Design, synthesis and anti-inflammatory activity of pyrimidine scaffold benzamide derivatives as epidermal growth factor receptor tyrosine kinase inhibitors

Article abstract of DOI:10.1016/j.molstruc.2018.06.003

Novel serious of pyrimidine scaffold benzamide derivatives (9 a-k) were synthesized and characterized by IR, HRMS, and NMR. Docking study of compounds 9 g, 9 h exhibited H-bonding interacts with Met769 into ATP binding site of EGFR-TK which showed similar binding mode to Lapitinib (PDB code: 1M17). Results indicated the ability to potent and selective inhibitors of the Epidermal Growth Factor Receptor tyrosine kinase (EGFR-TK). The molecular electrostatic potential (MEP), frontier molecular orbitals (FMOs) and HOMO-LUMO energy gap of the title compounds were investigated by using the B3LYP/6-31G method. The synthesized compounds were screened for in vitro anti-inflammatory activity.

Full text of DOI:10.1016/j.molstruc.2018.06.003

Accepted Manuscript
Design, synthesis and anti-inflammatory activity of pyrimidine scaffold benzamide
derivatives as epidermal growth factor receptor tyrosine kinase inhibitors
K. Thirumurugan, Sivalingam Lakshmanan, Dharman Govindaraj, D. Sam Daniel
Prabu, N. Ramalakshmi, S. Arul Antony
PII:
S0022-2860(18)30704-X
10.1016/j.molstruc.2018.06.003
MOLSTR 25290
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 16 March 2018
Revised Date: 12 May 2018
Accepted Date: 1 June 2018
Please cite this article as: K. Thirumurugan, S. Lakshmanan, D. Govindaraj, D.S. Daniel Prabu, N.
Ramalakshmi, S. Arul Antony, Design, synthesis and anti-inflammatory activity of pyrimidine scaffold
benzamide derivatives as epidermal growth factor receptor tyrosine kinase inhibitors, Journal of
Molecular Structure (2018), doi: 10.1016/j.molstruc.2018.06.003.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Design, Synthesis and Anti-inflammatory Activity of Pyrimidine Scaffold Benzamide
Derivatives as Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors †
K. Thirumurugan a, Sivalingam Lakshmanan a, Dharman Govindarajb, D. Sam Daniel
Prabu a, N. Ramalakshmi a,*, S. Arul Antony a
a *Post-Graduate and Research Department of Chemistry, Presidency College, Chennai-05,
Tamil Nadu, India.
b Biomaterials in Medicinal Chemistry Lab, Department of Natural Products Chemistry, School
of Chemistry, Madurai Kamaraj University, Madurai, 625021, India.
*Email address: lachuchem666@gmail.com; Tel: +91 9843632843
Abstract
Novel serious of pyrimidine scaffold benzamide derivatives (9 a-k) were synthesized and
characterized by IR, HRMS, and NMR. Docking study of compounds 9 g, 9 h exhibited H-
bonding interacts with Met769 into ATP binding site of EGFR-TK which showed similar
binding mode to Lapitinib (PDB code: 1M17). Results indicated the ability to potent and
selective inhibitors of the Epidermal Growth Factor Receptor tyrosine kinase (EGFR-TK). The
molecular electrostatic potential (MEP), frontier molecular orbitals (FMOs) and HOMO-LUMO
energy gap of the title compounds were investigated by using the B3LYP/6-31G method. The
synthesized compounds were screened for in vitro anti-inflammatory activity.
Keywords: Microwave method; Pyrimidine scaffold benzamide; Molecular docking; DFT;
MEPs; EGFR-inhibitors; Anti-inflammatory.
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1. Introduction
Cancer is continuing to be a major health problem in developed as well as undeveloped
countries [1]. The great cancer incidence worldwide increases the search for new, safer and
efficient anticancer agents, aiming the prevention or the cure of this illness. Generally, curing
of cancer is difficult because of the side effect of drugs on the normal cells and makes some
other abnormalities in our body. Lung cancers are malignant tumours with poor prognoses
and ranked as the top cause of cancer-related deaths [2, 3]. Inhibitors of the EGFR PTK are
therefore expected to have great therapeutic potential in the treatment of malignant and
nonmalignant epithelial diseases [4, 5]. One of the most prominent protein kinases is the
endothelial growth factor receptor (EGFR) because that kinase is known to be involved in the
various cancer-associated processes of uncontrolled cell growth [6, 7]. EGFR inhibitors are
well-known active targets and to be efficient in the drug development for the treatment of
cancer [8-10]. EGFR-TK inhibitors are the second most essential drug targets have been
approved for the therapy of non-small cell lung cancer and this motivated inhibition of EGFR
signaling may not only active in anti-proliferative effects and have also been increased
sensitivity to cytotoxic therapies [11]. In recent days, various approaches have been
developed to small molecule inhibitors of the intrinsic tyrosine kinase domain-like Lapitinib
(a), Erlotinib (b), Gefitinib (c), Figure 1 which has been approved for the chemotherapeutic
treatment of patients with advanced non-small lung cancer [12] and also Lapatinib (a) is a
potent dual EGFR/ErbB2 inhibitor, recently approved by US Food and Drug Administration
(FDA) for the breast cancer therapy [13]. The epidermal growth factor receptor (EGFR) is
cellular trans-membrane tyrosine kinases that are over-expressed in a significant number of
human tumors (e.g., Colon, breast, ovarian and NSC lung cancer) [14]. Therefore, EGFR
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inhibitors represent a sensible approach for the development of novel anticancer therapies
which would act by competing with ATP for binding at the catalytic domain of their target
enzyme [15-17].
