Discovery of potent, novel antibacterial hybrid conjugates from 4-aminoquinoline and 1,3,5-triazine: design, synthesis and antibacterial evaluation

Abstract

A series of hybrid 4-aminoquinoline-1,3,5-triazine conjugates 7a–g were synthesized and evaluated for their in vitro antibacterial activity against several Gram-positive and Gram-negative microorganisms. The entire set of target compounds displayed potent to excellent activity against human disease causing pathogens with reference to Levofloxacin as a standard drug.

---

Introduction

The rise of infection caused by the rapid development of bacterial resistance to most of the known antibiotics is a serious health problem.^1,2 Several factors are liable for mutations in microbial genomes and it has been generally established that incorrect use of antibiotics can greatly increase the development of resistant genotypes.³ Generations of multidrug-resistant (MDR) bacterial strains grow which further necessitates the discovery of novel antimicrobial molecules.⁴

The 1,3,5-triazine scaffold has provided the basis for the design of biologically significant molecules with diverse therapeutic profiles, e.g., as antifungal,^5,6 anticancer,⁷ antimalarial,^8,9 antiviral¹⁰ and antimicrobial agents.^11,12 As a part of our ongoing research on the discovery of economic and potential antimicrobial agents derived from 1,3,5-triazine,^13,14 herein, we investigate the antimicrobial potential of hybrid conjugates derived from 4-aminoquinoline and 1,3,5-triazine.

Results and discussion

Chemistry

The desired compounds 3, 5a–g, 6a–g and 7a–g were synthesised by the synthetic protocols as outlined in Scheme 1. The synthesis of compound (3) was achieved by reacting potassium thiocyanate (2) with 4-chloro of 4,7-dichloroquinoline (1) in the presence of anhydrous toluene along with a few granules of tin. The synthesis of di-substituted 1,3,5-triazines 5a–g was accomplished by the aromatic nucleophilic substitution (S_NAr) of the Cl atom of 2,4,6-trichloro-1,3,5-triazine (4) with different primary and secondary amines (a–g). Whereas, tri-substituted 1,3,5-triazines 6a–g were obtained by aromatic nucleophilic substitution (S_NAr) of the Cl atom of mono-chloro di-substituted 1,3,5-triazine (5a–g) with piperazine. Finally the target hybrid compounds 7a–g were synthesized by incorporating the tri-substituted 1,3,5-triazine skeleton 6a–g on to the 4-quinolyl isothiocyanates (3).

Scheme 1 Reagents and conditions: (a) Reflux with stirring for 18 h at 90–120 °C b) Dry acetone, 0–5 °C 1 h, 40–45 °C, for further 3 h, NaHCO₃ (c) Piperazine, 1,4-dioxane 120–130 °C, 6–7 h, K₂CO₃ (d) Dry acetone, 40–45 °C, 18 h.

From the structural investigation, FTIR spectra of compounds, peaks in the range of 1548.28–1387.38 cm⁻¹ attributable to aromatic C=N group of 1,3,5-triazine, where as the C=C aromatic group appears at 1475 cm⁻¹. Many strong absorption bands at 850–670 cm⁻¹ confirm the existence of the aromatic ring. The ¹H NMR spectra reveal a signal corresponding to quinoline at 7.27–8.85 ppm. The tri-substituted 1,3,5-triazine shielding for bridged NH was usually observed at 3.59 to 3.69 ppm but the chemical shift of the –NH bridge was lowered approximately by 0.55 ppm in the case of 6a–g. Moreover, all mass spectra and elemental analyses are in agreement with the proposed structures.

Antibacterial activity

As shown in Table 1 and 2 antibacterial screening of all the synthesized compounds 7a–g revealed potent to moderate activities against the tested Gram-positive and Gram-negative micro-organisms in comparison to Levofloxacin as a standard. Compounds with bromo substitution on the phenyl amine position 7a connected to the 1,3,5-triazine core exhibit significant activity against B. cereus, equipotent activity against E. coli and P. vulgaris and moderate activity against the S. aureus, B. subtilis, P. mirabilis. Replacement of a bromo by a methoxy in the case of compound 7b, yields significant activity against B. cereus, equipotent against B. subtilis, P. mirabilis, P. vulgaris and moderate against S. aureus, P. aeruginosa and E.coli. Compound 7c, formed by replacement of the methoxy by a chloro group disclosed the significant antibacterial activity against P. mirabilis and was equipotent against B. subtilis and B. cereus. Whereas, replacement of the chloro by a nitro in compound 7d, yields significant activity against S. aureus, B. subtilis, E. coli and moderate activity against B. cereus , P. aeruginosa, P. mirabilis and P. vulgaris. Moderate activity was observed in the case of compound 7e having a methyl group as substituent, towards all Gram-positive and Gram-negative strains. On the other hand, introduction of morpholine 7f caused a marked increase in activity against P. vulgaris and equipotent activity against P. aeruginosa, E. coli and moderate against S. aureus, B. subtilis, B. cereus. However, significant activity was observed in the case of the analogue that has an o-methyl substituent 7g aganist E.coli and moderate activity against S. aureus , B. cereus , B. subtilis respectively.

