Design, Facile Synthesis, and Antibacterial Activity of Hybrid 1,3,4-thiadiazole-1,3,5-triazine Derivatives Tethered via -S- Bridge

Article abstract of DOI:10.1111/j.1747-0285.2012.01433.x

Some hybrid 1,3,4-thiadiazole-1,3,5-triazine derivatives tethered via -S- bridge were synthesized and characterized with the aid of spectroscopic and elemental analysis. These hybrid conjugates were then investigated for their antibacterial activity against selected Gram-positive and Gram-negative bacteria. Excellent to moderate antibacterial activity was presented by the target compounds.

Full text of DOI:10.1111/j.1747-0285.2012.01433.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01433.x
Chem Biol Drug Des 2012
Research Article
Design, Facile Synthesis, and Antibacterial
Activity of Hybrid 1,3,4-thiadiazole-1,3,5-triazine
Derivatives Tethered via –S– Bridge
industries into the projects related to the development of new
antibioticsb. Thus, increased incidence of bacterial resistance to
currently available antibiotics together with financial constraints
necessitates the discovery and introduction of new, economic,
and effective drugs.
Vaibhav Dubey, Manish Pathak, Hans
R. Bhat and Udaya P. Singh*
Department of Pharmaceutical Sciences, Sam Higginbottom Institute
of Agriculture, Technology & Sciences (Formerly-Allahabad
Agricultural Institute), Deemed-to-be-University, Allahabad 211007,
India
Nowadays, combination therapies have emerged as a better
option to treat MDR pathogens than individual drugs, for exam-
ple, HAART and ACTs. However, combination therapies have cer-
tain disadvantages such as (i) increased expense; (ii) increased
risk of adverse effects; (iii) pharmacokinetic intolerability; and (iv)
superinfection (8). Molecular hybridization is an innovative
approach gaining attentionfrom medicinal chemists owing to
resemblance with combination therapies where two diverse phar-
*Corresponding author: Udaya P. Singh, udaysingh98@gmail.com
Some hybrid 1,3,4-thiadiazole-1,3,5-triazine deriv-
atives tethered via –S– bridge were synthesized
and characterized with the aid of spectroscopic
and elemental analysis. These hybrid conjugates
were then investigated for their antibacterial
activity against selected Gram-positive and Gram-
negative bacteria. Excellent to moderate antibac-
terial activity was presented by the target
compounds.
macophoric groups were joined covalently through
a linker.
Enlarged spectrums of activity, less prone to spontaneous muta-
tions and resistance development, dual drug targeting at more
than one site and privileged activity when compared to the indi-
vidual agent are deemed as main possible advantages of this
strategy, Figure 1 (9).
Key words: 1,3,4-thiadiazole, 1,3,5-triazine, antibacterial, hybrid
Received 26 March 2012, revised 18 May 2012 and accepted for publi-
cation 5 June 2012
As a part of our ongoing research on the discovery of economic
and potential antimicrobial agents, we are emphasizing on the
development of hybrid molecules containing diverse pharmacophoric
groups (10–16).
Since the discovery of penicillin by Sir Alexander Fleming some
eight decades ago, the use of antibiotics has made major contribu-
tions to public health. Surprisingly, just 4 years after the drug
started being mass-produced, over 50% of micro-organisms were
no longer susceptible (1). The rapid growth of antibiotic resistance
has resulted in the generation of multidrug-resistant (MDR) patho-
gens that produce infections that are increasingly difficult to treat
with antibiotics currently available.
In present study, we decided to enlarge our investigation by syn-
thesizing the hybrid analogues of 1,3,4-thiadiazole and 1,3,5-triazine
derivatives tethered via mercapto (–S–) bridge. The rationale
behind the study is to exemplify the versatility of substituent in
terms of antibacterial efficacy with the aim to confirm earlier
observations.
According to Centre for Disease Control Statistics, nearly 2 mil-
lion patients in the United States acquire an infection in the
hospital each year and about 90 000 of those patients die each
year as a result of their infectiona. Methicillin-resistant Staphylo-
coccus aureus (MRSA) alone account for the death of 19 000
people a year in the United States, which is more than the
death from AIDS (2). Now, we have recently moved into an era
of not just multiple resistant bacteria but of totally resistant
pathogens, which now include vancomycin-resistant enterococci
(3), carbapenem-resistant Acinetobacter baumannii (4,5), vancomy-
cin-resistant MRSA (6), and very recently, New Delhi metallo-
beta-lactamase-1 (7). Furthermore, heavy investment and far too
low returns compared with chronic disease and lifestyle medica-
tions has caused to prevent the investment of pharmaceutical
Experimental
Melting points of synthesized compounds were determined in open
capillary tubes Hicon melting point apparatus and are uncorrected.