<< Figure 1 >>
In recent years, various nitrogen heterocyclic rings are known to exhibit interesting biological
activities. Pyrimidine is an important class of heterocyclic ring is found in vitamins like thiamine,
riboflavin and folic acid. During the last two decades, several pyrimidine derivatives have been
developed as chemotherapy of AIDS [18]. The previously reported literature presented that
different structured fused pyrimidine ring systems are highly active in numerous
chemotherapeutic activities such as anti-cancer [19, 20], antibacterial [21, 22], antifungal [23,
24], and anti-viral [25, 26] applications. The substituted pyrimidine ring systems were also
reported to possess excellent antitumor activity [27-29]. Literature evaluations have pointed out
the inherited antitumor potency of compounds containing pyrimidine and fused quinazoline
moieties [30-32]. FDA has been approved for several quinazoline derivatives as anticancer drugs
(e.g., Gefitinib, Erlotinib, Lapatinib and Vandetanib). Nowadays, the bio-medicinal activity of
quinazoline derivatives and drug development of novel quinazoline derivatives for the anticancer
therapy is a promising field [33-35].
The present work, we have carried out the chemical modifications on the general features
of quinazoline-containing compounds. These modifications involve a replacement of the furan
moiety in the quinazoline skeleton by pyrimidine scaffold benzamide moiety to develop more
effective target molecules utilizing a fragment-based drug design approach and biologically
evaluated their anti-inflammatory activity Figure 2.
<< Figure 2 >>
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2. Experimental
2.1. Materials and methods
All the solvents and chemicals were used analytical grade from SRL and Merck Ltd.
Microwave reactions were performed with a Biotage®. Initiator with a continuous focused
microwave power delivery system in a pressure glass vessel (10 mL) sealed with a septum under
magnetic stirring. TLC was performed on commercial Merck silica gel 60 F 254. Column
chromatography was carried out using silica gel 60-120 mesh. 1HNMR spectra were recorded in
CDCl3 and DMSO-d6 using TMS an internal standard with Bruker 300 MHz NMR spectrometer.
High-resolution mass spectra (HRMS) were measured on a Finnigan spectrometer.
2.2.
3-(Dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one (3)
The mixture of N, N-dimethylformamide dimethylacetal (0.02 mol), acetylpyridine (0.01
mol), and toluene (3.5 mL) in a 10 mL glass vial well equipped. The reaction mixture was
irradiated in the microwave synthesizer at 120 °C for 15 mins. After completion of the reaction
(indicated by TLC), the mixture was cooled down to room temperature. Petroleum ether (2 mL)
was added to the reaction mixture and kept stirring for 20 min. The resultant solid was collected
by filtration and washed with hexane to get compound (3). Melting point 83-85 °C, ES-MS
(M+1) found (m/z): 177.1.
2.3.
4-(Pyridin-3-yl)pyrimidin-2-amine (4)
The obtained compound 3 followed by 3-(dimethylamino)-1-(pyridin-3-yl) prop-2-en-1-one
(0.1 mol), guanidine nitrate (0.1 mol), sodium hydroxide (0.1 mol), and n-butanol (12.5 mL) was
irradiated in the microwave synthesizer at 90 °C for 20 mins. After completion of the reaction
mixture poured into ice-cold water and the resulting solid was filtered and washed with water.
The obtained solid of compound 4 (1.18 g) was dried under vacuum. Melting point 190-192 °C,
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ES-MS (M+1) found (m/z): 173.1.
2.4. 2-Bromo-1-methyl-4-nitrobenzene (6)
The mixture of P-nitrotoluene (0.05 mol) and 0.1 g of iron powder in a 250 mL round bottom
flask with compound 4 was heated at 80 °C with vigorous stirring and 3.0 mL (0.06 mol) of
bromine was added over the period of 30-45 mins and heating continued for the course of 2 hrs.
