Synthesis and Cytotoxic Activity of Biphenylurea Derivatives Containing Indolin-2-one Moieties

In our endeavor towards the development of potent anticancer agents, two different sets of biphenylurea-indolinone conjugates, 5a–s and 8a,b were synthesized. The in vitro cytotoxicity of the synthesized compounds was examined in two human cancer cell lines, namely MCF-7 breast cancer and PC-3 prostate cancer cells using the sulforhodamine B (SRB) colorimetric assay. In particular, the MCF-7 cancer cell line was more susceptible to the synthesized compounds. Compound 5o (IC50 = 1.04 ± 0.10 μM) emerged as the most active member in this study against MCF-7, with 7-fold increased activity compared to the reference drug, doxorubicin (IC50 = 7.30 ± 0.84 μM). Compounds 5l, 5q and 8b also exhibited superior cytotoxic activity against MCF-7 with IC50 values of 1.93 ± 0.17, 3.87 ± 0.31 and 4.66 ± 0.42 μM, respectively. All of the tested compounds were filtered according to the Lipinski and Veber rules and all of them passed the filters. Additionally, several ADME descriptors for the synthesized compounds 5a–s and 8a,b were predicted via a theoretical kinetic study performed using the Discovery Studio 2.5 software.


Introduction
According to the World Health Organization (WHO) estimates, cancer is a disease responsible for major morbidity and mortality worldwide.As reported in 2012 it resulted in about 8.2 million deaths [1].Owning to its high incidence, prevalence, morbidity as well as mortality rates, cancer has been identified as a huge burden on the society.According to the statistics provided by GLOBOCAN in 2012, around 14.1 million newly diagnosed cancer cases were recorded.Among the newly diagnosed cases in females, breast cancer ranked in the first position worldwide and was regarded as the main cause of deaths attributed to cancer.On the other hand, prostate cancer was reported to be of the highest incidence among males in developed countries [2].Although extensive research work had been conducted to synthesize and evaluate novel anticancer agents [3][4][5][6][7][8], satisfactory progress, relative Molecules 2016, 21, 762; doi:10.3390/molecules21060762www.mdpi.com/journal/molecules to the speed of the disease progression, towards the development of anticancer agents of optimum activity and minimum side effects is not yet been accomplished.Biphenylurea derivatives showed interesting activity as anticancer agents.For example, sorafenib, (1, Figure 1) is a potent diphenylurea VEGFR-2 inhibitor in addition to its inhibitory action on c-Kit, PDGFRβ, and Raf kinases (Figure 1) [9].The U.S. FDA has approved it for the treatment of advanced renal cell carcinoma (RCC) and for hepatocellular carcinoma (HCC) [10].Regorafenib (2, Figure 1), a fluoro derivative of sorafenib, [11] inhibits angiogenic kinases VEGFR-1/3, PDGFRβ, FGFR1, and Tie-2.Furthermore, it showed anti-proliferative activities on different cancer cell lines [12].Regorafenib was approved for previously treated metastatic colorectal cancer (mCRC) and advanced gastrointestinal stromal tumors (GIST) [13].Linifanib, (3, Figure 1), a novel biphenylurea derivative in phase II clinical trials, has selective inhibitory action on VEGFR and PDGFRs in patients with locally advanced or metastatic non-small cell lung cancer [14].SLC-0111 ( 4) is a low molecular weight biphenylurea derivative in phase I clinical trials which has shown significant antitumor/ antimetastatic activity in animal models of the disease via the inhibition of tumor-associated human carbonic anhydrase (hCA) IX and XII isoforms (Ki = 4.5 and 45 nM, respectively) [15].Its thioureido analog 5 displayed selective inhibition against tumor-associated hCA IX and hCA XII isoforms with Ki = 39, 4 nM, respectively [16].
