Synthesis and in vitro anticancer activity of certain novel 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas as apoptosis-inducing agents

Abstract In connection with our research program on the development of novel anticancer candidates, herein we report the design and synthesis of novel series of 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas 5a–l. The target pyridins were evaluated for their in vitro anticancer activity against two cancer cell lines: non-small cell lung cancer A549 cell line and colon cancer HCT-116 cell line. Compound 5l emerged as the most active congener towards both A549 and HCT-116 cell lines with IC50 values equal to 3.22 ± 0.2 and 2.71 ± 0.16 µM, respectively, which are comparable to those of Doxorubicin; 2.93 ± 0.28 and 3.10 ± 0.22, respectively. Furthermore, compound 5l stood out as the most potent pyridine derivative (mean % GI = 40), at US-NCI Developmental Therapeutic Program anticancer assay, with broad-spectrum antitumor activity against the most tested cancer cell lines from all subpanels. Compound 5l was able to provoke apoptosis in HCT-116 cells as evidenced by the decreased expression of the anti-apoptotic Bcl-2 protein, and the enhanced expression of the pro-apoptotic proteins levels; Bax, cytochrome C, p53, caspase-3 and caspase-9. Moreover, 5l disrupted the HCT-116 cell cycle via alteration of the Sub-G1 phase and arresting the G2-M stage. Also, 5l showed a significant increase in the percent of annexinV-FITC positive apoptotic cells from 1.99 to 15.76%.


Introduction
Apoptosis, a self-automated cell death, represents the principal pathway in tissue homeostasis and in animal development; in addition, it is the main pathway for the clearance of aged or defective cells in the body. Mainly, two major signaling pathways for apoptotic cell death have been signified. The first one is the extrinsic cytoplasmic pathway that is triggered via pro-apoptotic ligands binding to the cell surface death receptor. Whereas, the second is the intrinsic mitochondrial apoptotic pathway that results from an intracellular cascade of events that are mainly produced by cellular stress, in which mitochondrial permeabilization plays a crucial role. Both extrinsic and intrinsic pathways converge onto the activation of effector caspases, resulting in apoptotic cell death program. During cancer pathogenesis, apoptosis deregulation has been widely recognized as a hallmark of cancer. Accordingly, induction of apoptosis in tumor cells has stood out as a successful tactic for combating different human malignancies, in the current medical era [1][2][3] .
On the other hand, non-fused pyridines have stood out as a promising class of anticancer agents with efficient pro-apoptotic activity. Regorafenib (StivargaV R , Figure 1), a pyridine-based biphenyl urea derivative developed by Bayer 4

, inhibits
Based on the aforementioned findings and as a part of our ongoing quest towards developing potent anticancer agents [12][13][14][15][16][17][18][19][20] , herein we report the synthesis and biological evaluation of novel series of 1-(2-methyl-6-arylpyridin-3-yl)-3-phenylureas 5a-l. Ten selected pyridines 5a, 5c-j and 5l were chosen to be in vitro evaluated for their antitumor activity at one dose (concentration 10 À5 M) primary anticancer assay towards a panel including 85 cancer lines according to US-NCI protocol. In addition, all pyridines 5a-l were examined for their potential anti-proliferative activity against non-small cell lung cancer A549 cell line and colon cancer HCT-116 cell line. Furthermore, apoptosis induction potential of the target pyridines was examined in HCT-116 cells, in order to acquire more mechanistic insights and to verify and enlighten the antitumor properties of the investigated pyridines.