The reaction mixture was poured into 75 mL ice-cold 10% sodium hydroxide solution and the
solid was heated with 25 mL of glacial acetic acid. The aqueous layer was decanted and the
resulting solid was filtered and dried under vacuum to obtained 8.9 g (82 %) compound (6).
Melting point 74-76 °C, ES-MS (M+1) found (m/z): 217.2.
2.5. N-(2-Methyl-5-nitrophenyl)-4-(pyridin-3-yl) pyrimidin-2-amine (7)
The mixture of 4-(Pyridin-3-yl) pyrimidin-2- amine (0.378 g, 2.2 mmol), copper iodide
(0.5 mmol) and anhydrous K2CO3 (4 mmol), Dioxane (5 mL), 2-bromo-1-methyl-4-
nitrobenzene (2 mmol), and DMEDA (0.5 mmol) was irradiated in the microwave
synthesizer and heated at 120 °C for 30 mins. After completion of the reaction, ammonium
hydroxide and brine solution was added and then extracted with ethyl acetate. The organic
layer was dried over anhydrous sodium sulphate, the solvent was evaporated in buchi rotary
evaporator and the resulting solid was dried under vacuum. The crude was purified by using
silica gel column chromatography using ethyl acetate: chloroform (50:50) to yield 0.498 g
(79.0 %) of the imatinib intermediate compound as yellowish powder. Melting point194-196
1
°C. ES-MS (M+1) found (m/z): 308.1. H-NMR (DMSO-d6): 2.49(S, 3H); 7.32-7.37(m,1H);
7.40-7.48(m,1H); 7.49-7.51(m,1H); 7.85-7.88(m,1H); 8.02(m,1H); 8.48-8.51(m,1H); 8.57-
8.59(m,1H); 9.20(S,1H); 9.29 (S,1H).
2.6. 6-Methyl-N-(4-(pyridin-3-yl)pyrimidin-2-yl)benzene-1,3-diamine (8)
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The imatinib intermediate of N-(2-methyl-5-nitrophenyl) - 4-(pyridin-3-yl) pyrimidin-2-
amine (1 mmol), was dissolved in THF followed by activated zinc (4 mmol) and ammonium
chloride (10 mmol) were added. The reaction mixture was irradiated in the microwave
synthesizer at 80 °C for 10 mins. After completion of the reaction filtered and washed with THF.
The filtrate was evaporated and crude product was stirred with crushed ice. The resulting pale
yellow precipitate was dried under vacuum (Yield, 0.208 g, 75.13 %); m.p. 142-144o C. ES-MS
(M+1) found (m/z): 278.1. 1H-NMR (DMSO-d6): 2.16 (S, 3H); 4.44 (S, 2H); 6.39-6.41 (m,1H);
6.91-6.93 (m,1H); 7.15-7.18 (m,1H); 7.22-7.23 (m,1H); 7.92-7.94 (m,1H); 8.12 8.13 (m,1H);
8.46-8.48 (m,1H); 8.68-8.69 (m,1H); 9.26 (S,1H).
2.7.
(9 a-k)
The final target compounds were synthesized from 6-methyl- N-(4-(pyridin-3-yl) pyrimidin-
General procedure for the synthesis of pyrimidine scaffold benzamide derivatives
2-yl) benzene-1,3-diamine 8 (2 mmol), DMF (10 mL), and DIPEA (4 mmol) followed by
substituted aromatic acid (2 mmol) was added and stirred at room temperature for 1 hr. After
completion of the reaction mixture was poured into ice-cold water. The obtained yellow
precipitate washed with water and dried to get target titled product pyrimidine scaffold
benzamide derivatives (9 a-k).
2.7.1. N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)picolinamide (9 a)
Yellow solid, Yield 90 %, FTIR (KBr, ν cm-1): 2360ꢀ(-C–H), 1644 (-C=O), 1585ꢀ(-N–H),
1
1549ꢀ(C=N), 1132 (C–N). H-NMR (300MHz, CDCl3) δ 9.80 (s, 1H), 9.01(s, 1H), 8.50-
8.45 (m, 2H), 8.37-8.30 (m, 2H), 8.29-8.27 (m, 2H), 8.07 (d, J = 7.8 Hz, 1H), 7.66 (t, J =
7.7 Hz, 1H), 7.24 - 7.16 (m, 2H), 7.00 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 5.2 Hz, 1H), 2.10
(s, 3H). ES-MS (M+1) Calculated (m/z) 382.15, Found 383.1. Anal. Calcd. For
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C22H18N6O: C, 69.10; H, 4.74; N, 21.98%; Found: C, 69.06; H, 4.72; N, 21.99%.