Thin-layer chromatographic analysis (TLC) was carried out to moni-
tor the completion of reaction. The different mobile phases were
selected according to the assumed polarity of the products. The
spots were visualized by exposure to iodine vapor and UV light.
The structures of the intermediate compounds were established on
the basis of spectral (FT-IR, mass) and elemental analysis, whereas
structures of the title compounds were ascertained on the basis of
1
FT-IR, H NMR, mass spectral and elemental analysis. FT-IR (in 2.0
1

Dubey et al.
A
B
C
Figure 1: Hybrid
molecules,
concept, and approaches.
per cm, flat, smooth, abex, KBr) spectra were recorded on Biored
FTs spectrophotometer. Proton magnetic resonance spectra (1H
NMR) were recorded on Bruker Model DRX-400 and 300 MHz NMR
spectrometer in CDCl3-d6 and DMSO using tetramethylsilane (TMS)
as an internal standard. Chemical shift is reported in parts per mil-
lion (ppm, d), and signals are described as s (singlet), d (doublet), t
(triplet), q (quartet), and m (multiplet). The FAB mass spectrum was
recorded on a THERMO Finnigan LCQ Advantage max ion trap mass
spectrometer; samples were introduced into ESI source through
Finnigan surveyor autosampler. Elemental analysis was carried out
on a Vario EL III CHNOS elemental analyzer, and results were found
within €0.4%.
Procedure of diazotization of 5-amino-1,3,4-
thiadiazole-2-thiol (4)
A mixture of 3 (0.01 mol) in concentrated HCl (3 mL) was cooled to
0–5 ꢀC under ice followed by dropwise addition of cooled sodium
nitrite solution (1.5 g in 10 mL of water) over a period of 10 min.
The reaction mixture was then stirred for 30 min to yield diazonium
salt intermediate 4.
Procedure for the preparation of 3-(5-mercapto-
1,3,4-thiadiazol-2-ylimino)pentane-2,4-dione
with active methylene (5)
To an ice-cold mixture of the active methylene compound, acetylac-
etone (0.01 mol), and sodium acetate (0.05 mol) in ethanol (50 mL)
was added dropwise with stirring a solution of diazonium salt com-
pound 4 (0.01 mol) over 15 min. The stirring was continued for
30 min and the reaction mixture then left for 2 h at room tempera-
ture, and the completion of reaction was indicated by TLC using tol-
uene ⁄ ethyl acetate ⁄ formic acid (5:4:1) as a mobile phase. Resulted
solid product was collected and recrystallized from ethanol to afford
the corresponding imino derivatives.
Step 1: Synthesis of 5-(3,5-dimethyl-1-phenyl-
4,5-dihydro-1H-pyrazol-4-ylamino)-1,3,4-
thiadiazole-2-thiol (6)
Procedure of synthesis of 5-amino-1,3,4-
thiadiazole-2-thiol (3)
Anhydrous sodium carbonate (24 g) with carbon disulfide 0.25 mol
was added in thiosemicarbazide (0.25 mol) suspended in absolute
ethanol. The mixture was warmed with stirring under reflux for 1 h
and then further heated on steam bath for 4 h. The completion of
reaction was indicated by TLC using toluene ⁄ ethyl acetate ⁄ formic
acid (5:4:1) as a mobile phase. The solvent was removed, and the
residue was dissolved in water and acidified further with concen-
trated HCl to afford solid product 3.
Brownish yellow crystals; yield: 82%; mp: 241 ꢀC; MW: 229; Rf :
0.48; FT-IR (mmax per cm, KBr): 2550 (S-H), 1250 (C-N), 1650, 1662
(C=O); elemental analysis for C7H7N3O2S2: Calculated: C, 36.67; H,
3.08; N, 18.33. Found: C, 36.52; H, 3.00; N 18.35.
Yellowish crystals; yield: 89%; mp: 233 ꢀC; MW: 133.19; Rf : 0.68
(hexane ⁄ ethyl acetate; 6:4); FT-IR (mmax per cm, KBr): 3410, 3300 (N-
H primary), 2555 (S-H broad thiol), 1000–1300 (C=N); elemental
analysis for C2H3N3S2: Calculated: C, 18.03; H, 2.37; N, 31.54.
Found: C, 18.12; H, 2.34; N, 31.62.
