Synthesis, characterization and molecular docking studies on some new

N-substituted 2-phenylpyrido[2,3-d]pyrimidine derivatives

Vivek B. Panchabhai*, Santosh R. Butle, Parag G. Ingole

Department of Pharmaceutical Chemistry, School of Pharmacy,

Swami Ramanand Teerth Marathwada University, Nanded-431606, Maharashtra, India.

*Corresponding Author E-mail: vivekpanchabhai82@gmail.com

ABSTRACT:

We report a novel scaffold of N-substituted 2-phenylpyrido(2,3-d)pyrimidine derivatives with potent antibacterial activity by targeting this biotin carboxylase enzyme. The series of eighteen N-substituted 2-phenylpyrido(2,3-d)pyrimidine derivatives were synthesized, characterized and further molecular docking studied to determine the mode of binding and energy changes with the crystal structure of biotin carboxylase (PDB ID: 2V58) was employed as the receptor with compounds 6a-r as ligands. The results obtained from the simulation were obtained in the form of dock score; these values represent the minimum energies. Compounds 6d, 6l, 6n, 6o, 6r and 6i showed formation of hydrogen bonds with the active site residues and van Der Walls interactions with the biotin carboxylase enzyme in their molecular docking studies. This compound can be studied further and developed into a potential antibacterial lead molecule.

KEYWORDS: Pyrido(2,3-d)pyrimidine, Docking score, Antibacterial, Biotin carboxylase, Molecular docking

INTRODUCTION:

In 2019, the World Health Organization (WHO) released an alarming report on microbial resistance to antibiotics especially in bacteria. The WHO report show a comprehensive picture of data obtained from 114 countries¹. According to it at least seven different common bacteria cause serious diseases like postoperative infections, blood stream infections, hospital acquired infections and gonorrhea have become drug resistant. Lot of information regarding the spread of drug resistant microorganisms are available still a big gap is observed in tracking antibiotic resistance. United States alone reported over two million deaths due to bacterial infection and 23,000 deaths due to drug resistant bacterial strains². One of the major reasons for rise of drug resistance is lack of new group of antibiotics. The penicillin was discovered during late 1920s, followed by cephalosporin in late 1940s. After a gap of two decades carbapenams and fluoroquinolones were discovered in the 80s.However, since then no such breakthrough discovery has been reported³. It is known that about 30-40 targets are generally harnessed for drug discovery process against the bacteria, thus there is an urgent need for smart molecules that target novel targets or multiple targets⁴.

The development of techniques like virtual screening, pharmacophore generation, and fragment-based drug discovery, advances in drug delivery has resulted in discovery of newer compounds and validation of newer targets and their proper administration to patients^5-6. One of the major metabolic pathways found in bacteria is the fatty acid synthesis pathway and has become a popular target among medicinal chemist. In a recent development, the enzyme biotin carboxylase (BC) was found to be a very attractive target for inhibiting the Acetyl CoA carboxylase mediated reaction. BC plays a vital role in Acetyl CoA carboxylase (ACC) catalysed reactions. Biotin carboxylases catalyse the carboxylation of biotin carboxyl carrier protein (BCCP)-biotin in the presence of bicarbonate to form the BCCP-biotin-CO₂. In the next step, BCCP-biotin-CO₂ transfer the carboxyl group to Acetyl-CoA and forms malonyl-CoA in presence of carboxyltransferase (CT) (Figure 1)⁷.

Figure 1: Reactions catalysed by biotin carboxylase and carboxyltransferase

The heterocyclic moieties like the pyridopyrimidines such as i to iv were reported to possess significant BC inhibitory activity (Figure 2)⁸. Mochalkin et al. showed that this class of compounds binds to the ATP binding site of bacterial BC did not bind or inhibit the human BC⁹. Several recent reports on the development of BC inhibitors have validated it as a potential drug target for the development of novel antibacterial compounds.

Figure 2: Pyridopyrimidine inhibitors of biotin carboxylase

Earlier, we have reported the development of various heterocyclic scaffolds like pyrimidines and benzazoles as anti-bacterial and anti-tubercular agents^10-12. In continuation to our efforts we aim to hit this newly validated target for the development of novel antibacterial agents on the basis of structural analogy and molecular docking studies. The pyridopyrimidine scaffolds provides for a wide range of activity like Bcr-abl inhibitors¹³, MexAB-OprM efflux pump inhibitor¹⁴, Adenosine kinase inhibitors¹⁵, PI3K/mTOR dual inihibition¹⁶, Jak1/2 inhibitor¹⁷, cell cycle inhibition¹⁸,antitubercular activity^19-20, antioxidant activity²¹ and antibacterial activity^22-27. Herein, we report the synthesis, characterization and molecular docking studies of some novel scaffold of N-substituted 2-phenyl pyridopyrimidine derivatives.