Chemistry
Melting points were measured with a Stuart melting point apparatus and were uncorrected. Infrared (IR) Spectra were recorded as KBr disks using Schimadzu FT-IR 8400S spectrophotometer. 1 H-NMR and 13 C-NMR experiments were carried out using Bruker NMR spectrometer (400/100 MHz). Chemical shifts (d H ) are reported relative to TMS as the internal standard. All coupling constant (J) values are given in hertz. Chemical shifts (d C ) were reported as follows: s, singlet; d, doublet; m, multiplet. High-resolution mass spectra were recorded using a Bruker MicroTOF spectrometer (Bruker Daltonics, Bremen, Germany). All reagents and solvents were dried and purified by the standard techniques. Compounds 2-methyl-6-arylnicotinohydrazides 2a-c 21-23 were previously prepared.
General procedures for preparation of the target pyridines 5a-l A solution of hydrazides 2a-c (10 mmol) and sodium nitrite (1 g, 14 mmol) in hydrochloric acid was stirred for 1 h in an ice bath, then stirring was continued for an additional 1 h at room temperature. The reaction mixture was poured over crushed ice. The precipitated solid was filtered off and air-dried to yield 2-methyl-6-arylnicotinoyl azides 3a-c, which were used in the next step without further purification. Azides 3a-c were heated in refluxing dry xylene for 1 h, then the appropriate aniline derivative was added to this xylene solution. The reaction mixture was heated under reflux temperature for 4 h. After cooling to room temperature, the formed precipitate was filtered, washed with ether and recrystallized from ethanol to afford the target pyridines 5a-l.    1-(4-Chlorophenyl)-3-(2-methyl-6-(thiophen-2-yl) pyridin-3yl)urea (5i)

Biological evaluation
In vitro antitumor activity towards 60 cancer cell lines (NCI, USA) The antitumor assay was performed according to the protocol of the Drug Evaluation Branch, NCI, Bethesda [24][25][26] . A 48 h drug exposure protocol was adopted, and sulforhodamine B (SRB) assay 27 was utilized to assess the cell growth and viability, as reported earlier 17,28 .
In vitro anti-Proliferative activity towards A549 and HCT-116 cell lines A549 (non-small cell lung cancer cell line) and HCT-116 (human colon cancer cell line), were obtained from American Type Culture Collection (Manassas, VA, USA). The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma-Aldrich, St. Louis, MO), and supplemented with 10% heat-inactivated FBS (Hyclone), 10 lg/mL of insulin (Manufacturer, Sigma, St. Louis, MO, USA), and 1% penicillin-streptomycin. MTT assay 29 was adopted to assess the in vitro antitumor activity of the newly synthesized pyridines 5a-l according to the reported procedures 30 , using Doxorubicin as a standard treatment. Experimental conditions were tested using three replicates (three wells of the 96-well plate per experimental condition) and all experiments were carried out in triplicates. IC 50 values were calculated by the use of the equation for Boltzman sigmoidal concentration-response curve using the nonlinear regression fitting models by Graph Pad, Prism version 5 (GraphPad Software Inc., La Jolla, CA).

ELISA immunoassay
The levels of the apoptotic markers Bax, cytochrome C, p53, caspase-3 and caspase-9 as well as the anti-apoptotic protein Bcl-2 were evaluated using ELISA colorimetric kits per the manufacturer's instructions, as reported earlier 31,32 .
Cell cycle analysis HCT-116 cells were treated with pyridine 5l at its IC 50 concentration (IC 50 ¼ 2.71 lM) for 24 h, then cells were washed with ice-cold phosphate-buffered saline (PBS). The treated cells were collected by centrifugation, fixed in ice-cold 70% (v/v) ethanol, washed with PBS, re-suspended with 100 lg/mL RNase, stained with 40 lg/mL PI, and analyzed by flow cytometry using FACS Calibur (Becton Dickinson, BD, USA). The cell cycle distributions were calculated using CellQuest software 5.1 (Becton Dickinson) 33 .
Annexin V-FITC apoptosis assay Phosphatidylserine externalization was assayed using Annexin V-FITC/PI apoptosis detection kit (BD Biosciences, USA) according to the manufacturer's instructions, as reported earlier 33,34 .

