New pyrazolylindolin-2-one based coumarin derivatives as anti-melanoma agents: design, synthesis, dual BRAF^V600E/VEGFR-2 inhibition, and computational studies

Abstract

Malignant melanoma is the most invasive skin cancer with the highest risk of death. The inhibition of BRAF^V600E appears relevant for overcoming secondary resistance developed during melanoma treatment. BRAF^V600E triggers angiogenesis via modification of the expression of angiogenic inducers, which play a crucial role in the metastasis of melanoma. Accordingly, the dual inhibition of the BRAF^V600E/VEGFR-2 signaling pathway is considered a rational approach in the design of anti-melanoma candidates. In this study, a new class of pyrazolylindolin-2-one linked coumarin derivatives as dual BRAF^V600E/VEGFR-2 inhibitors targeting A375 melanoma cells was designed. Target compounds were tailored to occupy the pockets of BRAF^V600E and VEGFR-2. Most of the synthesized compounds demonstrated potent mean growth inhibitory activity against A375 cells. Compound 4j was the most active cytotoxic derivative, displaying an IC₅₀ value at a low micromolar concentration of 0.96 μM with a significant safety profile. Moreover, 4j showed dual potent inhibitory activity against BRAF^V600E and VEGFR-2 (IC₅₀ = 1.033 and 0.64 μM, respectively) and was more active than the reference drug sorafenib. Furthermore, derivative 4j caused significant G0/G1 cell cycle arrest, induced apoptosis, and inhibited the migration of melanoma cells. Molecular docking showed that compound 4j achieved the highest ΔG value of −9.5 kcal mol⁻¹ against BRAF^V600E and significant ΔG of −8.47 kcal mol⁻¹ against VEGFR-2. Furthermore, the structure–activity relationship study revealed that TPSA directly contributed to the anticancer activity of the tested compounds.

New pyrazolylindolin-2-one linked coumarin derivatives were designed as dual BRAF^V600E/VEGFR-2 inhibitors targeting melanoma cells A375. Docking simulation showed various interactions with the binding residues in BRAF^V600E and VEGFR-2 active sites.

1. Introduction

Melanoma is one of the most aggressive forms of skin malignancy and considered the leading cause of death among other skin cancers.^1–3 Dysregulation of the RAS-RAF-MEK-ERK cascade, a mitogen-activated protein kinase (MAPK) pathway, is a critical step in melanoma pathogenesis.⁴

RAF family proteins involve serine–threonine kinases composed of A-RAF, B-RAF, and C-RAF, which regulate intracellular signal transduction. Among them, the BRAF oncogene shows better biochemical potencies than A-RAF and C-RAF isoforms owing to its higher basal kinase activity and ease of activation by RAS, which explains the observed frequent mutational activation of the BRAF gene in human tumors.^5–7 Approximately 90% of the detected BRAF mutations in human cancer are amino acid substitution at the nucleotide residue 600 (V600E) of valine by glutamic acid.⁸ The BRAF^V600E mutation leads to constitutive kinase activation, which is 500–700-fold greater than wild-type BRAF, resulting in the amplification of the MAPK signaling pathway and stimulation of vascular endothelial growth factor (VEGF), thus inducing metastasis.^9,10 This mutation is mostly associated with melanoma.^11–13

The successful inhibition of BRAF was a turning point in the development of anti-melanoma agents. Targeted therapy with BRAF inhibitors represents a milestone in the treatment of metastatic melanoma that harbors BRAF^V600E mutations. However, resistance to the treatment was identified in patients receiving these drugs within months, which makes the discovery of new targeted anti-melanoma therapeutics that can selectively block tumor signals in the RAS-RAF-MAPK complex network a substantial therapeutic challenge.^14–16

Various drug development programs have been initiated, such as the dual inhibition of BRAF^V600E and VEGFR-2, which has been considered a rational approach in the design of anti-melanoma candidates.^17,18 This approach aims to seek alternative chemical backbones that avoid the resistance and side effects acquired by current anti-melanoma FDA approved therapies such as vemurafenib,¹⁹ dabrafenib,²⁰ and encorafenib,²¹ which may be due to paradoxical MAPK activation in cells expressing wild-type BRAF.^22–25 Dual inhibitors such as sorafenib and RAF265, which are type II inhibitors, were approved for or entered clinical trials (Fig. 1).^26,27 Sorafenib, i.e., BAY 43e9006, is a multi-kinase inhibitor with dual inhibitory properties against VEGFR and RAF kinases. It suppresses MAPK signaling via RAF inhibition in various cancer cell lines including melanoma.²⁸ Regorafenib, i.e., BAY 73-4506, effectively inhibits a wide range of angiogenic kinases such as VEGFR-1/2/3 and intracellular signaling kinases, including BRAF and CRAF.²⁹ Furthermore, RAF265 is a strong dual RAF/VEGFR-2 inhibitor that efficiently suppresses the growth and survival of cancer cells by blocking tumor development and preventing blood supply to cancerous cells (Fig. 1).³⁰

Fig. 1. (A) Examples of FDA approved anti-melanoma drugs. (B) Dual inhibitors of BRAF/VEGFR-2.

Coumarin derivatives are known for their potent anticancer activity, displaying remarkable activity against human malignant melanoma and preventing its recurrence.^31–33 Moreover, coumarin analogues exhibit selective RAF inhibiting efficacies, making the coumarin nucleus a prominent bioactive structural motif in the search for new anti-melanoma cancer molecules.^34,35 Furthermore, 4-hydoxycoumarin derivatives have been extensively used as a valuable core in the synthesis of VEGFR-2 inhibitors.^36,37 Moreover, several FDA approved drugs and inhibitors in clinical trials containing pyrazole (e.g., encorafenib and GDC0879) and indole or indole isosteres (e.g., vemurafenib (azaindole), SB-590885 (indene) and XL281(indole)³⁸) have been investigated for potent and selective anti-BRAF activity. Alternatively, pyrazole and indole derivatives have also been reported for their potent VEGFR-2 inhibitory activity (Fig. 1 and 2).^30–43

Fig. 2. Rationale for the design of target compounds.

In this work, we aim to discover dual BRAF^V600E and VEGFR-2 inhibitors that were rationally designed based on the ABC triaryl pyrazole system,^44–46 simultaneously displaying the pharmacophoric features of BRAF^V600E and VEGFR-2 inhibitors (Fig. 2). Based on the above-mentioned studies, a new series of pyrazolyl indoline-based coumarin derivatives was designed, synthesized, and biologically assessed for their cytotoxic activity against melanoma cells. Furthermore, the promising derivatives were evaluated for dual inhibition towards BRAF^V600E and VEGFR-2, cell migration, cell cycle analysis and apoptosis. The molecular modeling study was carried out to investigate the binding mode of the promising candidates in the binding pockets of BRAF^V600E and VEGFR-2.

2. Results and discussion

2.1. Chemistry

The synthetic procedures for the synthesis of the target pyrazolylindoline-2-one-coumarin hybrids 4a–j, 5a–j and 6a–j are demonstrated in Scheme 1. Intermediate chalcone derivatives 2a–c were prepared through the Claisen–Schmidt condensation reaction of 3-acetyl-4-hydroxycoumarin 1 and the appropriate aromatic aldehydes, namely, 4-fluorobenzaldehyde, 4-chlorobenzaldehyde and/or 4-bromobenzaldehyde according to the reported procedures.^47,48 The cyclo–addition reaction of hydrazine hydrate with chalcone intermediates 2a–c furnished the corresponding pyrazolines 3a–c, respectively. The target pyrazolylindolin-2-one derivatives 4a–j, 5a–j and 6a–j were afforded by reacting pyrazoline compounds 3a–c with isatin derivatives via the elimination of a water molecule (Scheme 1). The structures of the newly synthesized compounds were assigned via elemental and spectral analyses including ¹H NMR and ¹³C NMR.

Scheme 1. Reagents and conditions: (i) POCl₃/AcOH/reflux. (ii) ArCHO/piperidine/EtOH/reflux. (iii) NH₂NH₂·H₂O/EtOH/reflux. (vi) Isatin derivatives/EtOH/reflux.

2.2. Biology

2.2.1. In vitro cytotoxic effect of synthesized compounds against melanoma cells A375

The target compounds 4a–j, 5a–j and 6a–j were assessed for their in vitro cytotoxic activity against the A375 human melanoma cell line via SRB assay using sorafenib as a positive control. The cytotoxic effects were expressed by median growth inhibitory concentration (IC₅₀), as shown in Table 1. The results revealed that among the derivatives, 4j, 4f, 4i, 5f and 5i were the most potent compounds with IC₅₀ values of 0.96, 1.114, 1.28, 1.09 and 1.02 μM, respectively, displaying higher cytotoxic activity than sorafenib and showing a significant safety profile, as presented in Table 2. Regarding the other compounds, most of them exerted promising cytotoxic activity against melanoma cells with IC₅₀ values of <10 μM, ranging from 8.99 μM (6e) to 3.8 μM (5c), while five derivatives, 4b, 4e, 4h, 5b, and 6h, exhibited weak cytotoxic activity with IC₅₀ values of 79.47, 82.63, 38.65, 33.76 and 47.22 μM, respectively.

Table 1. Cytotoxic activity of the target compounds on A375 cells via the SRB assay^a.

Compound	R	X1	X2	(IC50, μM)
4a	H	F	H	6.25 ± 0.75
4b	CH2CH3	F	H	79.47 ± 3.60
4c	CH2PH	F	H	4.6 ± 0.32
4d	H	F	Cl	4.3 ± 0.38
4e	CH2CH3	F	Cl	82.63 ± 2.65
4f	CH2Ph	F	Cl	1.114 ± 0.02
4g	H	F	Br	5.85 ± 0.95
4h	CH2CH3	F	Br	38.65 ± 2.60
4i	CH2Ph	F	Br	1.28 ± 0.02
4j	H	F	OCF3	0.96 ± 0.03
5a	H	Cl	H	7.13 ± 0.44
5b	CH2CH3	Cl	H	33.76 ± 1.80
5c	CH2Ph	Cl	H	3.8 ± 0.22
5d	H	Cl	Cl	5.43 ± 0.82
5e	CH2CH3	Cl	Cl	7.32 ± 0.69
5f	CH2Ph	Cl	Cl	1.09 ± 0.09
5g	H	Cl	Br	6.18 ± 0.73
5h	CH2CH3	Cl	Br	5.44 ± 0.41
5i	CH2Ph	Cl	Br	1.02 ± 0.07
5j	H	Cl	OCF3	4.17 ± 0.36
6a	H	Br	H	8.45 ± 0.60
6b	CH2CH3	Br	H	68.38 ± 3.50
6c	CH2Ph	Br	H	4.84 ± 0.30
6d	H	Br	Cl	6.25 ± 0.72
6e	CH2CH3	Br	Cl	8.99 ± 0.78
6f	CH2Ph	Br	Cl	4.36 ± 0.22
6g	H	Br	Br	7.32 ± 0.80
6h	CH2CH3	Br	Br	47.22 ± 1.90
6i	CH2Ph	Br	Br	5.47 ± 0.20
6j	H	Br	OCF3	4.37 ± 0.24
Sorafenib	—	—	—	3.2 ± 0.67

^aThe cytotoxic activity was determined after treatment of A375 with serial dilution of the target compounds for 72 h, where data are expressed as mean ± SD (n = 3).