2.7.2. 4-methyl-N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2yl)amino)phenyl)benzamide (9
b)
Yellow solid, Yield 88 %, FTIR (KBr, ν cm-1): 2361(-C–H), 1645 (-C=O), 1534 (-N–H),
1451(C=N), 1133 (C–N). 1H NMR (300 MHz, CDCl3) δ 8.98 (s, 1H), 8.44 (br, 1H), 8.34-
8.25 (m, 3H), 7.71 (s, 1H), 7.53 (d, J= 7.8 Hz, 1H), 7.19 -7.12 (m, 1H), 7.07-7.02 (m,
3H), 6.96-6.81 (m, 4H), 2.17 (s, 3H), 2.10 (s, 3H). ES-MS (M+1) Calculated (m/z)
395.17, Found 396.10. Anal. Calcd. For C24H21N5O:
Found: C, 72.86; H, 5.36; N, 17.73%.
C, 72.89; H, 5.35; N, 17.71%
2.7.3. 2-methyl-N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide
(9 c)
Yellow solid, Yield 92 %, FTIR (KBr, ν cm-1): 2361(-C–H), 1639 (-C=O), 1534 (-N–H),
1448 (C=N), 1133 (C–N). 1H NMR (300MHz, CDCl3) δ 8.91 (s, 1H), 8.39 (br, 1H),
8.33-8.23 (m, 3H), 7.66 (s, 1H), 7.24 (d, J = 7.4 Hz, 1H), 7.15-7.10 (m, 2H), 7.08-7.06
(m, 2H), 6.99-6.94 (m, 2H), 6.90-6.84 (m, 2H), 2.27 (s, 3H), 2.10 (s, 3H). ES-MS (M+1)
Calculated (m/z) 395.17, Found 396.12. Anal. Calcd. For C24H21N5O: C, 72.89; H, 5.35;
N, 17.71 % Found: C, 72.86; H, 5.36; N, 17.72 %.
2.7.4. 4-chloro-N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide (9
d)
Yellow solid, Yield 82 %, FTIR (KBr, ν cm-1): 2361(-C–H), 1644 (-C=O), 1535 (-N–H),
1
1449ꢀ(C=N), 1132 (C–N). H NMR (300 MHz, CDCl3) 8.99 (s, 1H), 8.44 (br,1H), 8.33-
8.21 (m, 3H), 7.96 (s, 1H), 7.56 (d, J = 8.1Hz, 2H), 7.16 (d, J = 6.9 Hz, 2H), 7.04-6.92
(m, 2H), 6.90-6.84 (m,2H). 2.10 (s, 3H). ES-MS (M+1) Calculated (m/z) 415.12, Found
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416.1. Anal. Calcd. For C23H18ClN5O: C, 66.43; H, 4.36; N, 16.84 % Found: C, 66.41; H,
4.35; N, 16.86 %.
2.7.5. 4-methoxy-N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide
(9 e)
Yellow solid, Yield 90%, FTIR (KBr, ν cm-1): 2361(-C–H), 1643 (-C=O), 1534 (-N–H),
1
1449 (C=N), 1119 (C–N). H NMR (300 MHz, CDCl3) δ 9.00 (s, 1H), 8.45 (br, 1H),
8.33-8.25 (m, 3H), 7.70 (s, 1H), 7.62 (d, J = 9.0 Hz, 2H), 7.20-7.15 (m, 1H), 7.08-7.02
(m, 2H), 6.96-6.91 (m, 2H), 6.82-6.70 (m, 2H), 3.62 (s, 3H), 2.10 (s, 3H). ES-MS (M+1)
Calculated (m/z) 411.17, Found 412.1. Anal. Calcd. For C24H21N5O2: C, 70.06; H, 5.14;
N, 17.02% Found: C, 70.09; H, 5.16; N, 17.01%.
2.7.6. 3-chloro-N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide (9
f)
Yellow solid, Yield 94 %, FTIR (KBr, ν cm-1): 2361(-C–H), 1662 (-C=O), 1549 (-N–H),
1444(C=N), 1127 (C–N). 1H NMR (300 MHz, CDCl3) δ 9.00 (s, 1H), 8.45 (br, 1H),
8.35-8.23 (m, 3H), 7.96 (s, 1H), 7.62 (s, 1H), 7.51 (d, J = 7.6 Hz, 1H), 7.26-7.15 (m, 3H),
7.11-7.04 (m, 2H), 6.97-6.86 (m, 2H), 2.10 (s, 3H). ES-MS (M+1) Calculated (m/z)
415.12, Found 416.1. Anal. Calcd. For C23H18ClN5O: C, 66.43; H, 4.36; N, 16.84%
Found: C, 66.41; H, 4.37; N, 16.82%.