Table 1 Antimicrobial screening results of the tested compounds

Compounds	Zone of Inhibition/mm
	S. aureus	B. subtilis	B. cereus	P. aeruginosa	E. coli	P. mirabilis	P. vulgaris
7a	15	14	16	14	16	14	15
7b	14	15	15	14	14	15	15
7c	13	15	14	14	15	16	13
7d	16	17	13	13	17	13	14
7e	10	11	09	12	11	12	11
7f	14	12	13	15	16	14	17
7g	12	11	10	14	17	14	14
Levofloxacin (standard)	15	15	14	15	16	15	15

---

Table 2 MIC (μg mL⁻¹) results of the tested compounds

Compounds	Minimum Inhibitory Concentration (μg mL^−1)
	S. aureus	B. subtilis	B. cereus	P. aeruginosa	E. coli	P. mirabilis	P. vulgaris
7a	6.25	12.5	12.5	12.5	6.25	12.5	6.25
7b	12.5	6.25	12.5	12.5	12.5	6.25	6.25
7c	12.5	6.25	6.25	12.5	12.5	3.125	12.5
7d	3.125	3.125	12.5	3.125	3.125	25	12.5
7e	25	25	50	25	50	25	25
7f	12.5	25	25	6.25	6.25	12.5	3.125
7g	25	25	50	12.5	3.125	12.5	12.5
Levofloxacin (standard)	6.25	6.25	6.25	6.25	6.25	6.25	6.25

---

It was inferred from the bioactivity profile of the target hybrid derivatives that compounds having a halogen group at the fourth position of phenyl amine viz., 7a and 7c showed activity against B. cereus and P. mirabilis and no activity was found against P. aeruginosa. Replacement of halogen with nitro 7d and methoxy 7b makes the compound prominent active against B. subtilis, E. coli but not against B. cereus, P. aeruginosa. In addition, it was also corroborated that, replacement of the halogen by a non-halogen substituent, leads to remarkable decreases in activity for B. cereus and P. mirabilis and a further decrease was reported for the rest of the strains. On the basis of these results, our hypothesis is that electron withdrawing groups on the para position of the R substituent are necessary to generate a potential antimicrobial compound.

Experimental

All commercially available solvents and reagents were of analytical grade and used without further purification. Melting points were determined on a Veego, MPI melting point apparatus and FTIR (2.0 cm⁻¹, flat, smooth, abex) were recorded on Perkin Elmer RX-I Spectrophotometer. ¹H NMR spectra were recorded on Bruker Avance II 400 NMR and ¹³C NMR spectra on Bruker Avance II 100 NMR spectrometer in DMSO-d₆ using TMS as internal standard. Mass spectra were obtained on VG-AUTOSPEC spectrometer equipped with electrospray ionization (ESI) sources. Elemental analysis was carried out on Vario EL-III CHNOS elemental analyzer.

Chloro-4-isothiocyanatoquinoline (3). A solution of 4,7-dichloroquinoline (1) (0.01 mol), potassium thiocynate (2) (0.02 mol) and few pieces of tin metal in anhydrous toluene was refluxed at 90–120 °C for 18 h. The completion of reaction was monitored by TLC using ethanol : acetone (1 : 1) as mobile phase. The reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane, washed with brine and dried over Na₂SO₄. The dried solution was concentrated under reduced pressure to obtain the title compound (3)

Brown crystal, Yield: 68%; M.p: 197–198 °C; MW: 220.68 ; R_f: 0.48; FT-IR (ν_max; cm⁻¹ KBr): 1275 (C–N), 1630 (C=C), 1690–1640 (C=N), 3000 (C–H), 1600 (C=C, aromatic ring), 1470 (C=C, aromatic ring); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 8.85 (d, 1H J = 4.97 Hz, quinoline ring), 7.27 (d, 1H J = 4.97 Hz, quinoline ring), 7.76 (d, 1H J = 8.60 Hz, quinoline ring), 8.40 (d, 1H J = 8.60 Hz, quinoline ring), 8.01 (d, 1H J = 1.96 Hz, quinoline ring); ¹³C NMR (100 MHz, CDCl₃); δ (ppm): 152.30, 118.30, 138.40, 124.10, 129.80, 128.60, 135.20, 129.10, 137.20; Mass: 221.60 (M+H)⁺; Elemental analysis for C₁₀H₅ClN₂S: Calculated: C, 54.43; H, 2.28; N, 12.69. Found: C, 54.41; H, 2.23; N, 12.58.

General procedure for the synthesis of di-substituted 1,3,5-triazine derivatives 5a–g. Various distinguished amines (a–g) (0.2 mol) were added into 100 mL of acetone at 40–45 °C. The solution of 2,4,6-trichloro-1,3,5-triazine (4) (0.1 mol) in 25 mL acetone was added constantly, stirred for 3 h followed by drop-wise addition of NaHCO₃ solution (0.1 mol), taking care that reaction mixture does not become acidic. The completion of reaction was monitored by TLC using benzene:ethyl acetate (9 : 1) as mobile phase. The product was filtered, washed with cold water and recrystallized with ethanol to afford pure compounds 5a–g.

N²,N⁴-Bis(4-bromophenyl)-6-chloro-1,3,5-triazine-2,4-diamine (5a). Brownish black crystal; Yield: 81%; M.p: 169–170 °C; MW: 455.53; R_f: 0.35; FT-IR (ν_max; cm⁻¹ KBr): 3350.62 (N–H secondary), 3015.43 (C–H broad), 1656.15 (C=C stretch); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.26 (d, 4H J = 8.72 Hz, 3,5-CH, Ar-H), 7.06 (d, 4H J = 8.53 Hz, 2,6-CH, Ar-H), 4.81 (s, 2H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.35, 167.85, 137.85, 118.50, 132.45, 116.80; Mass: 455.85 (M+H)⁺; Elemental analysis for C₁₅H₁₀Br₂ClN₅: Calculated: C, 39.55; H, 2.21; N, 15.37. Found: C, 39.53; H, 2.20; N, 15.34.

