Introduction

Pyrimidine derivatives were found to possess a wide range of biological activities such as antifungal, anti-tubercular, antibacterial (Ahluwalia and Madhu, 1996; Singh et al., 2005; Shamroukh et al., 2005; Pasha et al., 2005), antiviral (Lin et al., 1988; Holy et al., 2002; Rakesh et al., 2002; Joule et al., 1995), anticancer (Skibo et al., 2002), antioxidant (Vidal et al., 2000), and anti-inflammatory (Alam et al., 2013). Four-substituted and 4,6-disubstituted pyrimidines bonded to a saturated heterocyclic unit at C-2 position of pyrimidine ring and related compounds have attracted much attention as potent 5-HT2A receptor ligands, including atypical antipsychotic, antidepressants, anxiolytics (Mokrosz et al., 1997; Glennon et al., 1997; Miyamoto et al., 2000), dual EGFR/ErbB-2 kinase inhibitors (Li et al., 2012), as potent HIV-1 NNRTIs (Yu et al., 2009), and TRPV1 antagonists (Stec et al., 2008). Some of the anticancer agents such as fluorouracil, gemcitabine, capecitabine cytrabine, cladribine and bleomycin had pyrimidine ring (Fig. 1).

Fig. 1
figure 1

Reported structures of some anticancer agents having pyrimidine ring and the synthesized title compounds (5–16)

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Epidermal growth factor receptor tyrosine kinase (EGFR-TK) is one of the important therapeutic targets in human cancers. EGFR belongs to the ErbB family of TK proteins which includes four members: ErbB1/HER1, ErbB-2/Neu/HER2, ErbB-3/HER3 and ErbB-4/HER4 (Normanno et al., 2003). The participation of the EGFR family of TKs in cancer proliferation suggests that an inhibitor, which blocks the TK activity of the entire EGFR family, could have substantial therapeutic potential (Mendelsohn and Baselga, 2000). Gefitinib, erlotinib, lapatinib, canceratinib, PD 153035, AG 1295, AG 1385, etc. are examples of EGFR-TKIs, a biological target for human cancer. Owing to the significant biological activities of pyrimidine nucleus, we report herein the synthesis of some novel 4,6-disubstituted pyrimidine derivatives 5–16 (Scheme 1 ) derived starting from 2,4-dichloropyrimidine and subsequently screened for their cytotoxicity properties. Further, these compounds were evaluated for their anticancer activity on six cancer cells, including SIHA (cervical cancer), PANC-1 (pancreatic cancer), MDA-MB-231 (breast cancer), IMR 32 (neuroblastoma), DU 145 (prostate cancer) and A549 (lung adenocarcinoma). Although several EGFR-TKIs have been clinically validated for the treatment of cancer patients, yet the search for new active molecules against EGFR-TK is still continuing. Hence we have selected EGFR-TK as putative target for molecular docking studies and explored the binding mode of our ligands with active site of EGFR-TK.

Scheme 1
scheme 1

Synthesis of 4,6-disubstituted pyrimidine derivatives (5–16). Reagents and conditions i 4-N-Boc-Amio piperidine, DIPEA, THF, rt, 4h; ii a–d, Pd2(dba)3, X-Phos, DIPEA, RT, 4h; iii TFA, DCM, rt, 10 min; iv e–f, DIPEA, reflux, 10 min

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Results and discussion

Chemistry

The synthesis of 4,6-disubstituted pyrimidine derivatives 5–16 is shown in Scheme 1. The reaction of 2,4-dichloropyrimidine (1) with tert-butylpiperidin-4-ylcarbamate in the presence of diisopropylamine at room temperature afforded pyrimidine chloride derivative 2. Suzuki coupling of chloride intermediate 2 with various boronic acids (a–d) was carried out at room temperature in presence of Pd2(dba)3, X-Phos, diisopropylamine in tetrahydrofuran (THF) to obtain corresponding compounds 3a–d in 85–95 % yield. De-BOC of 3a–d was done by using trifluroacetic acid (TFA) at 0 °C for 10 min to isolate compounds 4a–4d as their respective TFA salts. The reaction of compounds 4a–d with isocyanates (e–f) in the presence of N,N-diisopropylethylamine (Hunig’s base) with reflux for 10 min, resulted in urea derivatives 5–16 (Table 1). The yield of the products varied from 90 to 95 %.

Table 1 Synthesis of 4,6-pyrimidine derivatives (5–16)
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All the synthesized compounds were well characterized by 1H & 13C-NMR, FT-IR and mass spectrometry. IR data of compounds 5–16 showed a strong –C=O stretching band around 1615–1657 cm−1 and a strong -NH stretching band at 3295–3338 cm−1. All final compounds have strong absorption around 3060–3435 cm−1 and around 1590 cm−1, which are evidence for aromatic C–H and aromatic C–C bonds respectively. All these compounds have shown a singlet in the range of δ 8.5–8.9 ppm of corresponding pyrimidine nuclei and another singlet in the range of δ 8.4–8.6 ppm of corresponding urea linkage (–NH proton), and signals for some protons in the range of δ 3.2–4.5 ppm, corresponding piperidine nuclei. These data clearly confirm the presence of specific functional groups present in final synthesized compounds.

In vitro biological activity

The compounds 5–16 (up to 300 μg/mL concentrations) were tested for cytotoxicity using brine shrimp lethality assay (Meyer et al., 1982) and compared with the activity of podophyllotoxin (Table 2). The compounds 6, 11, 14 and 15 were showed ED50 values of 3.50, 3.69, 3.78 and 4.49 µg/mL, respectively and displayed equipotent activity with respect to the standard Podophyllotoxin (ED50 = 3.69 µg/mL). The compounds 5 (ED50 = 27.86 µg/mL), compound 10 (ED50 = 39.49 µg/mL), compound 16 (ED50 = 39.64 µg/mL) were found to be exhibited moderate cytotoxicity. The remaining analogs in the series were less toxic.

