-c, which had GI 50 values of 35 nM, 31 nM, and 42 nM, indicating that cyclization has a significant decrease in antiproliferative action. Finally, the carboxylic acid derivatives 4a (R = H), 4b (R = p-pyrrolidin-1-yl), and 4c (R = p-piperidin-1-yl) were the least potent against the four cancer cell lines, with GI 50 values of 78 nM, 68 nM, and 72 nM, respectively, indicating the importance of the ethyl group at position two of indole nucleus for the antiproliferative action.

EGFR Inhibitory Assay
The most effective antiproliferative derivatives 3a-e were evaluated for their ability to inhibit EGFR using the EGFR-TK assay [37], and the findings are displayed in Table 2. The IC 50 range for compounds 3a-e inhibitions of EGFR were 68 to 89 nM. The m-piperidinyl derivative 3e (R = m-piperidin-1-yl) was found to be the most potent of all synthesized derivatives, with an IC 50 value of 68 nM, which was 1.2-fold more potent than erlotinib (IC 50 = 80 nM). Compound 3b (R = p-pyrrolidin-1-yl) is the second most active compound, with an IC 50 value of 74 nM, and it is also more potent than erlotinib. Compounds 3a, 3c, and 3d showed comparable inhibitory activity against EGFR to erlotinib, with IC 50 values of 85, 89, and 82 nM, respectively. These results are consistent with the antiproliferative assay results and show that EGFR-TK is a possible target for the antiproliferative effects of compounds 3a-e, and that compounds 3b and 3e were more potent against EGFR-TK than the reference erlotinib.

BRAF V600E Inhibitory Assay
The in vitro anti-BRAF V600E activity of compounds 3a-e was further investigated [38] using erlotinib and vemurafenib as reference compounds and results are shown in Table 2. The enzyme assay revealed that the five compounds tested significantly inhibited BRAF V600E , with IC 50 ranges from 35 to 67 nM, Table 2. Interestingly, all the tested compounds 3a-e had higher anti-BRAF V600E activity than the reference erlotinib (IC 50 = 60 nM) but were less potent than vemurafenib (IC 50 = 30 nM). Once again, compounds 3b (R = p-pyrrolidin-1-yl) and 3e (R = m-piperidin-1-yl) had nearly the same inhibitory efficacy as BRAF V600E , with IC 50 values of 39 and 35 nM, respectively, and were shown to be effective inhibitors of cancer cell proliferation (GI 50 = 31 and 29 nM) as well as potent inhibitors of EGFR (IC 50 = 74 and 68 nM). The unsubstituted derivative 3a (R = H) demonstrated promising BRAF V600E inhibitory activity, with an IC 50 value of 43 nM, which was more potent than erlotinib but 1.4-fold less potent than vemurafenib. The findings of the study show that the tested compounds have potent antiproliferative activity and are effective at inhibiting both EGFR and BRAF V600E .

EGFR T790M Inhibitory Assay
The HTRF KinEASE-TK assay [39] was used to assess the inhibitory activity of the most potent compounds, 3b and 3e, against mutant-type EGFR (EGFR T790M ). Osimertinib served as the positive control. As shown in Table 2, compounds 3b and 3e had excellent inhibitory activity against EGFR T790M , with IC 50 values of 8.6 ± 2 and 9.2 ± 2 nM, respec-tively, equivalent to the reference osimertinib (IC 50 = 8 ± 2 nM), which may explain their potent antiproliferative activity. Compounds 3b and 3e demonstrated 8-fold selectivity index toward EGFR T790M protein over wild-type EGFR. These findings suggested that the phenethyl moiety's m-piperidine and p-pyrrolidine substitutions are required for the inhibitory effect on EGFR T790M .

LOX-IMVI Melanoma Cell Line Cytotoxicity Assay
The MTT assay was used to assess the anticancer activity of 3b and 3e, the most potent BRAF V600E inhibitors, against the LOX-IMVI melanoma cell line, which has BRAF V600E kinase overexpression [40,41]. The IC 50 values of the test compounds were determined at 5-dose concentrations, with staurosporine serving as a control. Table 3 shows that the indole-2-carboxylate derivatives 3b and 3e have a high ability to reduce the viability of the LOX-IMVI cell line. Compound 3e showed potent antiproliferative activity against the LOX-IMVI melanoma cell line with an IC 50 value of 0.96 µM, followed by compound 3b with an IC 50 value of 1.12 µM in comparison to staurosporine (IC 50 = 7.10 µM). The results of this assay add to the evidence that these compounds have potent antiproliferative activity as BRAF V600E inhibitors.