Molecules 2016, 21, 762 2 of 11 relative to the speed of the disease progression, towards the development of anticancer agents of optimum activity and minimum side effects is not yet been accomplished.Biphenylurea derivatives showed interesting activity as anticancer agents.For example, sorafenib, (1, Figure 1) is a potent diphenylurea VEGFR-2 inhibitor in addition to its inhibitory action on c-Kit, PDGFRβ, and Raf kinases (Figure 1) [9].The U.S. FDA has approved it for the treatment of advanced renal cell carcinoma (RCC) and for hepatocellular carcinoma (HCC) [10].Regorafenib (2, Figure 1), a fluoro derivative of sorafenib, [11] inhibits angiogenic kinases VEGFR-1/3, PDGFRβ, FGFR1, and Tie-2.Furthermore, it showed anti-proliferative activities on different cancer cell lines [12].Regorafenib was approved for previously treated metastatic colorectal cancer (mCRC) and advanced gastrointestinal stromal tumors (GIST) [13].Linifanib, (3, Figure 1), a novel biphenylurea derivative in phase II clinical trials, has selective inhibitory action on VEGFR and PDGFRs in patients with locally advanced or metastatic non-small cell lung cancer [14].SLC-0111 ( 4) is a low molecular weight biphenylurea derivative in phase I clinical trials which has shown significant antitumor/ antimetastatic activity in animal models of the disease via the inhibition of tumor-associated human carbonic anhydrase (hCA) IX and XII isoforms (Ki = 4.5 and 45 nM, respectively) [15].Its thioureido analog 5 displayed selective inhibition against tumor-associated hCA IX and hCA XII isoforms with Ki = 39, 4 nM, respectively [16].On the other hand, the indolinone moiety proved its success in many compounds as antineoplastic [17,18].This success is supported by the FDA approval of the use of indolinone derivative, sunitinib maleate (20a) (Sutent ® ) in combating advanced renal carcinoma [19] and gastrointestinal stromal tumors [20].In addition, toceranib phosphate (20b) [21] (Palladia ® ), an orally bioavailable compound, is approved by FDA for the treatment of cutaneous (skin-based) mast cell tumors.On the other hand there are many compounds derived from indolinone are in clinical trials, for example, SU5416 (Semaxanib, 21a) is an indolinone derivative in clinical trials, it showed extreme potency with GI50 values of <10 nM against 46 out of 53 NCI cell lines [22] but the clinical trials at phase III stopped due to its high toxicity [23].Orantinib (21b) (SU6668), which is a more water soluble indolinone derivative than semaxanib, has significant anti-tumor activity in xenografts after daily oral treatment [24,25].SU5614 (21c), in phase II clinical trials, induces growth arrest and apoptosis in c-kit expressing Kasumi-1, UT-7 and M-07e cells [26].
Incorporation of biphenylurea with an indolin-2-one moiety was proved as a successful strategy to get active hits to treat hepatocellular carcinoma and to affect cancer cells by inhibition of tumor-associated carbonic anhydrases [4,27].As an extension of this work, new biphenylurea derivatives 5a-s and 8a,b (Figure 1) incorporating indolin-2-one moieties were investigated for their potential cytotoxic effect towards human breast (MCF-7) and prostate (PC-3) cancer cell lines.On the other hand, the indolinone moiety proved its success in many compounds as antineoplastic [17,18].This success is supported by the FDA approval of the use of indolinone derivative, sunitinib maleate (20a) (Sutent ® ) in combating advanced renal carcinoma [19] and gastrointestinal stromal tumors [20].In addition, toceranib phosphate (20b) [21] (Palladia ® ), an orally bioavailable compound, is approved by FDA for the treatment of cutaneous (skin-based) mast cell tumors.On the other hand there are many compounds derived from indolinone are in clinical trials, for example, SU5416 (Semaxanib, 21a) is an indolinone derivative in clinical trials, it showed extreme potency with GI 50 values of <10 nM against 46 out of 53 NCI cell lines [22] but the clinical trials at phase III stopped due to its high toxicity [23].Orantinib (21b) (SU6668), which is a more water soluble indolinone derivative than semaxanib, has significant anti-tumor activity in xenografts after daily oral treatment [24,25].SU5614 (21c), in phase II clinical trials, induces growth arrest and apoptosis in c-kit expressing Kasumi-1, UT-7 and M-07e cells [26].
Incorporation of biphenylurea with an indolin-2-one moiety was proved as a successful strategy to get active hits to treat hepatocellular carcinoma and to affect cancer cells by inhibition of tumor-associated carbonic anhydrases [4,27].As an extension of this work, new biphenylurea derivatives 5a-s and 8a,b (Figure 1) incorporating indolin-2-one moieties were investigated for their potential cytotoxic effect towards human breast (MCF-7) and prostate (PC-3) cancer cell lines.Moreover, physicochemical properties and prediction of ADME descriptors were employed to obtain useful data about their physicochemical interactions within the body.