Table 2 Brine shrimp lethality data of compounds 5–16
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Among the tested analogs, pyrimidine ring flanked by 4-phenoxyphenyl 6 (with R1=3-chlorophenyl on piperidine ring), thiophene 11 (with R1=4-methylbenzoate on piperidine ring) and 2-fluoro-4-methoxyphenyl 14 and 16 (with R1=4-methylbenzoate and 3-chloro phenyl on piperidine ring respectively) were exhibited cytotoxicity like Podophyllotoxin. The exact mechanism of cytotoxic activity between 4-phenoxyphenyl (with R1=3-chlorophenyl on piperidine ring), thiophene (with R1=4-methylbenzoate on piperidine ring) and 2-fluoro-4-methoxyphenyl (with R1=4-methylbenzoate and 3-chlorophenyl on piperidine ring) substitution on the pyrimidine/piperidine ring was not established earlier, so further we made an attempt to find out structural activity relationship (SAR) with cytotoxicity and anticancer activities.

The compounds were further evaluated for anticancer activity in six cancer cell lines (SIHA, PANC-1, MDA-MB-231, IMR-32, DU145, and A549). Three response parameters such as growth inhibitory activity (GI50), total growth inhibition (TGI), and lethal concentration (LC50) were calculated for each cell line, using Doxorubicin and Combretastatin as positive controls and data presented in Table 3. Different concentrations of compounds were studied for growth inhibition in cancer cell lines, and found to be exhibited various levels of growth arrest vary from cell line to cell line. The compound 14 showed better efficacy in inhibiting cell proliferation of cervical cancer and prostate cancer, followed by the compound 6 with GI50 range from 0.1 to 0.4 µM, the compound 14 required 1.3 µM for TGI in DU145 but more than 100 µM in SIHA. The compound 16 was showed maximum growth inhibition in PANC 1 as well A549 with GI50 of 0.47 and 0.11 µM, TGI of 1.6 and 4.3 µM respectively. The compound 7 was most effective inhibitor of MDA-MB-231 with GI50 of 0.2 and TGI of 3.1 µM whereas compound 6 was highly potent inhibitor of IMR 32 with GI50 of 0.65 and TGI of 4.6 µM correspondingly. Even though positive controls such as Doxorubicin and Combretastatin were highly potent growth inhibitors than tested compounds but exhibited severe toxicity with LC50 range of 1–5 µM. In the present study, all the compounds from 5 to 16 were less toxic than Doxorubicin and Combretastatin which might give a better therapeutic index in the treatment of cancer, which is very promising for this new group of compounds.

Table 3 In vitro anticancer activity of pyrimidine-piperidine derivatives (5–16)
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SAR developed from the anticancer data showed that pyrimidine derivatives having R (substitution) with 2-fluoro-4-methoxyphenyl responsible for maximum activity followed by 4-phenoxyphenyl and 2-benzthiazole while R1 (substitution) with 4-methoxyphenyl showed maximum activity followed by 4-methylbenzoate and 3-chlorophenyl.

Molecular docking studies

EGFR-TKs are believed to have an essential role in the instigation and the growth of the numerous cancers (Mostafa et al., 2013). Moreover compounds bearing pyrimidine rings were also reported as inhibitors of EGFR-TKs and few with molecular docking to understand the key interactions (Mao et al., 2013). Guided by these facts, we carried out the molecular docking studies to explore the possible mode of action of compounds 5–16 as EGFR-TKase inhibitors (Fig. 2). The X-ray crystallographic structure of EGFR-TK (PDB Code: 2J5F) was utilized for the current docking studies (Gangjee et al., 2013). The binding site of the co-crystallized ligand (N-[4-(3-bromophenylamino) quinazolin-6-yl] acrylamide) is well defined by the hydrophobic cavity lines by Lys745, Glu762, Met766, Leu788, Thr790, Met793, Cys797 and Thr854 (Blair et al., 2007; Ahsan et al., 2013). All the molecules were docked against EGFR-TKase (PDB Code: 2J5F) using XP-Glide-5.0 implemented in Maestro-8.5. Docked poses of the molecules were carefully analyzed to understand the crucial interaction responsible for the activity and found that the compound 5 and 6 were displayed interactions with EGFR-TK. Both 5 and 6 were exhibited hydrophobic interaction with Leu792 and Met793 and might be crucial for anticancer activity. The other interactions of compound 5 were as followed, H-bonding interaction with Cys797 and a π–π interaction with Phe723 (Fig. 3a), and compound 6, displayed two H-bonding interaction with Lys875 (Fig. 3b). The compounds 7, 8 and 9 were displayed hydrophobic interaction with Leu777 and Leu778 (Fig. 3c, d). The ATP binding pocket of EGFR-TK is considerably hydrophobic, thus hydrophobic interactions plays an important role in affinity toward EGFR-TK. Moreover it was proposed that at least two hydrogen bonds are required for small molecules to act as EGFR-TK inhibitor (Zuccotto et al., 2010). In the present study, compounds 5, 6, 7, 8 and 9 might be showed hydrophobic interactions towards ATP binding pocket to explored as EGFR-TK inhibitors. All the other compounds were exhibited unusual interactions with EGFR-TK and might be specific for other targets. Attempts are in progress to find out SAR with therapeutic targets in cancer.