Procedure for the synthesis of 5-(3,5-dimethyl-
1-phenyl-4,5-dihydro-1H-pyrazol-4-ylamino)-
1,3,4-thiadiazole-2-thiol (6)
To a solution of 5 (0.01 mol) in glacial acetic acid (30 mL), phenyl
hydrazine (0.012 mol) and anhydrous sodium acetate (0.01 mol)
2
Chem Biol Drug Des 2012

Antibacterial Activity of 1,3,5-triazines
were added. The reaction mixture was heated under reflux for 4 h.
Completion of reaction was monitored by TLC using toluene ⁄ ethyl
acetate ⁄ formic acid (5:4:1) as a mobile phase. Thus, mixture
obtained was poured into ice-cold water and stored in a refrigera-
tor. The obtained crude product was washed with water, dried and
recrystallized from methanol.
6-Chloro-N2,N4-di-p-tolyl-1,3,5-triazine-2,4-
diamine 9d
Pale-yellowish crystals; yield: 78%; mp: 212–214 ꢀC; MW: 325.80;
Rf: 0.72; FT-IR (mmax per cm, 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) d ppm: 7.03 (d, 1H,
J = 8.2 Hz, Ar-H), 7.06 (d, 1H, J = 5.4 Hz, Ar-H), 7.31 (d, 1H
J = 8.2 Hz, Ar-H), 7.23 (d, 1H, Ar-H), 5.24 (br, s, 1H, NH), 2.53 (s,
3H, CH3); elemental analysis for C17H16ClN5: Calculated: C, 62.67;
H, 4.95; N, 21.50. Found: C, 62.63; H, 4.98; N, 21.55.
Dark yellowish crystals; yield: 69%; mp: 253ꢀC; MW: 305; Rf : 0.46;
FT-IR (mmax per cm, KBr): 3305, 3300 (N-H secondary), 3010, 2998
(C-H), 2559 (S-H), 1156, 1685 (C=N), 1625 (C=C), 1610 (C=C aromatic
ring), 1450 (C=C aromatic ring), 1250 (C-N); elemental analysis for
C13H15N5S2: Calculated: C, 51.12; H, 4.95; N, 22.93. Found: C,
51.10; H, 4.92; N, 22.97.
6-Chloro-N2,N4-bis(4-methoxyphenyl)-1,3,5-
triazine-2,4-diamine 9e
Yellow crystals; yield: 88%; mp: 235 ꢀC; MW: 357.79; Rf: 0.69; FT-IR
(mmax per cm KBr): 3300 (N-H secondary), 3015 (C-H), 1670–1685
(C=O), 1630–1640 (C=C), 1585 (C=C aromatic ring), 1460 (C=C aromatic
Step 2: General procedure for the synthesis of
2,4-bis(substituted amine)-6-chloro-1,3,5-
triazine 9 (a–f)
1
ring), 1100–1230 (C-N). H NMR (400 MHz, CDCl3-d6, TMS) d ppm:
Various substituted anilines 8 (a–f) (0.2 mol) were added into
100 mL of acetone, maintaining temperature at 40–45 ꢀC. The solu-
tion of 2,4,6-tri chloro-1,3,5-triazine (7) (0.1 mol) in 25 mL acetone
was added constantly, stirred for 3 h followed by dropwise addition
of NaHCO3 solution (0.1 mol), taking care that reaction mixture does
not become acidic. Completion of the reaction was analyzed by TLC
utilizing benzene ⁄ ethyl acetate as a mobile phase (9:1). The product
was filtered and washed with cold water and recrystallized with
ethanol to afford pure compounds 9(a–f).
7.43 (d, 1H, J = 8.68 Hz, Ar-H), 7.52 (d, 1H J = 5.4 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). 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-nitrophenyl)-1,3,5-triazine-
2,4-diamine 9f
Black crystals; yield: 86%; mp: 143–145 ꢀC; MW: 387.74; Rf: 0.55; FT-
IR (mmax per cm KBr): 3289.56 (N–H secondary), 3055.70 (C–H broad),
1
1548.28–1446.06 (aromatic C=N); H NMR (400 MHz, CDCl3-d6, TMS)
6-Chloro-N2,N4-bis(4-chlorophenyl)-1,3,5-
triazine-2,4-diamine 9a
d ppm: 7.40 (t, 4H, 4 = CH aromatic), 7.32 (t, 4H, 4 = CH aromatic),
3.62 (d, 2H, 2-NH aromatic); elemental analysis for C15H10ClN7O4: Cal-
culated: C, 46.46; H, 2.60; N, 25.29. Found: C, 46.48; H, 2.65; N, 25.26.