Table 2. IC₅₀ values (μM) of the most active compounds on melanoma A375 and HSF cell lines and the selectivity index.

Compound	A375 IC50 (μM)	HSF IC50 (μM)	Selectivity index (SI)
4f	1.114 ± 0.02	4.18 ± 0.25	3.6
4i	1.28 ± 0.021	3.26 ± 0.29	2.5
4j	0.96 ± 0.03	9.58 ± 0.73	9.9
5f	1.09 ± 0.09	3.59 ± 0.32	3.3
5i	1.02 ± 0.07	3.63 ± 0.76	3.5

The cancerous specificity and the safety of the most active derivatives were evaluated on normal human skin fibroblast cells (HSF) and their selectivity index values were calculated and shown in Table 2. The five most potent derivatives (4j, 4f, 5i, 5f and 4i) showed relatively low toxicity with IC₅₀ values on HSF cells of 9.58, 4.18, 3.63, 3.59 and 3.26 μM, respectively. Among them, compound 4j was the safest hybrid (SI = 9.9), displaying the highest selectivity and potency towards melanoma cells A375.

2.2.2. In vitro BRAF^V600E and VEGFR-2 inhibitory activity of compound 4j

The most promising derivative 4j, which showed the most potent activity against A375 melanoma cells and the highest SI, was further evaluated for its inhibitory effect on both BRAF^V600E and VEGFR-2 kinases in A375 cells compared to sorafenib (Table 3). The results of the BRAF^V600E inhibition assay showed the pronounced inhibitory activity of 4j (IC₅₀ = 1.033 μM), which was 2-fold more active than the reference sorafenib (IC₅₀ = 2.86 μM). The selected derivative exhibited significant inhibition activity against VEGFR-2 with an IC₅₀ value of 0.64 μM versus 1.94 μM for sorafenib. These results are consistent with the cytotoxic effect of compound 4j against A375 cells, highlighting the postulation that BRAF^V600E and VEGFR-2 inhibition can be the molecular mechanisms of the potential cytotoxic effect of 4j, especially given that inhibitors of BRAF^V600E kinase have been reported to be antiproliferative agents against melanoma.⁴⁹ Therefore, 4j was selected for the following tests.

Table 3. In vitro BRAF^V600E and VEGFR-2 inhibitory activities.

Compound	BRAFV600E (IC50, μM)	VEGFR-2 (IC50, μM)
4j	1.03 ± 0.71	0.64 ± 0.08
Sorafenib	2.86 ± 0.45	1.94 ± 0.05

2.2.3. Effect of 4j on the cell cycle distribution in melanoma cells

The cell cycle distribution using DNA flow cytometry was used to assess the observed growth inhibition after treatment of the A375 cell line with 4j and sorafenib (Fig. 3). Treatment with 4j resulted in major changes in all cell cycle phases compared to control cells. Compound 4j caused a significant increase (P < 0.05) in the cell population of the G0/G1-phase (the non-proliferating cell proportion) from 55.71% to 74.14% compared to the control cells, indicating cell cycle arrest and implying that G0/G1 cell cycle arrest is a fundamental mechanism of the anticancer activities of coumarin derivatives, as previously reported.⁵⁰ Reciprocally, treatment with 4j showed a meaningful decrease in the cell percentage of the S phase (the DNA synthesis phase) after 48 h treatment from 17.62% to 9.86% concomitantly with a decrease in the cell percentage in the G2/M phase from 26.67% to 16.00% (Fig. 3A and B), respectively. Also, no significant changes were observed in the pre-G phase after treatment with 4j from 1.21% to 1.88% compared with the control cells.

Fig. 3. (A) Cell cycle distribution in A375 cells after treatment with 4j and sorafenib for 48 h determined using DNA cytometry analysis compared with a control (untreated cells). (B) Quantification of the percentage of cells in each phase of the cell cycle is depicted as a bar graph of mean ± SD; n = 3. (C) Sub-G1 phase was plotted alone as a proportion of total events. * Statistically significant difference from the control (P < 0.05).

Treatment with sorafenib resulted in observable cell cycle arrest at the G0/G1 phase from 55.71% to 60.43%, with almost the same alterations in the S phase from 17.62% to 16.55% and G2/M phase from 26.67% to 23.02% compared to the control cells (Fig. 3A and B), respectively. However, treatment with sorafenib resulted in significant cell death (P < 0.05) manifested by a considerable increase in the cell population of the Pre-G phase from 1.21% to 22.88% compared with the control cells (Fig. 3C). These results are consistent with that reported for sorafenib, which significantly inhibits the growth of cancer cells through the accumulation of cells in the pre-G1 phase.⁵¹

These findings shed light on the mode of action of coumarin derivatives as antiproliferative agents (inducing G0/G1 arrest), which differs from the cytotoxic effect of sorafenib (inducing pre-G1 arrest) and highlight the potential use of coumarins as effective anticancer agents in melanoma treatment. This can be explained by the fact that the G1 phase is a phase that precedes DNA replication, in which the cellular conditions influence cell cycle progression, causing DNA to either rebuild in a cell or initiate apoptosis, and several studies address the role of G0/G1 phase arrest in intensifying cellular apoptosis.⁵²

2.2.4. Modes of cell death in melanoma cells

To determine the exact mechanism of cell death (apoptosis/necrosis) induced by 4j and sorafenib, the treated A375 cells were evaluated using Annexin-V/FITC staining coupled with flow cytometry. The results are highlighted in Fig. 4A and B, showing the variations in apoptosis and necrosis detection between the groups.

Fig. 4. (A) Evaluation of cell death modality (apoptosis/necrosis) after treatment with 4j and sorafenib for 48 h in A375 cells, followed by double staining with Annexin-FITC/PI. (B) Different cell populations plotted as the percentage of total events. Data are presented as mean ± SD; n = 3. Total cell death calculated by combining apoptosis and necrosis. * Statistically significant difference from the control (P < 0.05).

Compound 4j induced significant (p < 0.05) total cell death of A375 cells (nearly 10.22%) by mainly apoptotic death with early apoptosis of 6.07% and late apoptosis of 2.3% compared with the control untreated A375 cells with 0.14% early and 0.7% late apoptosis, respectively (Fig. 4A). These findings show that most of the cells underwent cell death by the apoptotic mode, suggesting that 4j exerted its anticancer activity through the apoptosis route. This is consistent with the studies that reported apoptosis as a mechanism of cell death in cancer treatment by coumarin compounds.⁴⁸ However, treatment with 4j did not induce notable necrotic cell death compared to the control cells (2.52% and 1.85%, respectively). This indicates that 4j at its IC₅₀ concentration can induce apoptosis, mainly early apoptosis, of A375 melanoma cells but not necrosis. Furthermore, it arrested cells in the G1 phase, which promoted cell apoptosis in melanoma cells. Meanwhile, treatment with sorafenib showed a dramatic decrease in cell viability. Sorafenib treatment produced greater cell death than 4j in A375, which was mainly necrotic (16.6%) (p < 0.05), and nearly the same percentage of apoptotic cell death (9.6%) compared with the 4j-treated cells. This can be related to the sorafenib dose, which only modestly boosted apoptosis relative to necrosis.⁵³ It is also worth noting that the amount of necrosis did not correlate with apoptosis, indicating that distinct mechanisms are involved.⁵⁴

2.2.5. In vitro inhibition effect of 4j on the migration of melanoma cells

One of the main reasons for the increased mortality of melanoma is its rapid progression and metastasis to lymph nodes and distant organs.⁵⁵ We used the scratch/wound healing assay to investigate the effects of 4j and sorafenib on A375 melanoma cell migration (Fig. 5A and B), and we assessed the scratch closure daily until complete closure of the control untreated cells. At 0 h, the scratch line of all the groups revealed an insignificant change (P > 0.5) in the gap, indicating that the scratch technique was consistent.

Fig. 5. Effect of 4j and sorafenib on the migration of A375 cells. (A) Scratch widths were measured after 4j and sorafenib treatment at 24, 48, and 72 h. (B) Data are plotted as scratch closure % at each time interval and presented as triplicates. * Statistically significant difference from the control (P < 0.05).

Compound 4j displayed a significant (P < 0.05) anti-metastasis effect by inhibiting A375 cell migration at all time intervals compared with the control untreated cells. Also, it showed a comparable effect to sorafenib anti-migratory activity. After 24 h, 4j treatment postponed the migration tendency of A375 cancer cells, as evidenced by the 14.28% ± 2.13% delay in cell-free zone closure compared to 38.68% ± 0.89% for the untreated cells. After 48 h of 4j treatment, a similar pattern was observed, with a delayed scratch closure of 17.57% ± 1.72% compared to 62.77% ± 1.78% in the control cells, demonstrating that 4j may exert an anti-migratory effect in melanoma cancer cells. The following day (72 h), complete scratch closure was observed in the control group (100%); however, the scratch remained open in the 4j-treated cells, with the scratch closure of 35.16% ± 2.45%, indicating that 4j could exert a potent anti-migratory effect at this concentration (0.5 μM, nearly 1/2 IC₅₀ value). Similarly, compared to the untreated cells, the sorafenib-treated melanoma cells demonstrated delayed migration, which was slightly more potent than the 4j-treated cells, with scratch closure of 9.15% ± 0.64%, 12.07% ± 1.8%, and 24.17% ± 1.32% at 24, 48, and 72 h, respectively.

2.3. Molecular modeling study

2.3.1. Topological polar surface area (TPSA)

In this study we computed the topological polar surface area (TPSA) (Table 4) as a 2D descriptor that can help in the SAR study. TPSA can predict the drug-like properties of tested compounds and bioavailability properties such as transport and absorption. It was reported that any compound with a TPSA value of > 140 Ų will have poor absorption. Drug-like candidates will have a TPSA of <140 Ų.^56–58 Accordingly the tested compounds were classified into three groups according to their TPSA values (Fig. 6 and Table 4).