6-Chloro-N²,N⁴-bis(4-methoxyphenyl)-1,3,5-triazine-2,4-diamine (5b). Yellow crystal; Yield: 88%; M.p: 235 °C; MW: 357.79; R_f: 0.69; FT-IR (ν_max; cm⁻¹ KBr): 3300 (N–H secondary), 3015 (C–H), 1670–1685 (C=N), 1630–1640 (C=C), 1585 (C=C aromatic ring), 1460 (C=C aromatic ring), 1100–1230 (C–N); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.43 (d, 1H J = 8.68 Hz, Ar-H), 7.52 (d, 1H J = 5.49 Hz, Ar-H), 7.39 (d, 1H J = 8.76 Hz, Ar-H), 7.43 (d, 1H J = 8.51 Hz, Ar-H), 5.49 (br s, 1H, NH), 3.65 (s 3H, OCH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.35, 167.85, 131.10, 121.80, 115.10, 153.40, 55.90; Mass: 359 (M+H)⁺; Elemental analysis for C₁₇H₁₆ClN₅O₂: Calculated: C, 57.07; H, 4.51; N, 19.57. Found: C, 57.02; H, 4.45, N, 19.58.

6-Chloro-N²,N⁴-bis(4-chlorophenyl)-1,3,5-triazine-2,4-diamine (5c). White crystals; Yield: 75%; M.p: 135–137 °C; MW: 366.63; R_f: 0.48; FT-IR (ν_max; cm⁻¹ KBr): 3243.26 (N–H secondary), 2965.46 (C–H broad), 1387.38 (aromatic –C=N); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.32 (d, 4H J = 8.72 Hz, 3,5-CH, Ar-H), 7.08 (d, 4H J = 8.52 Hz 2,6-CH, Ar-H), 4.82 (s, 2H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.40, 167.85, 137.10, 122.25, 129.60, 127.70; Mass: 366 (M+H)⁺; Elemental analysis for C₁₅H₁₀Cl₃N₅: Calculated: C, 49.14; H, 2.75; N, 19.10. Found: C, 49.17; H, 2.77; N, 19.15.

6-Chloro-N²,N⁴-bis(4-nitrophenyl)-1,3,5-triazine-2,4-diamine (5d). Yellow crystals; Yield: 86%; M.p: 143–145 °C; MW: 387.74; R_f: 0.55; FT-IR (ν_max; cm⁻¹ KBr): 3289.56 (N–H secondary), 3055.70 (C–H broad), 1548.28–1446.06 (aromatic C=N); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.40 (t, 4H, 4xCH, Ar-H), 7.32 (t, 4H, 4xCH, Ar-H), 3.62 (d, 2H J = 13.03 Hz, 2XNH, Ar-H); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 126.23, 131.36, 143.16, 148.26, 168.85, 173.56; Mass: 388.10 (M+H)⁺; Elemental analysis for C₁₅H₁₀ClN₇O₄: Calculated: C, 46.46; H, 2.60; N, 25.29. Found: C, 46.48; H, 2.65; N, 25.26.

6-Chloro-N²,N⁴-di-p-tolyl-1,3,5-triazine-2,4-diamine (5e). Pale yellowish crystals; Yield: 78%; M.p: 212–214 °C; MW: 332.80; R_f: 0.72; FT-IR (ν_max; cm⁻¹ KBr): 3310 (N–H secondary), 3000 (C–H), 1605 (C=C aromatic ring), 1620–1650 (C=C), 1475 (C=C aromatic ring); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.03 (d,1H J = 8.27 Hz, Ar-H), 7.06 (d, 1H J = 5.43 Hz, Ar-H), 7.31 (d, 1H J = 8.21 Hz, Ar-H), 7.23 (d, 1H Ar-H), 5.24 (br, s, 1H, NH), 2.53 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.35, 167.85, 135.85, 120.40, 129.85, 131, 21.5; Mass: 326.20 (M+H)⁺; Elemental analysis for C₁₇H₁₆ClN₅: Calculated: C, 62.67; H, 4.95; N, 21.50. Found: C, 62.63; H, 4.98; N, 21.55.

6-Chloro-2,4-dimorpholino-1,3,5-triazine (5f). Buff white; Yield: 73.32%, M.p: 132–135 °C; FT-IR (ν_max; cm⁻¹ KBr): 2966 (C–H stretch), 1574–1451, 1362 (C–N stretch), 1116(C–O stretch); ¹H NMR (400 MHz, CDCl₃) δ (ppm): 3.70 (t, 8H J = 4.9 Hz, 4XCH₂–N), 3.78 (t, 8H, 4XCH₂–O); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 43.86, 66.56, 164.48, 169.69; Mass: 286.10 (M+H)⁺; Elemental analysis for C₁₁H₁₆ClN₅O₂: C, 46.24; H, 5.64; N, 24.51. Found: C, 46.28; H, 5.58; N, 24.56.