Yellow crystals; yield: 75%; mp: 135–137 ꢀC; MW: 366.63; Rf: 0.48;
FT-IR (mmax cm)1, KBr): 3243.26 (N–H secondary), 2965.46 (C–H broad),
1
1387.38 (aromatic –C=N); H NMR (400 MHz, CDCl3-d6, TMS) d ppm:
7.32 (d, 4H, 3,5-CH aromatic), 7.08 (d, 4H, 2,6-CH aromatic), 4.82 (s,
2H, NH); elemental analysis for C15H10Cl3N5: Calculated: C, 49.14; H,
2.75; N, 19.10. Found: C, 49.17; H, 2.77; N, 19.15.
Step 3: General procedure for synthesis hybrid
3,5-dimethyl-1-phenyl-4,5-dihydro-1H-pyrazole-
4ylamino-1,3,4-thiadiazole-1,3,5-triazine
derivatives tethered via –S– bridge 10 (a–f)
2,4-Bis(substituted amine)-6-chloro-1,3,5-triazine 9(a–f) (0.1 mol)
was added into 50 mL of 1,4-dioxane, maintaining temperature at
40–45 ꢀC. The solution of 5-(3,5-dimethyl-1-phenyl-4,5-dihydro-1H-
pyrazol-4-ylamino)-1,3,4-thiadiazole-2-thiol (6) (0.1 mol) in 35 mL of
1,4-dioxane was added constantly to above solution and stirred for
90 min followed by dropwise addition of K2CO3 (0.1 mol), and this
mixture was refluxed at 135–145 ꢀC for 9 h. The product was fil-
tered and washed with cold water and recrystallized with ethanol
to afford the corresponding pure compounds 10(a-f).
N2,N4-Bis(4-bromophenyl)-6-chloro-1,3,5-
triazine-2,4-diamine 9b
Brownish-black crystals; yield: 81%; mp: 169–170 ꢀC; MW: 455.53;
Rf: 0.35; FT-IR (mmax per cm, KBr): 3350.62 (N–H secondary), 3015.43
(C–H broad), 1656.15 (C=C stretch); 1H NMR (400 MHz, CDCl3-d6,
TMS) d ppm: 7.26 (d, 4H, 3,5-CH aromatic), 7.06 (d, 4H, 2,6-aromatic),
4.81 (s, 2H, NH); 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-fluorophenyl)-1,3,5-
triazine-2,4-diamine 9c
N2,N4-Bis(4-chlorophenyl)-6-((5-((3,5-dimethyl-1-
phenyl-4,5-dihydro-1H-pyrazol-4-yl)amino)-1,3,4-
thiadiazol-2-yl)thio)-1,3,5-triazine-2,4-diamine
10a
Reddish-orange crystals; yield: 66%; mp: 124–125 ꢀC; MW: 333.72;
Rf: 0.42; FT-IR (mmax per cm, KBr): 3054.72 (C–H broad), 1548.58
1
(C=N), 1448.25 (C–N aromatic); H NMR (400 MHz, CDCl3-d6, TMS)
Deep yellow crystals; yield: 92%; mp: 304–306 ꢀC; MW: 635.59; FT-
IR (mmax; per cm KBr): 3390–3318 (N–H secondary), 1610 (Ar, -C=C
d ppm: 7.68 (d, 4H, 2-CH, aromatic), 7.14 (d, 4H, 2-CH aromatic),
4.84 (t, 2H, -NH amino); elemental analysis for C15H10ClF2N5: Calcu-
lated: C, 53.99; H, 3.02; N, 20.99. Found: C, 53.96; H, 3.02; N,
20.95.
1
aromatic, Bz), 1540 (C=N stretching), 720, 680; H NMR (400 MHz,
CDCl3-d6, TMS) d ppm: 6.90 (m, 2H, Ar-Bz-H, 10,11), 7.16 (m, 2H,
Ar-Bz-H, 12,13), 9.15 (s, 1H, Ar-Bz-H, 8), 13.15 (s-1H, Ar-Bz-H, 6),
Chem Biol Drug Des 2012
3

Dubey et al.