Chemistry
The synthetic pathway adopted to synthesize the target indolinone derivatives is outlined in Schemes 1 and 2. Synthesis was initiated by reacting 4-nitrophenyl isocyanate with the appropriate anilines 1a-e to afford 1-(4-nitrophenyl)-3-phenylureas 2a-e, followed by hydrogenation using Pd/C in methanol to furnish the corresponding amines 3a-e.The target indolinone derivatives 5a-x were obtained in good yields (75%-81%) through the condensation of the amino intermediates 3a-e with indole-2,3-diones 4a-e in ethyl alcohol in the presence of a catalytic amount of glacial acetic acid reaction (Scheme 1).Moreover, physicochemical properties and prediction of ADME descriptors were employed to obtain useful data about their physicochemical interactions within the body.

Chemistry
The synthetic pathway adopted to synthesize the target indolinone derivatives is outlined in Schemes 1 and 2. Synthesis was initiated by reacting 4-nitrophenyl isocyanate with the appropriate anilines 1a-e to afford 1-(4-nitrophenyl)-3-phenylureas 2a-e, followed by hydrogenation using Pd/C in methanol to furnish the corresponding amines 3a-e.The target indolinone derivatives 5a-x were obtained in good yields (75%-81%) through the condensation of the amino intermediates 3a-e with indole-2,3-diones 4a-e in ethyl alcohol in the presence of a catalytic amount of glacial acetic acid reaction (Scheme 1).The structures of all newly synthesized derivatives were verified on the basis of spectral and elemental analyses which were in full agreement with the proposed structures.

Biological Evaluation
The in vitro cytotoxic activity of the prepared compounds 5a-s and 8a,b was examined against two different cell lines (MCF-7 and PC-3) utilizing the colorimetric assay (SRB) developed by Skehan et al. [28] using doxorubicin as a reference drug (Table 1).Moreover, physicochemical properties and prediction of ADME descriptors were employed to obtain useful data about their physicochemical interactions within the body.

Chemistry
The synthetic pathway adopted to synthesize the target indolinone derivatives is outlined in Schemes 1 and 2. Synthesis was initiated by reacting 4-nitrophenyl isocyanate with the appropriate anilines 1a-e to afford 1-(4-nitrophenyl)-3-phenylureas 2a-e, followed by hydrogenation using Pd/C in methanol to furnish the corresponding amines 3a-e.The target indolinone derivatives 5a-x were obtained in good yields (75%-81%) through the condensation of the amino intermediates 3a-e with indole-2,3-diones 4a-e in ethyl alcohol in the presence of a catalytic amount of glacial acetic acid reaction (Scheme 1).The structures of all newly synthesized derivatives were verified on the basis of spectral and elemental analyses which were in full agreement with the proposed structures.

