Structural optimization and evaluation of novel 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrrole derivatives as potential VEGFR-2/PDGFRβ inhibitors

Background Tumor angiogenesis, essential for tumor growth and metastasis, is tightly regulated by VEGF/VEGFR and PDGF/PDGFR pathways, and therefore blocking those pathways is a promising therapeutic target. Compared to sunitinib, the C(5)-Br derivative of 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrrole has significantly greater in vitro activities against VEGFR-2, PDGFRβ, and tube formation. Results and discussion The objective of this study was to perform further structural optimization, which revealed certain new products with even more potent anti-tumor activities, both cellularly and enzymatically. Of these, 15 revealed ten- and eightfold stronger potencies against VEGFR-2 and PDGFRβ than sunitinib, respectively, and showed selectivity against HCT116 with a favorable selective index (SI > 4.27). The molecular docking results displayed that the ligand–protein binding affinity to VEGFR-2 could be enhanced by introducing a hydrogen-bond-donating (HBD) substituent at C(5) of (2-oxoindolin-3-ylidene)methylpyrrole such as 14 (C(5)-OH) and 15 (C(5)-SH). Conclusions Among newly synthetic compounds, 7 and 13–15 exhibited significant inhibitory activities against VEGFR-2 and PDGFRβ. Of these, the experimental results suggest that 15 might be a promising anti-proliferative agent. Graphical abstract IC50 comparison of sunitinib, 14, and 15 against VEGFR-2 and PDGFRβ. Electronic supplementary material The online version of this article (doi:10.1186/s13065-017-0301-5) contains supplementary material, which is available to authorized users.


Introduction
Angiogenesis is a highly ordered process in which new capillaries are formed from pre-existing vessels in physiological conditions such as reproductive angiogenesis, pregnancy, and wound healing. Angiogenesis is up-regulated in many diseases, including rheumatoid arthritis and especially tumor angiogenesis, which is critical for tumor growth and metastasis [1,2]. New blood vessels are required for tumor tissues, when beyond 2 mm 3 , to provide oxygen, nutrients, and paths for metastasis, and to remove metabolic wastes [3]. In the absence of vascular support, tumor tissues would become necrotic or apoptotic [4,5]. Thus, anti-angiogenesis could be an effective therapeutic treatment for cancer.
Pro-angiogenic growth factors secreted by tumor cells, such as angiopoietin-2, epidermal growth factors (EGFs), fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), and platelet-derived growth factors (PDGFs) can stimulate angiogenesis around tumor tissue [6]. Among them, VEGFs, PDGFs, and their receptor tyrosine kinases (RTKs) are the keys of tumor Open Access *Correspondence: 416806@gmail.com 1 Graduate Institute of Medical Sciences, National Defense Medical Center, No. 161, Section 6, Mingchuan East Road, Taipei 11490, Taiwan Full list of author information is available at the end of the article angiogenesis signal transduction [7]. Specific binding of VEGFs and PDGFs to their RTKs triggers downstream signal pathways that induce proliferation, migration, and cell survival of endothelial cells, fibroblast, and vascular smooth muscle cells [8][9][10][11]. Therefore, targeting both VEGF and PDGF signal pathways is a promising approach for anti-angiogenesis drug development [9,10,12,13]. Many small-molecule anti-angiogenesis agents targeting VEGFRs and PDGFRs have been developed and approved for clinical use. Of these, sunitinib, an orally bioavailable indolinone-based RTK inhibitor, inhibits angiogenesis by targeting VEGFR-2 and PDGFRβ, and therefore triggers cancer cell apoptosis. The USFDA has approved the use of sunitinib for treating advanced renal cell carcinoma (RCC), gastrointestinal stromal tumors (GISTs) and pancreatic neuroendocrine tumors (pNETs) [7,14].
All the target compounds were isolated as free bases which were precipitated out during the synthesis. Compounds were purified by simply washing with EtOH. However, most cases required further purification by column chromatography (silica gel, 90:10:1 EtOAc-MeOH-TEA) with TEA to facilitate elution and to remove trace impurities with the exclusion of compound 7. Purification of 7 by column chromatography using various solvent systems only led to rapid decomposition and then a string of unidentifiable spots from the eluent appeared in TLC. Our experiment results showed that analytically pure 7 could be obtainable smoothly by recrystallization from tetrahydrofuran (THF). All the structures of synthetic intermediates and products were determined by spectroscopy and specific data of high-resolution mass analysis (Additional file 1).

