The synthesis of the novel substituted series of 7-morpholino-6-(5-ary-1,3,4-oxadiazol)-1,8-naphthyridine-2-carbonitrile derivatives (7a-h) has efficaciously synthesized by using cheap and readily available efficient oxidative cyclization catalyst Iron III chloride hexahydrate (FeCl3.6H2O) as catalyst the schematic representation shown in Scheme-1. In this reaction used a nontoxic and inexpensive reagent FeCl3.6H2O to create an environmentally friendly technology. One major advantage over the previous method is the remarkable simplicity of the experimental methods for these reactions. They also have quick reaction durations and do not need the use of costly catalysts. Initially the reaction continued by the condensation of the 6-amino-5-formylpyridine-2-carbonitrile 1 reactant with ethyl 3-morpholino-3-oxopropanoate 2, in the presence of the basic nature containing cyclic aliphatic amine catalyst Piperidine effectively formed the reaction intermediate ethyl 7-cyano-2-morpholino-1,8-naphthyridine-3-carboxylate 3. The 7-cyano-2-morpholino-1,8-naphthyridine-3-carbohydrazide 4 were formed by reacting the ester on reacted hydrazinolysis under the refluxing hydrazinehydrate in the polar solvent methanol. The (E)-N'-benzylidene-2-morpholino-1,8-naphthyridine-3-carbohydrazide 5 were obtained good yields with condensation of the 4 with different aromatic substituted aldehydes in the presence of catalyst the p-toluenesulphonic acid (PTSA) catalyst. Finally desired new compounds 7-morpholino-6-(5-aryl-1,3,4-oxadiazol-2-yl)-1,8-naphthyridine-2-carbonitrile derivatives were obtained with good yields in the presence of the mild and environmentally friendly FeCl3.6H2O catalyst occurred intramolecular oxidative cyclization reaction of (E)-N'-benzylidene-2-morpholino-1,8-naphthyridine-3-carbohydrazide 6. By using this catalyst smoothly synthesized final derivatives with good yields.
Biological studies
Antimicrobial activity; All of the newly prepared (7a-g) pure compounds Antibacterial studies checked against different Gram-positive pathogenic bacterial strains Staphylococcus aureus and Gram-negative bacteria strains Escherichia coli was tested using clinical reference drug Ampicillin. Among them compounds 7c and 7g shown potent antibacterial and antifungal properties against a wide range of species, including Aspergillus niger and Candida metapsilosis, when compared to ampicillin and grieseofulvin as a reference drug. Compounds 7c and 7g showed strong antifungal efficacy among all the species tested, compared to the standard drugs. 6-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol)-7-morpholino-1,8-naphthyridine-2-carbonitrile hydroxyl group containing molecule highest potent activity. Results of the all tested molecules data are shown in Table 1. The activity of the molecules was checked by the already established procedure agar well diffusion method according to the literature procedure [20, 21].
Table 1
In vitro studies screening data of the 7-morpholino-6-(5-Aryl-1,3,4-oxadiazol)-1,8-naphthyridines (7a-g) (conc.in µg/mL, ZOI in mm)
Molecule | Antibacterial | Antifungal |
Gram-positive strain | Gram-negative strain | A. niger | C. Metapsilosis |
S.aureus | E. coli |
100 µg/mL | 150 µg/mL | 100 µg/mL | 150µg/mL | 100 µg/mL | 150µg/mL | 100 µg/mL | 150µg/mL |
7a 7b 7c 7d 7e 7f 7g A G | 14 | 12 | 13 | 15 | 11 | 16 | 12 | 18 |
19 | 15 | 17 | 18 | 14 | 15 | 17 | 17 |
24 | 26 | 22 | 23 | 22 | 24 | 23 | 25 |
20 | 12 | 20 | 20 | 13 | 17 | 15 | 15 |
18 | 13 | 19 | 18 | 16 | 15 | 16 | 15 |
16 | 15 | 16 | 16 | 18 | 19 | 17 | 14 |
22 | 23 | 21 | 22 | 21 | 22 | 21 | 23 |
25 | 27 | 23 | 25 | -- | -- | -- | -- |
-- | | -- | | 25 | 27 | 24 | 26 |
Bacterial strains: (S.aureus = Bacteria Staphylococcus aureus, and E. coli = Escherichia coli |
Fungalstrains: A. niger = Aspergillus niger, C. Metapsilosis = Candida metapsilosis |
Positive control Drugs: A = Ampicillin, G = Grieseofulvin), ZOI = Zone of inhibition values (mm), -- = Not performed.