6-Chloro-N₂,N₄-di-o-tolyl-1,3,5-triazine-2,4-diamin (5g). Yellowish crystals; Yield: 72%; M.p: 225–226 °C; MW: 332.80; R_f: 0.67; FT-IR (ν_max; cm⁻¹ KBr): 3310 (N–H secondary), 3000 (C–H), 1605 (C=C aromatic ring), 1620–1650 (C=C), 1475 (C=C aromatic ring); ¹H- NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.05 (d,1H J = 7.89 Hz, Ar-H), 7.08 (d, 1H J = 7.50 Hz, Ar-H), 7.10 (d, 1H J = 8.68 Hz, Ar-H), 7.10 (s, 1H Ar-H), 5.24 (br, s, 1H, NH), 2.20 (s, 3H, CH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.35, 167.85, 141.50, 129, 123.85, 126.40, 123.60, 131.20,17.; Mass: 326.20 (M+H)⁺; Elemental analysis for C₁₇H₁₆ClN₅: Calculated: C, 62.67; H, 4.95; N, 21.50. Found: C, 62.63; H, 4.98; N, 21.55.

General procedure for the synthesis of tri-substituted 1,3,5-triazine derivatives. 6a–g. A solution of di-substituted 1,3,5-triazine compounds 5a–g (0.01 mol), piperazine (0.01 mol) and K₂CO₃ (0.01 mol) in 1,4-dioxane was refluxed for 6–7 h. The completion of reaction was monitored by TLC using benzene:ethyl acetate (9 : 1) as mobile phase. The reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was purified by ethanol to afford the desired product 6a–g.

N²,N⁴-Bis(4-bromophenyl)-6-(piperazin-1-yl)-1,3,5-triazine-2,4-diamine (6a). Brown crystals; Yield: 63%; M.p: 258–259 °C; MW: 505 ; R_f: 0.48; FT-IR (ν_max; cm⁻¹ KBr): 3350.62 (N–H secondary), 3015.43 (C–H broad), 1656.15 (C=C stretch), 1250 (C–N), 1650 (C=N), 1475(C=C); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.26 (d, 4H J = 8.80 Hz 3,5-CH, Ar-H), 7.06 (d, 4H , J = 5.50 Hz, 2,6-CH, Ar-H), 4.81 (s, 2H, NH), 3,28 (d, 4H J = 13.03 Hz, 2xCH₂, Ar-H), 2.91 (d,4H J = 13.29 Hz, 2xCH₂ Ar-H), 1.95 (s,1H NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.35, 167.85, 137.85, 118.50, 132.45, 116.80, 48.20, 45.50; Mass: 506.20(M+H)⁺; Elemental analysis for C₁₉H₁₉Br₂N₇: Calculated: C, 45.17; H, 3.79; N, 19.41. Found: C, 45.10; H, 3.82; N, 19.40.

N²,N⁴-Bis(4-methoxyphenyl)-6-(piperazin-1-yl)-1,3,5-triazine-2,4-diamine (6b). Light yellow crystal; Yield: 79%; M.p: 261–263 °C; MW: 407.47 ; R_f: 0.58; FT-IR (ν_max; cm⁻¹ KBr): 3300 (N–H secondary), 3015 (C–H), 1670–1685 (C=N), 1630–1640 (C=C), 1585 (C=C aromatic ring), 1460 (C=C aromatic ring), 1100–1230 (C–N). ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.43 (d, 1H J = 8.68 Hz, Ar-H), 7.52 (d, 1H J = 5.49 Hz, Ar-H), 7.39 (d, 1H J = 8.76 Hz, Ar-H), 7.43 (d, 1H Ar-H), 5.49 (br s, 1H, NH), 3.65 (s, 3H OCH3), 3.26 (d, 4H J = 13.03 Hz, 2xCH₂, Ar-H), 2.92 (d,4H J = 13.29 Hz, 2xCH₂, Ar-H), 1.94 (s,1H NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm):168.35, 167.85, 131.10, 121.80, 115.10, 153.40, 55.90, 47.70,45.10; Mass: 408.50 (M+H)⁺; Elemental analysis for C₂₁H₂₅N₇O₂: Calculated: C, 61.90; H, 6.18; N, 24.06. Found: C, 61.94; H, 6.19, N, 24.05.

N²,N⁴-Bis(4-chlorophenyl)-6-(piperazin-1-yl)-1,3,5-triazine-2,4-diamine (6c). White crystals; Yield: 76%; M.p: 234–236 °C; MW: 416.31; R_f: 0.52; FT-IR (ν_max; cm⁻¹ KBr): 3243.26 (N–H secondary), 2965.46 (C–H broad), 1387.38 (aromatic –C=N),1250 (C–N), 1475 (C=C aromatic ring), 3000 (C–H aromatic ring); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.32 (d, 4H J = 8.52 Hz, 3,5-CH ,Ar-H), 7.08 (d, 4H J = 5.52 Hz, 2,6-CH, Ar-H), 4.82 (s, 2H, NH), 3.25 (t, 4H, 2xCH₂ ,Ar-H),2.79(t, 4H, 2xCH₂, Ar-H), 1.95(s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 168.40, 167.85, 137.10, 122.25, 127.70, 48.30, 45.50; Mass: 438.20 (M+H)⁺; Elemental analysis for C₁₉H₁₉N₉O₄: Calculated: C, 52.17; H, 4.38; N, 28.82. Found: C, 51.90; H, 4.36; N, 28.81.

N²,N⁴-Bis(4-nitrophenyl)-6-(piperazin-1-yl)-1,3,5-triazine-2,4-diamine (6d). Yellow crystals; Yield: 74%; M.p: 261–263 °C; MW: 437.20; R_f: 0.69; FT-IR (ν_max; cm⁻¹ KBr): 3289.56 (N–H secondary), 3055.70 (C–H broad), 1548.28–1446.06 (aromatic C=N),1675 (C=N), 1250 (C–N), 1525(NO2 aromatic); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.40 (t, 4H, 4xCH, Ar-H), 7.32(t, 4H, 4xCH, Ar-H), 3.62 (d, 2H J = 13.03 Hz, 2xNH, Ar-H), 3.25 (t, 4H, 2xCH_2, Ar-H), 2.79(t, 4H, 2xCH₂,Ar-H), 1.95(s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 126.23, 131.36, 143.16, 148.26, 168.85, 173.56, 48.25, 45.80; Mass: 438.20 (M+H)⁺; Elemental analysis for C₁₉H₁₉N₉O₄: Calculated: C, 52.17; H, 4.38; N, 28.82. Found: C, 52.10; H, 4.39; N, 28.85.