8.20–8.18 (d, 1H, J = 8 Hz, Ar-H), 8.14–8.12 (d, 1H, J = 9.2 Hz, Ar-
H), 8.01–7.99 (d, 1H, J = 6.8 Hz, Ar-H); mass: 636 (M + H+); elemen-
tal analysis of C28H24Cl2N10S2: C, 52.51; H, 3.81; N, 22.04. Found:
C, 52.30; H, 3.95; N, 22.90.
stretching), 2870, 1645 (Ar, -C=C stretching, Bz), 1504 (C=N stretch-
ing), 810, 740. H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 3.84 (s,
1
3H, OCH3), 6.91 (d, 2H, J = 8.8 Hz, Ar-Bz-H 18,19), 7.33 (d, 1H,
J = 15.6 Hz, Ar-Bz-H, 9), 7.44 (d, 2H, J = 8.4 Hz, Ar-Bz-H 21,22),
7.57 (d, 1H, J = 8.8 Hz, Ar-Bz-H 12,13), 7.75 (d, 2H, J = 15.6 Hz Ar-
Bz-H 10), 7.92 (d, 2H, J = 8.8 Hz Ar-Bz-H 15,16); mass: 627 (M +
H+); elemental analysis of C30H30N10O2S2: C, 57.49; H, 4.82; N,
22.35. Found: C, 57.42; H, 4.80; N, 22.56.
N2,N4-Bis(4-bromophenyl)-6-((5-((3,5-dimethyl-1-
phenyl-4,5-dihydro-1H-pyrazol-4-yl)amino)-1,3,4-
thiadiazol-2-yl)thio)-1,3,5-triazine-2,4-diamine
10b
Yellowish brown crystals; yield: 89%; mp; 368–370 ꢀC; MW:
724.50; FT-IR (mmax per cm, KBr): 3129–3098 (N–H secondary), 1609
(Ar, -C=C aromatic, Bz), 1520 (C=N stretching), 1440, 660; H NMR
(400 MHz, CDCl3-d6, TMS) d ppm: 6.96 (d, 2H, J = 8.5 Hz Ar-Bz-H
11,13), 7.29 (dd, 1H, J = 8.5, 2.5 Hz Ar-Bz-H 10), 7.94–7.92 (d, 1H,
J = 8.4 Hz Ar-H), 7.76 (s, 1H, NH), 7.66–7.67 (d, 1H, J = 8.8 Hz, Ar-
H), 7.95 (dd, 1H, J = 8.5,2.5 Hz, Ar-Bz-H, 14), 8.64 (s, 1H, Ar-H 8);
mass: 725 (M + H+); elemental analysis of C28H24Br2N10S2: C, 46.42;
H, 3.34; N, 19.33. Found: C, 46.20; H, 3.48; N, 19.95.
6-((5-((3,5-Dimethyl-1-phenyl-4,5-dihydro-1H-
pyrazol-4-yl)amino)-1,3,4-thiadiazol-2-yl)thio)-N2,
N4-bis(4-nitrophenyl)-1,3,5-triazine-2,4-diamine
10f
1
Brown crystals; yield: 91%; mp; 221–223 ꢀC; MW: 656.70; FT-IR
(mmax per cm, KBr): 3390–3180 (N–H secondary), 1610 (Ar, -C=C
stretching, Bz), 1508 (C=N stretching), 1435 (C-H stretching, methyl),
1
769. H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 1.92 (s, 3H, CH3),
1.18 (d, 3H, J = 6.86 CH3), 6.93 (d, 2H, J = 8.7 Hz, Ar-Bz-H 18,19),
7.31 (d, 1H, J = 15.4 Hz, Ar-Bz-H, 9), 7.42 (d, 2H, J = 8.1 Hz, Ar-Bz-
H 21,22), 7.49 (d, 1H, J = 8.6 Hz, Ar-Bz-H 12,13), 7.68 (d, 2H,
J = 15.7 Hz Ar-Bz-H 10), 7.82 (d, 2H, J = 8.7 Hz Ar-Bz-H 15,16);
mass: 657 (M + H+). Elemental analysis of C28H24N12O4S2: C, 51.21;
H, 3.68; N, 25.59. Found: C, 51.02; H, 3.70; N, 25.56.