2.7.7. 4-fluoro-N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide (9
g)
Yellow solid, Yield 87 %, FTIR (KBr, ν cm-1): 2361(-C–H), 1654 (-C=O), 1538ꢀ(-N–H),
1446ꢀ(C=N), 1124 (C–N). 1H NMR (300 MHz, CDCl3) δ 9.01 (s, 1H), 8.44 (br, 1H),
8.34-8.23 (m, 3H), 7.82 (s, 1H), 7.65 (dd, J = 8.5, 5.3 Hz, 2H), 7.18-7.14 (m, 1H), 7.02-
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6.93 (m, 3H), 6.92- 6.83 (m, 3H), 2.10 (s, 3H). ES-MS (M+1) Calculated (m/z) 399.15,
Found 400.1. Anal. Calcd. For C23H18FN5O: C, 69.16; H, 4.54; N, 17.53% Found: C,
69.19; H, 4.52; N, 17.55 %.
2.7.8. N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)quinoline-2-
carboxamide (9 h)
Yellow solid, Yield 84 %, (KBr, ν cm-1): 2362ꢀ(-C–H), 1644 (-C=O), 1549 (-N–H), 1448
1
(C=N), 1176 (C–N). H NMR (300 MHz, CDCl3) δ 10.00 (s, 1H), 9.02 (s, 1H), 8.55 (s,
1H), 8.44 (br, 1H), 8.33-8.27 (m, 2H), 8.17 – 8.09 (m, 3H), 7.91 (d, 6.0 Hz, 1H), 7.64 (d,
J = 8.2 Hz, 1H), 7.52 (t, J = 6.0Hz, 1H), 7.37 (t, J = 7.8 Hz, 1H), 7.27-7.15 (m, 2H), 7.00-
6.96 (m, 1H), 6.92-6.86 (m, 1H), 2.10 (s, 3H). ES-MS (M+1) Calculated (m/z) 432.17,
Found 433.1. Anal. Calcd. For C26H20N6O: C, 72.21; H, 4.66; N, 19.43% Found: C,
72.25; H, 4.67; N, 19.45 %.
2.7.9. N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)pyrazine-2-
carboxamide (9 i)
Yellow solid, Yield 90 %, FTIR (KBr, ν cm-1): 2361(-C–H), 1644 (-C=O), 1587ꢀ(-N–H),
1
1450 (C=N), 1128 (C–N). H NMR (300 MHz, CDCl3) 9.38 (s, 1H), 9.11 (s, 1H), 8.66-
8.55 (m, 2H), 8.45-8.17 (m, 3H), 7.45 (s, 1H), 7.29-7.22(m, 2H), 7.00-6.94 (m, 2H),
6.88-6.83 (m, 2H), 2.10 (s, 3H). ES-MS (M+1) Calculated (m/z) 383.15, Found 384.1.
Anal. Calcd. For C21H17N7O: C, 65.79; H, 4.47; N, 25.57% Found: C, 65.81; H, 4.49; N,
25.55 %.
2.7.10. 2-chloro-N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide
(9 j)
Yellow solid, Yield 92 %, FTIR (KBr, ν cm-1): 2361(-C–H), 1644 (-C=O), 1549ꢀ(-N–H),
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1449ꢀ(C=N), 1157 (C–N). 1H NMR (300 MHz, CDCl3) 8.93 (s, 1H), 8.42 (br, 1H), 8.35 –
8.24 (m, 2H), 7.89 (s, 1H), 7.53 (d, J= 6.6 Hz, 1H), 7.15-7.07 (m, 3H), 7.01-6.97 (m, 2H),
6.93-6.91 (m, 2H), 6.87-6.83 (m, 2H), 2.10 (s, 3H). ES-MS (M+1) Calculated (m/z)
415.12, Found 416.1. Anal. Calcd. For C23H18ClN5O: C, 66.43; H, 4.36; N, 16.84%
Found: C, 66.45; H, 4.35; N, 16.82 %.
2.7.11. N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide (9 k)
Yellow solid, Yield 90 %, FTIR (KBr, ν cm-1): 2361(-C–H), 1671 (-C=O), 1531(-N–H),
1447 (C=N), 1129 (C–N). 1H NMR (300 MHz, CDCl3) δ 9.00 (s, 1H), 8.45-8.35 (m, 3H),
8.27 (br, 2H), 7.88 (s, 1H), 7.66-7.61 (m, 2H), 7.38-7.16 (m, 5H), 6.95-6.91 (m, 2H), 2.10
(s, 3H). ES-MS (M+1) Calculated (m/z) 381.16, Found 382.1. Anal. Calcd. For
C23H19N5O: C, 72.42; H, 5.02; N, 18.36% Found: C, 72.43; H, 5.01; N, 18.35 %.