6-(Piperazin-1-yl)-N²,N⁴-dip-tolyl-1,3,5-triazine-2,4-diamine (6e). Dark yellowish crystals; Yield: 65%; M.p: 284–286 °C; MW: 375.47; R_f: 0.63; FT-IR (ν_max; cm⁻¹ KBr): 3310 (N–H secondary), 3000 (C–H), 1605 (C=C aromatic ring), 1620–1650 (C=C), 1475 (C=C aromatic ring); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.03 (d,1H J = 8.27 Hz, Ar-H), 7.06 (d, 1H J = 5.43 Hz, Ar-H), 7.31 (d, 1H J = 8.21 Hz, Ar-H), 7.23 (d, 1H Ar-H), 5.24 (br, s, 1H, NH), 2.53 (s, 3H, CH₃) 3,26 (d, 4H J = 13.03 Hz, 2xCH₂, Ar-H), 2.92 (d,4H J = 13.29 Hz, 2xCH₂, Ar-H), 1.94 (s,1H NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm):168.35, 167.85, 135.85, 120.40, 129.85, 131, 21.5, 47.80, 45.30; Mass: 376.45 (M+H)⁺; Elemental analysis for C₂₁H₂₅N₇: Calculated: C, 67.18; H, 6.71; N, 26.11. Found: C, 66.97; H, 6.73; N, 26.10.

4,4′-(6-(Piperazin-1-yl)-1,3,5-triazine-2,4-diyl)dimorpholine (6f). White crystal; Yield: 79%: M.p: 281–283 °C; MW: 335.40; R_f: 0.72; FT-IR (ν_max; cm⁻¹ KBr): 2966 (C–H aromatic), 1574–1451 (C=C aromatic ring), 1362 (C–N) , 1116; ¹H NMR (400 MHz, CDCl₃) δ 3.70 (t, 8H J = 4.9 Hz, 4xCH₂–N), 3.78 (t, 8H, 4xCH₂–O), 3,26 (d, 4H J = 13.03 Hz, 2xCH₂, Ar-H), 2.92 (d, 4H J = 13.29 Hz, 2xCH₂, Ar-H), 1.94 (s,1H NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 43.86, 66.56, 164.48, 169.69, 47.60,45.20; Mass: 336.50 (M+H)⁺; Elemental analysis for C₁₅H₂₅N₇O₂: C, 53.71; H, 7.51; N, 29.23. Found: C, 53.64; H, 7.57; N, 29.26.

6-(Piperazin-1-yl)-N²,N⁴-dio-tolyl-1,3,5-triazine-2,4-diamine (6g). Brown crystals; Yield: 82%; M.p: 291–293 °C; MW:375.47; R_f: 0.46; FT-IR (ν_max; cm⁻¹ KBr): 3310 (N–H secondary), 3000 (C–H), 1605 (C=C aromatic ring), 1620–1650 (C=C), 1475 (C=C aromatic ring); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 7.05 (d,1H J = 7.89 Hz, Ar-H), 7.08 (d, 1H J = 7.50 Hz, Ar-H), 7.10 (d, 1H J = 8.68 Hz, Ar-H), 7.10 (s, 1H Ar-H), 5.24 (br, s, 1H, NH), 2.20 (s, 3H, CH₃), 3.26 (d, 4H J = 13.03 Hz, 2xCH₂, Ar-H), 2.92 (d,4H J = 13.29 Hz, 2xCH₂, Ar-H), 1.94 (s,1H NH); ¹³C NMR (100 MHz, CDCl₃) δ (ppm):168.35, 167.85, 141.50, 129, 123.85, 126.40, 123.60, 131.20,17, 47.5,44.97; Mass: 376.30 (M+H)⁺; Elemental analysis for C₂₁H₂₅N₇: Calculated: C, 62.67; H, 4.95; N, 21.50. Found: C, 62.69; H, 4.93; N, 21.51.

General procedure for the synthesis of titled compounds. 7a–g. A solution of compound (3) (0.01 mol.) and desired tri-substituted 1,3,5-triazine compounds 6a–g (0.01 mol.) in dry acetone was stirred at 40–45 °C for 18 h. The completion of reaction was monitored by TLC using ethanol : acetone (1 : 1) as mobile phase. The reaction mixture was filtered and concentrated under reduced pressure. The resulting residue was dissolved in dichloromethane, washed with brine and dried over anhydrous Na₂SO₄. The dried solution was concentrated under reduced pressure to obtain the titled compounds 7a–g

