Development of 4-aminoquinoline-1,3,5-triazine conjugates as potent antibacterial agent through facile synthetic route

Article abstract of DOI:10.1007/s00044-013-0521-8

A series of novel hybrid 4-aminoquinoline-1,3,5-triazine derivatives were developed and subsequently tested against representative Gram-positive and Gram-negative microorganisms for determination of their antibacterial activity. Screening results indicate that, title molecule exhibit moderate to potent activity in comparison to standard. These hybrid derivatives were synthesized through a facile synthetic routes and structure of reaction intermediates as well as target molecules were recognised with the aid of various spectroscopic techniques viz., FTIR, NMR, mass and elemental analysis.

Full text of DOI:10.1007/s00044-013-0521-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0521-8
ORIGINAL RESEARCH
Development of 4-aminoquinoline-1,3,5-triazine conjugates
as potent antibacterial agent through facile synthetic route
•
Hans Raj Bhat Pankaj Kumar Pandey
•
•
Surajit Kumar Ghosh Udaya Pratap Singh
Received: 15 October 2012 / Accepted: 29 January 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of novel hybrid 4-aminoquinoline-1,3,5-
triazine derivatives were developed and subsequently tested
against representative Gram-positive and Gram-negative micro-
organisms for determination of their antibacterial activity.
Screening results indicate that, title molecule exhibit moderate
to potent activity in comparison to standard. These hybrid
derivatives were synthesized through a facile synthetic routes
and structure of reaction intermediates as well as target mole-
cules were recognised with the aid of various spectroscopic
techniques viz., FTIR, NMR, mass and elemental analysis.
care system in both poor and developed countries (Alanis,
2005; Shapiro et al., 2011). Whereas, non-judicious use of
these drugs has contributed largely among the other factors
that brought up the mutation in microbial genome
(Martinez and Baquero, 2000). This has put a selective pressure
and necessitates the discovery of novel antimicrobial agents
that acted via a novel pathway (Davies and Davies, 2010).
The 1,3,5-triazine scaffold has provided the basis for the
design of biologically significant molecules with diverse
therapeutic profile, e.g. as antifungal (Singh et al., 2012a,
2013a), anticancer (Corbett et al., 1982), antimalarial (Bhat
et al., 2011, 2012a, 2013), antiviral (Lozano et al., 2011)
and antibacterial activity (Gahtori et al., 2012). In contin-
uation of our project (Junejo et al., 2011), till now, we had
reported numerous antibacterial hybrid conjugates of 1,3,5-
triazine with thiazole (Singh et al., 2011), piperazine
(Ghosh et al., 2012), 4-aminoquinoline (Bhat et al., 2012b)
and 1,3,4-thiadiazole (Dubey et al., 2012), thiazolidine-4-
one (Kumar et al., 2013) as potent antimicrobial com-
pounds. In our recent communication, inhibition of
bacterial translation was identified as mechanism of action
for these conjugates (Singh et al., 2012b). Concerning our
endeavour on discovery of antibacterial agents (Singh
et al., 2013b) and prompted by these results, herein, we
disclosed the facile synthesis and antibacterial activity of
hybrid conjugates derived from 4-aminoquinoline and
1,3,5-triazine.
Keywords 1,3,5-Triazine Á 4-Aminoquinoline Á
Antibacterial
Introduction
The emergence of multi-drug resistance (MDR) pathogens
in last decades significantly jeopardises the global health-
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0521-8) contains supplementary
material, which is available to authorized users.
H. R. Bhat (&) Á P. K. Pandey Á U. P. Singh
Department of Pharmaceutical Sciences, Sam Higginbottom
Institute of Agriculture Technology & Sciences, Formerly
Allahabad Agricultural Institute, Deemed to be University,
Allahabad 211007, India
e-mail: pharmahans@gmail.com
S. K. Ghosh
Department of Pharmaceutical Sciences, Dibrugarh University,
Dibrugarh 786004, Assam, India
Results and discussion
Chemistry
Present Address:
U. P. Singh
The synthesis of title hybrid 4-aminoquinoline 1,3,5-triazine
Archimedes DoRa5 Fellow, Division of Bioorganic Chemistry,
Institute of Chemistry, University of Tartu, Tartu, Estonia
derivatives 9 (a–i) were accomplished in five steps. The first
123

Med Chem Res
step corresponds to the synthesis 7-chloro-4-isothiocyanato-
quinoline (3) was achieved by the substitution of potassium
thiocynate (2) with 4-chloro of 4,7-dichloroquinoline (1) in
the presences of few pieces of tin granules, Scheme 1.
Whereas, in second step, synthesis of mono-substituted 1,3,5-
triazines viz., 4,6-dichloro-N-(4-nitrophenyl)-1,3,5-triazin-2-
amine (6) was accomplished by nucleophilic substitution of
the Cl atom of the 2,4,6-trichloro-1,3,5-triazine (4) with
p-nitro aniline (5) in the presence of saturated solution of
NaHCO3. The above-obtained compound (6) was subse-
quently treated with different primary and secondary amines
(a–i) as presented in Scheme 2 to afford di-substituted 1,3,5-
triazines 7(a–i) via nucleophilic substitution at one of Cl atom
in the presence of activating base. The synthesis of tri-substi-
tuted 1,3,5-triazine 8(a–i) was accomplished by the nucleo-
philic substitution of the remained Cl atom of di-substituted
1,3,5-triazine 7(a–i) with piperazine in the presence of
saturated solution of NaHCO3. Whereas, title compounds
9(a–i) were synthesized by incorporating tri-substituted 1,3,5-
triazine moiety 8(a–i) with 7-chloro-4-isothiocyanatoquinoline
pharmacophore (3) and serve as step five as showed in
Scheme 3.
The series of title hybrid analogues were synthesized in
moderate to excellent yields. Whereas, structure of the
intermediate as well as target molecules were ascertained
on the basis of spectroscopic analysis. FTIR spectra of
compounds 9(a–i) in which aromatic C=N group of 1,3,5-
triazine was found in range of 1,679.28–1,548.26 cm-1
.
Whereas quinoline ring aromatic C=C group appears in
range of 1,690–1,640 cm-1, C–N group appear in range of
1,275 cm-1 and many strong absorption bands in range of
894–628 cm-1 which confirm the existence of aromatic
1
ring. The H NMR spectrums of quinoline expose a signal
in the range of 7.27–8.85 ppm. The shielding for bridged
NH was usually observed at 3.69–3.59 ppm which is
attributable to tri-substituted 1,3,5-triazine but the chemical
shift of –NH2 bridge was found in range of 1.93 ppm in
case of 8b. Moreover, all mass spectra and elemental
analysis are in agreement of proposed structures.
Antibacterial activity
Antibacterial screening of all the synthesized compounds
9(a–i) (Table 1) revealed moderate to potent activities
against the tested gram-positive and gram-negative micro-
organisms in comparison to Ofloxacin as a standard drug.
The term significant, moderate and no activity were con-
sidered for the concentration range of 3.125, 6.25–25 and
50–100 lg mL-1, respectively. Compound with morpholine
substitution on 1,3,5-triazine 9a exhibit significant activity
= =
N
N
C S
Cl
N
i
2 KSCN
+
Cl
Cl
1
2
3
Scheme 1 i Reflux with stirring 18 h at 90–120 °C, anhydrous
toluene, tin metal
NO2
NO2
N
O2
Cl
NH
NH
iii
ii
iv
N
N
N
N
N
N
N
N
+
H2N
NO2
+
R-H
-HCl
-HCl
-HCl
N
R
Cl
Cl
N
N
R
N
Cl
(a-i)
N
Cl
Cl
NH
5
7 (a-i)
4
8 (a-i)
6
=
Where, R
H
3C
H
N
H
NH2
OH
N
a
b
O
d
e
N
g
HN
HN
h
NH NH2
H
2O
HN
.
