Introduction

The pyrimidine scaffold represents a widespread nucleus in a lot of pharmaceutically active compounds and suggestive of a broad range of pharmacological behavior; not only acting as kinase inhibitor but also stand for a well-designed option as initial material for the synthesis of pharmaceutical derivatives that possess diverse activities and show good protection profiles (Selvam, James, & Dniandev, 2012 a). One of the most important reports is that pyrimidine and fused pyrimidine derivatives acquire a variety of biological activities (Ismail, Ali, Ibrahim, Serya, & Ella, 2016). Various pyrimidine derivatives were tested for their anticancer activities in vitro and in vivo leading to hopeful lead motifs (Balbi et al., 2011). In the present study, we focus on the most significant aspect of pyrimidine, especially N₅-(3-substituted benzylidene)-N₄-phenyl pyrimidine-4,5-diamine [5A-5F] / N₅-(2-substituted benzylidene)-N₂,N₂-dimethyl-N₄-phenyl pyrimidine-2,4,5-triamine [5a-5f]. A large number of pyrimidine and their derivatives are reported to possess a wide range of biological activities such as anticancer (Ahmed, Mohamed, Ahmed, & Ahmed, 2009) , anxiolytic (Popik et al., 2006) , and antimicrobial (Selvam & Kumar, 2010 a; Selvam & Kumar, 2010 b) . Moreover, the pyrimidine derivatives are used as inhibitors of kinases, mainly to inhibit mediating signals of mitogenic and other events of cellular activities (Saravanan et al., 2018; Selvam, Karthik, Kumar, & Ali, 2012 b) such as differentiation, cell proliferation, migration, metabolism, and immune response. Also, many of the observed data indicate that these derivatives might block the proliferation of various cancer cell lines (Kamal et al., 2013; Prabhu, Panneerselvam, Shastry, Sivakumar, & Pande, 2015) . The present review spotlight on pyrimidines as c-Src kinase and p38 MAP Kinase complex inhibitors. The c-Src kinase and p38 MAP Kinase complex signaling is a major resource of cellular processes like migration, survival, proliferation, and succession of a cell. The deregulation processes of mutation or overexpression of kinases is experiential in an integer of disease condition plus cancer and immunological disorders (Frey et al., 2008). Based on their catalytic action, the regulation of varied physiological mechanisms depends on movement, cell creation, differentiation, and metabolism (Arora & Scholar, 2005) . The observed documents clearly showed that alterations in kinase activities such as mutations, hyperactivation, and hyperproduction lead to the disorder of cascades of cell signaling and enhance several diseases such as diabetes, inflammation, neurological disorders, and cancer (Arora et al., 2005) . Thus kinases and their role were considered very important and targeting these kinases may lead to the anticancer drug development regimen. Based on the aforementioned report, we had planned to synthesize novel N₅-(3-substituted benzylidene)-N₄-phenyl pyrimidine-4,5-diamine [5A-5F]

/ N₅-(2-substituted benzylidene)-N₂,N₂-dimethyl-N₄-phenyl pyrimidine-2,4,5-triamine [5a-5f] scaffolds, which may display as a clinically significant anticancer agent and the derivatives might direct towards the improvement in bioactivity.

Experimental Section

Materials and Methods

All laboratory-grade solvents used were purchased from SD Fine Chemicals and Merck, Mumbai, India. The Dr. Reddys laboratories Hyderabad, India had gifted the samples of standard Ciprofloxacin and Ketoconazole. Melting points were determined in open glass capillary tubes and were uncorrected. A thin layer chromatography plate was used for the analysis of purity and iodine chamber and UV lamp were used for visualization of TLC spots. The FT-IR spectrophotometers [KBr pellets] were used for the recording of IR spectra. Bruker DPX-300 NMR spectrometer in CDCl₃ was used for the recording of ¹H-NMR spectra [ppm scale] with internal standard tetramethylsilane. JEOL-SX-102 mass spectra were used for the impact of ionization of electron. Perkine Elmer 240C analyzer was used for elemental analyses within the theoretical values of ± 0.4 %.