MATERIALS AND METHODS:

We have successfully attempted to synthesis of some N-substituted 2-phenylpyridopyrimidine derivatives (Scheme 1).

Scheme 1: Process for synthesis of substituted 2-phenylpyridopyrimidine derivatives. Reagents and conditions: (i) SOCl₂, CH₃OH, (ii) benzoyl chloride, CHCl₃, (C₂H₅)₃N, (iii) 2M NH₃/CH₃OH, 28% NH₄OH (iv) POCl₃ (v) (C₂H₅)₃N, DMF.

Chemicals, assay kits, reagents and materials were obtained from Sigma Aldrich, Germany, USA and Alfa Aeser, UK. Analytical grade solvents were obtained from the E. Merck, India. Melting points (mp) were detected with open capillaries using Thermonik Precision Melting point cum Boiling point apparatus (C-PMB-2, Mumbai, India) and are uncorrected. Chromatography of the synthesized intermediates and title compounds were performed on silica gel pre-coated plates (Merck: 100–200 mesh). Thin layer chromatography (TLC) was used to monitor the progress and/or completion of the reactions. IR spectra (KBr) were recorded on FTIR-8400s spectrophotometer (Shimadzu, Japan). ¹H and ¹³C NMR was obtained using a Bruker Advance-II 400 Spectrometer on 400 MHz using tetramethylsilane (TMS) as internal standard. All chemical shift values were recorded as δ (ppm), coupling constant value J is measured in hertz, the peaks are presented as s (singlet), d (doublet), t (triplet), br s (broad singlet), dd (double doublet), m (multiplet). The purity of compounds was controlled by thin layer chromatography (Merck, silica gel, HF254-361, type 60, 0.25 mm, Darmstadt, Germany). Mass spectra (ESI-MS) were recorded at ESI-MS spectrometer (DPX-400, Bruker, USA). Shimadzu High Performance Liquid Chromatography (HPLC) system with SPD-20A prominence Photodiode Array (PDA) detector and LC-20AB prominence LC solution software were used in this study.

1. Formation of methyl 2-aminonicotinate (2):

2-Aminopyridine 3-carboxyllic acid (1) (13.8g, 0.1 mole) was dissolved in 400 mL of anhydrous methanol, to this solution thionyl chloride (11.8mL, 14.03g, 0.1 mole) was added dropwise for half hour with constant stirring. On complete addition, it was refluxed for 12 hours after which the reaction mixture was cooled to room temperature. The solvent from the mixture was evaporated under vacuum and remaining residue was dissolved in chloroform. The chloroform layer was collected and washed with 5% HCl and 10% NaHCO₃ followed by water. This layer was further dried over sodium sulphate and concentrated to give methyl 2-aminonicotinate.

Yield: 71%. mp: 126-129^°C (ethanol). IR (KBr) cm^-1: 3424.1, 3319.2, 1655.1, 1618.5, 1601.1. ¹H NMR (DMSO-d6, 400 MHz) δ: 3.86(s, 3H, CH₃), 7.0(d, 1H, J=7.01, Ar), 7.44(dd, 1H, J=7.45, Ar), 8.69(s, 1H, Ar), 12.19(bs, 2H, NH₂). ¹³C NMR (DMSO-d6, 100 MHz) δ: 51.5, 110.0, 111.6, 139.7, 155.4, 158.0, 167.5. HRMS (EI) m/z calcd for C₇H₈N₂O₂152.1530, found 152.1525. Anal: C (55.26/55.11), H (5.30/5.25), N (18.41/18.37), O (21.03/21.09).

2. Formation of methyl 2-benzamidonicotinate (3):

To a solution of compound 2 (7.60g, 0.5 mole) in 50 mL chloroform was added triethylamine (2.5 mL, 0.2 mole). To this solution benzoyl chloride (3.04 mL, 2.22g, 0.02 mole) in chloroform was added dropwise over a period of one hour with continues stirring. It was further stirred on room temperature for next 20 hours. After that the mixture was filtered to remove any residue formed, the solution was washed with 10% NaHCO₃ and 5% HCl. It was further treated with ethyl acetate in presence of hexane to give methyl-2-benzamidonicotinate as solid product.