Anti-proliferation activity
The in vitro anti-proliferation activity of synthetic compounds 4-15 and sunitinib (positive control) were Scheme 1 Synthesis of key intermediate 3 [19] and 5-substituted 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrrole derivatives evaluated in three different human cancer cell lines (human colon cancer cells HCT116, human non-small cell lung cancer cells NCI-H460, and human renal cell carcinoma 786-O) and a normal human fibroblast cell line Detroit 551. Table 1 summarizes the experimental results.
Since the proliferation of HCT116 cells is stimulated by HCT116-produced VEGF and VEGFR-1/2 via an autocrine mechanism, inhibiting VEGFR-1/2 of HCT116 cells with VEGFR-1/2 inhibitor AAL993 significantly decreases proliferation of HCT116 cells [36]. Table 1 shows that our experiments revealed a strong correlation between anti-proliferation activities of 4-15 against HCT116 cells and VEGFR-2 inhibition percentage at 80 nM.
Potential anticancer drug candidates should show greater selectivity for cancer cells compared with normal cells. Therefore, selectivity index (SI) values for synthetic products 4-15 as well as sunitinib were obtained in the three tested cancer lines (Table 1). For comparison, human normal fibroblast cells Detroit 551 were used as a control group. The SI values showed that all synthetic products except for 7 had high selectivity for tumor cells and, compared to sunitinib, even much lower toxicity to Detroit 551 cells. The toxic effects of C(5)-SO 2 N(CH 2 CH 2 Cl) 2 substituent of 7 on Detroit 551 Scheme 2 Synthesis of oxindoles 16 and 17 cells was evident and complex but nevertheless not yet completely understood. The likely explanation is that 7 contains a highly chemically reactive bis(2-chloroethyl) amino (-SO 2 N(CH 2 CH 2 Cl) 2 ) similar to chlorambucil, which has clinic applications as a non-specific alkylating agent. Thus, its cytotoxic effect probably resulted from DNA damage via the formation of cross-links. In this study, 15 had particularly high selectivity to HCT116 cells (SI > 4.27 for 15 vs. 1.32 for sunitinib), and 13 had particularly high selectivity to NCI-H460 cells (SI > 1.57 for 13 vs. 0.81 for sunitinib) and 786-O cells (SI > 1.27 for 13 vs. 0.76 for sunitinib).
Since our newly synthesized products generally showed high selectivity against HCT116 cancer cell proliferation, the next experiment was performed to determine whether the inhibitory response resulted from acute cellular toxicity. Compounds 7 and 13-15 were then chosen to subject to acute cytotoxicity test on HCT116 cells through the WST-8 cell viability assay. Figure 2 shows the experimental results, which confirmed that neither our compounds nor sunitinib had acute cytotoxicity in the two tested cell lines.
Our previous works apparently showed that C(5)-halogen substituents of 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrroles affected the potency and cell cycle profiles of HCT116 cell [19]. For an improved understanding of these effects, this study performed further cell cycle analyses of 7, 13-15, and sunitinib ( Fig. 3). The preliminary results showed that the cell cycle profiles of HCT116 cells incubated with 14 and sunitinib for 24 h caused G0/G1 cell cycle arrest. In contrast, the cell cycle profile of HCT116 cells incubated with 7 and 13 for 24 h displayed an increase in polyploid cells. Surprisingly, the cell cycle profile of HCT116 cells treated with 15 for 24 h showed an increase in tetraploid cells. Previous works had established that Inhibiting Aurora kinase obtained a polyploidal cell cycle profile [40][41][42]. Our previous studies proved that (2-oxoindolin-3-ylidene)methylpyrroles had great in vitro Aurora A kinase inhibition at 1.0 μM, and some of them revealed the inhibition of HCT116 cells proliferation via Aurora kinase inhibition. Our experiments again revealed a similar trend, i.e., 92.9% for 7, 94.4% for 13, and 93.6% for 15, and 50.7% for sunitinib at 1.0 μM, respectively (Table 2). Therefore, we hypothesized that using compounds 7, 13 and 15 to inhibit HCT116 cell proliferation might also inhibit Aurora kinase.
In summary, the experiments in this study suggested that substituents at C(5) markedly influenced the anti-proliferation activity and selectivity of synthetic derivatives of 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrrole. Additionally, hydrogen bond donor substituents at C(5) significantly affected the potency and selectivity of anti-proliferation activity.