Fig. 2
figure 2

The binding mode of ligands (5–16) with the EGFR-TK active site

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Fig. 3
figure 3

3D Binding mode of compounds against EGFR-TK active site: a Compound 5 b. Compound 6 c Compound 7, d Compound 9

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Conclusion

A series of novel 4, 6-disubstituted pyrimidine derivatives (5–16) were synthesized with good yields and were screened for their in vitro cytotoxicity and anticancer activity. The present study revealed that four compounds (5, 11, 14 and 15) were potent cytotoxic molecules in brine shrimp lethality assay. The compounds were also studied for in vitro anticancer properties using six different cancer cell lines viz SIHA, PANC-1, MDA-MB-231, IMR-32, DU145 and A549. The compound 14 was effective inhibitor of SIHA and DU145, whereas compound 16 in Panc 1 and A549, compound 7 in MDA-MB-231 and compound 6 in IMR 32 respectively. Molecular docking studies were carried out using an X-ray crystallographic structure of EGFR-TK to explore the possible mode of action of compounds as EGFR-TK inhibitors. Among potent anticancer compounds in the present study, compound 6 was found to be exhibited hydrophobic interaction with Leu792 and Met793 whereas compound 7 was with Leu777 and 778. This binding pattern with EGFR-TK might play a crucial role in its inhibition and anticancer activities. The present work provides strong incentive for further development of 4,6-disubstituted pyrimidine derivatives as potential antitumor agents for the treatment of metastatic cancer.

Materials and methods

All the chemicals and solvents used were purchased either from Fluka or Merck. All the reagents were of analytical grade. Thin layer chromatography (TLC) was performed on the E. Merck AL silica gel 60 F254 plates and visualized under UV light. The IR spectra were recorded on a Perkin Elmer FT-IR spectrometer. The 1H NMR spectra were recorded in CDCl3 on a Varian EM-360 spectrometer operating at 400 MHz. The 13C NMR spectra recorded in CDCl3 on a Varian EM-360 spectrometer operating at 100 MHz. The chemical shifts were reported in δ (ppm) using TMS as an internal standard. The mass spectra were recorded on Agilent ion trap MS. All the appropriate boronic acids (a–d) and isocyanates (e–f) used for the preparation of 5–16 were purchased from commercial sources. All computational analysis were carried out on a Dell Precision T3400n workstation with Intel core 2 quad processor, 8 GB RAM, 500 GB hard disk, running on the operating system Red Hat Enterprise 5.0 Linux (RHEL 5.0) platform. Molecular docking simulations were carried out using Protein preparation wizard, Ligprep and Glide module implemented in Maestro-8.5 (Schrödinger LLC).

Synthesis of tert-butyl 1-(6-chloropyrimidin-4-yl) piperidin-4-ylcarbamate (2)

To a solution of tert-butylpiperidin-4-ylcarbamate (4 g, 20 mmol) in THF (80 mL) was added N,N-diisopropylethylamine (3.87 g, 30 mmol) followed by 2,4-dichloropyrimidine (2.95 g, 20 mmol). The resulting mixture was stirred at room temperature for 4 h. The reaction mixture was evaporated under reduced pressure and then water was added. The resulted off-white solid was filtered and dried in vacuum to afford compound 2 (Yield: 5.6 g, 92 %, off-white solid); 1H NMR (400 MHz, DMSO-d 6): δ 8.30 (s, 1H), 6.96 (s, 1H), 6.87 (d, 1H, J = 8.0 Hz), 4.27 (brs, 2H), 3.55 (brs, 1H), 3.08 (t, 2H, J = 7.0 Hz), 1.79 (d, 2H, J = 6.0 Hz), 1.38 (s, 9H), 1.33–1.23 (m, 2H). EI MS: m/z (rel. abund.%): 313.0 (M+, 100).

General method for the synthesis of compounds (3a–d)

To a solution of compound 2 (1 g, 3.2 mmol) in THF (15 mL) was added sequentially corresponding boronic acid (a–d, 4.8 mmol), DIPEA (6.4 mmol) followed by Pd2(dba)3 (10 mol%) and X-Phos (10 mol%) under argon. The resultanting reaction mixture was stirred at room temperature for 4 h. TLC indicated complete consumption of starting material. The reaction mixture was diluted with water (5 mL) and extracted with ethyl acetate (15 mL), the combined organic layer was washed with water (3 × 5 mL) followed by brine solution. The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated under reduced pressure, to obtain the crude compounds. The crude compounds were purified by column chromatography (Silica gel: 100–200 mesh, elluant: 1:4 of ethyl acetate: n-hexane) to isolate compounds 3a–d (Yield: 95, 90, 85, 88 % respectively).

tert-Butyl 1-(6-(4-phenoxyphenyl)pyrimidin-4-yl)piperidin-4-ylcarbamate (3a)

1H NMR (400 MHz, DMSO-d 6): δ 1.33-1.28 (m, 2 H), 1.39 (s, 9H), 1.81 (d, 2H, J = 4.0 Hz), 3.07 (t, 2H, J = 5.0 Hz), 3.56 (brs, 2H), 4.48 (d, 2H, J = 12.0 Hz), 6.87 (d, 1H, J = 8.0 Hz), 7.10 (t, 4H, J = 2.0 Hz), 7.21 (t, 1H, J = 12.0 Hz), 7.28 (s, 1H), 7.45 (t, 2H, J = 4.0 Hz), 8.19 (d, 2H, J = 4.0 Hz), 8.53 (s, 1H); EI MS: m/z (rel. abund.%) 447.2 (M+, 100).