Table 4. Docking results against BRAF^V600E (PBD = 2FB8) and TPSA.

Compound	ΔG (kcal mol−1)	GBVI/WSA ΔG (kcal mol−1)	RMSD (Å)	TPSA (Å2)	Interacted residues
4a	−8.16	−38.3	1.6	93.4	Ile463, Asp594, Glu501
4b	−6.45	−30.75	2.75	84.6	Trp531, Asp594
4c	−8.77	−38.91	2.59	84.6	Asp594, Ile463
4d	−8.57	−42.5	1.2	93.4	Cys532
4e	−6.37	−30.89	2.7	84.6	Trp531
4f	−9.04	−41.35	1.44	84.6	Phe593, Cys532
4g	−8.82	−39.05	1.41	93.4	Cys532, Ala481, Lys483
4h	−7.54	−35.62	2.35	84.6	Trp531, Cys532
4i	−9.1	−41.39	1.37	84.6	Trp531, Phe583
4j	−9.50	−44.62	1.26	102.6	Asp594, Thr529
5a	−8.17	−37.08	1.26	93.4	Ile463, Glu501
5b	−7.66	−35.21	2.4	84.6	Asn581, Asp594
5c	−9.09	−41.45	1.79	84.6	Ile463
5d	−8.52	−38.46	1.04	93.4	Cys532, Trp531, Asp594
5e	−8.07	−37.5	2.5	84.6	Ile563, Ala481
5f	−9.08	−41.72	1.68	84.6	Cys532, Trp531
5g	−8.26	−38.02	1.86	93.4	Cys532, Ala481, Lys483
5h	−8.83	−38.55	1.41	84.6	Trp531, Cys532
5i	−9.11	−41.98	0.82	84.6	Phe593, Cys532
5j	−8.92	−41.36	2.72	102.6	Cys532, Ile463, Lys481
6a	−8.09	−34.05	1.6	93.4	Ile463, Asp594
6b	−6.85	−30.55	2.76	84.6	Asn581
6c	−8.59	−38.87	1.28	84.6	Ile463, Asp594
6d	−8.20	−38.42	1.52	93.4	Cys532, Ala481, Lys483
6e	−8.03	−34.35	1.44	84.6	Trp531, Cys532
6f	−8.75	−42.11	1.65	84.6	Trp531, Phe583
6g	−8.04	−37.66	1.27	93.4	Cys532, Ala481, Lys483
6h	−7.46	−34.79	2.5	84.6	Trp531, Cys532
6i	−8.72	−39.11	1.27	84.6	Trp531, Phe583
6j	−8.65	−38.87	1.22	102.6	Ile463, Lys481
SB-590885	−8.66	−43.54	0.95	86.6	Cys532, Ile463, Glu501
Sorafenib	−8.26	−42.93	0.97	92.36	Cys532, Asp594, Glu501

Fig. 6. Three main positions R1, R2, and R3 directly correlated with the anti-melanoma activity.

(i) Compounds with TPSA = 102.6 Ų, this group includes all compounds with trifluoromethoxy group at R2, halo atom (F, Cl, Br) at R3, and R1 H, e.g., 4j, 5j and 6j.

(ii) Compounds with TPSA = 93.4 Ų, this value is close to TPSA of sorafenib (92.36 Ų), e.g., compounds 4d, 5d, 4g, 5g, 4a, 6d, 5a, 6g and 6a. All these compounds showed potent anticancer activity.

(iii) Compounds with TPSA = 84.6 Ų, this value is close to that of the reference inhibitor SB-590885 (86.6 Ų), e.g., 5i, 5f, 4f, 4i, 5c, 6f, 4c, 6c, 5h, 6i, 5e and 6e. All these compounds also showed potent anticancer activity.

2.3.2. Structure activity relationship

According to the biological results, there are three main positions that are directly correlated with the activity of the tested compounds, i.e., position N1 at the indolin-2-one scaffold, position 5 at the indolin-2-one scaffold, and position 4 at the phenyl ring, which are represented as R1, R2, and R3, respectively, in Fig. 6. All the compounds can be classified into three classes.

2.3.2.1. Class I: compounds with no substitution at N1-indolin (R1 = H) and halo substitution at R2 and/or R3

It was observed that the number of fluoro atom substitutions at R2 and R3 is very important for cytotoxic activity. The most active compound 4j (IC₅₀ = 0.96 μM) with the highest number of F atoms (4 atoms) exhibited the highest TPSA value = 102.6 Ų among the compounds and the reference drugs given that the fluoro atom increases the lipophilicity and TPSA value. The replacement of F in compound 6 with Br or Cl yielded compound 6j and 5j with a decrease in cytotoxic activity (IC₅₀ = 4.37 and 4.17 μM, respectively). A further decrease in the number of F atoms resulted in a decrease in activity, for example, when the OCF₃ in compound 4j was replaced with Br it yielded compound 4g (IC₅₀ = 5.85 μM). This confirmed that the number of F atoms is directly proportional to the cytotoxic activity. The type of halo substitution at R2 and R3 can be ranked according to activity, following the order of (Cl&F) > (Cl&Cl) > (Br&Br) and this can be observed in compound 4d with Cl at R2 and F at R3 (IC₅₀ = 4.3 μM), which is more active than compound 5d with Cl substitution at R2 and R3 (IC₅₀ = 5.43 μM), while compound 6g with a Br atom at R2 and R3 was less active (IC₅₀ = 7.32 μM). In the case of compounds 4a, 5a, and 6a having only one halo atom at R3 in the phenylpyrazolyl moiety, it was deduced that the presence of F substitution increased the activity, following the order of compound 4a (IC₅₀ = 6.25 μM) > compound 5a (IC₅₀ = 7.13 μM) > compound 6a (IC₅₀ = 8.45 μM), which confirmed the importance of the presence of fluoro substitution, as discussed in the previous section. The compounds of this class (4a, 5a and 6a) exhibited the same TPSA value = 93.4 Ų, which is close to that of sorafenib (92.36 Ų).

2.3.2.2. Class II: compounds with benzyl group at N1-indolin (R1 = benzyl) and halo substitution at R2 and/or R3

The presence of a benzyl group in compounds 4c, 5c and 6c was very important due to the hydrophobic interactions provided by the phenyl ring, which were oriented between hydrophobic residues Trp531, Phe583, and Ile463. Also, the methylene –CH₂ of the benzyl group was important for van der Waals formation to support the fitting of these compounds. The benzyl group increased the lipophilicity and decreased the TPSA value of these derivatives to reach 84.6 Ų, which is close to that of SB-590885 = 86.6 Ų (Table 4) and may be correlated with their activity. The activity of 4c, 5c and 6c was improved compared to the compounds in the previous class with IC₅₀ values of 3.8, 4.6, and 4.84 μM, respectively. Moreover, the presence of bromo substitution at R2 and R3 decreased the activity, as observed in compound 6i (IC₅₀ = 5.47 μM), where the presence of fluoro substitution at the 4-position of the phenyl ring (R3) with either chloro or bromo substitution at the 5-position of the indolin-2-one scaffold (R2) improved the activity, as in compound 4f (IC₅₀ = 1.1 μM) and compound 4i (IC₅₀ = 1.28 μM). The substitution with two chloro atoms at both R2 and R3 retained the improved activity as in compound 5f (IC₅₀ = 1.09 μM). The presence of chloro substitution at R2 and bromo substitution at R3 decreased the activity as in compound 6f (IC₅₀ = 4.36 μM). The TPSA value of these compounds was 84.6 Ų, which is close to that of SB-590885 = 86.6 Ų (Table 4).

2.3.2.3. Class III: compounds with ethyl at N1-indolin (R1 = ethyl)

This class showed some deviation from the previous observations, where the best activity was achieved by compound 5h (R2 Br and R3 Cl) with an IC₅₀ value of 5.44 μM, which may need future investigation. In addition, dichloro substitution at R2 and R3 in compound 5e showed good activity (IC₅₀ = 7.32 μM). However, the TPSA of these compounds was 84.6 Ų, which is close to that of SB-590885.

2.3.3. Molecular docking study against BRAF^V600E

To determine the binding modes of the target compounds, molecular docking was performed within the BRAF^V600E binding active site (PBD = 2FB8). The docking results of the reference ligand SB590885 within the BRAF^V600E binding active site showed three hydrogen bonds formed between Cys532 and C N of the pyridine ring, between the dimethyl amino group in the side chain and Ile463 and between the hydroxyl group of oxime and Glu501 (Fig. 7A). Sorafenib also interacted by three hydrogen bonds, where one was formed between –NH of the urea group and Cys532, the other bond between the acetamide side chain and Asp594 and the last one with Glu501 (Fig. 7B). The molecular docking of 4j with the highest ΔG value of −9.50 kcal mol⁻¹ against BRAF^V600E, revealed that the 4-hydroxy group formed a hydrogen bond with the –COOH group of Asp594 and the –NH of indoline formed a hydrogen bond with C O of Thr529. The 5-(trifluoromethoxy) indolin-2-one scaffold was oriented and surrounded by hydrophobic residues, i.e., Phe583, Ile463, and Trp531, with the possible formation of hydrophobic interactions (Fig. 7C and Table 4).

Fig. 7. Docking poses against BRAF^V600E (A) SB590885, (B) Sorafenib, and (C) 4j.

Compound 5j showed the same orientation of the 5-(trifluoromethoxy) indolin-2-one scaffold surrounded by all the hydrophobic residues. It formed hydrogen bonds with Cys532, Ile463, and Lys481. Compound 6j also formed the same interactions as 5j with Ile463 and Lys481. Compound 4d formed one hydrogen bond with Cys532, while compound 5d formed three hydrogen bonds with Cys532, Trp531, and Asp594. Compound 6g showed three hydrogen bonds with Cys532, Ala481 and Lys483. The coumarin ring in compound 4a formed two hydrogen bonds with Asp594 and Glu501. The 4-bromophenyl ring in compound 5a showed hydrophobic interactions with Ile463. The –NH of the pyrazole in compounds 4a, 5a, and 6a interacted by a hydrogen bond with C O of Ile463 and the C O of coumarin in compound 6a showed a hydrogen bond with Asp594 (Fig. S1A–I†).