Kinase inhibitory assays
Next, the VEGFR-2 phosphorylation inhibitory activities of the newly synthesized compounds were evaluated. The experimental results in Table 1 show that the VEGFR-2 inhibitory activities of compounds 4, 8, 9, and 11 at concentrations of 80 nM did not differ from that of the 1% DMSO (control). However, compounds 5, 6, and 12 at the same concentration revealed 13-20% inhibition; 7 and 13 demonstrated approximately equal inhibition percentage to sunitinib; and 14 and 15 exhibited the most potent inhibitory activity. Therefore, IC 50 values of compounds 7 and 13-15 were further evaluated to assess their activities against VEGFR-2, PDGFRβ, and Aurora A kinase.
The effect of a C(5)-OMe substituent of indoline-2-one scaffold on kinase inhibitory activity and selectivity is highly dependent on the C(3) substituents of indoline-2-one [22,26]. Interestingly, our study showed As Table 2 shows, in comparison to sunitinib, compounds 7 and 13 had six-and threefold lower IC 50 values for VEGFR-2, respectively. Moreover, compared to their C(5)-OMe analog 13, compounds 14 (C(5)-OH) and 15 (C(5)-SH) even showed a two-and a fourfold decrease in IC 50 values, respectively. On the other hand, the inhibiting activities of 7 and 13 in PDGFRβ were slightly more potent than those of sunitinib; however, 14 and 15 had a three-and an eightfold decrease in IC 50 values for inhibiting PDGFRβ, respectively. Thus, both C(5)-OH and C(5)-SH substituents could significantly improve the activity of 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrroles in the inhibition of both VEGFR-2 and PDGFRβ. In accordance with Kammasud, we hypothesized that groups C(5)-OH and C(5)-SH probably produced favorable potency of 14 and 15 by providing additional hydrogen bonding interactions with ATP site of RTKs.
The results once again revealed a similar trend, i.e., different C(5) substitutions markedly affect the biochemical activities against VEGFR-2 and PDGFRβ. In summary, hydrogen-bond-donating (HBD) substituent at C(5) could greatly enhance inhibitory potency against both VEGFR-2 and PDGFRβ. These experimental results suggest that the influence of C(3) substituent to the C(5)-HBD substituted indoline-2-one scaffold needs further study.

In-vitro tube formation assay
In-vitro VEGF-induced tube formation inhibitory activity of 7, 13-15, and sunitinib were tested by Matrigel tube formation assay using ibidi μ-Slide angiogenesis kit. Figures 4 and 5 shows the photographs of Matrigel tube formation assays of control and tested compounds at 2.0, 1.0, 0.50 and 0.10 μM. Under these conditions, the density of tube-like structures was substantially reduced. Compounds 7 and 13-15 showed distinctly higher tube formation inhibitory activity than the reference drug sunitinib. Compared to sunitinib, 7 and 13 had twofold higher potency in inhibiting in vitro tube formation than sunitinib in terms of IC 50 ( Table 3). The more potent 14 and 15 were with IC 50 roughly 2.5-and threefold stronger than sunitinib, respectively. These experimental results agree well with those for VEGFR-2 kinase inhibitory assays, suggesting that our synthetic compounds inhibited the in vitro tube formation via VEGFR-2 inhibition.