Isopropyl 1-(6-(benzo[b]thiophen-2-yl)pyrimidin-4-yl)piperidin-4-ylcarbamate (3b)

1H NMR (400 MHz, DMSO-d 6): δ 1.35–1.30 (m, 2 H), 1.39 (s, 9H), 1.84 (d, 2H, J = 3.0 Hz), 3.10 (t, 2H, J = 6.0 Hz), 3.59 (brs, 1H), 4.46 (d, 2H, J = 12.0 Hz), 6.89 (d, 1H, J = 8.0 Hz), 7.44–7.39 (m, 2H), 7.50 (s, 1H), 7.90–7.87 (m, 1H), 8.01–7.99 (m,1H), 8.40 (s, 1H),8.50 (s, 1H); EI MS: m/z (rel. abund.%) 411.0 (M+, 100).

tert-Butyl 1-(6-(thiophen-2-yl)pyrimidin-4-yl)piperidin-4-ylcarbamate (3c)

1H NMR (400 MHz, DMSO-d 6): δ1.35-1.27 (m, 2H), 1.39 (s, 9H), 1.81 (d, 2H, J = 4.0 Hz), 3.07 (t, 2H, J = 6.0 Hz), 3.56 (brs, 1H), 4.42 (d, 2H, J = 6.0 Hz), 6.88 (d, 1H, J = 8.0 Hz), 7.20 (t, 1H, J = 8.0 Hz), 7.28 (s, 1H),7.69 (d, 1H, J = 4.0 Hz), 8.02 (d, 1 H, J = 4.0 Hz), 8.41 (s, 1H); EI MS: m/z (rel. abund.%) 361.2 (M+, 100).

tert-Butyl 1-(6-(2-fluoro-4-methoxyphenyl)pyrimidin-4-yl)piperidin-4-ylcarbamate (3d)

1H NMR (400 MHz, DMSO-d 6): δ 1.35–1.27 (m, 2 H), 1.38 (s, 9H), 1.81 (d, 2H, J = 4.0 Hz), 3.09 (t, 2H, J = 7.0 Hz), 3.56 (brs, 1H), 3.79 (s, 3H), 4.34 (d, 2H, J = 6.0 Hz), 6.87 (d, 1H, J = 8.0 Hz), 7.07–7.03 (m, 1H),7.11 (s, 1H), 7.28 (t, 1H, J = 10.0 Hz), 7.42 (m,1H), 8.58 (s, 1H); EI MS: m/z (rel. abund.%) 403.2 (M+, 100).

General method for the synthesis of compounds (4a–d)

A mixture of compound 3a–d (10 mmol), trifluroacetic acid (5 vol) was stirred at 0 °C to room temperature for 10 min. The reaction mixture was evaporated under vacuum and dried to obtain the compounds, 4a–d as TFA salts and were used in the next step without further purification.

1-(6-(4-Phenoxyphenyl)pyrimidin-4-yl)piperidin-4-amine (4a)

1H NMR (300 MHz, D2O): δ 1.76 (t, 2 H, J = 5.5 Hz), 2.23–2.20 (m, 3H), 3.24–3.16 (t, 1H, J = 12.0 Hz), 3.46 (t, 1H, J = 12.0 Hz), 3.68 (t, 1H, J = 14.0 Hz), 4.41 (d, 1H, J = 12.0 Hz), 5.16 (d, 1H, J = 18.0 Hz), 7.21 (t, 4H, J = 2.2 Hz), 7.32 (t, 1H, J = 7.5 Hz), 7.51 (t, 2H, J = 3.5Hz), 7.79 (d, 2H, J = 4.5 Hz), 8.61 (s, 1H); EI MS: m/z (rel. abund.%) 347.2 (M+, 100).

1-(6-(Benzo[b]thiophen-2-yl)pyrimidin-4-yl)piperidin-4-amine (4b)

1H NMR (300 MHz, D2O): δ 1.75–1. 68 (m, 2H), 2.30 (d, 2H, J = 4.5 Hz), 3.32–3.28 (m, 2H), 3.67 (brs, 1H), 3.67 (brs, 1H), 4-45–4.43 (m, 2H), 7.04 (s, 1H), 7.58–7.49 (m, 2H), 8.01–8.98 (m, 2H), 8.07 (s, 1H), 8.52 (s, 1H); EI MS: m/z (rel. abund.%) 311.2 (M+, 100).

1-(6-(Thiophen-2-yl)pyrimidin-4-yl)piperidin-4-amine (4c)

1H NMR (300 MHz, D2O): δ 1.51–1.40 (m, 2H), 2.01 (d, 2H, J = 4.5 Hz), 3.10 (t, 2H, J = 6.0 Hz), 3.38 (brs, 1H), 4.55 (d, 2H, J = 3.0 Hz), 7.24 (t, 1H, J = 4.5 Hz), 7.34 (s, 1H), 7.77 (d, 1H, J = 3.0 Hz), 8.04 (d, 1H, J = 3.0 Hz), 8.51 (s, 2H); EI MS: m/z (rel. abund.%) 261.2 (M+, 100).

1-(6-(2-Fluoro-4-methoxyphenyl)pyrimidin-4-yl)piperidin-4-amine (4d)

1H NMR (300 MHz, D2O): δ 1.78–1.63 (m, 2H), 2.25 (d, 2H, J = 6.0 Hz), 3.23 (t, 1H, J = 13.0 Hz), 3.40 (t, 1H, J = 6.0 Hz), 3.66–3.55 (m, 1H), 3.83 (s, 3H), 4.35 (d, 1H, J = 15.0 Hz), 5.15 (d, 1H, J = 6.0 Hz), 7.31–7.14 (m, 4.0 Hz), 8.62 (s, 1H); EI MS: m/z (rel. abund.%) 303.2.