Yield: 64%. mp: 122-124^°C (ethanol). IR (KBr) cm^-1: 3320.6, 1900.7, 1845.6, 1742.2, 1715.3, 1651.2, 1455.1. ¹H NMR (DMSO-d6, 400 MHz) δ: 3.77 (s, 3H, CH₃), 7.7 (d, 2H, J=7.78, Ar), 7.8 (s, 1H, Ar), 8.13 (d, 2H, J=7.12, Ar), 8.3 (d, 1H, J=7.26, Ar), 8.3 (dd, 2H, J=7.38, Ar), 12.73 (bs, 1H, NH). ¹³C NMR (DMSO-d6, 100 MHz) δ: 51.5, 110.4, 111.2, 127.1, 132.6, 134.7, 137.4, 148.2, 155.5, 167.2, 168.1. HRMS (EI) m/z calcd for C₁₄H₁₂N₂O₃ 256.0431, found 256.0459. Anal: C (65.62/65.68), H (4.72/4.60), N (10.62/10.67), O (18.73/18.39).

3. Formation of 2-phenylpyrido[2,3-d]pyrimidin-4(3H)-one (4):

Compound 3 (5.12g, 0.02 mole) was dissolved in 10 mL of methanol and to this a 28% solution of NH₃OH was added. This mixture was then heated for 5 hours and then 40 mL of NaOH was added with vigorous stirring followed by refluxing for 12 hours. It was allowed to cool down to room temperature leading to formation of a precipitate which was further filtered and dried to obtain white solid as 2-phenylpyrido[2,3-d]pyrimidin-4(3H)-one.

Yield: 78%. mp: 168-170^°C (ethanol). IR (KBr) cm^-1: 3420.1, 3318.2, 1715.3, 1657.4, 1655.1, 1618.5. ¹H NMR (DMSO-d6, 400 MHz) δ: 5.8 (bs, 1H, Ar-NH), 6.8 (m, 1H, Ar), 6.8 (m, 5H, Ar), 7.15 (t, 2H, J=7.60, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 115.4, 120.09, 128.2, 129.10, 130.18, 132.40, 136.7, 156.0, 157.5, 159.2, 161.0. HRMS (EI) m/z calcd for C₁₃H₉N₃O 233.0751, found 233.0772. Anal: C (69.95/69.80), H (4.06/4.03), N (18.82/18.67), O (7.17/7.14).

4. Formation of 4-chloro-2-phenylpyrido[2,3-d]pyrimidine (5):

Compound 4 (2.23 g, 0.01 mole) was refluxed with phosphorus oxychloride for five hour during which all the starting material was dissolved and utilised. Excess of phosphorus oxychloride was removed under vacuum and the remaining residue was poured in ice-cold water and chloroform. The chloroform layer was separated and treated with NaHCO₃. The chloroform layer was further washed with brine and then purified using silica gel column with chloroform as an eluent to provide 4-chloro-2-phenylpyrido[2,3-d]pyrimidine.

Yield: 67%. mp: 156-158^°C (ethanol). IR (KBr) cm^-1: 3423.1, 3376.2, 3319.2, 1657.4, 1655.1, 1619.8. ¹H NMR (DMSO-d6, 400 MHz) δ: 6.9 (s, 1H, Ar), 7.4 (m, 1H, Ar), 7.49 (m, 2H, Ar), 7.7 (m, 1H, Ar), 7.76 (m, 1H, Ar), 8.01 (t, 2H, J=8.50, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 118.4, 121.09, 128.8, 129.40, 130.13, 132.40, 134.7, 156.0, 157.5, 159.2, 160.10. HRMS (EI) m/z calcd for C₁₃H₈ClN₃ 241.0407, found 241.0410. Anal: C (64.61/64.50), H (3.34/3.39), Cl (14.65/14.67), N (17.39/17.30).

5. General Procedure for 2-phenyl-N-(substituted phenyl) pyrido[2,3-d] pyrimidin-4-amine (6a-r):

Compound 5 was reacted with various anilines in 4 mL of dry DMF, to this solution 86 microliters of diisopropylamine (DIPA) was added and heated over a period of 3-6 hours. This reaction mixture was cooled to room temperature and residue was filtered out and purified using column chromatography to yield respective derivatives. Total eighteen derivatives were synthesised, these compounds are enlisted as follows;

5.1. Compound 6a:- N-(4-Fluorophenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3422.1, 3373.2, 3315.2, 1652.4, 1654.1, 1618.8. ¹H NMR (DMSO-d6, 400 MHz) δ: 6.8(m, 1H, NH), 7.3(m, 2H, Ar), 7.36(m, 2H, Ar), 7.41(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33(t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₃FN₄ 316.1124, found 316.1126. Anal: C(72.14/72.17), H(4.14/4.12), F(6.01/6.03), N(17.71/17.67). Yield: 59%. mp: 152-154^°C.