Chemistry
The method adopted for preparation of the target pyridines 5a-l is depicted in Scheme 1. Firstly, esters 1a-c were hydrazinolyzed via reaction with hydrazine hydrate in methanol under reflux temperature to furnish 2-methyl-6-arylnicotinohydrazides 2a-c in 75, 71 and 80% yields, respectively. Treatment of hydrazides 2a-c with sodium nitrite in cold hydrochloric acid afforded 2-methyl-6-arylnicotinoyl azides 3a-c, which subsequently subjected to Curtius rearrangement upon heating in xylene to give the corresponding isocyanates derivatives 4a-c. The target hybrids 5a-l was obtained by reaction of isocyanates derivatives 4a-c with the appropriate aniline derivative in xylene with 55-73% yield (Scheme 1).
The structures of the newly prepared pyridines 5a-l were confirmed under the basis of spectral and elemental analyses which

Biological evaluation
In vitro antitumor activity towards 60 cancer cell lines (NCI, USA) The structures of all the newly synthesized pyridines 5a-l were submitted to the National Cancer Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.nih.gov). Ten pyridines 5a, 5c-j and 5l were chosen to be in vitro evaluated for their antitumor activity. The selected pyridines 5a, 5c-j and 5l were examined at one dose (concentration 10 À5 M) primary anticancer assay towards a panel including 85 cancer lines. Nine different types of cancer were tested in this assay: colon, ovarian, prostate, leukemia, melanoma, CNS, renal, breast and lung cancers. A 48 h drug exposure protocol was adopted, and sulforhodamine B (SRB) assay 27 was 37    -5  17  12  -22  48  UACC-257  25  20  23  -25  UACC-62  27  18 19 utilized to assess the cell growth and viability. The results were reported as mean-graph of the percentage growth of the treated cells, and displayed as percentage growth inhibition (GI%) caused by the test pyridines (Tables 1 and 2). Investigation of data in Tables 1 and 2 revealed that the examined pyridines exhibited distinctive patterns of sensitivity and selectivity against the different NCI cancer cell panels.
Further investigation of results in Tables 1 and 2 unveiled that all cell lines of the leukemia subpanel were sensitive to six tested pyridines 5a, 5e, 5f, 5h, 5j and 5l with GI ranging from 10% to 91%. It is noteworthy that only non-small cell lung cancer A549 and NCI-H522 cells were sensitive to all the tested pyridines with GI% range of 10-61% and 24-60%, respectively. Additionally, leukemia SR (except 5i), leukemia HL-60 (except 5d) and colon cancer HT29 (except 5i) cell line were susceptible to nine tested pyridines. The most susceptible cell lines towards the impact of pyridines 5a and 5l are displayed in Figure 2.
In vitro anti-proliferative activity against A549 and HCT-116 cell lines All newly synthesized pyridines 5a-l were examined for their antiproliferative activity towards two cancer cell lines: non-small cell lung cancer A549 cell line and colon cancer HCT-116 cell line. The MTT colorimetric assay was adopted to assess the anti-proliferative activity as described by Mosmann 29 . Doxorubicin was used as a control in this assay. The results were expressed as median growth inhibitory concentration (IC 50 ) values that represent the compound concentration required to produce a 50% inhibition of cell growth after 48 h of incubation ( Table 3).
The results of the MTT assay listed in Table 3 suggested that the examined pyridines 5a-l exhibited excellent to moderate growth inhibitory activity against the tested A549 and HCT-116 cancer cell lines. Also, HCT-116 cells were found to be more sensitive to the impact of the tested compounds than A549 cells, except compound 5j which is more effective towards A549 cells. Interestingly, compound 5l emerged as the most active one towards both A549 and HCT-116 cell lines with IC 50 values equal  3.22 ± 0.2 and 2.71 ± 0.16 mM, respectively, which are comparable to those of Doxorubicin: 2.93 ± 0.28and 3.10 ± 0.22, respectively.