The –CH₂ of benzyl moiety in compounds 4c and 6c formed van der Waals interactions with Ile463, while the same group in compound 4f formed hydrophobic interactions with Phe583. The phenyl ring of the benzyl moiety of compound 6i showed π–π interactions with Trp531 and Phe583. The –CH₂ of the benzyl moiety in compound 5f formed hydrophobic interactions with Trp531 and Cys532 (Fig. S2A–F†). The docking of compounds with R1 = ethyl and R3 = halo substitution showed that compound 5b formed two hydrogen bonds with Asn581 and Asp594. Compound 6b showed a hydrogen bond with Asn581. The ethyl group in compound 4b contributed to van der Waals interactions with Trp531 and the C O of coumarin formed a hydrogen bond with Asp594. The pyrazole ring in compound 5e contributed to van der Waals interactions with Ile463 and the C O of coumarin formed a hydrogen bond with Ala481. The ethyl group in compound 6h formed two van der Waals interactions with Trp531 and Cys532. Compound 4e showed one van der Waals interaction between the ethyl group at R1 and Trp531 (Fig. S3A–F†).

2.3.4. Molecular docking study against VEGFR-2

The synthesized compounds were also docked in the VEGFR-2 binding pocket (PBD = 3VO3). Sorafenib showed the best mode of binding, where it interacted with the important residues in the active site, i.e., Asp1046, Cys919, and Glu886 (Fig. 8A). The top ranked compounds 4j, 4i, 4f, 5f and 5i showed different binding interactions against VEGFR-2 and their binding mode was compared to that of sorafenib. Compound 4j achieved the ΔG of −8.47 kcal mol⁻¹ and ranked second after sorafenib, which showed ΔG of −9.44 kcal mol⁻¹ (Table 5). Compound 4j showed two hydrogen bonds with Cys919 and one hydrogen bond with Glu885 (Fig. 8B). The interaction with the Cys919 residue was a common feature among compounds 4j, 4f, 4i, and 5i and the reference drug. Compounds 4f and 4i formed hydrogen bonds with only Cys919. Compound 5f showed a hydrogen bond with Asp1046, while compound 5i has two hydrogen bonds with Cys919 and Lys868 (Fig. S4A–F†).

Fig. 8. Docking poses against VEGFR-2: (A) Sorafenib and (B) 4j.

Table 5. Docking results against VEGFR-2 (PBD = 3VO3).

Compound	ΔG (kcal mol−1)	GBVI/WSA ΔG (kcal mol−1)	RMSD (Å)	Interacted residues
4a	−7.65	−36.99	1.35	Glu885
4b	−7.04	−36.61	2.05	Lys868
4c	−8.23	−36.6	1.83	Cys919, Lys868
4d	−7.42	−39.21	1.57	Cys919
4e	−7.18	−36.57	1.74	Lys868
4f	−8.21	−33.3	1.95	Cys919
4g	−7.98	−38.44	1.92	Cys919
4h	−7.17	−35.63	1.82	Lys868, Glu885
4i	−7.86	−40.31	1.81	Cys919
4j	−8.47	−37.79	1.61	Cys919, Glu885
5a	−7.88	−39.58	1.05	Glu885
5b	−7.17	−37.35	2.33	Lys868
5c	−8.02	−34.73	1.31	Cys919, Lys868
5d	−7.53	−35.11	1.39	Cys919
5e	−7.71	−34.67	2.12	Lys868, Glu885
5f	−7.62	−37.01	1.70	Asp1046
5g	−7.27	−36.17	1.42	Cys919
5h	−7.44	−36.83	1.76	Lys868, Glu885
5i	−7.79	−35.65	1.57	Cys919, Lys868
5j	−7.75	−36.29	2.32	Cys919, Glu885
6a	−7.85	−37.38	1.44	Glu885
6b	−7.15	−32.09	1.54	Lys868, Glu885
6c	−7.10	−35.68	1.11	Cys919, Lys868
6d	−7.54	−38.08	2.77	Cys919
6e	−7.36	−37.52	1.87	Lys868, Glu885
6f	−7.18	−37.34	2.1	Cys919, Lys868
6g	−7.64	−32.51	1.44	Cys919
6h	−7.60	−37.66	1.29	Lys868, Glu885
6i	−7.95	−35.55	1.72	Cys919, Lys868
6j	−7.70	−35.81	1.93	Cys919, Glu885
Sorafenib	−9.44	−46.54	1.47	Asp1046, Cys919, Glu885

The aforementioned information showed that the results of the in silico studies were consistent with that of the biological assays, where the most active compound 4j with the IC₅₀ value of 0.96 μM against A375 cells and dual inhibition properties against BRAF^V600E/VEGFR-2 (IC₅₀ = 1.033 and 0.64 μM, respectively), revealed the highest ΔG value of −9.5 kcal mol⁻¹ against BRAF^V600E and showed a significant ΔG of −8.47 kcal mol⁻¹ towards VEGFR-2. Moreover, compound 4j displayed different types of interactions with essential catalytic sites in the active region of BRAF^V600E and VEGFR-2.

2.3.5. ADME prediction study

The pharmacokinetic properties of compounds 4j, 4f, 4i, 5f, and 5i were estimated, which showed the best anti-melanoma activity (Table S1†). The most promising compound 4j showed high skin permeability with no skin sensitization, which can be used to interpret its activity and show its appropriate use against melanoma skin cancer. The human colon adenocarcinoma cell line (Caco-2) was used to predict the in vivo drug transport through the intestinal epithelium, where 4j showed high predicted Caco-2 permeability and high intestinal absorption of 100%. Derivative 4j showed high plasma protein binding with poor clearance and very low fraction of unbound drug. Furthermore, compound 4j did not show any Ames toxicity or carcinogenicity.

3. Conclusion

In this study, a series of new coumarin derivatives bearing a pyrazolylindolin-2-one core was synthesized and evaluated for their anti-proliferative effects against human melanoma A375 cells as dual BRAF^V600E and VEGFR-2 inhibitors. Most of the compounds displayed potent antiproliferative activity against A375 cells with IC₅₀ values in the range of 0.96–8.99 μM. Among them, compounds 4j, 4f, 4i, 5f and 5i demonstrated potent cytotoxic activity (IC₅₀ = 0.96–1.28 μM) and good safety profile. Furthermore, compound 4j exerted greater potent dual inhibitory activity against BRAF^V600E/VEGFR-2 than the reference drug sorafenib. The anticancer effect of 4j was correlated with the inhibition of cell proliferation, boosting cell cycle arrest at the G0/G1 phase, induction of apoptosis, and inhibition of migration. Docking simulation was carried out for the active compounds to predict their binding modes in the BRAF^V600E and VEGFR-2 protein active sites, revealing various interactions with the binding residues in the active site. The SAR study showed the correlation between the number of fluoro atoms and both TPSA and cytotoxic activity.

4. Experimental

4.1. Chemistry

Melting points were determined on an Electrothermal IA 9000 apparatus and were uncorrected. Elemental analyses were carried out at the Micro-analytical Central Services Laboratory, Faculty of Science, Cairo University, Egypt. ¹H NMR and ¹³C NMR spectra were measured using a Bruker Avance II™ 400 MHz spectrometer (Bruker Biospin AG, Fällanden, Switzerland) in Prague, Czech Republic. ¹³C NMR spectra of compounds 4e, 4h and 5h could not be acquired due to their precipitation from the solvent used (DMSO). The reactions were monitored by TLC (silica gel, aluminium sheets 60 F254, Merck) using chloroform/methanol (9.5 : 0.5 v/v) as the eluent and sprayed with iodine–potassium iodide solution. The key intermediates 3a–c ^47,48 and isatin derivatives Ia–j ^59,60 and 4a ⁴⁷ were previously prepared.

4.1.1. General procedure for the preparation of pyrazolyl indolin-2-one derivatives 4a–j, 5a–j and 6a–j

A mixture of 1 mmol of pyrazoline compounds 3a (0.32 g), 3b (0.34 g) and/or 3c (0.38 g) and isatin derivatives Ia–j (1 mmol) in absolute ethanol (20 mL) was refluxed for 8–10 h. After completion of the reaction, the obtained product was filtered, washed with ethanol, and recrystallized from acetic acid to obtain the desired target compounds 4a–j, 5a–j and 6a–j, respectively.

4.1.1.1. 1-Ethyl-3-(5-(4-fluorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl) indolin-2-one 4b

Yellow crystals, m.p. 277–278 °C, yield (0.35 g, 73%). ¹H NMR (400 MHz, DMSO-d₆): δ = 1.15 (t, 3H, J = 7.2 Hz, CH₃), 3.72 (q, 2H, J = 6.4 Hz, CH₂), 6.32 (s, 1H, CH-indole), 7.06–7.09 (m, 1H, H–Ar), 7.18–7.28 (m, 4H, H–Ar), 7.34–7.44 (m, 5H, H–Ar), 7.64–7.70 (m, 2H, H–Ar), 7.84 (d, 1H, J = 8.4 Hz, H–Ar), 13.06 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 12.79, 34.95, 60.71, 95.10, 106.89, 109.85, 115.49, 116.44, 116.66, 116.78, 123.21, 124.04, 124.87, 125.06, 125.24, 130.59, 132.06, 132.15, 143.20, 145.46, 147.77, 152.43, 159.70, 162.10, 163.41, 164.56, 171.26. Anal. calcd for C₂₈H₂₀FN₃O₄ (481.47): C, 69.85; H, 4.19; N, 8.73. Found: C, 70.00; H, 4.37; N, 8.89.

4.1.1.2. 1-Benzyl-3-(5-(4-fluorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl) indolin-2-one 4c

Yellow crystals, m.p. 220–221 °C, yield (0.4 g, 74%). ¹H NMR (400 MHz, DMSO-d₆): δ = 4.91 (d, 1H, J = 15.6 Hz, CH_a-benzyl), 5.01 (d, 1H, J = 16.0 Hz, CH_b-benzyl), 6.47 (s, 1H, CH-indole), 6.99 (d, 1H, J = 8.0 Hz, H–Ar), (t, 1H, J = 7.6 Hz, H–Ar), 7.26 (s, 1H, H–Ar). 7.30–7.46 (m, 11H, H–Ar). 7.67–7.71 (m, 1H, H–Ar), 7.79 (brs, 2H, H–Ar), 7.87 (d, 1H, J = 6.8 Hz, H–Ar), 13.02 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 43.42, 60.42, 95.36, 106.83, 110.44, 115.52, 116.57, 116.79, 116.83, 123.48, 124.01, 124.99, 125.09, 125.13, 127.49, 127.99, 129.14, 130.48, 132.10, 132.19, 133.70, 136.15, 143.50, 145.86, 148.13, 152.55, 160.10, 162.17, 163.62, 164.62, 171.99. Anal. calcd for C₃₃H₂₂FN₃O₄ (543.15): C, 72.92; H, 4.08; N, 7.73. Found: C, 73.09; H, 4.22; N, 7.91.