6-((5-((3,5-Dimethyl-1-phenyl-4,5-dihydro-1H-
pyrazol-4-yl)amino)-1,3,4-thiadiazol-2-yl)thio)-
N2,N4-bis(4-fluorophenyl)-1,3,5-triazine-2,4-
diamine 10c
Deep yellow crystals; yield: 76%; mp; 391–393 ꢀC; MW: 602.68; FT-
IR (mmax per cm, KBr): 3310–3104 (N–H secondary), 3010 (C-H
stretching) 2490, 1652 (Ar, -C=C stretching, Bz), 1503 (C=N stretch-
Result and Discussion
1
ing), 810, 610 (Ar-C-F stretching, Bz); H NMR (400 MHz, CDCl3-d6,
Chemistry
TMS) d ppm: 7.14 (m, 3H, Ar-Bz-H 14, 15, 16), 7.24 (m, 2H, Ar-Bz-H,
17,18), 7.70 (d, 2H, J = 15.6 Hz, Ar-Bz-H 10,11), 7.89 (d, 2H,
J = 8.1 Hz, Ar-Bz-H 23,24), 7.94 (d, 1H, J = 15.6 Hz Ar-Bz-H 11,12),
8.14 (d, 2H, J = 8.4 Hz Ar-Bz-H 21,22); mass: 603 (M + H+); elemen-
tal analysis of C28H24F2N10S2: C, 55.80; H, 4.01; N, 23.24. Found: C,
55.25; H, 4.01; N, 23.01.
The synthesis of title 3,5-dimethyl-1-phenyl-4,5-dihydro-1H-pyrazole-
4ylamino-1,3,4-thiadiazole-1,3,5-triazine derivatives 10(a–f) tethered
via –S– bridge was accomplished by three different steps: First
step corresponds to synthesis of 5-(3,5-dimethyl-1-phenyl-4,5-dihy-
dro-1H-pyrazol-4-ylamino)-1,3,4-thiadiazole-2-thiol (6) by cyclo-con-
densation reaction in the presence of phenyl hydrazine from imino
derivative, 3-(5-mercapto-1,3,4-thiadiazol-2-ylimino)pentane-2,4-dione
(5). Compound 5 was obtained through the involvement of series of
steps started from the synthesis of 5-amino-1,3,4-thiadiazole-2-thiol
(3) by the reaction between thiosemicarbazide (1) and carbon disul-
fide (2) in the presence of alcoholic NaOH, in which first sodium
hydrazothiocarbonamide dithiocarboxylate (NH2CSNHNHCSSNa) is
formed and upon raising the temperature of the reaction, this
sodium salt yields the new closed-ring system, compound 3 (17).
Compound (3) was further subjected to diazotization to afford its
diazonium salt (4) which was again allowed to react with acetyl
acetone (active methylene) to generate imino derivative, 3-(5-merca-
pto-1,3,4-thiadiazol-2-ylimino)pentane-2,4-dione (5).
6-((5-((3,5-Dimethyl-1-phenyl-4,5-dihydro-1H-
pyrazol-4-yl)amino)-1,3,4-thiadiazol-2-yl)thio)-
N2,N4-di-p-tolyl-1,3,5-triazine-2,4-diamine 10d
Pale-yellow crystals; yield: 68%; mp; 227–229 ꢀC; MW: 594.76; FT-
IR (mmax per cm, KBr): 3389–3120 (NH stretching, Bz), 3010 (C-H
stretching), 1630 (Ar, -C=C stretching, Bz), 1503 (C=N stretching),
800; 1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.44 (d, 2H,
J = 8.5 Hz, Ar-Bz-H 12,13), 7.60 (d, 2H, J = 8.4 Hz, Ar-Bz-H, 15,16),
7.70 (d, 2H, J = 15.6 Hz, Ar-Bz-H 9), 7.80 (d, 2H, J = 3.1 Hz, Ar-Bz-
H 18,19), 7.89 (d, 1H, J = 15.6 Hz Ar-Bz-H 10), 8.14 (d, 2H,
J = 8.4 Hz Ar-Bz-H 21,22); mass: 595 (M + H+); elemental analysis
of C30H30N10S2: C, 60.58; H, 5.08; N, 23.55. Found: C, 60.25; H,
5.01; N, 23.59.
The synthesis of 2,4-bis(substituted phenyl amine)-6-chloro-1,3,5-tri-
azine derivatives was served as step 2. The synthesis was initiated
by using 2,4,6-trichloro-1,3,5-triazine (7) through consecutive aro-
matic nucleophilic substitution (SNAr) reactions under controlled
conditions by treating two equivalents of substituted amine deriva-
tives 8(a–f) in the presence of NaHCO3 as activating base.