General Procedure

The Figure 2 (Cui et al., 2018) to construct pyrimidine nucleus: In the first step, the equimolar quantity of formimidamide/ 4-(dimethylamino) benzimidamide [10 mmol], sodium ethoxide, (0.5 g in 5 mL water), and 25 ml of ethanol were stirred mechanically for 5 min. Then, ethyl 3-(dimethylamino)-2-nitroacrylate (10 mmol) was added and heated for 1 hr at 40 °C. It was recrystallized using ethyl alcohol to get pure 5-nitropyrimidin-4-ol/2-(4-(dimethylamino)phenyl)-5-nitropyrimidin-4-ol (1), and it was monitored by TLC. A mixture of 5-nitropyrimidin-4-ol/2-(4-(dimethylamino)phenyl)-5-nitropyrimidin-4-ol (1) underwent chlorination by using POCl₃ (10 mmol) and 10 mL of N, N-Diisopropylethylamine [DIEA]. Further, it was refluxed for 30 min on 80 °C, and it was recrystallized using ethyl alcohol to get pure product 4-chloro-5-nitropyrimidine/4-(4-chloro-5-nitropyrimidin-2-yl)-N,N-dimethylaniline. (2) A mixture of 4-chloro-5-nitropyrimidine/4-(4-chloro-5-nitro pyrimidin-2-yl)-N,N-dimethylaniline (2) (10 mmol) reacted with aniline (10 mmol) in 10 mL of DMF at 80 °C and quenched in ice water to get the precipitate. It was then filtered, washed with water, and recrystallized using ethyl alcohol to get 5-nitro-N-phenylpyrimidin-4-amine/2-(4-(dimethylamino)phenyl)-5-nitro-N-phenyl pyrimidin-4-amine (3) A mixture of 5-nitro-N-phenylpyrimidin-4-amine/2-(4-(dimethylamino)phenyl)-5-nitro-N-phenyl pyrimidin-4-amine (3) (10 mmol) reacted with Zn (3 mmol) and CuSO₄ (3 mmol) in the water on a magnetic stirrer for 3 h under room temperature to give N₄-phenylpyrimidine-4,5-diamine/ 2-(4-(dimethylamino)phenyl)-N₄-phenylpyrimidine-4,5-diamine (4). A mixture of 10mmol N₄-phenylpyrimidine-4,5-diamine/ 2-(4-(dimethylamino)phenyl)-N₄-phenylpyrimidine-4,5-diamine (4) It was added to a solution of an appropriate substituted aromatic aldehyde (10 mmol) in glacial acetic acid (20 mL) containing anhydrous sodium acetate (0.82 g, 10 mmol). It was heated under reflux for 2 h, and then under reduced pressure, the solvent was evaporated. The produced solid was dried to get a crystallized form of N₅-(3-substituted benzylidene)-N₄-phenyl pyrimidine-4,5-diamine [5A-5F]/ N₅-(2-substituted benzylidene)-N₂,N₂-dimethyl-N₄-phenyl pyrimidine-2,4,5-triamine [5a-5f].

Detection Method

N₅-(4-hydroxybenzylidene)-N₄-phenylpyrimidine-4,5-diamine [5A]

IR: 3524 (OH), 3121 & 3059 (NH), 3023 (Ar-CH), 1647 (C=N), 1591 (C=C); ¹H NMR: 3.52 (s, 1H, =CH linkage), 4.21 (s, 1H, OH), 5.36 (s, 1H, NH), 6.82-7.95 (m, 11H, Ar-H); Mass: C₁₇H₁₄N₄O; calcd, 290 [M+], found, 290 [M+]; Elemental Analysis: calcd, C, 70.33; H, 4.86; N, 19.30; O, 5.51; found, C, 70.32; H, 4.83; N, 19.31; O, 5.54

N₅-(4-hydroxybenzylidene)-N₂,N₂-dimethyl-N₄-phenylpyrimidine-2,4,5-triamine [5a]

IR: 3361 (OH), 3032 (NH), 2980 (Ar-CH), 1660 (C=N), 1608 (C=C); ¹H NMR: 2.48 (s, 6H, CH₃), 3.63 (s, 1H, =CH linkage), 4.21 (s, 1H, OH), 5.31 (s, 1H, NH), 6.52-7.79 (m, 10H, Ar-H) Mass: C₁₉H₁₉N₅O; calcd, 333 [M+], found, 333 [M+]; Elemental Analysis: calcd, C, 68.45; H, 5.74; N, 21.01; O, 4.80; found, C, 68.47; H, 5.73; N, 21.10; O, 4.82