O
S
CH3
i
HN NH
NH2
HN NH
NH2
c
f
Scheme 2 Reagents and conditions: ii p-nitro aniline, 1,4-dioxane 0–5 °C, KHCO3, iii R–H (a–i) various amines, 1,4-dioxane 40–45 °C, 3 h,
KHCO3, iv piperazine, 1,4-dioxane 120–130 °C, 5–6 h, K2CO3
123

Med Chem Res
= =
N C S
H
N
HN
N
NO2
S
N
N
N
Cl
N
HN
N
N
N
NO2
3
N
N
R
R
N
N
v
Cl
H
9 (a-i)
8 (a-i)
Scheme 3 Reagents and conditions: v dry acetone, 40-45 °C, 18 h
Table 1 Antibacterial activity
of target hybrid derivatives
9(a–i)
Compounds
Minimum inhibitory concentration (MIC) (lg mL-1
Gram ?ve Gram -ve
)
S. aureus B. subtilis B. cereus P. aeruginosa E. coli P. mirabilis P. vulgaris
9a
9b
9c
9d
9e
9f
3.125
25
6.25
6.25
6.25
12.5
12.5
3.125
25
3.125
6.25
3.125
12.5
3.125
6.25
12.5
25
12.5
12.5
6.25
6.25
6.25
12.5
12.5
25
6.25
25
12.5
6.25
12.5
12.5
6.25
6.25
12.5
50
3.125
12.5
12.5
25
25
6.25
6.25
12.5
12.5
12.5
50
25
12.5
12.5
25
12.5
6.25
25
9g
9h
9i
100
12.5
25
50
6.25
3.125
3.125
6.25
6.25
6.25
12.5
3.125
12.5
3.125
Ofloxacin (standard) 6.25
3.125 3.125
against Staphlococcos aureus, Bacillus cereus, Proteus
vulgaris and moderate activity against the Pseudomonas
aeruginosa and Bacillus subtilis. Whereas, on replacement of
morpholine to hydrazine in the case of compound 9b, pre-
sented moderate activities against B. subtilis, S. aureus,
P. aeruginosa, Proteus mirabilis, P. vulgaris and Escherichia
coli. Compound 9c, formed on replacement of hydrazine
by thiosemicarbazide showed the significant antibacterial
activity against B. cereus, equipotent to ofloxacin against
P. aeruginosa and moderate against the B. subtilis, P. vulgaris
and S. aureus. Reasonable activity was observed in case of
compound 9d having o-toluidine group as substituent,
towards entire microbial strains. Replacement of o-toluidine
to aromatic amine in the case of compound 9e, causes sig-
nificant shift in the activity against B. cereus and moderately
against the B. subtilis, S. aureus, P. vulgaris and P. aerugin-
osa. Compound 9f, resulted from introduction of semicarb-
azide in place of aromatic amine disclosed the significant
antibacterial activity against B. subtilis and moderate activity
against the P. aeruginosa, E.coli, S. aureus and P. vulgaris.
Moderate activity was observed in case of compounds 9g and
9h having 1,3-diaminopropane and p-aminophenol group as
substituent, towards all gram-positive and gram-negative
strains. However, substantial-to-significant activity was
observed in the case of analogue having p-toluidine 9i against
B. cereus and moderate activity against S. aureus, E. coli,
B. subtilis, P. vulgaris and P. mirabilis, respectively.
From the bioactivity profile of the target hybrid deriva-
tives, it was inferred that compounds having aliphatic group
at 1,3,5-triazine ring viz., 9b, 9c, 9f and 9g showed promi-
nent activity against B. cereus and B. subtilis and no activity
against P. aeruginosa, S. aureus, E. coli, P. vulgaris and
P. mirabilis. While, replacement of aliphatic group with
aromatic 9a, 9d, 9e, 9h and 9i makes the compound promi-
nent active against B. cereus, P. vulgaris and no activity
against B. subtilis, E. coli, S. aureus, P. mirabilis and
P. aeruginosa. In addition, it was also corroborated that,
replacement of aliphatic by aromatic substituent, was lead to
remarkable increase in activity for B. cereus and further
decrease was reported for rest of the strains.
Experimental
Chemistry
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)
123

Med Chem Res
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 CDCl3 using TMS as internal stan-
dard. Mass spectra were obtained on VG-AUTOSPEC
spectrometer equipped with electrospray ionisation (ESI)
sources. Elemental analysis was carried out on Vario
EL-III CHNOS elemental analyser.
NMR (100 MHz, CDCl3) d ppm: 118.25, 125.32, 139.26,
144.26, 168.85, 173.56; mass: 287.34 (M?H)?; elemental
analysis for C9H5Cl2N5O2: calculated: C, 37.79; H, 1.76;
N, 24.48. Found: C, 36.48; H, 1.75; N, 24.86.
General procedure for the synthesis of di-substituted
1,3,5-triazine derivatives 7(a–i)
Various distinguished amines (a–i) (0.1 mol) were added
into 100 mL of acetone maintaining temperature 40–45 °C.
The solution of mono substituted-1,3,5-triazine (6) (0.1 mol)
in 25 mL acetone was added constantly, stirred for 3 h fol-
lowed 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 and washed with cold water and recrystallized
with ethanol to afford pure products 7(a–i).
7-Chloro-4-isothiocyanatoquinoline (3)
A solution of 4,7-dichloroquinoline (1) (0.01 mol), potas-
sium 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 mix-
ture was filtered and concentrated under reduced pressure.
The resulted 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).
4-Chloro-6-morpholino-N-(4-nitrophenyl)-1,3,5-triazin-2-
amine (7a) Light yellow crystals; yield: 76 %; m.p.: 146–
148 °C; MW: 336.73; Rf: 0.56; FTIR (mmax; cm-1 KBr):
3,288.57 (N–H secondary), 3,055.79 (C–H broad), 1,679.28–
1,638.32 (aromatic C=N), 1,348.23–1,023.12 (aromatic C–N),
Brown crystals, yield: 68 %; m.p.: 197–198 °C; MW:
220.68; Rf: 0.48; FTIR (mmax; cm-1 KBr): 1,275 (C–N), 1,630
(C=C), 1,690–1,640 (C=N), 3,000 (C–H), 1,600 (C=C, aro-
matic ring), 1,470 (C=C, aromatic ring); 1H NMR (400 MHz,
CDCl3-d6, TMS) d 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,
1
1,528.24 (NO2), 1,662, 786; H NMR (400 MHz, CDCl3-d6,
TMS) d ppm: 7.28(d, 2H J = 8.57 Hz, 29 CH, Ar–H), 3.58
(br, s, 1H, NH), 3.68 (d, 1H J = 17.31 Hz, 29 CH2, Ar–H)
3.63 (d, 1H J = 18.37 Hz, 29 CH2, Ar–H); 13C NMR
(100 MHz, CDCl3) d ppm: 118.21, 124.22, 138.26, 144.25,
168.81, 173.55, 67.34, 46.67; mass: 337.79 (M?H)?; ele-
mental analysis for C13H13ClN6O3: calculated: C, 46.37; H,
3.89; N, 24.96. Found: C, 46.43; H, 3.97; N, 24.83.
quinoline ring), 8.01 (d, 1H J = 1.96 Hz, quinoline ring); 13
C
NMR (100 MHz, CDCl3); d 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.
4-Chloro-6-hydrazinyl-N-(4-nitrophenyl)-1,3,5-triazin-2-
amine hydrate (7b) Brown crystals; yield: 86 %; m.p.:
156–158 °C; MW: 299.67; Rf: 0.63; FTIR (mmax; cm-1
KBr): 3,315.32 (NH2), 3,288.53 (N–H secondary),
3,043.79 (C–H broad), 1,676.23–1,639.32 (aromatic C=N),
1,668, 1,347.13–1,002.19 (aromatic C–N), 1,525.27 (NO2),
757; 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.12 (d,
2H J = 8.67 Hz, 29 CH, Ar–H), 7.37 (d, 2H, J = 1.95 Hz
29 CH, Ar–H), 3.67 (br, s, 1H, NH), 1.87 (s, 2H, NH2);
13C NMR (100 MHz, CDCl3) d ppm: 183.24, 172.56,
168.37, 143.24, 137.25, 126.75, 117.28; mass: 301.36
(M?H)?; elemental analysis for C9H10ClN7O3: calculated:
C, 36.07; H, 3.36; N, 32.72. Found: C, 35.96; H, 3.28; N,
32.71.
4,6-Dichloro-N-(4-nitrophenyl)-1,3,5-triazin-2-amine (6)
p-Nitro aniline (5) (0.1 mol) was added into 100 mL of
acetone at temperature 0–5 °C. The solution of 2,4,6-tri-
chloro-1,3,5-triazine (4) (0.1 mol) in 25-mL acetone was
added to above mixture constantly. The resulting mixture
was then stirred for 2 h followed by drop-wise addition of
NaHCO3 solution (0.1 mol) taking care that reaction mix-
ture 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 and washed with
cold water and recrystallized with ethanol to afford pure
products (6).