Biological Evaluation
The in vitro cytotoxic activity of the prepared compounds 5a-s and 8a,b was examined against two different cell lines (MCF-7 and PC-3) utilizing the colorimetric assay (SRB) developed by Skehan et al. [28] using doxorubicin as a reference drug (Table 1).The structures of all newly synthesized derivatives were verified on the basis of spectral and elemental analyses which were in full agreement with the proposed structures.
Concerning activity against PC-3, the synthesized compounds showed moderate low activity against this cell line.In particular, compounds 8a,b were the most active analogues against PC-3 cell line through this study with IC50 values of 18.40 ± 1.33 and 13.79 ± 0.88 μM, respectively (Table 1).
Regarding the activity against MCF-7 SAR studies provided useful conclusions about the effects of substitutions on both the indolinone moiety and the terminal phenyl group of the biphenylurea moiety.Regarding our strategy, we have four different possibilities regarding substitution depending on whether the substituent is electron donating or electron withdrawing using two different positions.Herein, electron withdrawing substituents with different types and position were added to the terminal phenyl group of the biphenylurea moiety, while variable substituents were used on the indolinone moiety.Concerning substitution on the terminal phenyl group of the biphenylurea moiety, derivatives with chloro-substituents on either position 3 or 4 produced some derivatives with better cytotoxic activity than other substituents, as exemplified by compounds 5l, 5o, 5q and 8b (Table 1).It was noticed that the substitution on indolinone moiety should be either Cl or no substitution.The other derivatives such as 5j, 5k, 5m, 5n, 5p, 5r, 5s and 8a, with showed intermediate activity or no activity as it may be affected by the 5-substituents on indolinone moiety that are either with no substitution or with electron donating substitution except compound 5p with 5-F substitution.With no substituents on the terminal phenyl group of the biphenylurea moiety, compounds 5a-c showed intermediate cytotoxic activity, while 4-F or 3-CF3 substitution resulted in weak or no activity as in compounds 5d,5i.
Finally, exploration of the impact of the substitution on the N atom of the indolinone suggested that this was an advantageous approach for activity against PC-3, where the N-substituted members 8a and 8b (IC50 = 18.40 ± 1.33 and 13.79 ± 0.88 μM, respectively) showed enhanced activity over their unsubstituted counterpart 5o (IC50 = 83.54± 6.32 μM) towards PC-3.Nevertheless such an approach failed to improve the activity against MCF-7 cells (Table 1).

Physicochemical Properties and ADME Profiling
According to Table 2, it is worthy to mention that all of the synthesized compounds (5a-s and 8a,b) passed the filter of both Lipinski's rule of five [29] and the Veber rule [30].The previous fact suggested all the synthesized compounds possess good oral bioavailability.Additionally, several ADME descriptors for all the synthesized compounds 5a-s and 8a,b were estimated through a theoretical kinetic As shown in Table 1, MCF-7 cells were found to be more susceptible to the synthesized compounds than prostate cancer (PC-3) cells.The majority of the tested compounds exhibited a competitive cytotoxic activity against MCF-7 cells.In particular, compounds 5l, 5o, 5q and 8b exhibited potent cytotoxic activity against MCF-7, with IC 50 = 1.93 ˘0.17, 1.04 ˘0.10, 3.87 ˘0.31 and 4.66 ˘0.42 µM, or 3.8-, 7-, 1.88-and 1.56-fold increased activity as compared to the reference drug, doxorubicin (IC 50 = 7.3 ˘0.84 µM), respectively.Compound 5n possessed cytotoxic activity close to the activity of the reference drug doxorubicin, with IC 50 = 9.60 ˘1.08 µM.Compounds 5a-c, 5p, 5r and 8a were also moderately active towards MCF-7 with IC 50 values in the range of 12.16 ˘1.17-28.00˘2.93 µM.Other analogues showed weak or no activity against MCF-7 cell lines (Table 1).
Concerning activity against PC-3, the synthesized compounds showed moderate to low activity against this cell line.In particular, compounds 8a,b were the most active analogues against PC-3 cell line through this study with IC 50 values of 18.40 ˘1.33 and 13.79 ˘0.88 µM, respectively (Table 1).
Regarding the activity against MCF-7 cells, SAR studies provided useful conclusions about the effects of substitutions on both the indolinone moiety and the terminal phenyl group of the biphenylurea moiety.Regarding our strategy, we have four different possibilities regarding substitution depending on whether the substituent is electron donating or electron withdrawing using two different positions.Herein, electron withdrawing substituents with different types and position were added to the terminal phenyl group of the biphenylurea moiety, while variable substituents were used on the indolinone moiety.Concerning substitution on the terminal phenyl group of the biphenylurea moiety, derivatives with chloro-substituents on either position 3 or 4 produced some derivatives with better cytotoxic activity than other substituents, as exemplified by compounds 5l, 5o, 5q and 8b (Table 1).It was noticed that the substitution on indolinone moiety should be either Cl or no substitution.The other derivatives such as 5j, 5k, 5m, 5n, 5p, 5r, 5s and 8a, with showed intermediate activity or no activity as it may be affected by the 5-substituents on indolinone moiety that are either with no substitution or with electron donating substitution except compound 5p with 5-F substitution.With no substituents on the terminal phenyl group of the biphenylurea moiety, compounds 5a-c showed intermediate cytotoxic activity, while 4-F or 3-CF 3 substitution resulted in weak or no activity as in compounds 5d,5i.
Finally, exploration of the impact of the substitution on the N atom of the indolinone suggested that this was an advantageous approach for activity against PC-3, where the N-substituted members 8a and 8b (IC 50 = 18.40 ˘1.33 and 13.79 ˘0.88 µM, respectively) showed enhanced activity over their unsubstituted counterpart 5o (IC 50 = 83.54˘6.32 µM) towards PC-3.Nevertheless such an approach failed to improve the activity against MCF-7 cells (Table 1).