4.1.1.3. 5-Chloro-3-(5-(4-fluorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl) indolin-2-one 4d

Pale yellow powder, m.p. 294–295 °C, yield (0.38 g, 78%). ¹H NMR (400 MHz, DMSO-d₆): δ = 6.24 (s, 1H, CH-indole), 6.96 (d, 1H, J = 8.0 Hz, H–Ar), 7.22 (s, 1H, H–Ar), 7.36–7.44 (m, 6H, H–Ar), 7.66–7.70 (m, 1H, H–Ar), 7.76–7.79 (m, 2H, H–Ar), 7.88 (dd, 1H, J = 1.6 and 8.0 Hz, H–Ar), 10.98 (s, 1H, NH), 13.13 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 60.70, 95.56, 106.73, 112.17, 115.50, 116.47, 116.69, 116.81, 124.11, 124.90, 125.11, 124.15, 125.55, 126.69, 127.72, 130.36, 132.16, 132.32, 133.69, 142.09, 145.84, 148.04, 152.53, 160.09, 163.61, 173.09 Anal. calcd for C₂₆H₁₅ClFN₃O₄ (487.86): C, 64.01; H, 3.10; N, 8.61. Found: C, 64.19; H, 3.24; N, 8.77.

4.1.1.4. 5-Chloro-1-ethyl-3-(5-(4-fluorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one 4e

Yellow powder, m.p. 297–298 °C, yield (0.37 g, 72%). ¹H NMR (400 MHz, DMSO-d₆): δ = 1.13 (t, 3H, J = 7.2 Hz, CH₃), 3.71 (q, 2H, J = 7.2 Hz, CH₂), 6.32 (s, 1H, CH-indole), 7.22–7.26 (m, 2H, H–Ar), 7.36–7.45 (m, 4H, H–Ar), 7.48–7.51 (m, 2H, H–Ar), 7.67–7.75 (m, 3H, H–Ar), 7.88 (dd, 1H, J = 1.2 and 7.6 Hz, H–Ar), 13.03 (s, 1H, OH). Anal. calcd for C₂₈H₁₉ClFN₃O₄ (515.91): C, 65.18; H, 3.71; N, 8.14. Found: C, 65.36; H, 3.90; N, 8.32.

4.1.1.5. 1-Benzyl-5-chloro-3-(5-(4-fluorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl) indolin-2-one 4f

Violet crystals, m.p. 223–225 °C, yield (0.45 g, 78%). ¹H NMR (400 MHz, DMSO-d₆): δ = 4.90 (d, 1H, J = 16.0 Hz, CH-benzyl), 5.02 (d, 1H, J = 16.0 Hz, CH-benzyl), 6.47 (s, 1H, CH-indole), 7.01 (d, 1H, J = 8.8 Hz, H–Ar), 7.26 (s, 1H, H–Ar). 7.29–7.45 (m, 10H, H–Ar). 7.53 (brs, IH, H–Ar) 7.66–7.70 (m, 1H, H–Ar), 7.80–7.83 (m, 2H, H–Ar), 7.87 (dd, 1H, J = 1.6 and 8.0 Hz, H–Ar), 12.97 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 43.49, 60.17, 95.36, 106.89, 111.87, 115.47, 116.53, 116.74, 116.81, 124.03, 124.98, 125.07, 125.10, 125.49, 127.06, 127.45, 127.65, 128.06, 129.17, 130.33, 132.22, 132.30, 133.71, 135.81, 142.49, 146.10, 148.35, 152.54, 160.09, 162.21, 163.64, 164.66, 171.75. Anal. calcd for C₃₃H₂₁ClFN₃O₄ (577.12): C, 68.57; H, 3.66; N, 7.27. Found: C, 68.75; H, 3.83; N, 7.44.

4.1.1.6. 5-Bromo-3-(5-(4-fluorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl) indolin-2-one 4g

White powder, m.p. 295–296 °C, yield (0.36 g, 68%). ¹H NMR (400 MHz, DMSO-d₆): δ = 6.23 (s, 1H, CH-indole), 6.91 (d, 1H, J = 8.4 Hz, H–Ar), 7.21 (s, 1H, H–Ar), 7.35–7.43 (m, 4H, H–Ar), 7.52 (s, 2H, H–Ar), 7.65–7.69 (m, 1H, H–Ar), 7.77(brt, 2H, H–Ar), 7.87 (d, 1H, J = 7.6 Hz, H–Ar), 10.98 (s, 1H, NH), 13.14 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 60.61, 95.41, 106.72, 112.67, 114.30, 115.50, 116.46, 116.68, 116.81, 124.12, 124.90, 125.15, 128.09, 128.22, 132.16, 132.25, 133.21, 133.69, 142.51, 145.85, 148.05, 152.53, 162.16, 163.62, 172.97. Anal. calcd for C₂₆H₁₅BrFN₃O₄ (532.31): C, 58.66; H, 2.84; N, 7.89. Found: C, 58.85; H, 3.01; N, 8.09.

4.1.1.7. 5-Bromo-1-ethyl-3-(5-(4-fluorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl) indolin-2-one 4h

Yellow crystals, m.p. 287–289 °C, yield (0.4 g, 71%). ¹H NMR (400 MHz, DMSO-d₆): δ = 1.03 (t, 3H, J = 6.0 Hz, CH₃), 3.66 (q, 2H, J = 7.2 Hz, CH₂), 6.29 (s, 1H, CH-indole), 7.09 (s, 1H, H–Ar), 7.16–7.25 (m, 4H, H–Ar), 7.39–7.46 (m, 2H, H–Ar), 7.69 (d, 1H, J = 2.0 Hz, CH₂), 7.80–7.84 (m, 2H, H–Ar), 7.94–7.98 (m, 2H, H–Ar), 13.31 (s, 1H, OH). Anal. calcd for C₂₈H₁₉BrFN₃O₄ (559.05): C, 60.01; H, 3.42; N, 7.50. Found: C, 60.12; H, 3.51; N, 7.67.

4.1.1.8. 1-Benzyl-5-bromo-3-(5-(4-fluorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one 4i

Yellow powder, m.p. 247–249 °C, yield (0.41 g, 66%). ¹H NMR (400 MHz, DMSO-d₆): δ = 4.90 (d, 1H, J = 16.0 Hz, CH_a-benzyl), 5.02 (d, 1H, J = 16.00 Hz, CH_b-benzyl), 6.47 (s, 1H, CH-indole), 6.96 (d, 1H, J = 8.4 Hz, H–Ar), 7.26 (s, 1H, H–Ar), 7.29–7.45 (m, 9H, H–Ar), 7.54 (dd, 1H, J = 1.6 and 8.4 Hz, H–Ar), 7.63 (brs, 1H, H–Ar), 7.68–7.72 (m, 1H, H–Ar), 7.80–7.83 (m, 2H, H–Ar), 7.88 (dd, 1H, J = 1.8 and 8.0 Hz, H–Ar), 13.00 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 43.45, 60.09, 95.37, 106.87, 112.37, 115.31, 115.49, 116.53, 116.75, 116.84, 124.06, 125.01, 125.10, 127.39, 127.43, 128.07, 128.15, 129.18, 132.23, 132.32, 133.20, 133.75, 135.80, 142.92, 148.34, 152.56, 160.10, 162.21, 163.66, 164.66, 171.65. Anal. calcd for C₃₃H₂₁BrFN₃O₄ (621.06): C, 63.68; H, 3.40; N, 6.75. Found: C, 63.85; H, 3.58; N, 6.86.

4.1.1.9. 3-(5-(4-Fluorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-5 (trifluoromethoxy) indolin-2-one 4j

Yellow crystals, m.p. 293–294 °C, yield (0.4 g, 74%). ¹H NMR (400 MHz, DMSO-d₆): δ = 6.32 (s, 1H, CH-indole), 7.02 (d, 1H, J = 8.4 Hz, H–Ar), 7.22 (s, 1H, H–Ar), 7.35–7.44 (m, 6H, H–Ar), 7.66–7.70 (m, 1H, H–Ar), 7.76 (brs, 2H, H–Ar), 7.87 (dd, 1H, J = 0.8 and 7.6 Hz, H–Ar), 11.02 (s, 1H, NH), 13.11 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 60.96, 95.40, 106.88, 111.62, 115.49, 116.39, 116.61, 116.82, 119.56, 121.85, 123.78, 124.07, 124.92, 125.17, 127.30, 132.13, 132.22, 133.70, 142.38, 143.85, 147.94, 152.53, 160.09, 162.14, 163.60, 173.50. Anal. calcd for C₂₇H₁₅F₄N₃O₅ (537.41): C, 60.34; H, 2.81; N, 7.82. Found: C, 60.51; H, 2.87; N, 7.99.

4.1.1.10. 3-(5-(4-Chlorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one 5a

Rose crystals, m.p. 285–287 °C, yield (0.34 g, 72%). ¹H NMR (400 MHz, DMSO-d₆): δ = 6.21 (s, 1H, CH-indole), 6.92 (d, 1H, J = 7.6 Hz, H–Ar), 6.95(td, 1H, J = 0.8 and 6.8 Hz, H–Ar), 7.17 (d, 1H, J = 7.6 Hz, H–Ar), 7.20 (s, 1H, H–Ar), 7.28–7.39 (m, 3H, H–Ar), 7.58–7.65 (m, 3H, H–Ar), 7.70 (d, 2H, J = 8.0 Hz, H–Ar), 7.81 (dd, 1H, J = 1.6 and 8.0 Hz, H–Ar), 10.84 (s, 1H, NH), 13.11 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 60.76, 94.81, 106.75, 110.36, 115.02, 116.30, 122.56, 123.90, 124.73, 125.08, 125.56, 127.41, 129.47, 130.35, 131.33, 133.50, 134.94, 142.90, 145.28, 147.75, 152.27, 159.92, 163.41, 173.07. Anal. calcd for C₂₆H₁₆ClN₃O₄ (469.87): C, 66.46; H, 3.43; N, 8.94. Found: C, 66.62; H, 3.55; N, 9.08.