N₅-(4-fluorobenzylidene)-N₄-phenylpyrimidine-4,5-diamine [5B]

IR: 3126 (NH), 3086 (Ar-CH), 1656 (C=N), 1606 (C=C), 1166 (C-F); ¹H NMR: 3.26 (s, 1H, =CH linkage), 5.26 (s, 1H, NH), 7.36-7.96 (m, 11H, Ar-H); Mass: C₁₇H₁₃FN₄; calcd, 292 [M+], found, 292 [M+]; Elemental Analysis: calcd, C, 69.85; H, 4.48; F, 6.50; N, 19.17; found, C, 69.83; H, 4.46; F, 6.53; N, 19.14

N₅-(4-fluorobenzylidene)-N₂,N₂-dimethyl-N₄-phenylpyrimidine-2,4,5-triamine [5b]

IR: 3376 (NH), 3037 (Ar-CH), 1678 (C=N), 1619 (C=C), 1043 (C-F); ¹H NMR: 2.23 (s, 6H, CH₃), 3.24 (s, 1H, =CH linkage), 5.18 (s, 1H, NH), 6.61-7.83 (m, 10H, Ar-H); Mass: C₁₉H₁₈FN₅; calcd, 335 [M+], found, 335 [M+]; Elemental Analysis: calcd, C, 68.04; H, 5.41; F, 5.66; N, 20.88; found, C, 68.08; H, 5.43; F, 5.68; N, 20.87

N₅-benzylidene-N₄-phenylpyrimidine-4,5-diamine [5C]

IR: 3122 (NH), 3080 (Ar-CH), 1678 (C=N), 1641 (C=C); ¹H NMR: 3.32 (s, 1H, =CH linkage), 5.11 (s, 1H, NH), 7.34-8.12 (m, 12H, Ar-H); Mass: C₁₇H₁₄N₄; calcd, 274 [M+], found, 274 [M+] Elemental Analysis: calcd, C, 74.43; H, 5.14; N, 20.42; found, C, 74.45; H, 5.16; N, 20.41

N₅-benzylidene-N₂,N₂-dimethyl-N₄-phenylpyrimidine-2,4,5-triamine [5c]

IR: 3363 (NH), 3033 (Ar-CH), 1673 (C=N), 1594 (C=C); ¹H NMR: 2.23 (s, 6H, CH₃), 3.23 (s, 1H, =CH linkage), 5.13 (s, 1H, NH), 6.63-7.83 (m, 10H, Ar-H); Mass: C₁₉H₁₈ClN₅; calcd, 317[M+], found, 317 [M+]; Elemental Analysis: calcd, C, 71.90; H, 6.03; N, 22.07; found, C, 71.91; H, 6.05; N, 22.03

N₅-(4-nitrobenzylidene)-N₄-phenylpyrimidine-4,5-diamine [5D]

IR: 3118 (NH), 3080 (Ar-CH), 1641 (C=N), 1600 (C=C), 1519 & 1342 (NO₂); ¹H NMR: 3.17 (s, 1H, =CH linkage), 5.19 (s, 1H, NH), 6.98-7.94 (m, 11H, Ar-H); Mass: C₁₇H₁₃N₅O₂; calcd, 319 [M+], found, 319 [M+]; Elemental Analysis: calcd, C, 63.94; H, 4.10; N, 21.93; O, 10.02; found, C, 63.96; H, 4.12; N, 21.95; O, 10.07

N₅-(2-nitrobenzylidene)-N₂,N₂-dimethyl-N₄-phenylpyrimidine-2,4,5-triamine[5d]

IR: 3119 (NH), 3064 (Ar-CH), 1691 (C=N), 1644 (C=C), 1595 & 1366 (NO₂); ¹H NMR: 2.23 (s, 6H, CH₃), 3.39 (s, 1H, =CH linkage), 5.41 (s, 1H, NH), 6.54-7.83 (m, 10H, Ar-H); Mass: C₁₉H₁₈N₆O₂; calcd, 362 [M+], found, 362 [M+]; Elemental Analysis: calcd, C, 62.97; H, 5.01; N, 23.19; O, 8.83; found, C, 62.98; H, 5.05; N, 23.13; O, 8.83