Yellow crystals; yield: 83 %; m.p.: 123–124 °C; MW:
286.07; Rf: 0.48; FTIR (mmax; cm-1 KBr): 3,289.56 (N–H
secondary), 3,056.73 (C–H broad), 1,548.26– 1,446.16
2-(4-Chloro-6-((4-nitrophenyl)amino)-1,3,5-triazin-2-yl)
hydrazinecarbothioamide (7c) Yellow crystals; yield:
69 %; m.p.: 169–170 °C; MW:340.75; Rf: 0.68; FTIR
(mmax; cm-1 KBr): 3,321.35 (NH2), 3,287.47 (N–H secondary),
3,043.76 (C–H broad), 1,676.53–1,639.36 (aromatic C=N),
1
(aromatic C=N); H NMR (400 MHz, CDCl3-d6, TMS) d
ppm: 7.63 (d, 2H J = 8.63 Hz, 29 CH, Ar–H), 7.28(d, 2H
J = 8.57 Hz, 29 CH, Ar–H), 3.62 (br, s, 1H, NH); 13C
123

Med Chem Res
1,668, 1,525.27 (NO2), 1,347.13–1,002.19 (aromatic C–N),
1,273.78 (C=S),757; 1H NMR (400 MHz, CDCl3-d6, TMS) d
ppm: 8.08 (d, 2H J = 8.65 Hz, 29 CH, Ar–H), 7.48 (d, 2H,
J = 2.96 Hz, 29 CH, Ar–H), 3.67 (br, s, 1H, NH), 1.87
(s, 1H, NH), 8.77 (s, 1H, NH2); 13C NMR (100 MHz, CDCl3)
d ppm: 184.52, 180.43, 172.20, 165.73, 152.36, 136.32,
128.36, 117.65; mass: 341.76 (M?H)?; elemental analysis for
C10H9ClN8O2S: calculated: C, 35.25; H, 2.66; N, 32.88.
Found: C, 35.27; H, 2.72; N, 32.86.
6.77 (s, 1H, NH), 6.87 (s, 1H, NH2); 13C NMR (100 MHz,
CDCl3) d ppm: 183.35, 171.93, 168.24, 158.42, 147.28,
137.56, 125.35, 118.28; mass: 325.79 (M?H)?; elemental
analysis for C10H9ClN8O3: calculated: C, 36.99; H, 2.79; N,
34.51. Found: C, 37.02; H, 2.75; N, 34.57.
N2-(3-aminopropyl)-6-chloro-N4-(4-nitrophenyl)-1,3,5-
triazine-2,4-diamine (7g) Yellow white crystals; yield:
75 %; m.p.: 172–173 °C; MW:323.74; Rf: 0.54; FTIR
(mmax; cm-1 KBr): 3,319.56 (NH2), 3,284.57 (N–H sec-
ondary), 3,041.78 (C–H broad), 1,674.45–1,638.12 (aro-
matic C=N), 1,668, 1,347.13–1,023.19 (aromatic C–N),
1,527.27 (NO2), 759; 1H NMR (400 MHz, CDCl3-d6,
TMS) d ppm: 8.12 (d, 2H J = 8.66 Hz, 29 CH, Ar–H),
7.37 (d, 2H, J = 2.12 Hz, 29 CH, Ar–H), 3.67 (br,s, 1H,
NH), 3.25 (t, 2H J = 6.76 Hz, CH2,), 2.62 (t, 2H,
J = 6.85 Hz, CH2), 1.66 (t, 2H, J = 6.76 Hz, CH2), 6.87
(s, 1H, NH2); 13C NMR (100 MHz, CDCl3) d ppm: 173.25,
171.43, 165.73, 148.12, 136.43, 126.65, 119.28, 38.58,
31.63; mass: 324.83 (M?H)?; elemental analysis for
C12H14ClN7O2: calculated: C, 44.52; H, 4.36; N, 30.29.
Found: C, 45.03; H, 4.34; N, 30.28.
6-Chloro-N2-(4-nitrophenyl)-N4-(o-tolyl)-1,3,5-triazine-2,4-
diamine (7d) White crystals; yield: 73 %; m.p.: 152–
153 °C; MW:356.77; Rf: 0.47; FTIR (mmax; cm-1 KBr):
3,285.45 (N–H secondary), 3,045.72 (C–H broad), 1,672.51–
1,631.30 (aromatic C=N), 1,667–1,600 (aromatic C=C),
1,528.22 (NO2), 1,346.12–1,016.12 (aromatic C–N), 894, 635;
1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.12 (d, 2H
J = 8.66 Hz, 29 CH, Ar–H), 7.34 (d, 2H, J = 0.71 Hz, 29
CH, Ar–H), 3.67 (br, s, 1H, NH), 7.12(d, 1H J = 8.64 Hz, 19
CH, Ar–H), 7.03 (d, 1H J = 7.49 Hz, 19 CH, Ar–H), 6.72
(d, 1H J = 1.17 Hz, 19 CH, Ar–H), 2.21 (s, 3H, CH3); 13C
NMR (100 MHz, CDCl3) d ppm: 167.32, 164.62, 163.27,
146.23, 141.82, 138.21, 132.82, 129.25, 125.32, 123.73,
122.83, 118.56, 17.63; mass: 357.73 (M?H)?; elemental
analysis for C16H13ClN6O2: calculated: C, 53.86; H, 3.67; N,
23.56. Found: C, 54.02; H, 3.64; N, 23.58.
4-((4-Chloro-6-((4-nitrophenyl)amino)-1,3,5-triazin-2-yl)
amino)phenol (7h) Black crystals; yield: 63 %; m.p.:
184–185 °C; MW:358.74; Rf: 0.42; FTIR (mmax; cm-1
KBr): 3,416 (OH stretching), 3,286.51 (N–H secondary),
3,046.58 (C–H broad), 2,800–3,012 (CH2 stretching),
1,672.42–1,637.16 (aromatic C=N), 1,605 (C=C), 1,347.13–
6-Chloro-N2-(4-nitrophenyl)-N4-phenyl-1,3,5-triazine-2,4-
diamine (7e) Light brown crystals; yield: 81 %; m.p.:
182–183 °C; MW:342.74; Rf: 0.52; FTIR (mmax; cm-1
KBr): 3,285.45 (N–H secondary), 3,045.78 (C–H broad),
1,672.53–1,631.33 (aromatic C=N), 1,671–1,606 (aromatic
C=C), 1,524.32 (NO2), 1,345.10–1,016.14 (aromatic C–N),
1
1,023.19 (aromatic C–N), 1,527.27 (NO2), 753; H NMR
(400 MHz, CDCl3-d6, TMS) d ppm: 8.14 (d, 2H J = 8.65 Hz,
29 CH, Ar–H), 7.34 (d, 2H, J = 0.73 Hz, 29 CH, Ar–H),
3.67 (br,s, 1H, NH), 7.41 (d, 1H J = 8.73 Hz, Ar–H), 6.82
(d, 1H, J = 2.26 Hz, Ar–H), 5.67 (s, 1H, OH); 13C NMR
(100 MHz, CDCl3) d ppm: 173.25, 169.54, 167.43, 148.52,
143.85, 139.62, 132.21, 125.73, 122.82, 118.27, 114.75; mass:
359.65 (M?H)?; elemental analysis for C15H11ClN6O3: cal-
culated: C, 50.22; H, 3.09; N, 23.43. Found: C, 50.32; H, 3.07;
N, 23.46.
1
775, 628; H NMR (400 MHz, CDCl3-d6, TMS) d ppm:
8.14 (d, 2H J = 8.65 Hz, 29 CH, Ar–H), 7.34 (d, 2H,
J = 0.74 Hz, 29 CH, Ar–H), 3.67 (br,s, 1H, NH), 7.53(d,
1H J = 8.29 Hz, 19 CH, Ar–H), 7.48 (d, 1H J = 8.15 Hz,
19 CH, Ar–H), 6.89(d, 1H J = 1.18 Hz, 19 CH, Ar–H);
13C NMR (100 MHz, CDCl3) d ppm: 167.31, 164.52,
164.23, 143.52, 139.39, 137.25, 129.53, 124.73, 122.86,
119.37, 115.84; mass: 343.76 (M?H)?; elemental analysis
for C15H11ClN6O2: calculated: C, 53.86; H, 3.67; N, 23.56.