General method for the synthesis of compounds (5–16)

To a stirred mixture of compounds 4a–4d (10 mmol) in N,N-diisopropylethylamine (10 Vol) was added appropriate isocyanates (e–f, 10 mmol) and refluxed for 10 min. The reaction mixture was evaporated under reduced pressure and diluted with water to obtain crude compounds (5–16). The crude compounds were purified by column chromatography (Silica gel: 100–200 mesh, elluant: 1:1, ethylacetate: n-hexane) to obtain compounds 5–16 as off-white solids. Yields of the products varied from 90 to 95 %.

1-(4-Methyl benzoate)-3-(1-(6-(4-phenoxyphenyl)pyrimidin-4-yl)piperidin-4-yl)urea (5)

IR (KBr): ν max1651 (C=O), 3326 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.44–1.39 (m, 2H), 1.94 (d, 2H, J = 4.5 Hz), 3.22–3.16 (m, 2H), 3.79 (s, 3H), 3.82–3.81 (m, 1H), 4.40 (brs, 2H), 6.40 (d, 1H, J = 6.0 Hz), 7.10 (d, 3H, J = 2.0 Hz), 7.22 (t, 1H, J = 6.0 Hz), 7.30 (brs, 1H), 7.46 (t, 2H, J = 3.0 Hz), 7.52 (d, 2H, J = 4.5 Hz), 7.84 (d, 2H, J = 3.0 Hz), 8.18 (d, 2H, J = 3.0 Hz), 8.57–8.56 (m, 1H), 8.80 (s, 1H); 13C NMR (400 MHz, DMSO-d 6 + TFA): δ 165.96, 161.65, 159.55, 158.89, 157.00, 155.84, 154.06, 145.13, 131.11, 130.37, 130.18, 128.92, 124.11, 121.64, 119.27, 118.02, 116.69, 97.94, 61.65, 46.32, 42.58, 31.52; EI MS: m/z (rel. abund.%) 524.2 (M+, 100); analy. found for C30H29N5O4–C 68.86, H 5.54, N 13.58, O 12.02 (%).

1-(3-Chlorophenyl)-3-(1-(6-(4-phenoxyphenyl)pyrimidin-4-yl)piperidin-4-yl)urea (6)

IR (KBr): ν max1657 (–C=O), 3331 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.41–1.37 (m, 2H), 1.92 (d, 2H, J = 3.0 Hz), 3.21 (t, 2H, J = 4.5 Hz), 3.83–3.80 (m, 1H), 4.42 (d, 2H, J = 4.5 Hz), 6.31 (d, 1H, J =  6.0 Hz), 6.93(d, 1H, J = 3.0 Hz), 7.10 (t, 4H, J = 1.5 Hz), 7.25–7.16 (m, 3H), 7.32 (s, 1H), 7.46 (t, 2H, J = 3.7 Hz), 7.67 (t, 1H, J = 1.5 Hz), 8.21 (d, 2 H, J = 3.0 Hz), 8.57 (d, 2H, J = 4.5Hz); 13C NMR (400 MHz, DMSO-d 6): δ 161.88, 160.99 158.55, 158.02, 155.96, 154.23, 141.95, 133.06, 132.34, 130.19, 130.15, 128.71, 123.97, 120.58, 119.16, 117.99, 116.94, 115.97, 97.62, 46.42, 42.36, 31.52; EI MS: m/z (rel. abund.%) 500.1 (M+, 100); analy. found for C28H26ClN5O2–C 67.46, H 5.04, N 14.20, O 6.20, Cl 7.10 (%).

1-(4-Methoxybenzyl)-3-(1-(6-(4-phenoxyphenyl)pyrimidin-4-yl)piperidin-4-yl)urea (7)

IR (KBr): ν max1620 (–C=O), 3295 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.33–1.25 (m, 2H), 1.86 (d, 2H, J = 4.0 Hz), 3.16 (t, 2H, J = 6.0 Hz), 3.73 (s, 3H), 3.74–3.73 (m, 1H), 4.13 (d, 2H, J = 2.0 Hz), 4.38 (d, 2H, J = 6.0 Hz), 5.95 (d, 1H, J = 8.0 Hz), 6.15 (t, 1H, J = 6Hz), 6.87 (d, 2H, J = 4.0 Hz), 7.10 (t, 4H, J = 2.0 Hz), 7.21–7.15 (m, 3H), 7.27 (s, 1H), 7.45 (t, 2H, J = 4.0 Hz), 8.20 (d, 2H, J = 4.0 Hz), 8.53 (s, 1H); 13C NMR (400 MHz, DMSO-d 6): ð 161.07, 160.76, 159.39, 159.01, 158.63, 158.49, 158.26, 157.87, 155.48, 152.06, 151.70, 132.93, 130.59, 130.19, 128.64, 125.03, 124.72, 120.10, 118.35, 117.11, 114.22, 113.90, 99.49, 55.16, 46.13, 42.63, 32.32; EI MS: m/z (rel.abund.%) 510.1 (M+, 100); analy. found for C30H31N5O3–C 70.60, H 6.11, N 13.79, O 9.50 (%).