4.1.1.11. 3-(5-(4-Chlorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-1-ethylindolin-2-one 5b

Off white powder, m.p. 245–246 °C, yield (0.37 g, 74%). ¹H NMR (400 MHz, DMSO-d₆): δ = 1.15 (t, 3H, J = 7.2 Hz, CH₃), 3.72 (q, 2H, J = 8.0 Hz, CH₂), 6.34 (s, 1H, CH-indole), 7.06–7.09 (m, 1H, H–Ar), 7.19–7.29 (m, 5H, H–Ar), 7.35–7.45 (m, 4H, H–Ar), 7.60–7.71 (m, 2H, H–Ar), 7.85 (d, 1H, J = 7.2 Hz, H–Ar), 13.08 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 12.61, 35.15, 60.68, 95.49, 107.06, 110.07, 115.31, 116.18, 123.12, 124.07, 124.90, 125.10, 125.21, 127.51, 129.52, 131.54, 133.68, 135.12, 137.08, 143.40, 147.94, 152.51, 160.08, 163.60, 171.23. Anal. calcd for C₂₈H₂₀ClN₃O₄ (497.92): C, 67.54; H, 4.05; N, 8.44. Found: C, 67.72; H, 4.19; N, 8.55.

4.1.1.12. 1-Benzyl-3-(5-(4-chlorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one 5c

Yellow crystals, m.p. 235–237 °C, yield (0.41 g, 73%). ¹H NMR (400 MHz, DMSO-d₆): δ = 4.87 (d, 1H, J = 16.0 Hz, CH_a-benzyl), 4.99 (d, 1H, J = 15.6 Hz, CH_b-benzyl), 6.45 (s, 1H, CH-indole), 6.96 (d, 1H, J = 8.0 Hz, H–Ar), 7.00 (t, 1H, J = 7.6 Hz, H–Ar), 7.25 (s, 1H, H–Ar). 7.26–7.40 (m, 9H, H–Ar). 7.60–7.66 (m, 3H, H–Ar), 7.73–7.75 (m, 2H, H–Ar), 7.82 (dd, 1H, J = 1.6 and 8.0 Hz, H–Ar), 13.00 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 43.43, 60.45, 95.34, 106.89, 110.45, 115.48, 116.82, 123.50, 124.01, 124.99, 125.07, 125.12, 127.48, 127.52, 127.99, 129.14, 129.70, 130.50, 131.55, 133.71, 135.19, 136.12, 143.49, 145.72, 148.22, 152.54, 160.09, 163.61, 171.96. Anal. calcd for C₃₃H₂₂ClN₃O₄ (559.12): C, 70.78; H, 3.96; N, 7.50. Found: C, 70.91; H, 4.12; N, 7.66.

4.1.1.13. 5-Chloro-3-(5-(4-chlorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl) indolin-2-one 5d

Pale yellow powder, m.p. > 300 °C, yield (0.34 g, 67%). ¹H NMR (400 MHz, DMSO-d₆): δ = 6.26 (s, 1H, CH-indole), 6.97 (d, 1H, J = 8.0 Hz, H–Ar), 7.24 (s, 1H, H–Ar), 7.36–7.46 (m, 4H, H–Ar), 7.63–7.71 (m, 3H, H–Ar), 7.74 (d, 2H, J = 8.4 Hz, H–Ar), 7.88–7.90 (m, 1H, H–Ar), 10.99 (s, 1H, NH), 13.11 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 60.75, 95.40, 106.79, 112.19, 115.49, 116.82, 124.13, 124.92, 125.59, 126.70, 127.53, 127.68, 129.60, 130.38, 131.62, 133.72, 135.14, 142.10, 145.69, 148.13, 152.54, 160.09, 163.63, 173.06. Anal. calcd for C₂₆H₁₅Cl₂N₃O₄ (504.32): C, 61.92; H, 3.00; N, 8.33. Found: C, 62.09; H, 3.16; N, 8.45.

4.1.1.14. 5-Chloro-3-(5-(4-chlorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-1-ethylindolin-2-one 5e

Off white powder, m.p. 259–260 °C, yield (0.37 g, 69%). ¹H NMR (400 MHz, DMSO-d₆): δ = 1.13 (t, 3H, J = 6.8 Hz, CH₃), 3.72 (q, 2H, J = 7.2 Hz, CH₂), 6.34 (s, 1H, CH-indole), 7.25 (s, 1H, H–Ar). 7.36–7.40 (m, 1H, H–Ar), 7.42 (d, 1H, J = 8.0 Hz, H–Ar), 7.49–7.52 (m, 2H, H–Ar), 7.62–7.77 (m, 6H, H–Ar), 7.87 (dd, 1H, J = 1.6 and 8.0 Hz, H–Ar), 13.00 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 12.68, 35.20, 60.33, 95.39, 106.99, 111.36, 115.48, 116.82, 124.14, 124.91, 125.49, 127.15, 127.32, 127.48, 129.58, 130.41, 131.66, 133.73, 135.16, 142.45, 145.59, 148.17, 152.54, 160.10, 163.64, 171.00. Anal. calcd for C₂₈H₁₉Cl₂N₃O₄ (531.07): C, 63.17; H, 3.60; N, 7.89. Found: C, 63.26; H, 3.77; N, 8.05.

4.1.1.15. 1-Benzyl-5-chloro-3-(5-(4-chlorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one 5f

Violet crystals, m.p. 207–209 °C, yield (0.39 g, 66%). ¹H NMR (400 MHz, DMSO-d₆): δ = 4.91 (d, 1H, J = 15.6 Hz, CH-benzyl), 5.03 (d, 1H, J = 15.6 Hz, CH-benzyl), 6.49 (s, 1H, CH-indole), 7.02 (d, 1H, J = 8.8 Hz, H–Ar), 7.28 (s, 1H, H–Ar). 7.30–7.44 (m, 8H, H–Ar). 7.53(brs, 1H, H–Ar) 7.65–7.80 (m, 5H, H–Ar), 7.87 (dd, 1H, J = 1.6 and 8.0 Hz, H–Ar), 13.10 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 43.49, 60.21, 95.34, 106.95, 111.90, 115.49, 116.83, 124.06, 124.99, 125.54, 127.02, 127.38, 127.44, 127.65, 128.07, 129.13, 129.18, 129.66, 130.35, 131.68, 133.75, 135.22, 135.82, 142.50, 145.97, 148.44, 152.57, 160.09, 163.68, 171.72. Anal. calcd for C₃₃H₂₁Cl₂N₃O₄ (593.09): C, 66.68; H, 3.56; N, 7.07. Found: C, 66.79; H, 3.69; N, 7.22.

4.1.1.16. 5-Bromo-3-(5-(4-chlorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one 5g

Buff powder, m.p. 296–297 °C, yield (0.35 g, 64%). ¹H NMR (400 MHz, DMSO-d₆): δ = 6.26 (s, 1H, CH-indole), 6.92 (d, 1H, J = 8.8 Hz, H–Ar), 7.24 (s, 1H, H–Ar), 7.35 (t, 1H, J = 7.6 Hz, H–Ar), 7.41 (d, 1H, J = 8.4 Hz, H–Ar), 7.53 (s, 2H, H–Ar), 7.63–7.69 (m, 3H, H–Ar), 7.74 (d, 2H, J = 7.6 Hz, H–Ar), 7.87 (d, 1H, J = 7.6 Hz, H–Ar), 10.99 (s, 1H, NH), 13.09 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 60.65, 95.39, 106.79, 112.68, 114.32, 115.48, 116.80, 124.12, 124.89, 127.53, 128.04, 128.26, 129.04, 129.59, 131.62, 133.22, 133.70, 135.14, 142.51, 145.69, 148.13, 152.53, 160.07, 163.61, 172.93. Anal. calcd for C₂₆H₁₅BrClN₃O₄ (548.77): C, 56.90; H, 2.76; N, 7.66. Found: C, 57.10; H, 2.92; N, 7.77.

4.1.1.17. 5-Bromo-3-(5-(4-chlorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-1-ethylindolin-2-one 5h

Brown powder, m.p. > 300 °C, yield (0.45 g, 78%). ¹H NMR (400 MHz, DMSO-d₆): δ = 1.12 (t, 3H, J = 6.8 Hz, CH₃), 3.68 (q, 2H, J = 6.8 Hz, CH₂), 6.31 (s, 1H, CH-indole), 7.16–7.87 (m, 12H, H–Ar), 12.99 (s, 1H, OH). Anal. calcd for C₂₈H₁₉BrClN₃O₄ (575.02): C, 58.30; H, 3.32; N, 7.28. Found: C, 58.48; H, 3.51; N, 7.45.

4.1.1.18. 1-Benzyl-5-bromo-3-(5-(4-chlorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one 5i

Buff powder, m.p. 228–229 °C, yield (0.52 g, 82%). ¹H NMR (400 MHz, DMSO-d₆): δ = 4.90 (d, 1H, J = 15.6 Hz, CH_a-benzyl), 5.02 (d, 1H, J = 15.6 Hz, CH_b-benzyl), 6.49 (s, 1H, CH-indole), 6.97 (d, 1H, J = 8.8 Hz, H–Ar), 7.29 (s, 1H, H–Ar), 7.34–7.45 (m, 7H, H–Ar), 7.55 (d, 1H, J = 8.4 Hz, H–Ar), 7.64 (brs, 1H, H–Ar), 7.67–7.73 (m, 3H, H–Ar), 7.79–7.81 (m, 2H, H–Ar), 7.88 (d, 1H, J = 6.8 Hz, H–Ar), 12.93 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 43.46, 60.11, 95.35, 106.88, 112.39, 115.32, 115.47, 116.83, 123.97, 124.06, 125.00, 125.08, 127.34, 127.43, 127.85, 128.06, 128.20, 129.18, 131.77, 131.91, 132.58, 133.21, 133.76, 135.80, 142.93, 146.06, 148.46, 152.56, 160.08, 163.65, 171.61. Anal. calcd for C₃₃H₂₁BrClN₃O₄ (637.04): C, 62.04; H, 3.31; N, 6.58. Found: C, 62.19; H, 3.49; N, 6.77.