Molecular Modeling
The most active antiproliferative compounds (3a-e) were subjected to in silico docking study in order to investigate their binding modes within BRAF V600E active site. Molecular Operating Environment (MOE) software [42] was used as well as the crystal structure of the BRAF V600E in complex with vemurafenib (PDB: 3OG7) [43]. The accuracy of docking simulation within the protein binding site was validated via redocking the co-crystallized ligand showing S score of −11.78 kcal/mol with RMSD value of 0.96 Å, (S1), Table 4. Docking score analysis of the examined compounds revealed that compounds 3b and 3e showed the highest negative scores (−10.12 and −10.40 kcal/mol, respectively) which are compatible with their in vitro BRAF V600E inhibitory effects. Investigation of the compounds' binding mode revealed that merely compound 3e with (R = m-piperidin-1-yl) moiety extended comfortably along the large active site ( Figure 2C,D). The compound probes the space of the active site in a manner analogous to that of the co-crystalized ligand, vemurafenib. The ligand 5-chloro-indole moiety stacks between the amino acid residues Trp531 and Phe583 inside the hydrophobic pocket forming pi-H interaction with Val471 (4.09 Å) as well as hydrophobic interactions with Trp531, Phe583, Cys532, Ile463, Thr592, and Val471. In addition, the chlorine atom forms halogen bond interaction with the key amino acid residue Cys532 (3.27 Å) at the site gate. Moreover, the ligand indole-2carboxylate moiety forms ionic as well as H-bond interactions (3.13 Å) with the key amino acid Lys483. Additionally, the ligand stabilizes its complex within the active site by means of donating two H bond interactions with Thr529 (3.49Å), and Gly596 (3.41Å). On the other hand, the para-amino substitution in compounds 3b-d did not allow the ligand to bind deeply inside the pocket compared with the m-piperidine moiety in compound 3e. The latter finding confirms that the active site tolerates the meta substitution rather than the para one. Compound 3b with (R = p-pyrrolidin-1-yl) forms multiple interactions although exhibiting another bent conformation within the active site relative to compound 3e. The ligand indole-2-carboxylate moiety accepts a H-bond interaction from Lys483 (3.03 Å) as well as forming ionic interaction with Lys483 (3.03 Å). Moreover, the compound aminoethyl linker donates H bond to Thr529 (3.07 Å) while the (p-pyrrolidin-1-yl) moiety donates H-bond to Cys532 (3.28) at the gate of binding site. Furthermore, the (p-pyrrolidin-1-yl) moiety forms additional pi-H interaction with Cys532 (3.75 Å). (Figure 2A,B). The binding modes of compound 3c and 3d with R = p-piperidin-1-yl and R = p-2-methylpyrrolidin-1-yl, respectively, resemble that of compound 3b, however they are neither interacting with Cys532 at the gate of active site nor Lys483 at the pocket hinge. Furthermore, the unsubstituted derivative 3a probes the space of active site in an analogous pattern to that of compound 3e while missing interactions with the amino acid residues Cys532, Thr531, and Val471 at the binding site. Other ligands interactions within the active site include hydrophobic ones with Phe583, Cys532, Thr529, Val471, Lys483, and Leu514.
Moreover, the most potent compounds 3b and 3e were subjected to docking study within the active pocket of the EGFR mutant type T790M (PDB: 5J9Z) [44]. The docking protocol was validated by redocking the co-crystallized ligand that exhibiting S score of −10.42 kcal/mol with RMSD value of 0.88 Å, (S2), Table 5. Compounds 3b and 3e exhibited comparable binding modes within the protein binding site (Figure 3). The ligand indole-2-carboxylate moiety binds deeply inside the hydrophobic pocket forming multiple hydrogen bond interactions with Met790 and Lys745 as well as pi-H interactions with Val726. In addition, the compounds form ionic bond interactions with Lys745 and Asp855. In addition, the p-pyrrolidin-1-yl moiety of compound 3b forms ionic bond (3.74 and 3.62 Å) as well as H-bond interactions with Asp800 (3.62 Å) at the gate of the binding site. Moreover, the phenyl moiety of compound 3b forms additional pi-H with Arg841 (4.82 Å). (Figure 3A,B). The ligand protein complexes are stabilized via other hydrophobic interactions with Asp800, Phe723, Leu844, Cys797, Leu718, Val726, Met790, and Lys745. Results of the docking simulations attributed to explaining the biological effects of the compounds 3a-e relative to their binding affinity within the active site of BRAF V600E as well as EGFR mutant type T790M and confirmed the dual kinase targets for the anti-proliferative activity of compounds 3b and 3e.