Found: C, 54.02; H, 3.64; N, 23.58.
6-Chloro-N2-(4-nitrophenyl)-N4-(p-tolyl)-1,3,5-triazine-2,4-
diamine (7i) Light yellow crystals; yield: 65 %; m.p.:
158–159 °C; MW:356.77; Rf: 0.63; FTIR (mmax; cm-1
KBr): 3,285.45 (N–H secondary), 3,045.72 (C–H broad),
1,672.51–1,631.30 (aromatic C=N), 1,667–1,600 (aromatic
C=C), 1,528.22 (NO2), 1,346.12–1,016.12 (aromatic C–N),
2-(4-Chloro-6-((4-nitrophenyl)amino)-1,3,5-triazin-2-yl)
hydrazinecarboxamide (7f) Light yellow crystals; yield:
68 %; m.p.: 156–159 °C; MW:324.68; Rf: 0.46; FTIR
(mmax; cm-1 KBr): 3,321.35 (NH2), 3,287.47 (N–H second-
ary), 3,043.76 (C–H broad), 1,676.53–1,639.36 (aromatic
C=N), 1,668, 1,525.27 (NO2), 1,347.13–1,002.19 (aromatic
C–N), 1,753.62 (C=O),758; 1H NMR (400 MHz, CDCl3-d6,
TMS) d ppm: 8.05 (d, 2H J = 8.67 Hz, 29 CH, Ar–H), 7.65
(d, 2H, J = 0.74 Hz, 29 CH, Ar–H), 3.67 (br,s, 1H, NH),
1
894, 635; H NMR (400 MHz, CDCl3-d6, TMS) d ppm:
8.13 (d, 2H J = 8.64 Hz, 29 CH, Ar–H), 7.32 (d, 2H,
J = 0.74 Hz, 29 CH, Ar–H), 3.67 (br,s, 1H, NH), 7.35 (d,
1H J = 8.27 Hz, Ar–H), 6.97 (d, 1H J = 5.49 Hz, Ar–H),
2.21 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3) d ppm:
172.45, 168.32, 167.36, 152.65, 138.14, 134.53, 130.21,
128.72, 123.87, 118.52, 117.28, 24.37; mass: 357.53 (M?H)?;
123

Med Chem Res
2-(4-((4-Nitrophenyl)amino)-6-(piperazin-1-yl)-1,3,5-triazin-
2-yl)hydrazinecarbothioamide (8c) Brown crystals; yield:
71 %; m.p.: 197–198 °C; MW: 390.42; Rf: 0.58; FTIR
(mmax; cm-1 KBr):3,328.98 (N–H stretching in piperazine),
3,321.43 (NH2), 3,286.25 (N–H secondary), 3,048.83 (C–H
broad), 1,676.25–1,630.48 (aromatic C=N), 1,617.15 (N–H
bending piperazine), 1,525.27 (NO2), 1,349.13-1,007.19
elemental analysis for C16H13ClN6O2: calculated: C, 53.86; H,
3.67; N, 23.56. Found: C, 54.02; H, 3.64; N, 23.58.
General procedure for the synthesis of tri-substituted
1,3,5-triazine derivatives 8(a–i)
A solution of di-substituted 1,3,5-triazine compounds 7(a–i)
(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 8(a–i).
1
(aromatic C–N), 1,274.78 (C=S),753; H NMR (400 MHz,
CDCl3-d6, TMS) d ppm: 8.05 (d, 2H J = 8.67 Hz, 29 CH,
Ar–H), 7.94 (d, 2H, J = 0.96 Hz, 29 CH, Ar–H), 3.67 (br,s,
1H, NH), 1.97 (s, 1H, NH), 8.57 (s, 1H, NH2), 3.18 (d, 4H
J = 13.29 Hz, 29 CH2, Ar–H), 2.74 (d,4H J = 3.29 Hz, 29
CH2, Ar–H), 1.95 (s,1H NH); 13C NMR (100 MHz, CDCl3)
d ppm: 185.54, 181.32, 178.53, 186.72, 148,75, 139.36,
127.83, 121.59, 49.13, 44.57; mass: 391.56 (M?H)?; ele-
mental analysis for C14H18N10O2S: calculated: C, 43.07; H,
4.65; N, 35.88. Found: C, 43.11; H, 4.67; N, 35.89.
4-Morpholino-N-(4-nitrophenyl)-6-(piperazin-1-yl)-1,3,5-
triazin-2-amine (8a) White crystals; yield: 72 %; m.p.:
216–217 °C; MW: 386.41; Rf: 0.58; FTIR (mmax; cm-1
KBr): 3,323.98 (N–H stretching in piperazine), 3,287.37
(N–H secondary), 3,056.78 (C–H broad), 2,813–3,003 (CH2
stretching), 1,282–1,178 (C–N stretching), 1,676.25–1,637.
39 (aromatic C=N), 1,616.15 (N–H bending piperazine),
N2-(4-nitrophenyl)-6-(piperazin-1-yl)-N4-(o-tolyl)-1,3,5-
triazine-2,4-diamine (8d) Light yellow crystals; yield:
64 %; m.p.: 212–213 °C; MW:406.44; Rf: 0.47; FTIR
(mmax; cm-1 KBr): 3,328.98 (N–H stretching in piperazine),
3,286.43 (N–H secondary), 3,043.78 (C–H broad), 1,679.56–
1,637.35 (aromatic C=N), 1,663–1,602 (aromatic C=C),
1,617.15 (N–H bending piperazine), 1,525.29 (NO2), 1,455.38
(C–C stretching aromatic), 1,349.42–1,018.15 (aromatic
1
1,528.53 (NO2), 1,605 (C=C), 784; H NMR (400 MHz,
CDCl3-d6, TMS) d ppm: 8.12 (d, 2H J = 8.66 Hz, 29 CH,
Ar–H), 7.32 (d, 2H J = 0.71 Hz, 29 CH, Ar–H), 3.62 (br, s,
1H, NH), 3.69 (d, 1H J = 17.80 Hz, 29 CH2, Ar–H) 3.65
(d, 1H J = 15.61 Hz, 29 CH2, Ar–H), 3.12 (d, 4H J =
13.29 Hz, 29 CH2, Ar–H), 2.73 (d,4H J = 3.21 Hz, 29
CH2, Ar–H), 1.94 (s,1H NH); 13C NMR (100 MHz, CDCl3)
d ppm: 183.32, 178.65, 168.86, 147.42, 138.53, 126.21,
119.85, 68.36, 48.73, 46.63, 45.71; mass: 387.63 (M?H)?;
elemental analysis for C17H22N8O3: calculated: C, 52.84; H,
5.74; N, 29.00. Found: C, 52.82; H, 5.71; N, 28.85.
1
C–N), 892, 635; H NMR (400 MHz, CDCl3-d6, TMS) d
ppm: 8.15 (d, 2H J = 8.62 Hz, 29 CH, Ar–H), 7.38 (d, 2H,
J = 1.15 Hz, 29 CH, Ar–H), 3.64 (br,s, 1H, NH), 7.14(d, 1H
J = 8.42 Hz, 19 CH, Ar–H), 7.01 (d, 1H J = 7.89 Hz, 19
CH, Ar–H), 6.86 (d, 1H J = 1.18 Hz, 19 CH, Ar–H), 2.16
(s, 3H, CH3), 3.21 (d, 4H J = 13.26 Hz, 29 CH2, Ar–H), 2.82
(d,4H J = 3.11 Hz, 29 CH2, Ar–H), 1.98 (s,1H NH); 13C
NMR (100 MHz, CDCl3) d ppm: 184.56, 176.73, 168.63,
152.32, 143.21, 139.92, 134.36, 131.42, 128.57, 125.73,
124.83, 119.25, 52.31, 46.82, 18.64; mass: 407.47 (M?H)?;
elemental analysis for C20H22N8O2: calculated: C, 59.10; H,
5.46; N, 27.57. Found: C, 59.13; H, 5.47; N, 27.54.