3. Result and Discussion
3.1.
Chemistry
The synthetic route for the newly synthesized compounds (9 a-k) was exhibited and outlined
in Scheme 1. The microwave-assisted strategy with Ullmann reactions was performed by using a
scientific microwave synthesizer. All the compounds were prepared by Loiseleur’s method [36],
microwave irradiation of N,N-dimethylformamide dimethylacetal, acetyl pyridine in toluene
afforded 3-(Dimethylamino)-1-(pyridin-3-yl)prop-2-en-1-one (3). The cyclisation of compound
(3) with guanidine nitrate in the presence of sodium hydroxide provided 4-(Pyridin-3-yl)
pyrimidin-2-amine (4) was obtained in good yields. 2-Bromo-1-methyl-4-nitrobenzene (6) was
prepared by reaction with iron powder and bromine. This reaction was performed in a
conventional way, due to the evaporation of HBr as the by-product.
The Ullmann type reaction was performed and 4-(Pyridin-3-yl) pyrimidin-2-amine (4) reacts
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under microwave irradiation with 2-Bromo-1-methyl-4-nitrobenzene (6) in the presence of
copper iodide gave excellent yields. In this reaction, copper iodide used as a catalyst, potassium
iodide as accelerant and N, N′-dimethylethylenediamine as a ligand and the reaction has
proceeded in dioxane at 120 °C for 30 mins gave N-(2-Methyl-5-nitrophenyl)-4-(pyridin-3-
1
yl)pyrimidin-2-amine (7) with 79 % yield. The H-NMR spectrum of compound (7) showed a
singlet (3H) at 2.49 and aromatic protons assigned in the region of 7.32 - 9.29 ppm. Reduction
reaction of nitro compounds was performed by Fe and Zn in the presence of acids. The
Zn/NH4Cl method efficiently produced a good quantity of the expected amine (8). This was
confirmed by the appearance of -NH2 group at 4.44 ppm. The final product (9 a-k) was easily
obtained by peptide coupling reaction of amine using TBTU and diisopropylethylamine
corresponding acid gave excellent yield. All target synthesized pyrimidine scaffold benzamide
derivatives (9 a-k) listed in Figure 3.
<< Scheme 1 >>
<< Figure 3 >>
4. Molecular docking
Molecular docking methodology was used to identify the structural features required for
EGFR receptor bearing quinazoline inhibitor (PDB ID: 1M17(Lapitinib)) were studied Figure 2.
It was found that a hydrogen bond acceptor of quinazoline ring interacts with Met769 [37]. The
crystal structure of epidermal growth factor receptor with erlotinib (Tarceva™) (PDB code:
1M17) [38-41].
The docking study of synthesized compounds with receptor RTK exhibited well-
established bonds with one or more amino acids in the receptor active pocket. The compounds 9
a, 9 d, and 9 h show very high binding energy with the RTK (1m17) receptor in Figure 4. The
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compound 9 a exhibits binding energy value -8.20 kcal/mol showed two H-bonding with
Glu734, Lys721 as well as the strong affinity of Asp831, Val702, which results from two
hydrophobic interaction its reduced the hydrogen bond interaction of compound 9 a with RTK
receptor. The quinoline containing compound 9 h also exhibits strong binding energy -
9.64kcal/mol which result four H-bonding with Asp831, Cys773, Met769, Met742 and as well as
Asp776, Asp831, Arg817, Leu649, Cys773, Val702 and Lys721 amino acids, which results
hydrophobic and electrostatic interactions to responsible for more activity of RTK receptor and
its indicated that the presence of the bi-cyclic hetero nucleus has higher affinity for RTK protein.
The electron donating group of compound 9 e shows binding energy -9.41 kcal/mol
exhibited H-bonding with Asp831, Asn818, Arg817, Cys773 showed four hydrogen bond. The
molecule was deeply embedded in the hydrophobic pocket formed by Phe771, Gly772, Glu780
exhibits hydrophobic bond with a chlorine atom, which result shows highest anti-inflammatory
activity. The electron withdrawing compound 9 f shows very low binding energy value -5.31
kcal/mol interact with amino acids Asp776, Cys773 shows two H-bonding compare to electron
donating compound 9 g. The compound 9 g exhibits highest binding energy value -8.18k cal/mol
interact with four hydrogen-bonded amino acid residue Met769, Cys773, Asp831, Thr766 and
inside of strong hydrophobic interaction with Arg817, Asp831, Cys773, Leu694, Val702 and
Lys721 amino acid residue, due to the presence of electron donating group in phenyl ring at para
position. The order of binding affinity of docked benzamide derivatives against the RTK receptor
is 9 h > 9 d > 9 a > 9 g > 9 e > 9 c > 9 i > 9 k > 9 j > 9 b and 9 f with the range of binding
energy being -9.64 to -5.96 kcal/mol. Generally, the functional groups (amine and ketone) are
forming the hydrogen bond with amino acid residues Figure 5. The free energy of binding (FEB)
of all compounds was calculated Table 1.