6-((5-((3,5-Dimethyl-1-phenyl-4,5-dihydro-1H-
pyrazol-4-yl)amino)-1,3,4-thiadiazol-2-yl)thio)-N2,
N4-bis(4-methoxyphenyl)-1,3,5-triazine-2,4-
diamine 10e
Deep yellow crystals; yield: 82%; mp; 236–238 ꢀC; MW: 626.76; FT-
IR (mmax per cm, KBr): 3117–2990 (N–H secondary), 2930 (C-H
The last step corresponds to the clubbing of 3,5-dimethyl-1-phenyl-
4,5-dihydro-1H-pyrazole-4ylamino-1,3,4-thiadiazole (6) with di-substi-
tuted monochloro-1,3,5-triazines 9(a–f) by means of the nucleophilic
4
Chem Biol Drug Des 2012

Antibacterial Activity of 1,3,5-triazines
substitution (SNAr) reaction between SH of 1,3,4-thiadiazole (6) and
monochlorine of di-substituted-1,3,5-triazines 9(a–f) in the presence
of base under vigorous condition to yield title hybrid analogues 10
(a–f), Scheme 1 (Parts 1–3) (10).
adiazole-2-thiol was optimally tolerated as pendant substituent on
1,3,5-triazine core. The compounds having halogen atoms on fourth
position of phenyl amine 10(a–c) showed prominent activities
against P. aeruginosa and E. coli. However, no activity was reported
against B. subtilis and B. cereus by the same test compounds. In next
instance, replacement of halogen by non-halogen substituents causes
a dramatic decrease in the activity for P. aeruginosa, and a further
decrease was reported for the rest of strains. Though, it is noteworthy
to consider here the effect of halogen atom on the modulation of anti-
bacterial activity, which is further found in agreement with the study
carried out by Singh et al. (10). These results suggest the necessity of
electron-withdrawing group for the generation and escalation of anti-
bacterial activity. However, ease of penetration of these molecules
across the bacterial cell wall because of the modulation of the steric
effect on phenyl ring could be acted as a plausible cause of variation
in antibacterial profile.
Both analytical and spectral data of all the compounds are found in
full agreement with the proposed structures.
Antibacterial activity
As depicted from the Table 1, entire set of target compounds
10(a–f) showed moderate to least activity against the tested
Gram-positive and Gram-negative micro-organisms in comparison
with cefixime as standard. Compounds with chloro-substitution on
phenyl amine position (10a) connected to 1,3,5-triazine core exhibit
significant activity against Pseudomonas aeruginosa (6.25 lg ⁄ mL),
moderate activity against Bacillus cereus and Escherichia coli
(12.5 lg ⁄ mL) and presented no activity at highest test concentra-
tion of 100 lg ⁄ mL against the Bacillus subtilis. Upon replacement
of chloro to bromo (10b), no significant shift in activity was
observed, except against B. cereus (100 lg ⁄ mL). However, consider-
able increase in the activity was observed in the case of compound
10c having fluoro as substitution against the E. coli, whereas no
change in the activity for P. aeruginosa and no activity against the
rest of the strains were revealed by the same test compound. Intro-
duction of methyl group (10d) markedly results in decrease and
increase in the activity for P. aeruginosa and B. cereus, respectively.
Substantial to moderate activity was observed in the case of com-
pound 10e having methoxy as substituent, toward P. aeruginosa
and E. coli, respectively. However, substantial to significant activity
was observed in the case of analogue having nitro as substituent
(10f) for P. aeruginosa and E. coli, respectively. The resultant MIC
value for the title compounds was found in good agreement with
the results of zone of inhibition (Table 1).
Conclusion
As a concluding remark, the present study demonstrates the utility
of hybrid 1,3,4-thiadiazole-1,3,5-triazine as a potential lead for
ongoing antimicrobial lead discovery program on the basis of con-
siderable bioactivity, less reaction time, cheap, and facile synthesis.
Additionally, it was confirmed that the presence of 5-(3,5-dimethyl-
1-phenyl-4,5-dihydro-1H-pyrazol-4-ylamino)-1,3,4-thiadiazole-2-thiol
as a pendent substituent was optimally tolerated on 1,3,5-triazine
core. Nevertheless, further studies are needed to obtain novel
insight into their structure–activity relationship and confirmation of
molecular mechanism through additional experiments. Our studies
are in progress and reported subsequently in the future.
Antibacterial Screening
Minimum inhibitory concentration
It was disclosed from the antibacterial screening that the presence of
5-(3,5-dimethyl-1-phenyl-4,5-dihydro-1H-pyrazol-4-ylamino)-1,3,4-thi-
All synthesized compounds were screened for their minimum inhibi-
tory concentration (MIC, lg ⁄ mL) against selected Gram-positive
Scheme 1: Part 1, Synthesis of 5-(3,5-dimethyl-1-phenyl-4,5-dihydro-1H-pyrazol-4-ylamino)-1,3,4-thiadiazole-2-thiol (6). Reagents and condi-
tions: (a) Ethanol, Na2CO3, reflux. (b) NaNO2 ⁄ HCl. (c) Acetyl acetone, CH3COONa, ethanol, stirring. (d) Phenyl hydrazine, CH3COONa; Part 2,
Synthesis of di-substituted monochloro-1,3,5-triazines 9 (a–f). (e) NaHCO3, 40–45 ꢀC; Part 3, Synthesis of hybrid 3,5-dimethyl-1-phenyl-4,5-di-
hydro-1H-pyrazole-4ylamino-1,3,4-thiadiazole and 1,3,5-triazine derivatives tethered via –S– bridge 10 (a–f) f) Reflux, 120–135ꢀC, K2CO3.