N₅-(4-methoxybenzylidene)-N₄-phenylpyrimidine-4,5-diamine[5E]

IR: 3147 (NH), 3059 (Ar-CH), 1643 (C=N), 1591 (C=C), 1076 (C-O-C); ¹H NMR: 3.12 (s, 3H, OCH₃), 3.74 (s, 1H, =CH linkage), 5.15 (s, 1H, NH), 6.43-7.91 (m, 11H, Ar-H); Mass: C₁₈H₁₆N₄O; calcd, 304 [M+], found, 304 [M+]; Elemental Analysis: calcd, C, 71.04; H, 5.30; N, 18.41; O, 5.26; found, C, 71.06; H, 5.32; N, 18.40; O, 5.23

N₅-(4-methoxybenzylidene)-N₂,N₂-dimethyl-N₄-phenylpyrimidine-2,4,5-triamine[5e]

IR: 3119 (NH), 3062 (Ar-CH), 1698 (C=N), 1645 (C=C), 1071 (C-O-C); ¹H NMR: 2.03 (s, 6H, CH₃), 2.47 (s, 3H, OCH₃), 3.43 (s, 1H, =CH linkage), 5.05 (s, 1H, NH), 6.51-7.54 (m, 10H, Ar-H); Mass: C₂₀H₂₁N₅O; calcd, 347 [M+], found, 347 [M+]; Elemental Analysis: calcd, C, 69.14; H, 6.09; N, 20.16; O, 4.61; found, C, 68.43; H, 5.72; N, 21.07; O, 4.82

N₅-(4-methylbenzylidene)-N₄-phenylpyrimidine-4,5-diamine [5F]

IR: 3122 (NH), 3081 (Ar-CH), 1642 (C=N), 1623 (C=C); ¹H NMR: 2.31 (s, 3H, CH₃), 3.82 (s, 1H, =CH linkage), 5.34 (s, 1H, NH), 6.51-7.13 (m, 11H, Ar-H); Mass: C₁₈H₁₆N₄; calcd, 288 [M+], found, 288 [M+]; Elemental Analysis: calcd, C, 74.98; H, 5.59; N, 19.43; found, C, 74.96; H, 5.57; N, 19.44

N₅-(4-methylbenzylidene)-N₂,N₂-dimethyl-N₄-phenylpyrimidine-2,4,5-triamine[5f]

IR: 3365 (NH), 3035 (Ar-CH), 1682 (C=N), 1611 (C=C);¹H NMR: 2.32 (s, 6H, CH₃), 2.72 (s, 3H, CH₃), 3.12 (s, 1H, =CH linkage), 5.42 (s, 1H, NH), 6.52-7.32 (m, 10H, Ar-H); Mass: C₂₀H₂₁N₅; calcd, 331[M+], found, 331[M+]; Elemental Analysis: calcd, C, 72.48; H, 6.39; N, 21.13; found, C, 72.47; H, 6.35; N, 21.12

In Silico Screening Methods

The insilico modelling (Kunjiappan et al., 2019) of N₅-(3-methoxy benzylidene)-N₄-phenyl pyrimidine-4,5-diamine [S1-25]/ N₅-(2-bromo benzylidene)-N₂,N₂-dimethyl-N₄-phenyl pyrimidine-2,4,5-triamine [R1-25] docked with the active site of c-Src kinase, Human p38 MAP Kinase complex. The crystal structures of c-Src kinase, p38 MAP Kinase complex (PDB ID: 2OIQ and 3LFF) were obtained from the Protein Data Bank. The preparation of protein file and SKETCH module and all other parameters were assigned by Surflex-Dock program software.

Anticancer Activity

MTT test

In the MTT assay, HeLa cell line was used, and it was obtained from the National Cancer Institute. Cisplatin was used as a standard and the proceeding was followed by the standard literature protocol (Kunjiappan et al., 2020). Various concentrations of cells were used to treat with the synthesized compounds. The microplate reader was used to measure the absorbance at 570 nm.