4.1.1.19. 3-(5-(4-Chlorophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-5-(trifluoromethoxy)indolin-2-one 5j

Yellow powder, m.p. 299–300 °C, yield (0.34 g, 61%). ¹H NMR (400 MHz, DMSO-d₆): δ = 6.34 (s, 1H, CH-indole), 7.03 (d, 1H, J = 8.4 Hz, H–Ar), 7.24 (s, 1H, H–Ar), 7.36–7.44 (m, 4H, H–Ar), 7.61–7.70 (m, 3H, H–Ar), 7.74–7.83 (m, 2H, H–Ar), 7.86 (dd, 1H, J = 1.2 and 8.0 Hz, H–Ar), 11.04 (s, 1H, NH), 13.00 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 60.98, 95.38, 106.94, 111.65, 115.47, 116.82, 119.32, 119.60, 121.85, 123.80, 124.08, 124.92, 127.25, 127.56, 129.53, 131.59, 133.71, 135.11, 142.39, 143.86, 145.61, 148.02, 152.53, 160.09, 163.60, 173.46. Anal. calcd for C₂₇H₁₅ClF₃N₃O₅(553.87): C, 58.55; H, 2.73; N, 7.59. Found: C, 58.68; H, 2.91; N, 7.78.

4.1.1.20. 3-(5-(4-Bromophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one 6a

Rose powder, m.p. 291–292 °C, yield (0.42 g, 81%). ¹H NMR (400 MHz, DMSO-d₆): δ = 6.25 (s, 1H, CH-indole), 6.96 (d, 1H, J = 8.0 Hz, H–Ar), 6.99 (td, 1H, J = 0.4 and 3.6 Hz, H–Ar), 7.21 (d, 1H, J = 7.6 Hz, H–Ar), 7.24 (s, 1H, H–Ar), 7.32–7.39 (m, 2H, H–Ar), 7.41 (d, 1H, J = 8.0 Hz, H–Ar), 7.65–7.69 (m, 3H, H–Ar), 7.76 (d, 2H, J = 8.4 Hz, H–Ar), 7.86 (dd, 1H, J = 1.6 and 8.0 Hz, H–Ar), 10.87 (s, 1H, NH), 13.11 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 60.92, 95.37, 106.69, 110.73, 115.49, 116.81, 122.72, 123.85, 124.07, 124.90, 125.21, 125.73, 127.93, 130.51, 131.71, 132.56, 133.67, 143.03, 145.52, 147.93, 152.52, 160.03, 163.57, 173.29. Anal. calcd for C₂₆H₁₆BrN₃O₄ (514.32): C, 60.72; H, 3.14; N, 8.17. Found: C, 60.92; H, 3.33; N, 8.32.

4.1.1.21. 3-(5-(4-Bromophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-1-ethylindolin-2-one 6b

Yellow powder, m.p. 273–275 °C, yield (0.41 g, 76%). ¹H NMR (400 MHz, DMSO-d₆): δ = 1.11 (t, 3H, J = 6.8 Hz, CH₃), 3.68 (q, 2H, J = 7.2 Hz, CH₂), 6.30 (s, 1H, CH-indole), 7.02–7.06 (m, 1H, H–Ar), 7.15–7.24 (m, 3H, H–Ar), 7.30–7.41 (m, 3H, H–Ar), 7.58–7.64 (m, 3H, H–Ar), 7.69 (d, 2H, J = 8.0 Hz, H–Ar), 7.80 (dd, 1H, J = 1.2 and 8.0 Hz, H–Ar), 12.97 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 12.74, 35.00, 60.54, 95.33, 106.86, 109.83, 115.42, 116.75, 123.20, 123.82, 124.02, 124.85, 125.06, 125.17, 127.82, 129.61, 130.58, 131.70, 132.47, 133.63, 143.36, 145.34, 147.93, 152.46, 160.04, 163.51, 171.17. Anal. calcd for C₂₈H₂₀BrN₃O₄ (542.38): C, 62.00; H, 3.72; N, 7.75. Found: C, 62.12; H, 3.90; N, 7.89.

4.1.1.22. 1-Benzyl-3-(5-(4-bromophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one 6c

Off white powder, m.p. 239–241 °C, yield (0.47 g, 78%). ¹H NMR (400 MHz, DMSO-d₆): δ = 4.91 (d, 1H, J = 16.0 Hz, CH_a-benzyl), 5.02 (d, 1H, J = 16.0 Hz, CH_b-benzyl), 6.49 (s, 1H, CH-indole), 7.00 (d, 1H, J = 8.0 Hz, H–Ar), 7.04 (t, 1H, J = 6.8 Hz, H–Ar), 7.29 (s, 1H, H–Ar). 7.30–7.45 (m, 9H, H–Ar). 7.67–7.72 (m, 3H, H–Ar), 7.78–7.80 (m, 2H, H–Ar), 7.86 (dd, 1H, J = 1.2 and 8.0 Hz, H–Ar), 12.97 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 43.42, 60.44, 95.33, 106.84, 110.46, 115.51, 116.83, 121.65, 123.49, 123.92, 124.02, 124.99, 125.09, 125.12, 127.48, 127.89, 127.99, 128.86, 129.14, 130.49, 131.77, 132.63, 133.72, 136.13, 140.34, 143.50, 145.79, 148.25, 152.56, 160.10, 163.63, 171.95. Anal. calcd for C₃₃H₂₂BrN₃O₄ (603.07): C, 65.57; H, 3.67; N, 6.95. Found: C, 65.70; H, 3.84; N, 7.12.

4.1.1.23. 3-(5-(4-Bromophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-5-chloroindolin-2-one 6d

White powder, m.p. > 300 °C, yield (0.42 g, 77%). ¹H NMR (400 MHz, DMSO-d₆): δ = 6.26 (s, 1H, CH-indole), 6.92 (d, 1H, J = 8.8 Hz, H–Ar), 7.24 (s, 1H, H–Ar), 7.36–7.44 (m, 3H, H–Ar), 7.52–7.55 (m, 2H, H–Ar), 7.66–7.69 (m, 2H, H–Ar), 7.77 (d, 2H, J = 8.4 Hz, H–Ar), 7.87–7.89 (m, 1H, H–Ar), 11.00 (s, 1H, NH), 13.09 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 60.69, 95.26, 106.46, 114.34, 115.38, 116.52, 123.88, 124.12, 124.90, 127.89, 128.03, 128.24, 131.83, 132.17, 132.52, 133.18, 133.71, 142.54, 148.15, 152.44, 160.01, 163.58, 173.00. Anal. calcd for C₂₆H₁₅BrClN₃O₄ (548.77): C, 56.90; H, 2.76; N, 7.66. Found: C, 57.01; H, 2.92; N, 7.79.

4.1.1.24. 3-(5-(4-Bromophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-5-chloro-1-ethylindolin-2-one 6e

Off-white powder, m.p. 275–277 °C, yield (0.44 g, 76%). ¹H NMR (400 MHz, DMSO-d₆): δ = 1.10 (t, 3H, J = 7.2 Hz, CH₃), 3.68 (q, 2H, J = 6.8 Hz, CH₂), 6.30 (s, 1H, CH-indole), 7.20–7.22 (m, 2H, H–Ar). 7.31–7.35 (m, 1H, H–Ar), 7.37–7.39 (m, 1H, H–Ar), 7.44–7.48 (m, 2H, H–Ar), 7.57–7.66 (m, 3H, H–Ar), 7.72 (d, 2H, J = 8.8 Hz, H–Ar), 7.82 (dd, 1H, J = 1.2 and 8.0 Hz, H–Ar), 12.97 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 12.65, 35.17, 60.27, 69.51, 95.35, 106.89, 111.32, 115.41, 116.77, 123.88, 124.08, 124.87, 125.45, 127.09, 127.30, 127.79, 130.38, 131.82, 132.46, 133.68, 142.40, 145.61, 148.15, 152.48, 160.04, 163.56, 170.94. Anal. calcd for C₂₈H₁₉BrClN₃O₄ (576.82): C, 58.30; H, 3.32; N, 7.28. Found: C, 58.48; H, 3.50; N, 7.44.

4.1.1.25. 1-Benzyl-3-(5-(4-bromophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-5-chloroindolin-2-one 6f

Violet crystals, m.p. 222–223 °C, yield (0.42 g, 66%). ¹H NMR (400 MHz, DMSO-d₆): δ = 4.91 (d, 1H, J = 16.0 Hz, CH_a-benzyl), 5.03 (d, 1H, J = 16.00 Hz, CH_b-benzyl), 6.49 (s, 1H, CH-indole), 7.02 (d, 1H, J = 8.4 Hz, H–Ar), 7.29 (s, 1H, H–Ar), 7.34–7.45 (m, 8H, H–Ar), 7.53–7.54 (m, 1H, H–Ar), 7.67–7.74 (m, 3H, H–Ar), 7.79–7.81 (m, 2H, H–Ar), 7.88 (dd, 1H, J = 1.2 and 7.6 Hz, H–Ar), 12.96 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 43.49, 60.21, 95.35, 106.89, 111.90, 115.47, 116.83, 123.97, 124.06, 125.00, 125.54, 127.02, 127.44, 127.64, 127.85, 128.06, 129.18, 129.68, 130.35, 131.90, 132.59, 133.75, 135.82, 142.50, 146.04, 148.46, 152.56, 160.08, 163.65, 171.71. Anal. calcd for C₃₃H₂₁BrClN₃O₄ (637.04): C, 62.04; H, 3.31; N, 6.58. Found: C, 62.22; H, 3.51; N, 6.77.

4.1.1.26. 5-Bromo-3-(5-(4-bromophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl) indolin-2-one 6g

Off-white powder, m.p. 298–299 °C, yield (0.36 g, 60%). ¹H NMR (400 MHz, DMSO-d₆): δ = 6.27 (s, 1H, CH-indole), 6.97 (d, 1H, J = 8.4 Hz, H–Ar), 7.25 (s, 1H, H–Ar), 7.36–7.44 (m, 4H, H–Ar), 7.66–7.70 (m, 3H, H–Ar), 7.77 (d, 2H, J = 8.4 Hz, H–Ar), 7.87 (dd, 1H, J = 1.2 and 8.0 Hz, H–Ar), 10.99 (s, 1H, NH), 13.09 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 60.74, 95.39, 106.75, 112.19, 115.48, 116.81, 123.89, 124.12, 124.91, 125.59, 126.70, 127.67, 127.89, 130.38, 131.83, 132.53, 133.72, 142.10, 145.75, 148.15, 152.54, 160.08, 163.62, 173.05. Anal. calcd. For C₂₆H₁₅Br₂N₃O₄ (593.22): C, 52.64; H, 2.55; N, 7.08. Found: C, 52.75; H, 2.69; N, 7.22.