1-(4-Methyl benzoate)-3-(1-(6-(benzo[b]thiophen-2-yl)pyrimidin-4-yl)piperidin-4-yl) urea (8)

IR (KBr): νmax1641 (–C=O), 3310 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.45–1.37 (m, 2H), 1.95 (d, 2H, J = 4.0 Hz), 3.26 (t , 2H, J = 6.0 Hz), 3.79 (s, 3H), 3.85–3.83 (m, 1H), 4.43–4.40 (d, 2H, J = 3.0 Hz), 6.40–6.38 (d, 1H, J = 8.0 Hz), 7.43–7.40 (m, 2H), 7.54–7.50 (t, 2H, J = 4.0 Hz), 7.84–7.82 (d, 2H, J = 4.0 Hz), 7.90–7.88 (m, 1H), 8.01–7.99 (m, 1H), 8.42 (s, 1H), 8.52 (s, 1H), 8.81 (s, 1H); 13C NMR (400 MHz, DMSO-d 6): δ 165.49, 161.41, 158.07, 156.84, 154.03, 145.08, 143.33, 140.06, 139.89, 130.33, 125.57, 124.76, 124.34, 123.44, 122.69, 121.66, 116.69, 97.18, 51.65, 46.40, 42.41, 31.50; EI MS: m/z (rel. abund.%) 488.1 (M+, 100); analy. found for C26H25N5O3S–C 64.15, H 5.06, N 14.37, O 9.88, S 6.54 (%).

1-(1-(6-(Benzo[b]thiophen-2-yl)pyrimidin-4-yl)piperidin-4-yl)-3-(3-chlorophenyl)urea (9)

IR (KBr): ν max1639 (–C=O), 33140 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.44–1.36 (m, 2H), 1.95 (d, 2H, J = 6.0 Hz), 3.24 (t, 2H, 6.0 Hz), 3.83–3.82 (m, 1H), 4.43 (d, 2H, J = 4.0 Hz), 6.32 (d, 1H, J = 8.0 Hz), 6.94 (d, 1H, J = 8.0 Hz), 7.25–7.16 (m, 2H), 7.43–7.40 (m, 2H), 7.54 (s, 1H), 7.67 (s, 1H), 7.90–7.88 (m,1H), 8.01–7.99 (m, 1H), 8.41 (s, 1H), 8.52 (s, 1H), 8.58 (s, 1H); 13C NMR (400 MHz, DMSO-d 6): δ 161.44, 158.15, 156.97, 154.26, 143.49, 141.98, 140.07, 139.90, 133.09, 130.2, 125.54, 124.74, 124.34, 123.37, 122.67, 120.60, 116.97, 115.99, 97.15, 46.41, 42.42, 31.56; EI MS: m/z (rel. abund.%) 464.1 (M+, 100); analy. found for C24H22ClN5OS–C 62.14; H 4.76; N 15.11; O 3.43; S 3.93; Cl 7.63 (%).

1-(4-Methoxybenzyl)-3-(1-(6-(benzo[b]thiophen-2-yl)pyrimidin-4-yl)piperidin-4-yl)urea (10)

IR (KBr): ν max1625 (–C=O), 3293 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.35–1.27 (m, 2H), 1.89 (d, 2H, J = 3.0 Hz), 3.20 (t, 2H, J = 6.0 Hz), 3.72 (s, 3H), 3.74 (m, 1H), 4.14 (d, 1H, J = 4.0 Hz), 4.40 (d, 2H, J = 6.0 Hz), 5.97 (d, 1H, J = 8.0 Hz), 6.17 (t, 1H, J = 6.0 Hz), 6.88 (d, 2H, J = 4.0 Hz), 7.18 (d, 2H, J = 4.0 Hz),7.42 (d, 2H, J = 4.0 Hz), 7.51 (s, 1H), 7.89–7.87 (m, 2H), 8.01–7.99 (m, 1H), 8.40 (s, 1H), 8.50 (s, 1H); 13C NMR (400 MHz, DMSO-d 6): δ 161.50, 158.22, 158.11, 157.40, 156.99, 143.50, 140.11, 139.96, 132.77, 128.40, 125.63, 124.83, 124.42, 123.42, 122.75, 113.67, 97.19, 55.07, 46.50, 42.59, 32.00; EI MS: m/z (rel. abund.%) 474.1 (M+, 100); analy. found for C26H27N5O2S–C 65.91, H 5.78, N 14.80, O 6.72, S 6.80 (%).

1-(4-Methyl benzoate)-3-(1-(6-(thiophen-2-yl)pyrimidin-4-yl)piperidin-4-yl)urea (11)

IR (KBr): ν max1650 (–C=O), 3316 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.43–1.35 (m, 2H), 1.94 (d, 2 H, J = 6.0 Hz), 3.21 (t, 2H, J = 6.0 Hz), 3.80 (s, 3H), 3.83–3.82 (m, 1H), 4.39 (d, 2H, J = 6.0 Hz), 6.39 (d, 1H, J = 4.0 Hz), 7.20–7.18 (t, 1H, J = 8.0 Hz), 7.31 (s, 1H), 7.52 (d, 2H, J = 4.0 Hz), 7.70–7.69 (d, 1H, J = 4.0 Hz), 7.84 (d, 2H, J = 4.0 Hz), 8.04 (d, 1H, J = 4.0 Hz), 8.44 (s, 1H), 8.90 (s, 1H); 13C NMR (400 MHz, DMSO-d 6): δ 165.96, 161.54, 158.03, 156.99, 154.02, 145.08, 143.22, 130.33, 129.37, 128.34, 126.78, 121.67, 116.71, 95.95, 51.65, 46.41, 42.33, 31.51. EI MS: m/z (rel. abund.%) 438.1 (M+, 100); analy found for C22H23N5O3S - C 60.36, H 5.33, N 15.98, O 10.96, S 7.37 (%).