Results and Discussion

Chemistry

The series of heterocycles, N₅-(3-substituted benzylidene)-N₄-phenyl pyrimidine-4,5-diamine [5A-5F]/ N₅-(2-substituted benzylidene)-N₂,N₂-dimethyl-N₄-phenyl pyrimidine-2,4,5-triamine [5a-5f], were synthesized by the reaction of formimidamide/4-(dimethylamino)benzimidamide with an appropriate solution of sodium ethoxide as presented in theFigure 2. The novel compounds were characterized by FTIR, ¹H-NMR, and mass spectroscopy. The IR spectrum of compounds [5A-5F]/ [5a-5f] showed bands of NH group at 3118-3385 cm^-1. In [5A-5F]/ [5a-5f], Ar-CH stretching bands appears at 2951-3089 cm^-1. The appearance of a strong intensity band in the IR spectra of compounds [5A-5F]/ [5a-5f] in the range of 3118-3385 cm^-1 attributable to -NH stretching and provides strong evidence for the conformation of the conversion chlorine to -NH. The proton magnetic resonance spectra of [5A-5F]/ [5a-5f] and their corresponding derivatives have been recorded in CDCl₃. In this spectra [5A-5F]/ [5a-5f], =CH linkage signals appear at 3.01-3.53 ppm. The presence of =CH linkage proton signals in the ¹H-NMR spectra of the final compounds confirms the formation of benzylidine moiety. All these observed facts clearly envisage the formation of N₅-(3-substituted benzylidene)-N₄-phenyl pyrimidine-4,5-diamine [5A-5F]/ N₅-(2-substituted benzylidene)-N₂,N₂-dimethyl-N₄-phenyl pyrimidine-2,4,5-triamine [5a-5f] as indicated in the Figure 2 and confirms the proposed structure [5A-5F]/ [5a-5f].

Molecular docking study

The PDB-ID: 2OIQ and 3LFF were utilized for molecular docking study, and it exposed that compounds 5A and 5c were active as an inhibitor of c-Src kinase [2OIQ] and 5f and 5b were active as an inhibitor of p38 MAP Kinase complex [3LFF]. The results are given in Table 1 and Figure 1. The title molecules of N₅-(3-substituted benzylidene)-N₄-phenyl pyrimidine-4,5-diamine [S1-25]/ N₅-(2-substituted benzylidene)-N₂,N₂-dimethyl-N₄-phenyl pyrimidine-2,4,5-triamine [R1-25] were screened for their anticancer activity against cervical HeLa (ME 180) cells. To test the anticancer activity, different concentrations (5, 12.5, 25, and 40µmol L^-1) were used, and drug concentration was plotted after a specified time. IC₅₀ was calculated between Cisplatin and synthesized compounds. The results are shown in Table 2. The data from Table 2 reveal that the compounds 5A, 5a, 5B, and 5b exhibited significant activity compared to that of Cisplatin towards the HeLa cell lines and 5c, 5C, 5D and 5d were inactive in the concentration. In comparison with NO_2, unsubstituted pyrimidine compounds such as 5E, 5e, 5F, and 5f showed reasonable activity. In the observed results, among all the compounds, 5A and 5b with IC₅₀ values of 12.41 and 12.15 showed that both electron-donating -OH and withdrawing -F group with para position enhanced anticancer activity. Thus, nitro and unsubtitutions in pyrimidine showed inactive and other derivatives are considered as a most potent analogue against cancer.

Conclusions

In this review, we have compiled and discussed specifically the anticancer potential of N₅-(3-substituted benzylidene)-N₄-phenyl pyrimidine-4,5-diamine [5A-5F]/ N₅-(2-substituted benzylidene)-N₂,N₂-dimethyl-N₄-phenyl pyrimidine-2,4,5-triamine [5a-5f] derivatives through the inhibition of dissimilar kinase enzymes and their synthetic strategies. The conformation and orientation supplies for kinase binding site through molecular modeling studies indicate the importance of substituent's [4-Hydroxy, 4-Fluoro, Unsubstituted, 4-Nitro, 4-Methoxy and 4-Methyl] effect. The finding of current studies could afford insight to a medicinal chemist and for clinical development of possible pyrimidine-based anticancer drugs.

Conflict of interest statement

The authors declare no competing financial interest.

Funding Support

None.