4-Hydrazinyl-N-(4-nitrophenyl)-6-(piperazin-1-yl)-1,3,5-
triazin-2-amine hydrate (8b) Light yellow crystals; yield:
59 %; m.p.: 187–188 °C; MW: 349.35; Rf: 0.46; FTIR
(mmax; cm-1 KBr): 3,323.98 (N–H stretching in piperazine),
3,312.34 (NH2), 3,287.56 (N–H secondary), 3,046.72 (C–H
broad), 2,803–3,012 (aromatic CH2) 1,676.23–1,639.32
(aromatic C=N), 1,616.15 (N–H bending piperazine), 1,606
(C=C), 1,349.13-1,008.19 (aromatic C–N), 1,523.27 (NO2),
767; 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.13 (d,
2H J = 8.67 Hz, 29 CH, Ar–H), 7.35 (d, 2H, J = 1.01 Hz
29 CH, Ar–H), 3.65 (br,s 1H, NH), 1.97 (s, 2H, NH2), 3.15
(d, 4H J = 13.25 Hz, 29 CH2, Ar–H), 2.76 (d,4H
J = 3.11 Hz, 29 CH2, Ar–H), 1.97 (s,1H NH); 13C NMR
(100 MHz, CDCl3) d ppm: 184.25, 178.54, 171.75, 152.12,
142.65, 128.73, 121.51, 52.48, 47.14; mass: 351.12
(M?H)?; elemental analysis for C13H19N9O3: calculated:
C, 44.69; H, 5.48; N, 36.08. Found: C, 44.71; H, 5.49; N,
36.12.
N2-(4-nitrophenyl)-N4-phenyl-6-(piperazin-1-yl)-1,3,5-tri-
azine-2,4-diamine (8e) Brown crystals; yield: 79 %; m.p.:
217–218 °C; MW: 392.41; Rf: 0.59; FTIR (mmax; cm-1
KBr): 3,328.98 (N–H stretching in piperazine), 3,285.45
(N–H secondary), 3,045.78 (C–H broad), 1,672.53–
1,631.33 (aromatic C=N), 1,617.15 (N–H bending pipera-
zine), 1,590.27 (C=C stretching aromatic) 1,529.38 (NO2),
1,349.15–1,015.19 (aromatic C–N), 779, 625; 1H NMR
(400 MHz, CDCl3-d6, TMS) d ppm: 8.13 (d, 2H J = 8.67 Hz,
29 CH, Ar–H), 7.32 (d, 2H, J = 0.69 Hz, 29 CH, Ar–H),
3.68 (br,s, 1H, NH), 7.55 (d, 1H J = 8.15 Hz, 19 CH, Ar–H),
7.46 (d, 1H J = 8.13 Hz, 19 CH, Ar–H), 6.91 (d, 1H
J = 1.21 Hz, 19 CH, Ar–H), 3.18 (d, 4H J = 13.29 Hz,
123

Med Chem Res
29 CH2, Ar–H), 2.91 (d,4H J = 3.16 Hz, 29 CH2, Ar–H),
1.97 (s,1H NH); 13C NMR (100 MHz, CDCl3) d ppm: 182.35,
173.42, 168.36, 153.63, 142.23, 138.73, 132.46, 128.92,
124.24, 119.53, 52.85, 47.84; mass: 393.49 (M?H)?; ele-
mental analysis for C19H20N8O2: calculated: C, 58.15; H,
5.14; N, 28.55. Found: C, 58.17; H, 5.13; N, 28.45.
(aromatic C=N), 1,618.13 (N–H bending piperazine), 1,605
(C=C), 1,347.13–1,023.19 (aromatic C–N), 1,527.27 (NO2),
1
753; H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 8.13 (d,
2H J = 8.66 Hz, 29 CH, Ar–H), 7.37 (d, 2H, J = 0.68 Hz,
29 CH, Ar–H), 3.68 (br,s, 1H, NH), 7.37 (d, 1H J = 8.74 Hz,
Ar–H), 6.87 (d, 1H, J = 3.22 Hz, Ar–H), 5.48 (s, 1H, OH),
3.24 (d, 4H J = 13.24 Hz, 29 CH2, Ar–H), 2.91 (d,4H
J = 3.11 Hz, 29 CH2, Ar–H), 1.98 (s,1H NH); 13C NMR
(100 MHz, CDCl3) d ppm: 183.42, 173.23, 167.84, 153.57,
147.32, 139.24, 134.47, 124.63, 123.21, 119.56, 117.82,
52.31, 46.73; mass: 409.45 (M?H)?; elemental analysis
for C19H20N8O3: calculated: C, 55.88; H, 4.94; N, 27.44.
Found: C, 55.90; H, 4.92; N, 27.43.
2-(4-((4-Nitrophenyl)amino)-6-(piperazin-1-yl)-1,3,5-tria-
zin-2-yl)hydrazinecarboxamide (8f) Yellow crystals;
yield: 64 %; m.p.: 234–235 °C; MW:374.16; Rf: 0.62; FTIR
(mmax; cm-1 KBr): 3,328.98 (N–H stretching in piperazine),
3,323.38 (NH2), 3,289.42 (N–H secondary), 3,046.72 (C–H
broad), 1,672.56–1,634.38 (aromatic C=N), 1,663, 1,617.15
(N–H bending piperazine) 1,527.29 (NO2), 1,337.17–
1
1,008.29 (aromatic C–N), 1,759.66 (C=O),753; H NMR
N2-(4-nitrophenyl)-6-(piperazin-1-yl)-N4-(p-tolyl)-1,3,5-
triazine-2,4-diamine (8i) Yellow crystals; yield: 73 %;
m.p.: 218–219 °C; MW:406.44; Rf: 0.54; FTIR (mmax; cm-1
KBr): 3,329.97 (N–H stretching in piperazine), 3,283.48
(N–H secondary), 3,045.71 (C–H broad), 1,674.53–1,633.31
(aromatic C=N), 1,668–1,609 (aromatic C=C), 1,613.19
(N–H bending piperazine), 1,527.28 (NO2), 1,456.35 (C–C
stretching aromatic), 1,344.41–1,015.12 (aromatic C–N),
(400 MHz, CDCl3-d6, TMS) d ppm: 8.08 (d, 2H J = 8.63 Hz,
29 CH, Ar–H), 7.48 (d, 2H, J = 0.78 Hz, 29 CH, Ar–H),
3.65 (br,s, 1H, NH), 6.73 (s, 1H, NH), 6.82 (s, 1H, NH2),
3.21 (d, 4H J = 13.25 Hz, 29 CH2, Ar–H), 2.96 (d,4H
J = 3.12 Hz, 29 CH2, Ar–H), 1.93 (s,1H NH); 13C NMR
(100 MHz, CDCl3) d ppm: 185.93, 178.32, 168.34, 158.63,
148.75, 138,72, 128.42, 121.63, 52.36, 45.83; mass: 375.28
(M?H)?; elemental analysis for C14H18N10O3: calculated:
C, 44.92; H, 4.85; N, 37.42. Found: C, 44.95; H, 4.82; N,
37.47.
1
897, 632; H NMR (400 MHz, CDCl3-d6, TMS) d ppm:
8.14 (d, 2H J = 8.66 Hz, 29 CH, Ar–H), 7.34 (d, 2H,
J = 0.69 Hz, 29 CH, Ar–H), 3.67 (br,s, 1H, NH), 7.38 (d,
1H J = 8.09 Hz, Ar–H), 7.05 (d, 1H J = 5.42 Hz, Ar–H),
2.25 (s, 3H, CH3), 3.28 (d, 4H J = 13.29 Hz, 29 CH2, Ar–
H), 2.87 (d,4H J = 3.08 Hz, 29 CH2, Ar–H), 1.97 (s,1H
NH); 13C NMR (100 MHz, CDCl3) d ppm: 183.42, 172.53,
168.75, 153.47, 142.76, 134.21, 129.72, 126.42, 121.62,
118.82, 52.12, 46.35; mass: 407.47 (M?H)?; elemental
analysis for C20H22N8O2: calculated: C, 59.10; H, 5.46; N,
27.57. Found: C, 59.13; H, 5.47; N, 27.54.
N2-(3-aminopropyl)-N4-(4-nitrophenyl)-6-(piperazin-1-yl)-
1,3,5-triazine-2,4-diamine (8g) White crystals; yield: 65 %;
m.p.: 241–242 °C; MW: 373.41; Rf: 0.43; FTIR (mmax
;
cm-1 KBr): 3,327.95 (N–H stretching in piperazine),
3,319.56 (NH2), 3,284.57 (N–H secondary), 3,041.78 (C–H
broad), 2,964.65 (C–H stretching in aliphatic chain)
1,674.45–1,638.12 (aromatic C=N), 1,668, 1,618.13 (N–H
bending piperazine), 1,347.13–1,023.19 (aromatic C–N),
1
1,527.27 (NO2), 1,455.38 (C–C stretching), 756; H NMR
General procedure for the synthesis of titled
compounds 9(a–i)
(400 MHz, CDCl3-d6, TMS) d ppm: 8.16 (d, 2H J = 8.68 Hz,
29 CH, Ar–H), 7.35 (d, 2H, J = 1.03 Hz, 29 CH, Ar–H),
3.68 (br,s, 1H, NH), 3.27 (t, 2H J = 6.78 Hz, CH2,), 2.74
(t, 2H, J = 6.83 Hz, CH2), 1.69 (t, 2H, J = 6.74 Hz, CH2),
6.74 (s, 1H, NH2), 3.19 (d, 4H J = 13.29 Hz, 29 CH2, Ar–
H), 2.84 (d,4H J = 3.14 Hz, 29 CH2, Ar–H), 1.96 (s,1H NH);
13C NMR (100 MHz, CDCl3) d ppm: 184.52, 172.65, 148.72,
142.83, 128.93, 121.53, 53.42, 46.75, 39.45, 32.21; mass:
374.56 (M?H)?; elemental analysis for C16H23N9O2: calcu-
lated: C, 51.46; H, 6.21; N, 33.76. Found: C, 51.48; H, 6.20;
N, 33.76.
A solution of compound (3) (0.01 mol) and desired tri-
substituted 1,3,5-triazine compounds 8(a–i) (0.01 mol) in
dry acetone was stirred at 40–45 °C for 8–9 h. The com-
pletion of reaction was monitored by TLC using etha-
nol: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 9(a–i).
4-((4-((4-Nitrophenyl)amino)-6-(piperazin-1-yl)-1,3,5-tria-
zin-2-yl)amino)phenol (8h) Black crystals; yield: 72 %;
N-(7-chloroquinolin-4-yl)-4-(4-morpholino-6-((4-nitrophenyl)
amino)-1,3,5-triazin-2-yl)piperazine-1-carbothioamide (9a)
Light black crystals; yield: 57 %; m.p: 256–257 °C; MW:
607.09; Rf: 0.42; FTIR (mmax; cm-1 KBr): 3,397.12 (C–O
stretching), 2,933.32(C–H stretching), 2,344.21 (N–H stretching
m.p.: 236–238 °C; MW:408.41; Rf: 0.59; FTIR (mmax
;
cm-1 KBr): 3,328.93 (N–H stretching in piperazine), 3,416
(OH stretching), 3,286.51 (N–H secondary), 3,046.58 (C–H
broad), 2,800–3,012 (CH2 stretching), 1,672.42–1,637.16
123

Med Chem Res
N-(7-chloroquinolin-4-yl)-4-(4-((4-nitrophenyl)amino)-6-
(o-tolylamino)-1,3,5-triazin-2-yl)piperazine-1-carbothio-
amide (9d) Light yellow crystals; yield: 48.83 %; m.p.:
274–276 °C; MW: 627.12; Rf: 0.46; FTIR (mmax; cm-1
KBr): 3,397.83 (N–H stretching), 2,371.13 (N–H stretching
in secondary amine), 1,500.63 (N=O stretching), 1,418.14
(C=N stretching), 1,333.82 (C=C stretching aromatic),
1,222.21 (C–N stretching), 771.63 (C–Cl stretching),
secondary amine), 1,602.13(N=O stretching), 1,325.63–
1,489.96 (C=C stretching), 1,219.82 (C–N stretching),
1
771.13 (Cl), 685.7 (C=S stretching); H NMR (400 MHz,
CDCl3-d6, TMS) d ppm: 8.36 (d, 1H J = 6.60 Hz, quino-
line ring), 7.35 (d, 1H J = 6.65 Hz, quinoline ring), 4.07
(m, 4H, 29 CH2, Ar–H), 2.54 (m, 4H, 29 CH2, Ar–H),
6.90 (m,2H C–H, Ar–H), 4.13 (br, s, 1H, NH); 13C NMR
(100 MHz, CDCl3); d ppm:185.53, 181.25, 174.31, 168.93,
152.73, 151.26, 145.44, 137.51, 134.83, 129.44, 125.62,
124.74, 121.68, 119.21, 68.35, 58.72, 52.14, 48.94; mass:
608.18 (M?H)?; elemental analysis for C27H28ClN11O3S:
calculated: C, 53.42; H, 4.48; N, 23.07. Found: C, 53.46;
H, 4.46; N, 23.09.
1
662.85 (C=S stretching); H NMR (400 MHz, CDCl3-d6,
TMS) d ppm: 8.81 (d, 1H J = 8.23 Hz, quinoline ring),
7.90 (d, 1H J = 6.78 Hz, quinoline ring), 4.23 (m, 4H, 29
CH2), 6.93 (m,2H C–H, Ar–H), 3.66–3.85 (d, 4H J = 48.6 Hz,
Ar–H), 2.24 (s, 1H NH), 2.51 (s, 3H, CH3), 4.19 (br, s, 1H,
NH); 13C NMR (100 MHz, CDCl3); d ppm: 186.83, 178.64,
169.41, 164.62, 154.12, 149.53, 143.55, 142, 137.92, 136.35,
132.32, 131.96, 129.21, 126.83, 126.12, 123.97, 121.63,
119.26, 58.71, 53.37; mass: 628.35 (M?H)?; elemental anal-
ysis for C30H27ClN10O2S: calculated: C, 57.46; H, 4.34; N,
22.33. Found: C, 57.49; H, 4.31; N, 22.36.
N-(7-chloroquinolin-4-yl)-4-(4-hydrazinyl-6-((4-nitrophenyl)
amino)-1,3,5-triazin-2-yl)piperazine-1-carbothioamide hydrate
(9b) Light yellow crystals; yield: 73.32 %; m.p.: 230–
236 °C; MW: 570.03; Rf: 0.27; FTIR (mmax; cm-1 KBr):
3,396.21 (O–H stretching), 2,372.23 (N–H stretching sec-
ondary amine), 1,592.25 (N=O stretching), 1,432.81 (C=N
stretching), 1,324.41–1,488.73 (C=C stretching aromatic),
1,220.62 (C–N stretching), 771.73 (C–Cl stretching),
N-(7-chloroquinolin-4-yl)-4-(4-((4-nitrophenyl)amino)-6-
(phenylamino)-1,3,5-triazin-2-yl)piperazine-1-carbothio-
amide (9e) Brown crystals; yield: 49.23 %; m.p.: 247–
248 °C; MW: 613.09; Rf: 0.54; FTIR (mmax; cm-1 KBr):
3,020.93 (C–H stretching aromatic), 2,336.21–2,402.35 (N–
H stretching secondary amine), 1,500,24 (N=O stretching),
1,417.68 (C=N stretching), 1,330.26–1,493.47 (C=C stretc-
hing), 1,217.48 (C–N stretching), 771 (C–Cl stretching),
1
676.32 (C=S stretching); H NMR (400 MHz, CDCl3-d6,
TMS) d 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, 29
CH2, Ar–H), 6.90 (m,2H C–H, Ar–H), 6.70 (s, 1H, NH),
4.13 (br, s, 1H, NH),; 13C NMR (100 MHz, CDCl3); d
ppm: 186.73, 181.35, 176.42, 153.92, 151.52, 151.17,
150.06, 139.13, 135.58, 126.83, 124.80, 124.36, 119.39,
117.83, 40.74, 39.94; mass: 572.13 (M?H)?; elemental
analysis for C23H24ClN11O3S: calculated: C, 48.46; H,
4.24; N, 27.03. Found: C, 48.50; H, 4.21; N, 27.13.
1
670.85 (C=S stretching); H NMR (400 MHz, CDCl3-d6,
TMS) d ppm: 8.13 (d, 1H J = 8.76 Hz, quinoline ring), 7.78
(d, 1H J = 6.36 Hz, quinoline ring), 4.12 (m, 4H, 29 CH2),
6.85 (m,2H C–H, Ar–H), 3.78 (m. 4H, 49 CH, Ar–H), 2.50
(s, 1H NH), 4.19 (br, s, 1H, NH),; 13C NMR (100 MHz,
CDCl3); d ppm: 186.85, 181.65, 173.42, 168.96, 158.47,
151.28, 149.36, 146.73, 139.96, 138.21, 136.24, 129.43,
128.94, 126.72, 125.93, 121.67, 119.85, 117.85, 113.38,
57.71, 53.12; mass: 614.17 (M?H)?; elemental analysis for
C29H25ClN10O2S: calculated: C, 56.81; H, 4.11; N, 22.85.