Physicochemical Properties and ADME Profiling
According to Table 2, it is worthy to mention that all of the synthesized compounds (5a-s and 8a,b) passed the filter of both Lipinski's rule of five [29] and the Veber rule [30].The previous fact suggested all the synthesized compounds possess good oral bioavailability.Additionally, several ADME descriptors for all the synthesized compounds 5a-s and 8a,b were estimated through a theoretical kinetic study carried out using the Discovery Studio 2.5 software (Table 3, Accelrys, San Diego, CA, USA).All of the synthesized compounds showed good predicted levels of human intestinal absorption except 8a,b that showed moderate intestinal absorption.Regarding aqueous solubility, all of the tested compounds ranged from very low to low solubility.Concerning blood-brain barrier penetration, all of the tested compounds were discovered to possess medium penetrability except compounds 5i, and 8a,b that were undefined.Compounds 5f, 5g, 5h and 5p were predicted to be CYP2D non-inhibitors.a The upper limit of the number of hydrogen bond donors is 5; b The upper limit of the number of hydrogen bond acceptors is 10; c The upper limit of the molecular weight is 500; d The upper limit of the AlogP (the log value of octanol-water partition coefficient) is 500; e The upper limit of the number of rotatable bonds is 10; * No. of violation allowed is 1 and AlogP value should not be ě 6; f The upper limit of the polar surface area is 140 Å; # The upper limit of the sum of hydrogen bond donor and acceptors is 12.  (0; ě 0.7 = very high penetrant, 1; 0 ď logBB < 0.7) = High penetrant, 2; ´0.52 < LogBB < 0 = low, 3; logBB ď ´0.52 = low, 4; NA = undefiend); e CYP2D inhibition.(0 = non inhibitor, 1 = inhibitor).

General Information
Melting points were measured with a Stuart melting point apparatus and are uncorrected.Infrared (IR) spectra were recorded as potassium bromide disks on a FT-IR 8400S spectrophotometer (Shimadzu, Kyoto, Japan) and values are expressed in wavenumber (cm ´1) NMR spectra were recorded on an AV-400 MHz NMR spectrophotometer (Bruker, Karlsruhe, Germany). 1 H-NMR spectra were run at 400 MHz and 13 C spectra were run at 100 MHz in deuterated dimethylsulfoxide (DMSO-d 6 ).Chemical shifts are expressed in δ values (ppm) using the solvent peak as internal standard.All coupling constants (J) values are given in Hertz.The abbreviations used to indicate peak multiplicity are as follows: s, singlet; d, doublet; m, multiplet.Elemental analyses were carried out at the Regional Center for Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt.Reaction courses and product mixtures were routinely monitored by thin layer chromatography (TLC) on silica gel precoated F 254 plates Merck (Merck KGaA, Darmstadt, Germany).Unless otherwise noted, all solvents and reagents were commercially available and used without further purification.Compounds 2a-e, 3a-e, 5a-l and 5o-q [4,31] and 7a,b [32] were all previously prepared.