3.2 Molecular Docking studies
A molecular modeling study is a most important computational study to understand the binding interactions of ligand target protein 3D dimensional structures of structural biology. To know the relationship between the protein and active molecules docking analysis of the 4-Hydroxyphenyl 1,3,4-oxadiazol 8-morpholino 1,8-naphthyridine analogues against the microbial epidermal well growth factor receptor was conducted using the Auto Dock reference module. It is commonly known that proteins are effectively targets for the newly development of antimicrobial effective drugs. Furthermore, protein is a unique cell-surface receptor that is activated by the interaction with ligands. The binding process of the synthesized 4-Hydroxyphenyl 1,3,4-oxadiazol 8-morpholino 1,8-naphthyridine hybrids with their corresponding receptors was investigated using molecular docking analysis. Using the cancer protein receptor (PDB ID: 8g4d) from the PDB file, molecular docking was utilized to investigate the most successful antibacterial hybrids [22-26]. The binding energies, interacted groups, and interacted amino acids are displayed in Table 2. Compound 6c, one of the most successful hybrids in these studies, exhibited two H-bonding contacts with the amino acids O and NH group of PHE106, TYR 67, in addition to other hydrophobic interactions, such as stacking with aromatic rings. Its binding affinity was likewise -12.04 kcal/mol The compounds 7g, which have two hydrogen bond interactions PHE106-O, TYR67-HN and a value of -11.67 kcal/mole have the lowest binding energies.
Table 2 Molecular docking interactions with active synthesized molecules 7c and 7g with protein receptor (PDB ID: 8g4d)
Compounds
|
Lowest binding energy,Kcal/mole
|
H-bonding
|
Interacting groups
|
7a
|
-10.04
|
2
|
Active site region with electron density
|
7b
|
-9.48
|
1
|
LEU 119-HN, TYR87-HN
|
7c
|
-12.04
|
3
|
THR147-O, TRO 105-HN, LEU 103-HN
|
7d
|
-8.67
|
1
|
TRO 95-HN, PHE106-O
|
7e
|
-10.17
|
2
|
Active site NO2 group
|
7f
|
-10.17
|
3
|
Active site region with electron density
|
7g
|
-11.67
|
2
|
PHE106-O, TYR67-HN
|
Experimental section
All the required materials like chemicals and solvents were get from Aldrich and utilized without additional purification. The prepared compounds melting points were dignified by using a Buchi melting point instrument. Synthesized compounds’ purity was checked by using F254 silica-gel precoated material TLC plates with a hexane and ethyl acetate solvent ratio (7:3). 1H NMR, 13C spectrums were captured using DMSO-d6 solvent on a Bruker 400 MHz spectrometer. An ESI mass spectrometer used to record the mass data. A Bruker Tensor 27 series for recorded the KBr pellets. Elemental analysis studies done by using a Carlo-Erba model EA1108
Reaction procedure of ethyl 7-cyano-2-morpholino-1,8-naphthyridine-3-carboxylate reaction intermediate (3)
Initially in the round bottom flask take 6-amino-5-formylpicolinonitrile 1 (1 mmol, 147.04 mg) and 6-amino-5-formylpicolinonitrile 2 (1 mmol, 201.10 mg) and catalytic amount of the piperidine catalyst added reactants taken an equimolar amount taken then heated conventional reflux for 4–5 hours. Molecules formation the reaction progress checked by TLC, after the completetion reaction stop the reaction condition then poured in to cold water filtered and washed with cold methanol solvent. Analytically pure reaction intermediate (3) obtained with good yield (75-86%).
Synthesis of 7-cyano-2-morpholino-1,8-naphthyridine-3-carbohydrazide (4)
The starting material ethyl 7-cyano-2-morpholino-1,8-naphthyridine-3-carboxylate (1 mmol, 312.12mg) reacted with hydrazine hydrate under the reflux condition mixture heated (3-5 hours) progress of the reaction checked by TLC, after finish the reaction condition cooled to RT and the solid obtained filtered and washed with cold Ethanol and Hexane obtained desired products with good yields (81-87%).