5.2. Compound 6b:- N-(2-Fluorophenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3421.1, 3376.4, 3319.5, 1657.6, 1655.3, 1619.1. ¹H NMR (DMSO-d6, 400 MHz) δ: 7.12(m, 2H, Ar), 7.27(m, 2H, Ar), 7.5(m, 2H, Ar), 7.8(m, 2H, Ar), 8.11(s, 2H, Ar), 8.13(d, 2H, Ar), 12.63(bs, 1H, NH). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₃FN₄ 316.1124, found 316.1126. Anal: C (72.14/72.17), H (4.14/4.12), F (6.01/6.03), N (17.71/17.67). Yield: 48%. mp: 150-152^°C.

5.3. Compound 6c:- N-(4-Chlorophenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3420.4, 3375.4, 3317.5, 1656.7, 1656.3, 1618.8. ¹H NMR (DMSO-d6, 400 MHz) δ: 6.8(m, 1H, NH), 7.31(m, 2H, Ar), 7.36(m, 2H, Ar), 7.41(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33(t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₃ClN₄ 332.0829, found 332.0835. Anal: C(68.50/68.57), H(3.94/3.90), Cl (10.65), N(16.84/16.88). Yield: 64 %. mp: 149-151^°C.

5.4. Compound 6d:- N-(2-Chlorophenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3423.6, 3376.3, 3319.5, 1657.3, 1655.2, 1619.7. ¹H NMR (DMSO-d6, 400 MHz) δ: 6.8(m, 1H, NH), 7.31(m, 2H, Ar), 7.36 (m, 2H, Ar), 7.4(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33(t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₃ClN₄ 332.0829, found 332.0835. Anal: C (68.55/68.57), H (3.90/3.94), Cl (10.65), N (16.84/16.80). Yield: 60 %. mp: 147-149^°C.

5.5. Compound 6e:- N-(4-Bromophenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3422.4, 3375.4, 3317.1, 1654.5, 1656.2, 1615.4. ¹H NMR (DMSO-d6, 400 MHz) δ(m, 1H, NH), 7.31(m, 2H, Ar), 7.36 (m, 2H, Ar), 7.41(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33(t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₃BrN₄ 376.0324, found 376.0320. Anal: C (60.49/60.50), H (3.47/3.50), Br (21.18/21.14), N (14.85/14.87). Yield: 51%. mp: 156-158^°C.

5.6. Compound 6f:- N-(2-Bromophenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3422.4, 3375.4, 3317.1, 1654.5, 1656.2, 1615.4. ¹H NMR (DMSO-d6, 400 MHz) δ: 6.8(m, 1H, NH), 7.31(m, 2H, Ar), 7.36(m, 2H, Ar), 7.41(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33(t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar).¹³ C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₃BrN₄ 376.0324, found 376.0321. Anal: C (60.47/60.49), H (3.48/3.51), Br (21.11/21.15), N (14.82/14.86). Yield: 50%. mp: 159-161^°C.

5.7. Compound 6g:- 2-Phenyl-N-(4-(trifluoromethyl) phenyl)pyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3423.1, 3378.2, 3317.2, 1659.4, 1654.1, 1620.8. ¹H NMR (DMSO-d6, 400 MHz) δ: 6.8(m, 1H, NH), 7.31(m, 2H, Ar), 7.36(m, 2H, Ar), 7.41(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33(t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar).¹³ C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₂₀H₁₃F₃N₄ 366.3472, found 366.3211. Anal: C (65.57/65.60), H (3.58/3.61), F (15.56/15.67), N (15.29/15.40). Yield: 65%. mp: 158-160^°C.

5.8. Compound 6h:- 2-Phenyl-N-(p-tolyl)pyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3423.3, 3377.4, 3318.5, 1656.9, 1656.2, 1617.4.¹ H NMR (DMSO-d6, 400 MHz) δ: 2.18(s, 3H, CH₃), 6.8(m, 1H, NH), 7.31 (m, 2H, Ar), 7.36(m, 2H, Ar), 7.41(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33(t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar).¹³ C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₂₀H₁₆N₄ 312.1375, found 312.1390. Anal: C (76.90/76.85), H (5.16/5.19), N (17.94/17.90). Yield: 55 %. mp: 165-167^°C.