In vitro cytotoxicity towards non-tumorigenic human WI-38 cells
The cytotoxic activity of all synthesized pyridines 5a-l were assessed against non-tumorigenic human lung fibroblast WI-38 cell line to investigate their safety, using the MTT colorimetric assay 29 . The results were expressed as IC 50 values and the calculated selectivity index are presented in Table 4.
The examined pyridines 5a-l displayed non-significant cytotoxic impact towards human lung fibroblast WI-38 cell line with IC 50 values spanning from 63.48 to 151.08 mM, thereby providing a good safety profile as anticancer agents with selectivity index range (6.3-17.6).

Induction of apoptosis in colorectal cancer HCT-116 cells
To investigate the mechanism of antitumor activity of the target pyridines and in continuation of our efforts to develop potent pro-apoptotic anticancer agents [35][36][37][38][39] , the ability of sulfonamide 5l to provoke apoptosis in HCT-116 cells was evaluated.

Effects on mitochondrial apoptosis pathway proteins Bcl-2 and Bax
Bcl-2 and Bax are two discrete members of a gene family involved in the regulation of cellular apoptosis known as BcL-2 family, which finely tune the apoptotic switch on/off mechanism and considered as an important gatekeeper to the apoptotic response. While Bcl-2 protein is functionally characterized as an apoptosissuppressing factor, the Bax protein is more functionally characterized as an apoptosis-promoting factor. So, the intracellular Bax/ Bcl-2 ratio can profoundly influence the ability of a cell to respond to an apoptotic signal 40,41 .
In this study, treatment of HCT-116 cells with the IC 50 of pyridine 5l (IC 50 ¼ 3.22 ± 0.25 mM) resulted in a significant up-regulation of the expression level of the pro-apoptotic Bax protein by 6fold compared to untreated control, with a concomitant significant decrease in the expression level of the anti-apoptotic Bcl-2 protein by approximately 75% compared to control (Table 5). These results revealed that pyridine 5l significantly boosted the Bax/Bcl-2 ratio 25-fold in compared to control.
Effect on the level of cytochrome C The interplay between the pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins triggers the activated Bax to bind to the mitochondrial outer membrane which induces the opening of the mitochondrial voltage-dependent anion channel (VDAC), resulting in the release of cytochrome C from mitochondria into cytosol where it activates the caspase-dependent signaling and subsequent apoptosis. Involvement of cytochrome C release from mitochondria is an indicator of activation of the intrinsic apoptotic pathway 42 .
Herein, we assessed the expression level of cytochrome C to assure the adoption of the intrinsic pathway. As shown in Table 6, the level of cytochrome C was induced significantly higher (12folds) in HCT-116 cells treated with pyridine 5l, compared to untreated control (Table 6).

Effect on the level of p53
One of the major apoptosis signaling pathways involves the p53 tumor suppressor. The ability of p53 to control apoptosis in response to abnormal proliferative signals and stress is crucial for its tumor suppression role. p53 tumor suppressor protein is a nuclear transcription factor that regulates the expression of a wide variety of genes involved in apoptosis. p53 is able to induce Bax oligomerization and cytochrome c release from mitochondria 43 .
The effect of pyridine 5l on p53 expression in HCT-116 cells was evaluated in this study. Results in Table 5 highlighted that treatment of HCT-116 cells with pyridine 5l led to 21-fold enhanced expression levels of p53, compared to control (Table 6).
Effects on the levels of active caspase-3 and caspase-9 Caspases, cysteine-dependent aspartate-directed proteases, are key factors in apoptotic cell death that have been shown to play an important role in cleavage of vital structural and regulatory  proteins important for cells survival, so activation of caspases is a hallmark for apoptosis induction 44 . The leading upstream caspases are caspase-9 in the intrinsic pathway and caspase-8 in the extrinsic pathway, where both converge to caspase-3 which is the key executioner of apoptosis 45 .
In comparison with the untreated control, the expression levels of active caspase-3 and caspases-9 in HCT-116 cells were 5.1-and 2.5-fold increased, respectively, in response to pyridine 5l treatment with its IC 50 concentration (Table 6).