In Silico ADME/Pharmacokinetics Studies
The most active antiproliferative compounds 3a-e were subjected to in silico ADME studies using the web tools SwissADME [45] as well as ADMETlab [46] by entering a list of two compounds' SMILES (Simplified Molecule Input Line Entry Specification) provided by ChemDraw software. The in silico pharmacokinetic data (Table 6) showed that all compounds are orally active as they obey Lipinski's rules of five with zero violation. All compounds are more likely to be a P-gp non-substrate. They exhibit high intestinal absorbance in the range of 88.9-90.5 %. They are capable of crossing BBB with logBB ranging from 0.22 to 0.31. According to Lipinski's rules, logP should be ≤5. Thus, all compounds exhibited good permeability as indicated by logP values in the range of 4.23-4.82. Regarding CYP inhibition, all compounds are considered inhibitors with probability exceeding 0.5 as shown in Table 7. The results predict that compounds 3a-e exhibit good pharmacokinetics and ADME properties (Tables 6 and 7). Probability: 0-0.1 (−−−); 0.5-0.7 (+); 0.7-0.9 (++); 0.9-1.0 (+++).

Chemistry
General Details: refer to Appendix SA (Supplementary Materials).

General Method for the Synthesis of Compounds 3a-e
A mixture of compound 1 (0.73 g, 2.90 mmol, 1 equiv) and 2a-e (1.2 equiv) in absolute ethanol (35 mL) was refluxed overnight with stirring. After cooling, NaBH 4 (0.13 g, 1.2 equiv) was added portion wise over a period of 20 min with stirring for further 30 min at rt. H 2 O (30 mL) was added and the reaction mixture was concentrated in vacuo to a minimum, extracted with EtOAc (80 mL), dried over MgSO 4 , and concentrated in vacuo to give oil which was re-dissolved in EtOAc (30 mL) and treated with 3 M HCl till formation of white precipitate. The precipitate formed was filtered, washed with EtOAc, and dried to give secondary amine as its hydrochloride salt. The hydrochloride salt was dissolved in water/methanol 1:1 (70 mL) and treated with saturated solution of 5% NaOH till alkaline to liberate free amine. The resulting mixture was concentrated in vacuo to a minimum and extracted twice with EtOAc. The organic layer was dried under MgSO 4 , and evaporated under reduced pressure to give 3a-e.    13  The MTT assay was used to determine how the synthesized compounds affected the viability of mammary epithelial cells (MCF-10A) [33,34]. See Appendix SA (Supplementary Materials).

Antiproliferative Test
To investigate the antiproliferative potential of 3a-e, 4a-c, and 5a-c, the MTT assay was carried out using various cell lines in accordance with previously reported procedures [35,36]. See Appendix SA (Supplementary Materials).

EGFR Inhibitory Assay
The EGFR-TK assay was used to evaluate the EGFR inhibitory effectiveness of 3a-e [37]. See Appendix SA (Supplementary Materials).

BRAF Kinase Assay
The activity of 3a-e against BRAF was investigated using a V 600E mutant BRAF kinase assay [38]. See Appendix SA (Supplementary Materials).

In Vitro Cytotoxicity of LOX-IMVI Melanoma Cell Line
The anticancer activity of the synthesized derivatives was determined using the MTT cytotoxicity assay on LOX-IMVI melanoma cell line [40,41]. See Appendix SA (Supplementary Materials).

Conclusions
A new series of 5-chloro-indole-2-carboxylate and pyrrolo [3,4-b]indol-3-one was synthesized and structurally characterized using various spectroscopic methods. The new compounds had no cytotoxic effects on human normal cell lines but demonstrated potent antiproliferative activities against four human cancer cell lines. Some of the compounds tested were found to be dual inhibitors of both wild type and mutant type EGFR and BRAF V600E . Molecular docking attempted to investigate the binding mode of the most active antiproliferative compounds 3a-e within the binding site of BRAF V600E in comparison with vemurafenib. Results proved that compound 3e, with m-piperidinyl substitution at the phenethyl amine moiety, was found to fit more tightly within the active site than the other derivatives with para-amine substituents. Moreover, docking results of compounds 3b and 3e against EGFR T790M concludes that the ligand indole-2-carboxylate scaffold binds intensely forming a combination of H-bond as well as hydrophobic interactions at the hydrophobic pocket of active site. In silico ADME and pharmacokinetic prediction revealed that compounds 3a-e have good pharmacokinetic and ADME properties. Compounds 3b and 3e may act as anticancer agents targeting the EGFRT 790M and BRAF V600E pathways after structural modifications, but more in vitro and in vivo testing is needed. Data Availability Statement: The data will be provided upon request.