5.9. Compound 6i:- 2-Phenyl-N-(m-tolyl)pyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3423.1, 3376.2, 3319.2, 1657.4, 1655.1, 1619.8. ¹H NMR (DMSO-d6, 400 MHz) δ: 2.4(s, 3H, CH₃), 6.8(m, 1H, NH), 7.11 (t, 1H, J=7.82, Ar), 7.31(m, 2H, Ar), 7.36(m, 2H, Ar), 7.41(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33(t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar).¹³ C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₂₀H₁₆N₄ 312.1375, found 312.1399. Anal: C (76.90/76.84), H (5.16/5.18), N (17.94/17.91). Yield: 50 %. mp: 164-166^°C.

5.10. Compound 6j:- 2-Phenyl-N-(o-tolyl)pyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3423.4, 3377.8, 3318.6, 1656.5, 1656.3, 1618.7.¹ H NMR (DMSO-d6, 400 MHz) δ: 2.12(s, 3H, CH₃), 6.8(m, 1H, NH), 7.11 (t, 1H, J=7.82, Ar), 7.31(m, 2H, Ar), 7.36(m, 2H, Ar), 7.41(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33(t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₂₀H₁₆N₄ 312.1375, found 312.1299. Anal: C (76.90/76.89), H (5.16/5.22), N (17.94/17.87). Yield: 58 %. mp: 160-162^°C.

5.11. Compound 6k:- N-(4-Methoxyphenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3425.3, 3378.2, 3319.7, 1656.5, 1656.3, 1618.8. ¹H NMR (DMSO-d6, 400 MHz) δ: 3.81(s, 3H, CH₃), 6.8(m, 1H, NH), 7.01 (t, 1H, J=7.82, Ar), 7.21(m, 2H, Ar), 7.36(m, 2H, Ar), 7.41(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33 (t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₂₀H₁₆N₄O 328.1324, found 328.0259. Anal: C (73.15/73.34), H (4.91/5.02), N (17.06/17.13), O (4.87/4.18). Yield: 66%. mp: 167-169^°C.

5.12. Compound 6l:- N-(3-Methoxyphenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3423.1, 3376.2, 3319.2, 1657.4, 1655.1, 1619.8. ¹H NMR (DMSO-d6, 400 MHz) δ: 2.8(s, 3H, CH₃), 6.5(m, 2H, Ar), 6.02(m, 3H, Ar), 7.4(s, 2H, Ar), 7.6(s, 3H, Ar), 2.5(d, 2H, J=7.2, Ar), 9.0(bs, 1H, NH). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₂₀H₁₆N₄O 328.1324, found 328.0259. Anal: C (73.15/73.34), H (4.91/5.02), N (17.06/17.13), O (4.87/4.18). Yield: 66%. mp: 167-169^°C.

5.13. Compound 6m:- N-(2-Methoxyphenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3423.5, 3375.3, 3318.1, 1656.3, 1656.2, 1618.7. ¹H NMR (DMSO-d6, 400 MHz) δ: 3.82(s, 3H, CH₃), 6.8(m, 1H, NH), 7.01 (t, 1H, J=7.82, Ar), 7.21(m, 2H, Ar), 7.36(m, 2H, Ar), 7.41(m, 1H, Ar), 7.59(m, 2H, Ar), 8.33 (t, 3H, J=7.76, Ar), 8.59(dd, 1H, J=7.26, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₂₀H₁₆N₄O 328.1324, found 328.0259. Anal: C (73.15/73.34), H (4.91/5.02), N (17.06/17.13), O (4.87/4.18). Yield: 66%. mp: 167-169^°C .

5.14. Compound 6n:- 4-((2-Phenylpyrido[2,3-d]pyrimidin-4-yl)amino)phenol

IR (KBr) cm^-1: 3612.1, 3423.6, 3376.3, 3319.3, 1657.6, 1655.5, 1619.7. ¹H NMR (DMSO-d6, 400 MHz) δ: 6.7(m, 2H, Ar), 7.0(m, 2H, Ar), 7.21(m, 2H, Ar), 7.85(t, 2H, J=7.8, Ar), 7.9(m, 2H, Ar), 8.30(m, 2H, Ar), 8.91(d, 2H, J=7.75, Ar), 10.89(s, 1H, Ar-OH). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₄N₄O 314.1168, found 314.1100. Anal: C (72.60/72.69), H (4.49/4.56), N (17.85/17.67), O (5.09/5.11). Yield: 65 %. mp: 130-132^°C.