12

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<< Figure 4 >>
<< Table 1 >>
<< Figure 5 >>
5. Density Functional Theory
Theoretical calculations were performed by the density functional theory (DFT) method
at the B3LYP/ 6-31G level of theory in the Gaussian 03 package of programs [42].
Density functional theory (DFT) which is one of these methods has been widely used in
literature because of its efficiency and accuracy with respect to the evaluation of a number of
molecular properties. The highest occupied molecular orbital (HOMO) and the lowest
unoccupied molecular orbital (LUMO) reveal the biological potency of a molecule. Extending
the concept to binding in drug-receptor systems. The extents of these stabilizing interactions are
inversely related to the energy gap between the interacting orbitals. Higher HOMO energy and
lower LUMO energy in the drug molecule result in larger stabilizing interactions and hence
binding with the receptor. The orbital energies of both HOMO and LUMO and their gap were
calculated for all the molecules and are reported in Table 2. It is remarkable that compounds 9 c,
9 d, 9 g and 9 h having the lowest energy gap (∆E) of 1.3938, 0.8131, 0.9532, and 0.3913eV,
respectively, exhibit the highest anti-inflammatory activity [43-47].
We also obtained a plot of the HOMO and LUMO of the molecules of each group to analyze
the main atomic contributions for these orbitals. The importance of observing these plots was to
determine which atoms were located at the possible sites of electronic transfer between the
molecule under study and its biological target. The plots of the HOMO and LUMO of some
molecules obtained from DFT calculations are displayed in Figure 6. The results illustrated that
13

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HOMO molecular orbital of 9 a, 9 d, 9 g, and 9 h is mainly located in pyrimidine ring, indicating
the existence of possible reactive sites; consequently, electrophilic attacks might take place on
these sites. On the other hand, the LUMO is primarily concentrated on benzamide rings in which
the negatively charged polar residues of the receptor are favorable, but HOMO of compound 9 g
presents similar characteristic, while the LUMO changes significantly. In this case, the LUMO is
primarily located in Pyridine ring comparing with other compounds. This also implies that the
orbital interaction between the target compound and the aromatic ring or some other side of
residue chains of receptors is dominated by π–π or hydrophobic interaction. Moreover, anti-
inflammatory activity is dependent on the low-lying ELUMO because compound 9c has the lowest
ELUMO value [48]. The compound 9j size of the EHOMO lobes located on the pyridine ring was
significantly little smaller than that of compounds 9c which result compound 9c are likely to
exhibit strong charge transfer, and good photosensitizers and therefore more active than 9j.
<< Figure 6 >>
<< Table 2 >>
6. Molecular electrostatic potential surface (MEPs)
Molecular electrostatic potential (MEP) is related to the electronic density which is a very
useful descriptor in understanding sites for nucleophilic reactions or electrophilic attack. In other
words, they have been contemplated as an indicator of the reactivity regions of a target molecule;
hence they have been employed in order to study electron-donator and electron-acceptor
interactions between a drug and the amino acid residues of the target receptor [49].
As a result, this property was calculated for the target molecules at the B3LYP/6-31G
level of theory, and indicated by a colour range from -2.420 X 10-3 (deepest blue) to +2.420 X
10-3 (deepest red) in the corresponding maps displayed. MEP is created by mapping of the
14

ACCEPTED MANUSCRIPT
electrostatic potential on the total electron density of the molecules. For most active compounds
9 d, 9 g and 9 h which shows in red the nucleophilic sites (negative potential), located at the
oxygen atoms which result in H-bond interaction with the amino acid of target receptor. The
large electrophilic sites (positive potential) appeared on the hydrogen attached to aromatic ring
nitrogen consequence the blue cloud which is symbolized for electron deficient region. Due to
the accumulation of positive potential, these moieties exhibited hydrophobic interactions with the
aromatic residues of the active site in Figure 7. The other parts of molecule seem to potentially
be less active in chemical point of view.
<< Figure 7 >>
7. Anti-inflammatory activity
Heterocycles containing pyrimidine scaffold benzamide derivatives moiety are of great
interest because they exhibit useful biological activities and clinical applications.