Chem Biol Drug Des 2012
5

Dubey et al.
Table 1: Antibacterial activity of hybrid 1,3,4-thiadiazole-1,3,5-triazine derivatives
Minimum inhibitory concentration (lg ⁄ mL)
Zone of Inhibition (in mm)
Percentage of inhibition (in comparison with
standard)
Pseudomonas Bacillus Bacillus Escherichia Pseudomonas Bacillus Bacillus Escherichia Pseudomonas Bacillus Bacillus Escherichia
Compounds aeruginosa
cereus subtilis coli
aeruginosa
cereus subtilis coli
aeruginosa
cereus subtilis coli
10a
6.25
6.25
6.25
12.5
12.5
12.5
12.5
100
100
50
100
100
6.25
100
100
100
100
100
100
12.5
12.5
6.25
6.25
50
11
14
12
15
10
13
20
8
10
10
11
6
1
4
2
1
3
8
3
6
5
2
58.3
69.8
62.0
77.4
50.6
68.4
100
41.6
50.3
51.7
56.0
33.3
60.5
6.5
25.5
13.6
9.3
19.2
21.4
50.7
21.1
36.6
30.4
13.0
6.0
10b
10c
10d
10e
10f
Cefixime
6.25
3.125
12
20
3
20
1
18
3.125
6.25
100
100
100
organisms viz. Bacillus subtilis (NCIM-2063), B. cereus (NCIM-2156)
and Gram-negative organism viz. Pseudomonas aeruginosa (NCIM-
2036), E. coli (NCIM-2065), and by the broth dilution method as
recommended by the National Committee for Clinical Laboratory
Standards (18) with minor modifications. Cefixime was used as
standard antibacterial agent. Solutions of the test compounds and
reference drug were prepared in dimethyl sulfoxide (DMSO) at con-
centrations of 100, 50, 25, 12.5, 6.25, 3.125 lg ⁄ mL. 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.
agar plate is inoculated by streaking the swab over the entire ster-
ile agar surface (19).
The plates containing bacteria were inoculated a disk of cefixime
(20 lg) and synthesized compound (20 lg), while the control plate
was inoculated with DMSO which shows no inhibition of bacterial
growth. Each disk must be pressed down to ensure complete con-
tact with the agar surface. Then, the plates are inverted and
placed in an incubator set to 35 ꢀC within 15 min after the disks
are applied. They were then incubated at 37 ꢀC for 24 h, after
which the inhibition halo was measured with a milimetric ruler
(zone of inhibition). This qualitative screening was performed to
verify positive antimicrobial activity of the synthesized compound.
These results are further quantified in terms of percentage of inhi-
bition in reference to cefixime as standard and results are shown
in Table 1.
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 because it contained bac-
teria but not antibiotic. The test compounds were dissolved in
DMSO (100 lg ⁄ mL); 0.1 mL of increasing concentration of the pre-
pared test compounds is serially diluted from tube 2 to tube 7 from
highest (100 lg ⁄ mL) to lowest (3.125 lg ⁄ mL) concentration (tubes
2–7 containing 100, 50, 25, 12.5, 6.25, 3.125 lg ⁄ mL). After this
process, each tube was inoculated with 0.1 mL of the bacterial sus-
pension whose concentration corresponded to 0.5 McFarland scale
(9 · 108 cells ⁄ mL), 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, that is, no growth of
inoculated bacteria.
Acknowledgments
Authors are gratified to SAIF, Central Drug Research Institute, Luc-
know, India, for providing spectral data of compounds synthesized
herein and SHIATS for providing basic facilities to carry out the pro-
ject. One of the author, UPS, is also pleased to acknowledge Prof.
Ray C. F. Jones, Professor of Organic and Biological Chemistry,
Loughborough University, UK, for his critical discussion and sugges-
tions on the chemistry of 1,3,5-triazines.
Conflict of Interest
None.
Disk diffusion along with percentage of
inhibition in comparison with standard
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