1-(3-Chlorophenyl)-3-(1-(6-(thiophen-2-yl)pyrimidin-4-yl)piperidin-4-yl)urea (12)

IR (KBr): ν max1633 (–C=O), 3296 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.41–1.34 (m, 2H), 1.93 (d, 2H, J = 6.0 Hz), 3.20 (t, 2H, J = 6.0 Hz), 3.81 (brs, 1H), 4.39 (d, 2H, J = 6.0 Hz), 6.31 (d, 1H, J = 8.0 Hz), 6.94 (d, 1H, J = 8.0 Hz), 7.25–7.16 (m, 3H), 7.31 (s, 1H), 7.70–7.66 (m, 3H), 8.03 (d, 1H, J = 4.0 Hz), 8.43 (s, 1H), 8.58 (s, 1H); 13C NMR (400 MHz, DMSO-d 6): δ 161.62, 158.10, 157.06, 154.35, 143.24, 142.00, 133.16, 130.30, 129.46, 128.44, 126.86, 120.73, 117.05, 116.09, 96.02, 46.47, 42.46, 31.61; EI MS: m/z (rel. abund.%) 414.1 (M+, 100); analy. found for C20H20ClN5OS–C 58.05, H 4.87, N 16.90, O 3.89, S 7.74, Cl 8.55 (%).

1-(4-Methoxybenzyl)-3-(1-(6-(thiophen-2-yl)pyrimidin-4-yl)piperidin-4-yl)urea (13)

IR (KBr): ν max1622 (–C=O), 3295 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.32–1.24 (m, 2H), 1.86 (d, 2H, J = 4.0 Hz), 3.15 (t, 2H, J = 5.0 Hz), 3.72 (s, 3H), 3.73 (brs, 1H), 4.13(d, 2H, J = 2.0 Hz), 4.36 (d, 2H, J = 6.0 Hz), 5.95 (d, 1H, J = 8.0 Hz), 6.15 (t, 1H, J = 6.0 Hz), 6.88–6.85 (d, 2H, J = 6.0 Hz), 7.20–7.15 (m, 3H), 7.29 (s, 1H), 7.69 (d, 1H, J = 4.0 Hz), 8.03 (d, 1H, J = 4.0Hz), 8.42 (s, 1H); 13C NMR (400 MHz, DMSO-d 6): δ 161.55, 158.03, 158.00, 157.27, 156.94, 143.21, 132.73, 129.35, 128.31, 126.75, 113.59, 95.92, 55.00, 46.42, 42.46, 31.93. EI MS: m/z (rel. abund.%) 424.1 (M+, 100); analy. found for C22H25N5O2S–C 62.31, H 2.01, N 16.54, O 7.57, S 7.60 (%).

1-(4-Methylbenzoate)-3-(1-(6-(2-fluoro-4-methoxyphenyl)pyrimidin-4-yl)piperidin-4-yl)urea (14)

IR (KBr): ν max1582 (–C=O), 3338 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.43–1.36 (m, 2H), 1.94 (d, 2H, J = 6.0 Hz), 3.22 (t, 2H, J = 5.0 Hz), 3.79 (s, 3H), 3.91–3.90 (m, 1H), 4.31 (d, 2H, J = 4.0 Hz), 6.41 (d, 1H, J = 8.0 Hz), 7.07–7.04 (m, 1H), 7.15 (s, 1H), 7.29 (t, 1H, J = 10 Hz), 7.43–7.41 (m, 1H), 7.52 (d, 2H, J = 4.0 Hz), 7.84 (d, 2 H, J = 4.0 Hz), 8.60 (s, 1H), 8.81 (s, 1H); 13C NMR (400 MHz, DMSO-d 6): δ 166.14, 160.36, 159.30, 158.80, 158.29, 157.79, 155.82, 155.03, 154.28, 151.79, 151.58, 148.18, 145.23, 130.47, 121.86, 119.48, 117.38, 117.29, 116.77, 115.60, 113.46, 103.39, 56.08, 51.70, 45.69, 31.68; EI MS: m/z (rel. abund.%) 480.1 (M+, 100); analy. found for C25H26FN5O4–C 62.63, H 5.48, N 14.61, O 13.36, F 3.95 (%).

1-(3-Chlorophenyl)-3-(1-(6-(2-fluoro-4-methoxyphenyl)pyrimidin-4-yl)piperidin-4-yl) urea (15)

IR (KBr): ν max1634 (–C=O), 3302 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.43–1.35 (m, 2H), 1.93 (d, 2H, J = 6.0 Hz), 3.22 (t, 2H, J = 5.0 Hz), 3.80 (s, 3H), 3.82–3.81 (m, 1H), 4.32 (d, 2H, J = 6.0 Hz), 6.32 (d, 1H, J = 8.0 Hz), 6.94–6.91 (m, 1H), 7.09–7.05 (m, 1H), 7.30–7.16 (m, 4H), 7.42–7.27 (m, 1H), 8.61 (s, 1H), 7.66–7.65 (m, 1H), 8.57 (s,1H); 13C NMR (400 MHz, DMSO-d 6): ð 161.28, 157.73, 155.92, 155.44, 154.23, 152.70, 141.95, 133.05, 130.19, 126.13, 120.58, 117.30, 116.98, 115.97, 114.47, 102.54, 102.42, 55.68, 46.27, 42.45, 31.42; EI MS: m/z (rel. abund.%) 456.1 (M+, 100); analy. found for C23H23ClFN5O2 C 60.60, H 5.07, F 4.21, N 15.33, O 7.05, Cl 7.74 (%).