5.15. Compound 6o:- 2-((2-Phenylpyrido[2,3-d]pyrimidin-4-yl)amino)phenol

IR (KBr) cm^-1: 3651.5, 3423.5, 3378.2, 3319.4, 1657.5, 1655.5, 1619.7. ¹H NMR (DMSO-d6, 400 MHz) δ: 7.2(m, 7H, Ar), 7.32(t, 2H, J=7.58, Ar), 7.5(m, 2H, Ar), 8.0(d, 2H, J=7.8, Ar), 12.52(bs, 1H, Ar-OH). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₄N₄O 314.1168, found 314.1110. Anal: C (72.60/72.65), H (4.49/4.57), N (17.85/17.69), O (5.09/5.10). Yield: 67 %. mp: 129-131^°C.

5.16. Compound 6p:- N-(4-Nitrophenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3423.1, 3376.2, 3319.7, 1658.4, 1655.1, 1619.1. ¹H NMR (DMSO-d6, 400 MHz) δ: 6.89(bs, 1H, NH), 7.44(m, 4H, Ar), 7.50 (m, 3H, Ar), 8.03(d, 2H, J=7.16, Ar), 8.36(t, 2H, J=7.32, Ar), 8.59(t, 1H, J=7.75, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₃N₅O₂ 343.1069, found 343.1259. Anal: C (66.47/66.32), H (3.82/3.90), N (20.40/20.67), O (9.32/9.44). Yield: 66%. mp: 172-174^°C.

5.17. Compound 6q:- N-(3-Nitrophenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3425.2, 3377.1, 3318.1, 1657.7, 1655.8, 1619.2. ¹H NMR (DMSO-d6, 400 MHz) δ: 6.89(bs, 1H, NH), 7.2(m, 1H, Ar), 7.44(m, 3H, Ar), 7.50(m, 3H, Ar), 8.03(d, 2H, J=7.16, Ar), 8.36(t, 2H, J=7.32, Ar), 8.59(t, 1H, J=7.75, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₃N₅O₂ 343.1069, found 343.1123. Anal: C (66.47/66.34), H (3.82/3.95), N (20.40/20.45), O (9.32/9.40). Yield: 60%. mp: 177-179^°C .

5.18. Compound 6r:- N-(2-Nitrophenyl)-2-phenylpyrido[2,3-d]pyrimidin-4-amine

IR (KBr) cm^-1: 3423.9, 3376.2, 3319.5, 1657.1, 1656.6, 1617.2. ¹H NMR (DMSO-d6, 400 MHz) δ: 6.89 (bs, 1H, NH), 7.2 (m, 1H, Ar), 7.44 (m, 3H, Ar), 7.50 (m, 3H, Ar), 8.03 (d, 2H, J=7.16, Ar), 8.36 (t, 2H, J=7.32, Ar), 8.59 (t, 1H, J=7.75, Ar). ¹³C NMR (DMSO-d6, 100 MHz) δ: 103.6, 114.0, 116.1, 120.7, 124.1, 126.5, 127.1, 129.2, 130.1, 131.5, 136.7, 153.0, 155.1, 157.9, 159.0. HRMS (EI) m/z calcd for C₁₉H₁₃N₅O₂ 343.1060, found 343.1089. Anal: C (66.47/66.32), H (3.82/3.94), N (20.40/20.44), O (9.32/9.39). Yield: 65%. mp: 172-174 ^°C.

6. Molecular docking studies:

All the in silico designing and docking studies were performed on the Schrodinger modelling suite (Version 15.3) the compounds designed were subjected to energy minimisation by Ligprep module of the software²⁸. All the protein preparation work was carried out on the protein preparation wizard of the Glide module, the crystal structure obtained from the protein data bank contains the ligands, cofactors, water molecules, they may also be deficient of certain loops, and these anomalies are refined by the protein preparation wizard. The cofactor unless essential are removed the ligand is replaced and the water molecules are also eliminated. The missing loops are filled and the caps are inserted. Thus, when the protein is ready energy minimisation program is run to optimise the protein for molecular docking studies. Glide module was employed for the molecular docking; compounds were docked in the grid designed in the module. The docking was carried out with flexible ligands and the protein with the simulations made on the OPLS5.0 force field. All the results were based on 10 conformations for each molecule in the extra precision (XP) mode. The crystal structure (PDB 2V58)⁸ was employed as the receptor with compounds 6a-r as ligands. The results obtained from the simulation were obtained in the form of dock score; these values represent the minimum energies. Interactions between the ligand and residues were presented in the form of H-bond, van der Waals forces and the pi bonds. The results in the form of 3D and 2D representation were obtained for simplified understanding.