Cell cycle analysis
Targeting the cell cycle of cancer cells has emerged as a promising approach for cancer therapy 46 . In the current study, pyridine 5l was examined for its effect on the cell cycle distribution in HCT-116 cells (Figure 3). The results of the DNA flow cytometric assay showed that treatment of HCT-116 cells with pyridine 5l at its IC 50 concentration for 24 h resulted in a significant 7.3-fold increased percentage of HCT-116 cells at Sub-G 1 , with concurrent significant reduction in the G 2 -M phase by approximately 2.2-fold. Both arrest of G 2 -M phase and alteration of the Sub-G 1 phase are considered significant remarks for pyridine 5l to induce apoptosis in HCT-116 cells.
AnnexinV-FITC/propidium iodide analysis of apoptosis Translocation of phosphatidylserine (PS) from the inner to the outer membrane leaflet of the cell is an early apoptotic event, which could be detected by fluorescein-labeled annexinV (annexinV-FITC), a Ca 2þdependent phospholipid-binding protein with high affinity for PS. Combined with propidium iodide PI (an indicator of cell integrity), a measure of percentage cell population in early apoptosis can be achieved. Cells displaying increased annexinV-FITC fluorescence without a concurrent increase in PI fluorescence are considered to be in early apoptosis, whereas an increase is seen in both fluorescence channels, signifies a late apoptosis 47 .
In this study, AnnexinV-FITC/PI dual staining assay was performed to evaluate the effect of compound 5l on both early and late apoptosis percentages in HCT-116 cells (Figure 4, Table 7). As presented in Figure 4, the assay outcomes clearly indicate that the treatment of HCT-116 cells with 5l resulted in a significant increase in the percentage of annexinV-FITC-positive apoptotic cells, including both the early and late apoptotic phases (LR; from 1.18% to 6.79%, and UR; from 0.81% to 8.97%), that represents about eightfold total increase in comparison with control (Table 7).

Conclusion
In summary, herein we report the synthesis of novel series of 1-(2methyl-6-arylpyridin-3-yl)-3-phenylureas 5a-l. All the prepared pyridins were evaluated for their in vitro anticancer activity against two cancer cell lines: non-small cell lung cancer A549 cell line and colon cancer HCT-116 cell line. Compound 5l was found to be the most active congener towards both A549 and HCT-116 cell lines with IC 50 values equal to 3.22 ± 0.2 and 2.71 ± 0.16 mM, respectively, which are comparable with those of Doxorubicin: 2.93 ± 0.28 and 3.10 ± 0.22, respectively. Furthermore, compound 5l stood out as the most potent pyridine derivative (mean % GI ¼ 40), at US-NCI Developmental Therapeutic Program anticancer assay, with broad-spectrum antitumor activity against the most tested cancer cell lines from all subpanels. The ability of sulfonamide 5l to provoke apoptosis in HCT-116 cells was evaluated. Results revealed that pyridine 5l significantly boosted the Bax/Bcl-2 ratio 25-fold compared to control. Also, the expression levels of cytochrome C, p53, active caspase-3 and caspases-9 in HCT-116 cells were 12-, 21-, 5.1-and 2.5-fold increased, respectively, in response to pyridine 5l treatment. Furthermore, treatment of HCT-116 cells with pyridine 5l at its IC 50 concentration resulted in a significant 7.3fold increased percentage of HCT-116 cells at Sub-G 1 , with concurrent significant reduction in the G 2 -M phase by approximately 2.2-fold, in addition to a significant increase in the percentage of annexinV-FITC-positive apoptotic cells, including both the early and late apoptotic phases (LR; from 1.18% to 6.79%, and UR; from 0.81% to 8.97%) that represent about eightfold total increase in comparison with control.

Disclosure statement
No potential conflict of interest was reported by the authors.