All the synthesized compounds were screened for their in vitro anti-inflammatory activity
summarized in (Table 1 and Figure 8). The tested compounds, 9 a, 9 d, 9 g, 9 h, and 9 i were
found significant activity when compared to the standard drug Diclofenac at their lower
concentrations (10 µM, 50 µM, 100 µM). The substitution of electron donating group
Compounds 9 b, 9 c and 9 e decreases the activity compared with the standard. The compound 9
h much more active against anti-inflammatory in relation to Diclofenac sodium 89 % activity at
the minimum concentration of 10 µM. This may be the presence of bi-cyclic hetero nucleus of
quinoline moiety [50]. Whereas compound 9 a shows lower activity due to the presence of
mono-heterocyclic of pyridine moiety. From the present study, it was noted that compound 9 d, 9
g, and 9 h exhibited good anti-inflammatory.
<< Figure 8 >>
15

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8. Conclusion
In conclusion, novel pyrimidine scaffold benzamide derivatives have been synthesized
and characterized. Theoretical investigation some electronic parameters including energies of
HOMO-LUMO energy gaps and MEPs has exposed very clear trends between the molecular
descriptor and inhibitory activity. SAR for analysis of nature of the substituent present on
benzamide ring of lipophilic H-bond acceptor type functionalities like F, Cl at para position and
the aromatic ring of lipophilic hydrophobic region (i.e. electron withdrawing group, pyridine,
quinoline) of compounds 9 d, 9 g, and 9 h significant structural motives leading to the
moderately potent anti-inflammatory compounds. Moreover, molecular docking studies
confirmed that compounds 9 d, 9 g, and 9 h shared the similar binding pattern with Lapitinib in
the binding pocket of EGFR. The obtained results suggested that compounds 9 d, 9 g, and 9 h
may be a promising lead for further modifications towards epidermal growth factor receptor
(EGFR).
Acknowledgment
Authors have no finical interest.
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TABLE
Table.1. Docking results of novel biological active pyrimidine scaffold benzamide derivatives
against tyrosine kinases inhibitor [1m17.pdb] protein.
Table.2. Energies of both HOMO and LUMO and their gaps (in eV) calculated for all
synthesized compounds (9 a - k).
Table.1.
Binding energy No. of Hydrogen anti-inflammatory
Compounds
(kcal/mol)
-8.20
-6.28
-7.36
-9.41
-7.63
-5.96
-8.18
-9.64
-6.72
-6.35
-6.58
Bonding
(%) inhibition at 100µM
2
1
2
2
4
2
4
4
1
1
2
54.27
46.26
58.56
74.62
38.16
40.18
76.13
86.75
36.22
59.26
34.46
82.57
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
Diclofenac
sodium
-
-

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Table.2.
Compounds
E total (kcal) EHF
-1252.2638
-1275.5163
-1274.8248
-1695.3577
-1350.7017
-1695.7481
-1334.8580
-1267.9391
-1405.9406
-1695.5669
-1682.7321
ꢀ (Debye)
1.9632
2.2611
10.2240
8.2076
3.6058
4.8668
4.8082
8.5707
3.0235
7.6481
4.6209
EHOMO
-5.3747
-5.4395
-3.6240
-3.1938
-5.4512
-5.6518
-3.1249
-3.4621
-5.7211
-3.7769
-5.7089
ELUMO
-2.2362
-1.8773
-2.2302
-2.3807
-1.9037
-2.1997
-2.1717
-3.0708
-2.1331
-2.5486
-2.2204
∆E
3.1385
3.5622
1.3938
0.8131
3.5475
3.4521
0.9532
0.3913
3.594
9a
9b
9c
9d
9e
9f
9g
9h
9i
1.2283
3.4885
9j
9k

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FIGURE CAPTIONS
Fig.1. 4-Anilinoquinazolines EGFR-TK inhibitors, Lapitinib (a), Erlotinib (b), Gefitinib (c)
Fig.2. Design of target pyrimidine scaffolds benzamide compounds
Fig.3. Structure of synthesized pyrimidine scaffold benzamide derivatives (9 a-k)
Fig.4. Binding energy of synthesized compounds against tyrosine kinases inhibitor [1m17.pdb]
protein
Fig.5. Molecular docking and amino acids interaction of the most active compounds 9 a, 9 d, 9 g
and 9 h with RTK receptor. The amino acids involved in hydrogen (blue dashed line),
hydrophobic (white) and electrostatic (red dashed line) interactions are highlighted.
Fig.6. Schematic representation of HOMO and LUMO coefficient distribution of 9 a, 9 d, 9 g
and 9 h
Fig.7. Structure active relationships of Molecular electrostatic potential surface and amino acid
interaction against target EGFR receptor.
Fig.8. Anti-inflammatory activity of synthesized compounds (9 a-k)

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Fig.1.
b
a
c

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Fig.2.
Target compound
Lipophilic Aromatic insersion
Small Lipophilic fragment insersion
Lapitinib