1-(4-Methoxybenzyl)-3-(1-(6-(2-fluoro-4-methoxyphenyl)pyrimidin-4-yl)piperidin-4-yl) urea (16)

IR (KBr): ν max1615 (–C=O), 3319 (–NH) cm−1; 1H NMR (400 MHz, DMSO-d 6): δ 1.34–1.23 (m, 2H), 1.86 (d, 2H, J = 4.0 Hz), 3.17 (t, 2H, J = 5.0 Hz), 3.75 (s, 4H), 3.79 (s, 3H), 4.13 (d, 2H, J = 4.0 Hz), 4.29 (d, 2H, J = 6.0 Hz), 5.95 (d, 1H, J = 8.0 Hz), 6.15 (t, 1H, J = 6.0 Hz), 6.88 (d, 2H, J = 6.0 Hz), 7.08–7.04 (m, 1H), 7.17 (t, 3H, J = 2.5 Hz), 7.29–7.24 (dd, 1H, J = 7.0, 3.8 Hz), 7.42–7.40 (m, 1H), 8.59 (s, 1H); 13C NMR (400 MHz, DMSO-d 6): δ 161.38, 158.03, 157.29, 155.96, 155.88, 154.95, 153.51, 133.76, 128.67, 117.48, 117.24, 116.51, 116.43, 115.43, 113.59,101.62, 55.00, 53.51, 46.40, 42.28, 41.76, 31.84; EI MS: m/z (rel. abund.%) 466.1 (M+, 100); analy. found for C25H28FN5O3–C 64.48, H 6.07, N 15.02, O 10.33, F 4.09 (%).

Brine shrimp lethality assay

Brine shrimp (Artemia Salina) nauplii were hatched using brine shrimp eggs in a conical shaped vessel (1 L), filled with sterile, artificial sea water of 38 g  L−1 of sea salt and adjusted to pH 8.5 using 1 N NaOH and kept under constant aeration for 48 h. After hatching, ten nauplii were drawn through a pipette and placed in each vial containing 4.5 mL brine solution and added 300 µg mL−1 concentration of compounds and the final volume was made up to 5 mL using brine solution and maintained 37 °C for 24 h under the light of incandescent lamps. Assays were carried out in duplicates. The percentage lethality was determined by comparing the mean surviving larvae of test and control tubes. Podophyllotoxin was used as a positive control.

In vitro anticancer activity

The cancer cell lines, SIHA, PANC 1, MDA MB 231, IMR 32, DU 145 and A549 used in this study were procured from the American Type Culture Collection (ATCC), USA. The synthesized test compounds were evaluated for their in vitro antiproliferative activity on these six different human cancer cell lines. All the cell lines were grown in Dulbecco’s modified Eagle’s medium (containing 10 % FBS in a humidified atmosphere of 5 % CO2 at 37 °C). Cells were trypsinized when sub-confluent and seeded in 96-well plates in 100 μL aliquots at plating densities depending on the doubling time of individual cell lines. A protocol of 48 h continuous drug exposure was used, and a sulforhodamine B (SRB) cell proliferation assay was used to estimate cell viability or growth. The microtiter plates were incubated at 37 °C, 5 % CO2, 95 % air, and 100 % relative humidity for 24 h prior to addition of experimental drugs and were incubated for 48 h with different doses (0.01, 0.1, 1, 10, 100 µM) of prepared derivatives. After 48 h incubation at 37 °C, cell mono layers were fixed by the addition of 10 % (wt/vol) cold trichloroacetic acid and incubated at 4 °C for 1 h and were then stained with 0.057 % SRB dissolved in 1 % acetic acid for 30 min at room temperature. Unbound SRB was washed with 1 % acetic acid. The protein bound dye was dissolved in 10 mM tris base solution for OD determination at 510 nm using a microplate reader (Enspire, Perkin Elmer, USA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth was calculated at each of the drug concentration levels. Percentage growth inhibition was calculated as:

$$\left[{\frac{{\left( {{\rm{Ti}}\,{\rm{ - }}\,{\rm{Tz}}} \right)}}{{\left( {{\rm{C}}\,{\rm{ - }}\,{\rm{Tz}}} \right)}}} \right]{\rm{ \,\times\, }}{\rm{100}}\,{\rm{for}}\,{\rm{concentrations}}\,{\rm{for}}\,{\rm{which}}\,{\rm{Ti >\!/= Tz}}$$
$$\left[ {\frac{{\left( {{\rm{Ti}}\,{\rm{ - }}\,{\rm{Tz}}} \right)}}{{{\rm{Tz}}}}} \right]\, \times \,{\rm{100}}\,{\rm{for}}\,{\rm{concentrations}}\,{\rm{for}}\,{\rm{which}}\,{\rm{Ti}}\,{\rm{ < }}\,{\rm{Tz}}{\rm{.}}$$

Three dose response parameters were calculated for each experimental agent (Boyd and Paul, 1995). Growth inhibition of 50 % (GI50) was calculated from [(Ti−Tz)/(C−Tz)] ×100 = 50, which is the drug concentration resulting in a 50 % reduction in the net growth increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration of the drug resulting in a 50 % reduction in cell growth at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells (toxicity) following treatment was calculated from [(Ti − Tz)/Tz] × 100 = −50. Values were calculated for each of these three parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested. Standard error (±) values were also presented in the Table 3.

Molecular docking

Molecular docking simulations were carried out using Maestro-8.5 (Schrodinger LLC) installed on RHEL-5 operating system. The structures of molecules were sketched using build module implemented in Maestro-8.5. All the sketched molecules were prepared for docking using Ligprep module. The X-ray crystal structure of EGFR-TKase (PDB Code: 2J5F) was downloaded from protein data bank (http://www.rcsb.org/pdb/explore.do?structureId=2j5f). The protein was prepared for docking using the Protein Preparation Wizard, keeping OPLS2001 Force field for minimization. The grid was constructed, keeping the reference ligand (34-JAB) as the center of the grid using GLIDE (software v5.5) Receptor Grid Generation module. The ligands were then docked with the receptor grid generated using XP-Docking implemented in Glide-5. The docked conformers were analyzed through XP-Visualizer.