4-(4,6-Bis(4-bromophenylamino)-1,3,5-triazin-2-yl)-N-(7-chloroquinolin-4-yl)piperazine-1-carbothioamide (7a). Yellowish brown crystals; Yield: 71%; M.p: 315–317 °C; MW: 725.89 ; R_f: 0.76; FT-IR (ν_max; cm⁻¹ KBr): 1647 (C=C), 1236 (C–N), 1671 (C=N), 3025(C–H aromatic ring), 1475 (C=C aromatic ring), 3435 (N–H secondary), 1072, 769, 672; ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 8.20 (d, 1H J = 6.60 Hz, quinoline ring), 7.65 (d, 1H J = 6.45 Hz, quinoline ring), 3.87 (m, 4H, 2xCH₂, Ar-H), 3.33 (m, 4H, 2xCH₂, Ar-H), 7.53 (m,2H, C–H, Ar-H),7.79 (m, 2H, C–H, Ar-H), 4.10 (br, s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃); δ,ppm:179.30, 160.50, 146.70, 142.40, 140.40, 130.50, 129.80, 125.30, 118.70, 50.20; Mass: 726.90 (M+H)⁺; Elemental analysis for C₂₉H₂₄Br₂ClN₉S: Calculated: C, 46.67; H, 3.09; N, 19.40. Found: C, 46.60; H, 3.10; N, 19.56.

4-(4,6-Bis(4-methoxyphenylamino)-1,3,5-triazin-2-yl)-N-(7-chloroquinolin-4-yl)piperazine-1-carbothioamide (7b). Yellowish crystal, Yield: 66%; M.p: 320–322 °C; MW: 625.15 ; R_f: 0.73; FT-IR (ν_max; cm⁻¹ KBr): 1650 (C=C), 1236 (C–N), 1678 (C=N), 3018 (C–H aromatic ring), 1463 (C=C aromatic ring), 3410 (N–H secondary), 1570, 1176, 1033, 671; ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 8.79 (d, 1H J = 7.6 Hz, quinoline ring), 7.61 (d, 1H J = 6.20 Hz, quinoline ring), 3.98 (m, 4H, 2xCH₂, Ar-H), 3.34 (m, 4H, 2xCH₂, Ar-H), 7.55 (m, 2H, C–H, Ar-H), 7.10 (m, 2H, C–H, Ar-H), 3.83 (t, 3H, OCH₃), 4.20 (br, s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃); δ,ppm: 181.20, 162.10, 149.20, 140.70, 139.40, 133.50, 128.70, 127.60, 119.60, 51.20; Mass: 626.10 (M+H)⁺; Elemental analysis for C₃₁H₃₀ClN₉O₂S: Calculated: C, 59.22; H, 6.57; N, 21.42. Found: C, 59.24; H, 6.56; N, 21.47.

4-(4,6-Bis(4-chlorophenylamino)-1,3,5-triazin-2-yl)-N-(7-chloroquinolin-4-yl)piperazine-1-carbothioamide (7c). Whitish yellow crystals, Yield: 68%; M.p: 213–215 °C; MW: 636.99; R_f: 0.75; FT-IR (ν_max; cm⁻¹ KBr): 1645 (C=C), 1232 (C–N), 1675 (C=N), 3010 (C–H aromatic ring), 1470 (C=C aromatic ring), 3420 (N–H secondary), 1007, 671 (Cl aromatic); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 8.20 (d, 1H J = 6.62 Hz, quinoline ring), 7.33 (d, 1H J = 6.4 Hz, quinoline ring), 4.20 (m, 4H, 2xCH₂, Ar-H), 3.34 (m, 4H, 2xCH₂, Ar-H), 6.90 (m,2H C–H, Ar-H),8.03 (m, 2H, C–H, Ar-H), 4.13 (br, s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃); δ (ppm): 181.80, 176, 167.30, 152.70, 149.50, 149.30, 134.9, 129, 127, 122, 119.70, 113, 56.70, 52; Mass: 637.90 (M+H)⁺; Elemental analysis for C₂₉H₂₄Cl₃N₉S: Calculated: C, 55.35; H, 5.13; N, 17.23. Found: C, 54.68; H, 4.97; N, 19.79.

4-(4,6-Bis(4-nitrophenylamino)-1,3,5-triazin-2-yl)-N-(7-chloroquinolin-4-yl)piperazine-1-carbothioamide (7d). Yellow crystals, Yield: 58%; M.p: 230–232 °C; MW: 658.09 ; R_f: 0.81; FT-IR (ν_max; cm⁻¹ KBr): 1640 (C=C), 1235 (C–N), 1675 (C=N), 3000 (C–H aromatic ring), 1475 (C=C aromatic ring), 3300 (N–H secondary), 1525 (NO₂ aromatic); ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 8.36 (d, 1H J = 6.60 Hz, quinoline ring), 7.35 (d, 1H J = 6.65 Hz, quinoline ring), 4.07 (m, 4H, 2xCH₂, Ar-H), 3.18 (m, 4H, 2xCH₂, Ar-H), 6.90 (m,2H C–H, Ar-H),8.03 (m, 2H, C–H, Ar-H), 4.13 (br, s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃); δ (ppm): 152.70, 124.70, 121.60, 134.90, 129.40, 176.10, 167, 119, 124.70, 136.80, 48.5, 56.70, 52.70; Mass: 659.09 (M+H)⁺; Elemental analysis for C₂₉H₂₄ClN₁₁S: Calculated: C, 51.90; H, 5.50; N, 21.68. Found: C, 51.85; H, 5.53; N, 21.70.