General construction method for the (E)-N'-benzylidene-2-morpholino-1,8-naphthyridine-3-carbohydrazide (6)
In the round bottom flask (E)-N'-benzylidene-2-morpholino-1,8-naphthyridine-3-carbohydrazide 4 (1 mmol, 298.31 mg) and various aromatic aldehyde 5 (1 mmol) in the presence of the 10 mol% PTSA was taken and reflux condition obtained good yields under conventional method.
Synthesis of 7-morpholino-6-(5-aryl-1,3,4-oxadiazol)-1,8-naphthyridine-2-carbonitrile derivatives (7a-g)
(E)-N'-benzylidene-2-morpholino-1,8-naphthyridine-3-carbohydrazide (6) (1 mmol, 361.15mg mg) and FeCl3.6H2O (1.5 mmol, 405.36 mg) taken in the round bottom flask under the heating condition oxidative cyclization occurred and obtained desired final products 7-morpholino-6-(5-aryl-1,3,4-oxadiazol-2-yl)-1,8-naphthyridine-2-carbonitrile derivatives (7a-g) with good yields. Improvement of the reaction condition was checked by TLC after competition of the reaction transpired into cold water and faltered it then recrystallization from methanol solvent.
7-morpholino-6-(5-phenyl-1,3,4-oxadiazol-2-yl)-1,8-naphthyridine-2-carbonitrile (7a)
Light Yellowish solid, mp. 190-192oC, IR (KBr): 2130 (CN) cm-1. 1H NMR (DMSO-d6) δ ppm: 3.39 (m, 4H-ArCH2), 3.94 (m, 4H-ArCH2), 7.49 (m, 5H-ArH), 8.37 (s, 1H, ArH), 8.28 (s, 2H, ArH). 13C NMR (DMSO-d6) δ ppm: 46.22, 65.89, 113.01, 117.93, 118.36, 120.24, 126.66, 127.37, 129.35, 131.57, 132.31, 135.83, 138.83, 146.97, 150.19, 161.61, 165.57. MS m/z: 384.13. Anal. Calcd: C21H16N6O2; C, 65.62; H, 4.20; N, 21.86%. Found: C, 65.65; H, 4.24; N, 21.89%.
6-(5-(4-chlorophenyl)-1,3,4-oxadiazol-2-yl)-7-morpholino-1,8-naphthyridine-2-carbonitrile (7b)
Yellowish solid, mp. 195-197oC, IR (KBr): 2300 (CN) cm-1. 1H NMR (DMSO-d6) δ ppm: 3.48 (m, 4H-ArCH2), 3.87 (m, 4H-ArCH2), 7.61 (m, 4H-ArH), 8.22 (s, 1H, ArH), 8.45 (m, 2H, ArH). 13C NMR (DMSO-d6) δ ppm: 46.12, 65.79, 113.01, 117.93, 118.36, 120.34, 18.20, 128.39, 129.65, 132.36, 135.83, 137.31, 138.83, 146.97, 150.19, 161.61, 165.47. MS m/z: 418.09. Anal. Calcd: C21H15ClN6O2; C, 60.22; H, 3.61; N, 20.07%. Found: C, 60.25; H, 3.65; N, 20.06%.
6-(5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-yl)-7-morpholino-1,8-naphthyridine-2-carbonitrile (7c)
Pale brown solid, mp. 192-194oC, IR (KBr): 2320 (CN), 3584 (OH) cm-1. 1H NMR (DMSO-d6) δ ppm: 3.41 (m, 4H-ArCH2), 3.83 (m, 4H-ArCH2), 7.51 (m, 4H-ArH), 8.19 (m, 2H, ArH), 8.37 (s, 1H, ArH), 8.58 (s, 1H, ArH). 13C NMR (DMSO-d6) δ ppm: 46.03, 65.70, 113.01, 115.88, 117.41, 118.36, 120.24, 127.65, 132.31, 135.83, 138.83, 146.97, 150.19, 160.80, 161.61, 165.47. MS m/z: 400.03. Anal. Calcd: C21H16N6O3; C, 60.97; H, 4.03; N, 20.99%. Found: C, 60.99; H, 4.05; N, 21.02%.