Molecular modeling
The kinase inhibitory assays revealed that the VEGFR-2 and in vitro tube formation inhibitory activities of the synthetic compounds 7 and 13-15 exceeded that of sunitinib, and the C(5) substituents of 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrrole were critical for VEGFR-2 inhibitory activity. For further clarification of these results, sunitinib, 7, and 13-15 were examined and compared by docking into the ATP-binding site of VEGFR-2 (PDB ID: 4AGD) using Discovery Studio Lib-Dock [43]. LibDock is a method placing the generated ligand conformations into the protein active site based on polar and apolar interaction sites (hotspot). Figure 6 shows the predicted binding modes of sunitinib, 7, and 13-15. Interestingly, the modeling results for compound 7 differed from those of compounds 13-15 in that 7 formed four hydrogen bonds with VEGFR-2: the Cl of C(5)-SO 2 N(CH 2 CH 2 Cl) 2 and the NH of the oxindole  Cys1045, and the oxygen atom of pyrrolidone (C(4′)) with Asn923 (Fig. 6b). The docking results further showed that C(5)-SO 2 N(CH 2 CH 2 Cl) 2 of 7 was laid in the hydrophobic pocket of the VEGFR-2 active site (Fig. 6c). The above experimental results might explain why compound 7 had the most potent VEGFR-2 inhibiting effects among 4-9.
In Fig. 6d-f, the predicted binding modes of highly active compounds 13-15 reveal that each of them formed three hydrogen bonds with Lys868, Glu917, and Cys919, respectively. Additionally, compounds 13-15 all formed pi-pi interactions between their pyrrole-scaffolds and Phe918 of VEGFR-2 ( Fig. 6d-f

Conclusions
The novel series of 2-pyrrolidone-fused (2-oxoindolin-3-ylidene)methylpyrrole derivatives with various C(5) substitutions synthesized in our laboratory showed notable cellular and enzymatic anti-tumor activities. Several of these derivatives had superior inhibitory activity against VEGFR-2 and PDGFRβ compared to sunitinib.   studies are in progress and will be reported in future works.

Chemistry
All the chemicals were purchased from Aldrich-Sigma Chemical Company (St. Louis, MO, USA) and Alfa-Aesar Chemical Company (Lancashire, Heysham, England) and used without further purification. All reactions were routinely monitored by TLC on Merck F 254 silica gel plates. Silica gel (70-230 mesh, Silicacycle) was used for column chromatography. The 1 H-and 13 C-NMR spectra were determined on an Agilent Varian-400 NMR (Agilent Technologies, Santa Clara, CA, USA) instrument in CDCl 3 , acetone-d 6 , methanol-d 4 , or acetic acid-d 6 unless otherwise noted. Chemical shifts (δ) were expressed as parts per million (ppm) downfield from tetramethylsilane (TMS) as the internal standard (σ 0.00), and coupling constants (J) were given in hertz (Hz). High-resolution mass spectra (HRMS) using a Bruker Impact HD (ESI) were performed in the Instrument Center of the Ministry of Science and Technology at the National Chiao-Tung University, Taiwan. Dry tetrahydrofuran (THF) was freshly distilled from lithium aluminum hydride (LAH) before use. All the other solvents were obtained from commercial sources and purified before use if necessary. Images were acquired with a Leica DM1000 LED microscope (Leica Microsystems, Wetzlar, Hessen, Germany). UV-VIS spectra were recorded on a Thermo Multiskan Go Microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). IR spectra were registered on a Thermo Nicolet iS5 FT-IR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with attenuated total reflection (ATR) method. The purities of the final compounds were all greater than 95% as determined by analytical reverse-phase HPLC.