4-(4,6-Bis(p-tolylamino)-1,3,5-triazin-2-yl)-N-(7-chloroquinolin-4-yl)piperazine-1-carbothioamide (7e). Yellowish crystals, Yield: 64%; M.p: 342–343 °C; MW: 596.15 ; R_f: 0.83; FT-IR (ν_max; cm⁻¹ KBr): 1556(C=C), 1261 (C–N), 1693 (C=N), 3020 (C–H aromatic ring), 1421 (C=C aromatic ring), 3405 (N–H secondary), 1220, 1000.6, 671 ; ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 8.86 (d, 1H J = 7.44 Hz, quinoline ring), 7.75 (d, 1H J = 6.30 Hz, quinoline ring), 4.05 (m, 4H, 2xCH₂, Ar-H), 3.17 (m, 4H, 2xCH₂, Ar-H), 7.06 (m, 2H, 2xCH, Ar-H), 7.45 (m, 2H, 2xCH, Ar-H), 2.43 (t, 3H, CH₃),4.10 (br, s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃); δ (ppm):179.30, 176, 160.50, 146.90, 142.50, 140.40, 135.90, 130.50, 129.80, 125.30, 56.70, 52, 21.30; Mass: 597.20 (M+H)⁺; Elemental analysis for C₃₁H₃₀ClN₉S: Calculated: C, 61.96; H, 6.94; N, 20.65. Found: C, 61.98; H, 7.01; N, 20.64.

N-(7-Chloroquinolin-4-yl)-4-(4,6-dimorpholino-1,3,5-triazin-2-yl)piperazine-1-carbothioamide (7f). Brown crystal, Yield: 77%; M.p: 333–334 °C; MW: 557.08 ; R_f: 0.82; FT-IR (ν_max; cm⁻¹ KBr): 1579 (C=C), 1243 (C–N), 1689 (C=N), 3042 (C–H aromatic ring), 1444 (C=C aromatic ring), 3347 (N–H secondary), 1301, 1218, 699; ¹H NMR (400 MHz, CDCl₃-d₆, TMS) δ (ppm): 8.82 (d, 1H J = 7.40 Hz, quinoline ring), 7.68 (d, 1H J = 6.40 Hz, quinoline ring), 4.10 (m, 4H, 2xCH₂), 3.10 (m, 4H, 2xCH₂), 3.78 (m,4H, 2x CH₂), 3.58 (m, 4H, 2xCH₂),4.25 (br, s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃); δ,ppm: 181.30, 179.40, 160.50, 146.90, 142.40, 140.40, 130.50, 129.80, 125.30, 66.30, 50.10, 48.70; Mass: 558.10 (M+H)⁺; Elemental analysis for C₂₅H₃₀ClN₉O₂S: Calculated: C, 46.12; H, 6.38; N, 24.33. Found: C, 46.10; H, 6.32; N, 24.30.

4-(4,6-Bis(o-tolylamino)-1,3,5-triazin-2-yl)-N-(7-chloroquinolin-4-yl)piperazine-1-carbothioamide (7g). Yellowish crystals, Yield: 71%; M.p: 396–398 °C; MW: 596.15 ; R_f: 0.78; FT-IR (ν_max; cm⁻¹ KBr): 1556(C=C), 1261 (C–N), 1693 (C=N), 3020 (C–H aromatic ring), 1421 (C=C aromatic ring), 3404 (N–H secondary), 1225, 1020, 672,; ¹H NMR (400 MHz, CDCl₃–d₆, TMS) δ (ppm): 8.85 (d, 1H J = 7.44 Hz, quinoline ring), 7.73 (d, 1H J = 6.32 Hz, quinoline ring), 4.06 (m, 4H, 2xCH₂, Ar-H), 3.17 (m, 4H, 2xCH₂, Ar-H), 7.15 (m, 2H, 2xCH, Ar-H), 6.69 (m, 2H, 2xCH, Ar-H), 2.15 (t, 3H, CH₃),4.05 (br, s, 1H, NH); ¹³C NMR (100 MHz, CDCl₃); δ (ppm): 179.30, 176, 160.50, 146.90, 142.50, 140.40, 135.90, 130.50, 129.80, 126.50, 123.70, 17.70; Mass: 597.20 (M+H)⁺; Elemental analysis for C₃₁H₃₀ClN₉S: Calculated: C, 61.96; H, 6.94; N, 20.65. Found: C, 61.92; H, 7.05; N, 20.58.

Antibacterial screening

Disc diffusion. The inoculum can be prepared by making a direct broth or saline suspension of isolated colonies of the same strain from 18 to 24 h Müeller-Hinton agar plate. The suspension is adjusted to match the 0.5 McFarland turbidity standard, using saline and a vortex mixer. Optimally, within 15 min after adjusting the turbidity of the inoculum suspension, a sterile cotton swab is dipped into the adjusted suspension and then the dried surface of an agar plate is inoculated by streaking the swab over the entire sterile agar surface. Any surface moisture to be absorbed before applying the drug impregnated disk.

The plates containing bacterial inoculums received a disc of levofloxacin (5 μg) and synthesized compound (5 μg), while the control plate was inoculated with DMSO which shows no inhibition of bacterial growth. Each disc must be pressed down to ensure complete contact with the agar surface. Then plates are inverted and placed in an incubator set to 35 °C within 15 min after the discs are applied. They were then incubated at 37 °C for 24 h, after which the inhibition halo was measured with a milimetric ruler. This qualitative screening was performed to verify positive antimicrobial activity of the synthesized compound. Each test was carried out in triplicate.¹⁵ Results were shown in Table 1 and 2.