6-(5-(4-methoxyphenyl)-1,3,4-oxadiazol-2-yl)-7-morpholino-1,8-naphthyridine-2-carbonitrile (7d)
Light green solid, mp. 194-195oC, IR (KBr): 2330 (CN), 3564 (OH) cm-1. 1H NMR (DMSO-d6) δ ppm: 3.42 (m, 4H-ArCH2), 3.77 (s, 3H, CH3), 3.83 (m, 4H-ArCH2), 7.65 (m, 4H-ArH), 8.20 (m, 1H, ArH), 8.37 (s, 2H, ArH). 13C NMR (DMSO-d6) δ ppm: 46.03, 56.04, 65.70, 113.01, 113.94, 117.93, 118.36, 120.24, 121.55, 127.14, 132.31, 135.83, 138.83, 146.97, 150.19, 161.61, 162.19, 165.47. MS m/z: 414.14. Anal. Calcd: C22H18N6O3; C, 63.76; H, 4.38; N, 20.28%. Found: C, 63.78; H, 4.40; N, 22.30%.
7-morpholino-6-(5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl)-1,8-naphthyridine-2-carbonitrile (7e)
White solid, mp. 193-195oC, IR (KBr): 2330 (CN), 1532 (NO2) cm-1. 1H NMR (DMSO-d6) δ ppm: 3.52 (m, 4H-ArCH2), 3.83 (m, 4H-ArCH2), 7.92 (m, 2H-ArH), 8.19 (s, 1H, ArH), 8.44 (m, 3H, ArH), 8.58 (s, 1H, ArH). 13C NMR (DMSO-d6) δ ppm: 46.03, 65.70, 113.01, 117.93, 118.36, 120.24, 124.75, 127.52, 132.08, 132.31, 135.83, 135.83, 146.97, 147.39, 150.19, 161.61, 165.47. MS m/z: 429.12. Anal. Calcd: C21H15N7O4; C, 58.74; H, 3.52; N, 22.83%. Found: C, 58.78; H, 3.55; N, 22.87%.
6-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)-7-morpholino-1,8-naphthyridine-2-carbonitrile (7f)
Pale yellow solid, mp. 191-193oC, IR (KBr): 2330 (CN) cm-1. 1H NMR (DMSO-d6) δ ppm: 3.44 (m, 4H-ArCH2), 3.71 (m, 4H-ArCH2), 7.20 (m, 2H-ArH), 7.64 (m, 2H, ArH), 8.19 (s, 1H, ArH), 8.38 (s, 2H, ArH). 13C NMR (DMSO-d6) δ ppm: 46.03, 65.70, 113.01, 117.19, 118.36, 120.24, 123.55, 129.93, 132.31, 135.83, 138.83, 146.97, 150.19, 161.61, 172.04. MS m/z: 402.12. Anal. Calcd: C21H15FN6O2; C, 62.68; H, 3.76; N, 20.89%. Found: C, 62.70; H, 3.79; N, 20.92%.
6-(5-(4-bromophenyl)-1,3,4-oxadiazol-2-yl)-7-morpholino-1,8-naphthyridine-2-carbonitrile (7g)
Brown solid, mp. 195-197oC, IR (KBr): 2340 (CN) cm-1. 1H NMR (DMSO-d6) δ ppm: 3.50 (m, 4H-ArCH2), 3.83 (m, 4H-ArCH2), 7.56 (m, 2H-ArH), 7.71 (m, 2H, ArH), 8.24 (s, 1H, ArH), 8.48 (s, 2H, ArH). 13C NMR (DMSO-d6) δ ppm: 46.03, 65.70, 113.01, 117.93, 118.36, 120.24, 125.31, 127.74, 130.21, 132.31, 132.87, 135.83, 138.83, 146.97, 150.19, 150.19, 161.61, 165.47. MS m/z: 462.04. Anal. Calcd: C21H15BrN6O2; C, 54.44; H, 3.26; N, 18.44%. Found: C, 54.48; H, 3.30; N, 18.46%.