RESULTS AND DISSCUSSION:

Biotin carboxylase acts as an important catalyst in the synthesis of malonyl-CoA which is an important precursor for the synthesis of fatty acids. These are essential for formation of the bacterial cell wall. With the advent of drug resistant bacterial strains and urgent need for drugs with potential for inhibiting drug resistant bacteria was felt. Herein, we are reporting the development of some new pyridopyrimidine derivatives with their potential for inhibiting the drug resistant bacterial strains. Earlier, Chakravarty et al., has reported several quinazolines as TGF-β inhibitors²⁹. Pyridopyrimidines were designed on the basis of structural analogy to the compounds previously reported with antibacterial potential; these consisted of some common features like the pyridopyrimidine nucleus as shown in figure 2. Eighteen pyridopyrimidine derivatives were synthesised following a five-step synthetic protocol as shown in scheme 1.

2-Amino pyridine carboxylic acid (1) undergoes Fischer-Speier esterification³⁰and provides with methyl 2-aminonicotinate (2).The free primary amine reacts with 3-chlorobenzoyl chloride in the presence of triethyl amine and forms the methyl 2-benzamidonicotinate (3). Compound (3) undergoes cyclisation in the presence of ammonia leading to formation of 2-phenylpyrido[2,3-d]pyrimidin-4(3H)-one (4). On treatment with phosphorus oxychloride, compound (4) forms the 4-chloro-2-phenylpyrido[2,3-d]pyrimidine (5). The chloro group of compound five is substituted with various anilines in basic medium to give eighteen final derivatives. The structural confirmation was made on the basis of IR, NMR and mass spectra.

The spectral details of compounds are mentioned in the experimental section with spectra of selected compounds in the supplementary material available with the online version of this article. Compound (2) shows the characteristic amine absorption spectra around 3424.1 cm-1 and the absorption for ester around 1655.1 cm-1. Proton NMR spectra of this compound shows a broad singlet corresponding to the amine at 12.19 ppm and a singlet at 3.86 ppm with three protons of the methyl group. The compound (3) shows absorption around 1455.1 cm-1 for the secondary amine group, the proton NMR spectra shows a singlet of the methyl group at 3.77 ppm with three protons, aromatic protons around 7.7 to 8.3 ppm whereas the amide proton is seen at 12.73 ppm. In case of compound (4) the methyl group seen in compound (3) disappears as a result of cyclisation and a single proton of secondary nitrogen is observed at 5.8 ppm as abroad singlet.

The compound (5) shows all the protons in the aromatic region because chlorination of compound (4) leads to disappearance of amide proton. In the series of compounds (6a-r), the characteristic properties of derivatives involved singlets at 2.4 ppm corresponding to the methyl group of the tolyl derivative (6i). singlet at 2.12 ppm of methyl group of the tolyl derivative (6j). The methoxy derivatives (6k), (6l) and (6m) showed a methyl proton at 3.81, 2.8 and 3.82 ppm respectively. In case of compounds (6n) and (6o) showed characteristic -OH broad singlets at 10.89 and 12.52 ppm respectively. All the compounds displayed the number of carbon atoms as per the molecular formula and the characteristic signals were observed for the compounds like in (2) the carboxyl carbon signals at 167.5 ppm, in compound (3) there are two carboxyl carbons at 167.1 and 168.1 respectively, in compound (4) the amide carbons show signal at 161.0 ppm. All compounds were subjected to determination of molecular weight following a mass spectral analysis; all compounds were found to be in agreement with the theoretical values.