General Procedure for Preparation of Target Compounds 5a-x
Indoline-2,3-diones 4a-e (0.001 mol) were added to a suspension of the appropriate 1-(4-aminophenyl)-3-phenyl/substituted phenylurea 3a-e (0.001 mol) in ethyl alcohol (10 mL) containing acatalytic amount of glacial acetic acid.The reaction mixture was heated under reflux for 3 h.The precipitate formed was collected by filtration while hot, washed with hot ethanol, dried and crystallized from ethanol/DMF to afford target compounds 5a-x. 1 H-NMR of 5m, 5n, 5p, 5s, and 8a,b can be found in the Supplementary Materials.-7) and prostate cancer (PC-3) cells were purchased from National Cancer Institute (Cairo, Egypt).Maintenance of cells was carried out using Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% heat-inactivated fetal bovine serum, 100 Units/mL of penicillin and 100 µg/mL streptomycin.Cells were kept as a monolayer culture by serial passaging under standard culture conditions of 37 ˝C, 95% humidified air and 5% CO 2 .

In vitro Cytotoxic Activity
Investigated compounds were dissolved in DMSO and kept at a stock concentration of 100 mM.Cytotoxicity was assessed via SRB method as indicated by Skehan et al. [28] In brief, exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeded in 96-well plates at a 3000 cells/well density in culture medium and incubated for 24 h till the formation of a monolayer.Subsequently, cells were incubated with different concentrations (0-10 3 µM) of the compounds under investigation as well as doxorubicin as a reference drug for further 72 h.At the end of the incubation, the treatment was terminated through fixing the cells with 10% trichloroacetic acid for 1 h at 4 ˝C.This was followed by a staining procedure using 0.4% SRB (1% acetic acid) for 10 min at room temperature.Finally, microplates were left to dry for 24 h and Tris-HCl was added afterwards for 5 min on a shaker at 1600 rpm for solubilization.Using an ELISA microplate reader (ChroMate-4300, Palm City, FL, USA), optical density was determined at 545 nm.The IC 50 values were calculated according to the equation for Boltzman sigmoidal concentration-response curve using the nonlinear regression fitting models (Graph Pad Prism Version 5; GraphPad Software, Inc. La Jolla, CA, USA).The results reported are means of at least three separate experiments.Statistical analyses were carried out using one-way analysis of variance (ANOVA) followed by the Tukey-Kramer test for post-hoc analysis.Statistical difference was considered to be significant at p < 0.05.Statistical testing was performed using GraphPad InStat software, version 3.05.

Physicochemical Properties and ADME Profiling
Physicochemical properties and ADME profiling for all the synthesized compounds were performed using Discovery Studio 4 (Accelrys, San Diego, CA, USA).All the tested compounds were drawn as a small library and prepared using prepare ligand protocol to find the suitable orientation in 3D.For studying physicochemical properties, the prepared library was filtered using the Lipinski and Veber rules protocols.ADME profiling was predicted for the designed library using ADME descriptors protocol.

Conclusions
We reported herein the synthesis, in vitro cytotoxic activity and in silico ADME prediction studies of twenty one biphenylurea derivatives 5a-s and 8a,b that incorporated an indolinone moiety.All the synthesized compounds were evaluated for their in vitro cytotoxicity against two human cancer cell lines, namely MCF-7 breast cancer and PC-3 prostate cancer.Compounds 5l, 5o, 5q and 8b exhibited

Figure 1 .
Figure 1.Biphenylurea derivatives and indolinone derivatives as anticancer agents and the proposed structures 5a-s and 8a,b.

Figure 1 .
Figure 1.Biphenylurea derivatives and indolinone derivatives as anticancer agents and the proposed structures 5a-s and 8a,b.

Table 1 .
In vitro cytotoxic activities of the synthesized derivatives against MCF-7 and PC-3 cell lines.

Table 1 .
In vitro cytotoxic activities of the synthesized derivatives against MCF-7 and PC-3 cell lines.

Table 2 .
Lipinski rule of five parameters results for all the synthesized compounds.

Table 3 .
ADME studies results for all the synthesized compounds.
a Human intestinal absorption level.