Found: C, 56.78; H, 4.15; N, 22.87.
4-(4-(2-Carbamothioylhydrazinyl)-6-((4-nitrophenyl)amino)-
1,3,5-triazin-2-yl)-N-(7-chloroquinolin-4-yl)piperazine-1-car-
bothioamide (9c) Dark yellow crystals; yield: 64.16 %;
m.p.: 270–271 °C; MW: 611.10; Rf: 0.49; FTIR (mmax
;
cm-1 KBr): 3,417.21 (N–H stretching of primary amine),
2,373.52 (N–H stretching of secondary amine), 1,595.72
(N–O stretching), 1,429.31 (C=N stretching), 1,330.32–
1,485.72 (C=C aromatic), 1,220.12 (C–N stretching), 771.62
2-(4-(4-((7-Chloroquinolin-4-yl)carbamothioyl)piperazin-1-
yl)-6-((4-nitrophenyl)amino)-1,3,5-triazin-2-yl)hydrazin-
ecarboxamide (9f) Dark yellow crystals; yield: 46.91 %;
1
(C–Cl stretching), 677.14 (C=S stretching); H NMR (400
MHz, CDCl3-d6, TMS) d ppm: 8.39 (d, 1H J = 6.62 Hz,
quinoline ring), 7.32 (d, 1H J = 6.64 Hz, quinoline ring),
4.09 (m, 4H, 29 CH2), 6.88 (m,2H C–H, Ar–H), 6.78 (s, 2H,
NH2), 2.50 (s, 1H NH), 4.19 (br, s, 1H, NH),; 13C NMR
(100 MHz, CDCl3); d ppm: 185.32, 183.12, 179.72, 175.68,
171.65, 152.81, 146.92, 138.96, 135.32, 126.83, 124.80,
121.47, 120.38, 119.39, 58.94, 54.23; mass: 612.12
(M?H)?; elemental analysis for C24H23ClN12O2S2: calcu-
lated: C, 47.17; H, 3.79; N, 27.50. Found: C, 47.16; H, 3.76;
N, 27.53.
m.p.: 214–215 °C; MW: 595.04; Rf: 0.52; FTIR (mmax
;
cm-1 KBr):3,116.63–3,411.87 (N–H stretching of primary
amine), 3,020.21 (C–H stretching), 2,402.23 (N–H stretching
secondary amine), 1,782.83 (C=O stretching), 1,488.53 (N=O
stretching), 1,437.13 (C=N stretching), 1,322.64–1,363.52
(C=C stretching), 1,217.15 (C–N stretching), 770.37 (C–Cl
1
stretching), 670.49 (C=S stretching); H NMR (400 MHz,
CDCl3-d6, TMS) d ppm: 8.47 (d, 1H J = 6.24 Hz, quinoline
ring), 7.61 (d, 1H J = 5.12 Hz, quinoline ring), 4.21 (m, 4H,
123

Med Chem Res
29 CH2), 6.82 (m, 2H C–H, Ar–H), 6.54 (s, 2H, NH2), 2.60
(s, 1H NH), 4.32 (br, s, 1H, NH),; 13C NMR (100 MHz,
CDCl3); d ppm: 185.63, 182.36, 176.32, 171.92, 163.41,
154.48, 151.52, 149.32, 146.12, 138.81, 136.94, 129.42,
124.81, 123.42, 121.65, 119.72, 118.54, 57.71, 53.48; mass:
597.07 (M?H)?; elemental analysis for C24H23ClN12O3S:
calculated: C, 48.44; H, 3.90; N, 28.25. Found: C, 48.47; H,
3.93; N, 28.24.
3,376.63–3,490.13 (N–H stretching), 2,934.23–3,078.51 (C–H
stretching), 2,343.65 (N–H stretching secondary amine),
1,489.73 (N=O stretching), 1,414.57 (C=N stretching), 1,327.12
(C=C stretching), 1,218.57 (C–N stretching), 769.18(C–Cl
1
stretching), 673.37 (C=S stretching); H NMR (400 MHz,
CDCl3-d6, TMS) d ppm: 8.84 (d, 1H J = 7.42 Hz, quinoline
ring), 7.43 (d, 1H J = 5.38 Hz, quinoline ring), 4.09 (m, 4H, 29
CH2,Ar–H),3.67(m,4H,29CH2,Ar–H),7.08(m,2H,29CH,
Ar–H), 7.43 (m, 2H, 29 CH, Ar–H), 2.49 (t, 3H, CH3),4.17 (br,
s, 1H, NH); 13C NMR (100 MHz, CDCl3); d ppm: 186.56,
182.38, 172.94, 168.36, 161.24, 153.58, 148.36, 145.58, 141.27,
138.69, 136.94, 132.26, 131.86, 129.47, 126.64, 123.74, 121.87,
118.26, 58.73, 51.54, 23.47; mass: 628.10 (M?H)?; elemental
analysis for C30H27ClN10O2S: calculated: C, 57.46; H, 4.34; N,
22.33. Found: C, 57.49; H, 4.32; N, 22.35.
4-(4-((3-Aminopropyl)amino)-6-((4-nitrophenyl)amino)-1,3,5-
triazin-2-yl)-N-(7-chloroquinolin-4-yl)piperazine-1-carbothio-
amide (9g) Dark brown crystals; yield: 88.83 %; m.p.:
196–197 °C; MW: 594.09; Rf: 0.24; FTIR (mmax; cm-1
KBr):3,371.43 (N–H stretching primary amine), 3,020.71
(C–H stretching), 2,369.83 (N–H secondary amine), 1,485.47
(N=O stretching), 1,419.52 (C=N stretching), 1,326.12 (C=C
stretching), 1,217.57 (C–N stretching), 768.82 (C–Cl
1
stretching), 671.75 (C=S stretching); H NMR (400 MHz,
Antibacterial screening
CDCl3-d6, TMS) d ppm: 8.67 (d, 1H J = 5.87 Hz, quinoline
ring), 7.70 (d, 1H J = 5.37 Hz, quinoline ring), 4.78 (m, 4H,
29 CH2), 6.895 (m, 2H C–H, Ar–H), 6.68 (s, 2H, NH2), 3.67
(m, 2H, CH2) 2.50 (s, 1H NH), 4.38 (br, s, 1H, NH),; 13C
NMR (100 MHz, CDCl3); d ppm: 184.53, 178.91, 171.20,
164.52, 154.38, 151.21, 149.62, 148.36, 138.57, 136.29,
131.53, 129.78, 126.85, 123.62, 119.83, 118.38, 58.21, 53.28,
41.81, 32.17; mass: 596.12 (M?H)?; elemental analysis for
C26H28ClN11O2S: calculated: C, 52.56; H, 4.75; N, 25.93.
Found: C, 52.52; H, 4.77; N, 25.98.
Minimum inhibitory concentration
Entire target compounds were screened for their minimum
inhibitoryconcentration(MIC, lg/mL) againstselectedgram-
positive organisms viz. B. subtilis (NCIM-2,063), B. cereus
(NCIM-2,156), S. aureus (NCIM-2079) and gram-negative
organism viz. P. aeruginosa (NCIM-2036), E. 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. Ofloxacin was used as standard antibacterial
agent. Solutions of the test compounds and reference drug
were prepared in dimethyl sulfoxide (DMSO) at concentra-
tions of 100, 50, 25, 12.5, 6.25 and 3.125 lg mL-1. Eight
tubes were prepared in duplicate with the second set being
usedasMICreferencecontrols(16–24 hvisual).Aftersample
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 pipette (tubes
2–7), tube 1 (negative control) received 1.0 mL of nutrient
broth and tube 8 (positive control) received 0.9 mL of nutri-
ent. 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 lg/mL),
0.1 mL of increasing concentration of the prepared test
compounds which are serially diluted from tube 2 to tube 7
N-(7-chloroquinolin-4-yl)-4-(4-((4-hydroxyphenyl)amino)-
6-((4-nitrophenyl)amino)-1,3,5-triazin-2-yl)piperazine-1-
carbothioamide (9h) Brown yellow crystals; yield:
81.84 %; m.p.: 310–311 °C; MW: 629.09; Rf: 0.46; FTIR
(mmax; cm-1 KBr): 3,439.83 (O–H stretching), 3,020.73 (C–H
stretching), 2,335.52–2,375.81 (N–H stretching in secondary
amine), 1,625.38 (C=C stretching), 1,488.57 (N=O stretch-
ing), 1,414.32 (C=N stretching), 1,218.31 (C–N stretching),
770.83 (C–Cl stretching), 672.35 (C=S stretching); 1H NMR
(400 MHz, CDCl3-d6, TMS) d ppm: 8.92 (d, 1H J = 7.54
Hz, quinoline ring), 7.45 (d, 1H J = 5.32 Hz, quinoline ring),
4.09 (m, 4H, 29, Ar–H), 7.53 (m, 4H, 49 CH, Ar–H), 6.75
(m, 4H, 49 CH, Ar–H), 5.40 (s, 1H Ar-OH),4.05 (br, s, 1H,
NH); 13C NMR (100 MHz, CDCl3); d ppm: 185.62, 178.41,
171.28, 167.76, 161.78, 154.53, 149.51, 147.12, 139.96,
134.26, 132.54, 131.29, 128.92, 125.74, 123.15, 121.64,
118.63, 117.28, 115.38, 58.26, 53.42; mass: 630.21 (M?H)?;
elemental analysis for C29H25ClN10O3S: calculated: C, 55.37;
H, 4.01; N, 22.26. Found: C, 55.38; H, 4.00; N, 22.32.