General procedure for the preparation of compounds 4-15
The key intermediate 3 for preparation of target compounds was synthesized according to the method reported previously [19]. To each stirred solution containing 3 (263 mg, 1.00 mmol) in EtOH (15 ml) was added dropwise a solution of 5-substituted oxindole (1.00 mmol) in EtOH (2 ml) and then piperidine (0.1 ml) was added. After stirring at room temperature for 6 h, the precipitate formed was filtrated, washed with EtOH, and purified by column chromatography (silica gel, 90:10:1 EtOAc-MeOH-TEA).

Cell proliferation assay
The cells incubated as above were plated at a density of 2000 cells/well (cancer cells) [41,44,45] or 10,000 cells/ well (Detroit 551) [46] on a 96-well plate for 24 h. Serial dilutions of indicated compounds were added and incubated for additional 72 h. At the end of the incubation, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The MTT formazan crystals formed were dissolved in DMSO, and the absorbance at 570 nm was recorded using a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [46].

Acute cytotoxicity
The acute cytotoxicity effect of compounds 7 and 13-15, and sunitinib was determined by Cell-Counting-Kit-8 (Dojindo, Rockville, MD, USA) assay on HCT116, NCI-460, 786-O, and Detroit 551 cells according to the manufacturer's protocol. Cells were seeded at 5000 cells/well on a 96-well plate for 24 h. The indicated compounds in different concentrations (100 μl) were added to cells. After 6 h, old medium was aspirated, and the cells were washed three times with PBS. WST-8 (Dojindo, Rockville, MD, USA) (10 µl) was added to each well, and the absorbance of the plate was recorded at 450 nm on a microplate spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

In-vitro tube formation assay
The in vitro tube formation assay was assessed using ibidi μ-Slides (15-well, ibidi GmbH, Martinsried, Germany) in accordance with manufacturer's protocol. Briefly, growth factor reduced Matrigel (10 μl) (Sigma, St. Louis MO) was added to the inner well of ibidi μ-Slides, and incubated at 37 °C for 1 h. HUVEC cells were harvested by centrifugation, and the cell suspension was adjusted to 200,000 cells/ml by 10 ng/ml VEGF contained growth medium (M199) with or without indicated compounds 7, 13, 14, 15, or sunitinib in different concentrations (1.0, 0.50 and 0.10 μM). 10,000 HUVEC cells in 50 μl of above growth medium was added to Matrigel (Sigma, St. Louis MO, USA) coated ibidi μ-Slides. After 6 h of incubation at 37 °C, the supernatant was discarded, and 50 μl of serum-free medium with diluted calcein AM (6.25 μg/ ml) was added to above ibidi μ-Slides. After incubation in the dark at room temperature for 30 min, the μ-Slides were washed with PBS (50 μl) and fluorescence pictures were taken at 485 nm with a Leica DM1000 LED microscope (Leica Microsystems, Wetzlar, Hessen, Germany).

In-vitro kinase assay
The Reaction Biology Corporation (http://www.reactionbiology.com) HotSpot assay platform was used to determine the inhibitory activity of 7, 13, 14, 15, and sunitinib against VEGFR-2, PDGFRβ, and Aurora A, measured by quantifying the amount of 33 P incorporated into the substrate in the presence of the test compound [47]. Briefly, specific kinase and substrate and required cofactors were prepared in reaction buffer. Test compounds were added to the reaction and after 20 min a mixture of ATP (Sigma, St. Louis MO, USA) and 33 P ATP (Perkin Elmer, Waltham MA, USA) was added to make a final concentration of 10.0 μM. Reactions were stood at room temperature for 120 min, and then the reactions were spotted onto a P81 ion exchange filter paper (Whatman Inc., Piscataway, NJ, USA). Unbound phosphate was removed by extensive washing of filters in 0.1% phosphoric acid. Kinase activity data was reported as the percent remaining kinase activity in test compounds compared to the solvent control dimethyl sulfoxide (DMSO).

Molecular modeling
Ligands-receptor docking calculation was carried out in accordance with the LibDock protocol. Briefly, receptor