All the compounds were subjected to molecular docking studies to determine the mode of binding and energy changes. The crystal structure of biotin carboxylase (PDB: 2V58) was used for this study and ligands were prepared for docking by method described elsewhere. The biotin carboxylase is co-crystallised with 6-(2,6-dibromophenyl)pyrido[2,3-d]pyrimidine-2,7-diamine, which we have used as reference ligand for molecular docking studies. It was observed that compound 6d, 6l, 6n, 6o, 6r and 6i showed highest interaction with the enzyme in their molecular docking studies, the details of docking studies are mentioned in the table 1 (Supplementary material). The bound ligand in the active site forms covalent nitration with Glu201A, Lys202A, aromatic interactions with Leu204A and Lys159A, it shows weaker interactions with Ile437A and His438. The docked compound 6d, show a high docking score of -7.58 and Glide energy of -44.50. It forms a salt bridge between pyridopyridine nucleus and HIS438 (Figure 3a and b). It forms an aromatic interaction with the Lys159, the presence of electronegative substituent like chlorine on the phenylpyridopyrimidine nucleus contributes to its biological activity. Compound 6l, showed a high docking score of -7.34 and Glide energy of -45.06, the pyridopyridine nucleus forms a salt bridge with the residue HIS430 and the phenyl ring formed a salt bridge with HIS209 (Figure 3c and d), this may be due to the presence of electron donating group like methoxy on the phenylpyridopyrimidine nucleus. Compound 6n, show a docking score of -7.76 and Glide energy of -47.34. It forms a salt bridge between pyridopyridine nucleus and HIS438, the phenyl ring of substituent forms another salt bridge with HIS209 and a hydrogen bond is formed between hydroxy group with GLU276 (Figure 4a and b). Compound 6o, showed a high docking score of -7.96 and Glide energy of -46.71, the pyridopyridine nucleus forms a salt bridge with the residue HIS438 and the OH group forms a hydrogen bond with GLY165 (Figure 4c and d). In case of compounds 6l and 6n there is hydroxyl group (-OH) on the 4^th and 2^nd positions respectively. Their biological activity, high dock score and hydrogen bond formation with Glu276 and Gly165 suggest importance of strong electron donating character of hydroxyl functional group on the phenylpyridopyrimidine nucleus. Compound 6r, showed a high docking score of -7.62 and Glide energy of -45.89, the pyridopyrimidine nucleus forms a salt bridge with the residue HIS438 and the phenyl ring formed a salt bridge with HIS209, this may be due to the presence of electron withdrawing group like nitro on the phenylpyridopyrimidine nucleus. Compound 6i, showed a high docking score of -7.34 and Glide energy of -46.30, the pyridopyrimidine nucleus forms a salt bridge with the residue HIE438 and the phenyl ring formed a salt bridge with HIS236, this may be due to the presence of electron donating group like methyl on the phenylpyridopyrimidine nucleus.

Figure 3: (a, b) Molecular interaction between the biotin carboxylase and compound 6d, (c, d) Molecular interaction between the biotin carboxylase and compound 6l

Figure 4: (a, b) Molecular interaction between the biotin carboxylase and compound 6n, (c, d) Molecular interaction between the biotin carboxylase and compound 6o

Table 1: Molecular docking study data for docking score, glide score and energy and ecoulomb score for compounds 6a-r.

Compound No.	Functional group	Glide Energy	Docking score	Glide score	Ecoul. score
6a	4-F	-41.07	-6.78	-6.78	-3.53
6b	2-F	-40.56	-6.32	-6.32	-0.84
6c	4-Cl	-42.63	-6.65	-6.65	-3.33
6d	2-Cl	-44.50	-7.58	-7.58	-2.07
6e	4-Br	-43.03	-6.38	-6.38	-3.61
6f	2-Br	-46.18	-7.48	-7.48	-2.26
6g	4-CF₃	-41.09	-6.45	-6.45	-1.89
6h	4-CH₃	-41.34	-6.59	-6.59	-3.24
6i	3-CH₃	-46.30	-7.56	-7.56	-1.19
6j	2-CH₃	-45.40	-7.48	-7.48	-1.20
6k	4-OCH₃	-45.39	-6.89	-6.89	-2.24
6l	3-OCH₃	-45.06	-7.34	-7.34	-5.78
6m	2-OCH₃	-46.30	-7.56	-7.56	-2.83
6n	4-OH	-47.34	-7.76	-7.76	-7.37
6o	2-OH	-46.71	-7.96	-7.96	-3.34
6p	4-NO₂	-45.08	-7.07	-7.07	-4.08
6q	3-NO₂	-46.66	-7.11	-7.11	-4.94
6r	2-NO₂	-45.89	-7.62	-7.626	-2.54

CONCLUSION:

A synthesis of novel scaffold of N-substituted 2-phenylpyrido (2,3-d) pyrimidine derivatives following a five-step synthetic protocol as shown in scheme1. Characterization of N-substituted 2-phenylpyrido(2,3-d)pyrimidine derivatives are mentioned in the experimental section with spectra of selected compounds and their molecular docking studied to determine the mode of binding and energy changes with the crystal structure of biotin carboxylase (PDB ID: 2V58). Promising compounds 6d, 6l, 6n, 6o, 6r and 6i showed formation of hydrogen bonds with the active site residues and van Der Walls interactions with the biotin carboxylase enzyme in their molecular docking studies. This compound can be studied further and developed into a potential antibacterial lead molecule.

ACKNOWLEDGEMENTS:

All authors are gratefully thanks to UGC, New Delhi for providing grants to purchase Drug Designing software (Schrodinger modeling suite) sanctioned under Major Research Project from School of Pharmacy, Swami Ramanand Teerth Marathwada University, Nanded.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 23.07.2020 Modified on 24.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3846-3854.

DOI: 10.52711/0974-360X.2021.00667