from highest (100 lg mL-1) to lowest (3.125 lg mL-1
)
concentration (tube 2–7 containing 100, 50, 25, 12.5, 6.25,
3.125 lg mL-1). After this process, each tube was inoculated
with 0.1 mL of the bacterial suspension, concentration of
which corresponded to 0.5 McFarland scale (9 9 108 cells/
mL) and each bacterium was incubated at 37 °C for 24 h. The
final volume in each tube was 1.0 mL. The incubation
N-(7-chloroquinolin-4-yl)-4-(4-((4-nitrophenyl)amino)-6-
(p-tolylamino)-1,3,5-triazin-2-yl)piperazine-1-carbothio-
amide (9i) Brown crysals; yield: 78.82 %; m.p.: 145–148
°C; MW: 627.12; Rf: 0.64; FTIR (mmax; cm-1 KBr):
123

Med Chem Res
Davies J, Davies D (2010) Origins and evolution of antibiotic
resistance. Microbiol Mol Biol 74:417–733
Dubey V, Pathak M, Bhat HR, Singh UP (2012) Design, facile
synthesis and antibacterial activity of hybrid 1,3,4-thiadiazole-
1,3,5-triazine derivatives tethered via S-bridge. Chem Biol Drug
Des 80:598–604
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, (National Committee for Clinical Labo-
ratory Standards, 1982) results were shown in Table 1.
Gahtori P, Ghosh SK, Singh B, Singh UP, Bhat HR, Uppal A (2012)
Synthesis, SAR and antibacterial activity of hybrid chloro,
dichloro-phenylthiazolyl-s-triazines. Saudi Pharm J 20:35–43
Ghosh SK, Saha A, Hazarika B, Singh UP, Bhat HR, Gahtori P (2012)
Design, facile synthesis, antibacterial activity and structure-
activity relationship of novel di- and tri-substituted 1,3,5-
triazines. Lett Drug Des Dis 9:329–335
Junejo JA, Ghosh SK, Shaikh M, Gahtori P, Singh UP (2011) Facile
synthesis, antibacterial activity and molecular properties predic-
tion of Some new 1,3-dihydroimidazol-2-thione derivatives. Lett
Drug Des Dis 8:763–768
Conclusion
As a concluding remark, we had developed a new series of
hybrid 4-aminoquinoline-1,3,5-triazine conjugates as potent
antibacterial agents through facile and economical route. In
addition,thisstudysuggeststhepotentialutilityofthishybrid
skeleton to develop newer antibacterial agents. Our studies
are in progress towards the development of newer entities of
this skeleton and reported subsequently in future.
Kumar S, Bhat HR, Kumawat MK, Singh UP (2013) Design and one-
pot synthesis of hybrid thiazolidin-4-one-1,3,5-triazines as potent
antibacterial agent against human disease causing pathogens. New
J Chem. doi:10.1039/C2NJ41028A
Lozano V, Aguado L, Hoorelbeke B, Renders M, Camarasa MJ,
Acknowledgments Authors are gratified to SAIF, Central Drug
Research Institute, Lucknow, India for providing spectral data of
compounds synthesized herein and SHIATS for providing basic
facilities to carry out the project.
´
´
´
Schols D, Balzarini J, San-Felix A, Perez-Perez MJ (2011)
Targeting HIV entry through interaction with envelope glyco-
protein 120 (gp120): synthesis and antiviral evaluation of 1,3,5-
triazines with aromatic amino acids. J Med Chem 54:5335–5348
Martinez JL, Baquero F (2000) Mutation frequencies and antibiotic
resistance. Antimicrob Agents Chemother 44:1771–1777
National Committee for Clinical Laboratory Standards (1982) Stan-
dard methods for dilution antimicrobial susceptibility test for
bacteria which grow aerobically. NCCLS, Villanova, p 242
Shapiro RS, Robbins N, Cowen LE (2011) Regulatory circuitry
governing fungal development, drug resistance, and disease.
Microbiol Mol Biol Rev 75:213–267
Conflict of interest Authors declare no conflict of interest.
References
Alanis AJ (2005) Resistance to antibiotics: are we in the post-
antibiotic era. Arch Med Res 36:697–705
Bhat HR, Singh UP, Yadav PS, Kumar V, Das A, Chetia D, Prakash
A, Mahanta J (2011) Synthesis, characterization and antimalarial
activity of hybrid 4-aminoquinoline-1,3,5-triazine derivatives.
Arabian J Chem. doi:10.1016/j.arabjc.2011.07.001
Bhat HR, Ghosh SK, Prakash A, Gogoi K, Singh UP (2012a) In vitro
antimalarial activity and molecular docking analysis of 4-ami-
noquinoline-clubbed 1,3,5-triazine derivatives. Lett Appl Micro-
biol 54:483–486
Bhat HR, Gupta SK, Singh UP (2012b) Discovery of potent, novel
antibacterial hybrid conjugates from 4-aminoquinoline and
1,3,5-triazine: design, synthesis and antibacterial evaluation.
RSC Adv 2:12690–12695
Bhat HR, Singh UP, Gahtori P, Ghosh SK, Gogoi K, Prakash A, Singh
RK (2013) Antimalarial activity and docking studies of novel bi-
functional hybrids derived from 4-aminoquinoline and 1,3,5-
triazine against wild and mutant malaria parasites as pf-DHFR
inhibitor. RSC Adv. doi:10.1039/C2RA21915H
Corbett TH, Leopold WR, Dykes DJ, Roberts BJ, Griswold DP Jr,
Schabel FM Jr (1982) Toxicity and anticancer activity of a new
triazine antifolate (NSC 127755). Cancer Res 42:1707–1715
Singh UP, Singh RK, Bhat HR, Subhaschandra YP, Kumar V,
Kumawat MK, Gahtori P (2011) Synthesis and antibacterial
evaluation of a series of novel trisubstituted-s-triazines derivatives.
Med Chem Res 20:1603–1610
Singh UP, Bhat HR, Gahtori P (2012a) Antifungal activity, SAR and
physicochemical correlation of some thiazole-1,3,5-triazine
derivatives. J Mycol Med 22:134–141
Singh UP, Pathak M, Dubey V, Bhat HR, Gahtori P, Singh RK
(2012b) Design, synthesis, antibacterial activity and molecular
docking studies on novel hybrid 1,3-thiazine-1,3,5-triazine deriv-
atives as potential bacterial translation inhibitor. Chem Biol Drug
Des 80:572–583
Singh UP, Bhat HR, Gahtori P, Singh RK (2013) Hybrid thiazole-
1,3,5-triazines target cytosolic Leucyl-tRNA synthetase for anti-
fungal action revealed by molecular docking studies. In Silico
Pharmacology, In press
Singh UP, Bhat HR, Singh RK (2013b) Ceric ammonium nitrate
catalysed expeditious one-pot synthesis of 1,3-thiazine as IspE
kinase inhibitor of gram-negative bacteria using polyethylene
glycol (PEG-400) as an efficient recyclable reaction medium.
C. R. Chime. doi:10.1016/j.crci.2012.11.019
123