4.1.1.27. 5-Bromo-3-(5-(4-bromophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-1-ethylindolin-2-one 6h

Buff powder, m.p. 276–278 °C, yield (0.41 g, 66%). ¹H NMR (400 MHz, DMSO-d₆): δ = 1.14 (t, 3H, J = 7.2 Hz, CH₃), 3.66 (q, 2H, J = 4.8 Hz, CH₂), 6.34 (s, 1H, CH-indole), 7.16–7.29 (m, 1H, H–Ar). 7.39–7.44 (m, 3H, H–Ar), 7.54–7.59 (m, 2H, H–Ar), 7.62–7.70 (m, 3H, H–Ar), 7.87 (d, 2H, J = 8.4 Hz, H–Ar), 7.98 (dd, 1H, J = 1.2 and 7.6 Hz, H–Ar), 12.97 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 12.60, 34.83, 95.17, 102.50, 116.45, 116.75, 120.77, 124.13, 124.70, 125.98, 127.13, 127.48, 127.92, 128.04, 129.52, 129.57, 129.64, 131.65, 133.26, 133.89, 140.20, 142.69, 148.02, 152.58, 156.48, 160.51, 164.54, 171.13. Anal. calcd for C₂₈H₁₉Br₂N₃O₄ (618.97): C, 54.13; H, 3.08; N, 6.76. Found: C, 54.29; H, 3.22; N, 6.92.

4.1.1.28. 1-Benzyl-5-bromo-3-(5-(4-bromophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)indolin-2-one 6i

Violet crystals, m.p. 218–220 °C, yield (0.44 g, 64%). ¹H NMR (400 MHz, DMSO-d₆): δ = 4.73 (d, 1H, J = 16.0 Hz, CH-benzyl), 4.86 (d, 1H, J = 16.0 Hz, CH-benzyl), 6.32 (s, 1H, CH-indole), 6.80 (d, 1H, J = 8.4 Hz, H–Ar), 7.12 (s, 1H, H–Ar), 7.14–7.27 (m, 7H, H–Ar), 7.38 (d, 1H, J = 8.4 Hz, H–Ar), 7.48–7.54 (m, 4H, H–Ar), 7.62–7.64 (m, 2H, H–Ar), 7.71 (d, 1H, J = 6.8 Hz, H–Ar), 12.81 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 43.46, 60.11, 95.35, 106.93, 112.38, 115.32, 115.46, 116.82, 124.05, 124.99, 127.34, 127.43, 127.49, 128.06, 128.19, 129.18, 129.65, 131.55, 131.68, 133.21, 133.74, 135.22, 135.78, 142.92, 145.98, 148.44, 152.55, 160.08, 163.64, 171.61, Anal. calcd for C₃₃H₂₁Br₂N₃O₄ (683.34): C, 58.00; H, 3.10; N, 6.15. Found: C, 58.17; H, 3.25; N, 6.33.

4.1.1.29. 3-(5-(4-Bromophenyl)-3-(4-hydroxy-2-oxo-2H-chromen-3-yl)-1H-pyrazol-1-yl)-5-(trifluoromethoxy)indolin-2-one 6j

Yellow crystals, m.p. 298–300 °C, yield (0.46 g, 77%). ¹H NMR (400 MHz, DMSO-d₆): δ = 7.06 (d, 1H, J = 8.4 Hz, H–Ar), 7.25–7.28 (m, 3H, H–Ar), 7.46–7.49 (m, 1H, H–Ar), 7.59–7.69 (m, 4H, H–Ar), 7.78–7.82 (m, 3H, H–Ar), 7.84 (dd, 1H, J = 1.6 and 8.0 Hz, H–Ar), 10.89 (s, 1H, NH), 13.03 (s, 1H, OH). ¹³C NMR (101 MHz, DMSO-d₆): δ = 69.95, 89.80, 92.82, 112.45, 116.68, 119.29, 120.03, 120.69, 121.83, 124.09, 125.86, 125.98, 126.14, 129.63, 130.90, 132.44, 132.59, 134.40, 141.34, 144.23, 153.38, 156.54, 163.38, 174.13. Anal. calcd for C₂₇H₁₅BrF₃N₃O₅ (598.32): C, 54.20; H, 2.53; N, 7.02. Found: C, 54.39; H, 2.71; N, 7.17.

4.2. Biology

4.2.1. Cell lines and culture conditions

The human melanoma cell line (A375) and normal human skin cell fibroblast cells (HSF) were obtained from Nawah Scientific Inc. (Mokattam, Cairo, Egypt) and cultured in DMEM (Dulbecco's modified Eagle's medium), Gibco, USA, supplemented with fetal bovine serum (FBS) at a concentration of 10% and 100 U mL⁻¹ of penicillin and streptomycin (PS). The cells were incubated at 37 °C in a humidified environment containing 5% CO₂.⁶¹

4.2.2. Assessment of cytotoxicity by SRB assay

Cells were seeded in 96-well plates as aliquots of 100 μL cell suspension and incubated in complete medium for 24 h. Afterward, the cells were treated with all the synthesized derivatives 4a–j, 5a–j, and 6a–j (from 0.01 μM to 100 μM) for 72 h. Then, the cells were fixed for one hour at 4 °C with 150 μL of 10% trichloroacetic acid (TCA). After washing the cells five times with distilled water, 70 μL of sulforhodamine (SRB) solution (0.4% w/v) was added and incubated for 10 min at room temperature in the dark. The cells were washed with 1% acetic acid three times and allowed to air-dry. Then, 150 μL of Tris pH 10.5 (10 mM) was added, and the absorbance was measured at 540 nm. The dose–response curves of the derivatives were analyzed by applying the Emax model using the Sigma plot software. The half-maximum inhibitory concentrations (IC₅₀) values are reported as mean ± SD. The selectivity index (SI), which denotes the cytotoxic selectivity for the proposed treatments, was determined as SI IC₅₀ of normal cells/IC₅₀ of tumor cells.⁶²

4.2.3. BRAF^V600E and VEGFR-2 inhibition assay

Aliquots of 100 μL cell suspension (5 × 10³ cells) were seeded in 96-well plates and incubated in complete media for 24 h. The cells were treated with another aliquot of 100 μL media containing 10 μM of the most potent derivative 4j. After drug exposure for 24 h, the media were collected for the measurement of the concentrations of BRAF^V600E and VEGFR-2 using the Human BRAF (B-Raf Proto Oncogene Serine/Threonine Protein Kinase) ELISA Kit (Elabscience) and Human VEGFR-2/KDR (vascular endothelial growth factor receptor 2) ELISA Kit (Elabscience), respectively, according to the instructions. The absorbance was measured at 450 nm using an Infinite F50 microplate reader (TECAN, Switzerland). Data are presented as percentage inhibition. The experiment was carried out at various concentrations (0.1, 0.5, 1, 5, and 10 μM) to determine the IC₅₀ of BRAF and VEGFR2 for derivative 4j and sorafenib.⁶³

4.2.4. Cell cycle analysis

Melanoma cells (A375) were seeded with 1 × 10⁵ cells per well in a 6-well plate, incubated for 24 h, and treated with derivative 4j and sorafenib for 48 h. Then, adherent and floating cells were collected from the cultures, washed twice with PBS (phosphate buffered saline), fixed in ice-cold 60% ethanol at 40 °C, and re-washed in PBS. Subsequently, the cells were resuspended in 500 μL propidium iodide (PI) with RNase staining buffer, BD (Franklin Lakes, NJ, USA), and incubated for 30 min. Lastly, FACS analyses were performed utilizing an ACEA Novocyte™ flow cytometer (ACEA Biosciences Inc., San Diego, USA). For every sample, the data from 12 000 cells were collected and the distribution of cell cycle phases was analyzed using the ACEA Novo Express™ software (ACEA Biosciences Inc., San Diego, USA).⁶⁴

4.2.5. Apoptosis assay

Apoptosis detection was done through a flow cytometer using the Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences, San Diego, USA) following the manufacturer's instructions to differentiate among living, apoptotic, and necrotic cells. With a seeding density of 1 × 10⁵ cells per well on a 6-well plate, A375 cells were treated with the IC₅₀ of derivative 4j and sorafenib for 48 h. Adherent and floating cells were collected from the cultures, resuspended in 0.5 mL of binding buffer, and then staining solution. Annexin V-FITC (5 μL) and PI (5 μL) were added for 15 min at room temperature in the dark. Finally, the cells were subjected within one hour of staining to FACS analysis using an ACEA Novocyte™ flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA).⁶⁵

4.2.6. Scratch wound healing assay (cell migration assay)

A375 cells were grown in 6-well culture plates at a seeding density of 1 × 10⁵/well to achieve the confluent monolayer. Then, the cell monolayer was gently scratched with a sterile 200 μL pipette tip to make one straight cell-free line. After washing with PBS, the cells were treated with 4j and sorafenib. Scratch healing was recorded at 0, 24, 48, and 72 h. The scratch images were captured at a magnification of ×100 using an inverted microscope (Olympus, Japan). The horizontal distance of the wound gap was measured using Image J (version 1.53C, NIH, US) and the percentage of wound closure calculated according to the following equation: %Wound closure = 100 − [(W_t/W₀) × 100], where W_t is the wound width at time t and W₀ is its initial width.⁶⁶

4.2.7. Data analysis

All data are presented as mean ± SD (n = 3). Analysis of variance (ANOVA) with Tukey's post-hoc test (P < 0.05) was applied using GraphPad Prism Software version 6.

4.3. Molecular modeling

The molecular docking studies were conducted using the Molecular Operating Environment (MOE 2022.02)⁶⁷ package license purchased from Chemical Computing Group Inc. and a Sherbrooke St, Montreal, QC, Canada. Triangle matcher was used as a placement method. The London ΔG scoring method was applied to estimate the free energy of binding (kcal mol⁻¹). Validation of docking was done by computing (GBVI/WSA) dG, which is a force field-based scoring function to estimate the free energy of binding (kcal mol⁻¹). Redocking of the co-crystalized ligand SB-5902885 was done as a type of validation as well. The root mean square deviation (RMSD) of the docked ligands was computed to measure the deviation from the co-crystalized ligand. The crystal structures of BRAF (PBD = 2FB8) and VEGFR-2 (PBD = 3VO3) were downloaded from the Protein Data Bank (https://www.rcsb.org).

4.4. ADME prediction

ADME prediction of all pharmacokinetic properties was performed and validated using two platforms, i.e. ADMETLab 2.0 ⁶⁸ and pKCSM.⁶⁹

Ethical approval

This study was conducted according to the appropriate ethical standards as required by guidance from The Medical Research Ethics Committee (MREC), Egypt, Ethical Clearance Certificate No. 9446072023.

Conflicts of interest

There are no conflicts of interest to declare.