Minimum inhibitory concentration. All synthesized compounds were screened for their minimum inhibitory concentration (MIC, μg mL⁻¹) against selected Gram-positive organisms viz. Bacillus subtilis (NCIM-2063), Bacillus cereus (NCIM-2156), S.aureus (NCIM-2079) and Gram-negative organism viz. Pseudomonas aeruginosa (NCIM-2036), Escherichia coli (NCIM-2065), P.mirabilis (NCIM-2241), P.vulgaris (NCIM-2027) by the broth dilution method as recommended by the National Committee for Clinical Laboratory Standards with minor modifications. Levofloxacin was used as the standard antibacterial agent. Solutions of the test compounds and reference drug were prepared in dimethyl sulfoxide (DMSO) at concentrations of 100, 50, 25, 12.5, 6.25, 3.125 μg mL⁻¹. Eight tubes were prepared in duplicate with the second set being used as MIC reference controls (16–24 h visual). After sample preparation, the controls were placed in a 37 °C incubator and read for macroscopic growth (clear or turbid) the next day. Into each tube, 0.8 mL of nutrient broth was pipetted (tubes 2–7), tube 1 (negative control) received 1.0 mL of nutrient broth and tube 8 (positive control) received 0.9 mL of nutrient. Tube 1, the negative control, did not contain bacteria or antibiotic. The positive control, tube 8, received 0.9 mL of nutrient broth since it contained bacteria but not antibiotic. The test compound were dissolved in DMSO (100 μg mL⁻¹), 0.1 mL of increasing concentration of the prepared test compounds which are serially diluted from tube 2 to tube 7 from highest (100 μg mL⁻¹) to lowest (3.125 μg mL⁻¹) concentration (tube 2–7 containing 100, 50, 25, 12.5, 6.25, 3.125 μg mL⁻¹). After this process, each tube was inoculated with 0.1 mL of the bacterial suspension whose concentration corresponded to 0.5 McFarland scale (9 × 10⁸ cells mL⁻¹) and each bacterium was incubated at 37 °C for 24 h at 150 rpm. The final volume in each tube was 1.0 mL. The incubation chamber was kept humid. At the end of the incubation period, MIC values were recorded as the lowest concentration of the substance that gave no visible turbidity, i.e. no growth of inoculated bacteria. Disc diffusion along with percentage of inhibition in comparison to standard The inoculum can be prepared by making a direct broth or saline suspension of isolated colonies of the same strain from 18 to 24 h Müeller-Hinton agar plate. The suspension is adjusted to match the 0.5 McFarland turbidity standard, using saline and a vortex mixer. Optimally, within 15 min after adjusting the turbidity of the inoculum suspension, a sterile cotton swab is dipped into the adjusted suspension and then afterwards the dried surface of a agar plate is inoculated by streaking the swab over the entire sterile agar surface. Any excess surface moisture to be absorbed before applying the drug impregnated disk.¹⁶

Conclusions

A series of hybrid 4-aminoquinoline-1,3,5-triazine derivatives were developed through facile synthetic routes. These compounds showed excellent to significant antibacterial activity against a panel of Gram-positive and Gram-negative micro-organisms. We have started to develop advanced analogues of this hybrid skeleton which will be reported in the future.

Acknowledgements

Authors acknowledge S.A.I.F. Punjab University Chandigarh for providing spectroscopic data.

References

1. B. Beovic, Int. J. Food Microbiol., 2006, 112, 280.
2. R. Finch and P. A. Hunter, J. Antimicrob. Chemother., 2006, 58, i3.
3. P. F. Chan, R. Macarron, D. J. Payne, M. Zalacain and D. J. Holmes, Curr. Drug Targets: Infect. Disord., 2002, 2, 291.
4. J. Davies and D. Davies, Microbiol. Mol. Biol. Rev., 2010, 74, 417.
5. U. P. Singh, H. R. Bhat and P. Gahtori, J. Mycol. Med., 2012, 22, 134.
6. V. Milata, L. Reinpreicht and J. Kizlink, Acta. Chim. Slov., 2012, 5, 95.
7. T. H. Corbett, W. R. Leopold, D. J. Dykes, B. J. Roberts, D. P. Jr Griswold and F. M. Schabel Jr., Cancer. Res., 1982, 42, 1707.
8. H. R. Bhat, S. K. Ghosh, A. Prakash, K. Gogoi and U. P. Singh, Lett. Appl. Microbiol., 2012, 54, 483.
9. H. R. Bhat, U. P. Singh, P. S. Yadav, V. Kumar, A. Das, D. Chetia, A. Prakash and J. Mahanta, Arabian. J. Chem. In press, 2011 DOI:10.1016/j.arabjc.2011.07.001O.
10. V. Lozano, L. Aguado, B. Hoorelbeke, M. Renders, M. J. Camarasa, D. Schols, J. Balzarini, A. San-Félix and M. J. Pérez-Pérez, J. Med. Chem., 2011, 54, 5335.
11. U. P. Singh, R. K. Singh, H. R. Bhat, Y. P. Subhaschandra, V. Kumar, M. K. Kumawat and P. Gahtori, Med. Chem. Res., 2011, 20, 1603.
12. U. P. Singh, M. Pathak, V. Dubey, H. R. Bhat, P. Gahtori and R. K. Singh, Chem. Biol. Drug Des., 2012, 80, 572–583.
13. P. Gahtori, S. K. Ghosh, B. Singh, U. P. Singh, H. R. Bhat and A. Uppal, Saudi Pharm. J., 2012, 20, 35.
14. S. K. Ghosh, A. Saha, B. Hazarika, U. P. Singh, H. R. Bhat and P. Gahtori, Lett. Drug Des. Discovery, 2012, 9, 329.
15. M. K. Lalitha, C.M.C. Vellore, India, 2004.
16. National Committee for Clinical Laboratory Standards. NCCLS, Villanova, 1982, p. 242.

---