Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of Novel Hybrid 1,3-Thiazine-1,3,5-Triazine Derivatives as Potential Bacterial Translation Inhibitor

Article abstract of DOI:10.1111/j.1747-0285.2012.01430.x

Some novel hybrid 1,3-thiazine-1,3,5-triazine derivatives were synthesized and tested for antibacterial activity. Compounds 8c and 8f were found active against Gram positive and Gram negative microorganisms. Molecular docking studies have been performed o

Full text of DOI:10.1111/j.1747-0285.2012.01430.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01430.x
Chem Biol Drug Des 2012
Research Article
Design, Synthesis, Antibacterial Activity, and
Molecular Docking Studies of Novel Hybrid 1,3-
Thiazine-1,3,5-Triazine Derivatives as Potential
Bacterial Translation Inhibitor
Udaya P. Singh1*, Manish Pathak1,
people die each year from infections because of antibiotic-resistant
bacteria, which also result in around 2.5 million extra hospital days
(2). As a result, increased incidence of bacterial resistance to cur-
rently available antibiotics altogether increases healthcare cost
Vaibhav Dubey1, Hans R. Bhat1, Prashant
Gahtori2 and Ramendra K. Singh3
1Department of Pharmaceutical Sciences, Sam Higginbottom
associated with it. According to recent study by Robert and cowork-
ers, it has been calculated that annual costs of antibiotic-resistant
infections to the U.S. Healthcare system comes to be in excess of
$20 billion (3).
Institute of Agriculture Technology and Sciences, Formerly
Allahabad Agricultural Institute, Deemed to be University, 211007
Allahabad, India
2Faculty of Pharmacy, Uttarakhand Technical University, Dehradun,
Uttarakhand 248007, India
In an our effort to design effective antimicrobial compound derived
from 1,3,5-triazine, we had earlier reported a novel heterocyclic
hybrid skeleton encompassing thiazole and 1,3,5-triazine connected
through –NH– linker (4,5). Structure-activity relationship suggested
that, the thiazole amine pendant is well tolerated along with the
presence of electron withdrawing groups on phenyl amine on either
side of 1,3,5-triazine core. Prompted by the results, we further
devised a series of analogues as potential antimicrobials by keeping
thiazole fragment rigid and having diverse substitution pattern on
both wings of 1,3,5-triazine by amine and aromatic, aliphatic frag-
ments tethered via amine and mercapto (-S-) bridge (6). Results
revealed that analogues having amine bridge were more effective
than their respective mercapto equivalents and further explicated
the critical structural requirement of electron withdrawing groups.
3Nucleic Acids Research Laboratory, Department of Chemistry,
University of Allahabad, 211002 Allahabad, India
*Corresponding author: Udaya P. Singh, udaysingh98@gmail.com
Some novel hybrid 1,3-thiazine-1,3,5-triazine deriv-
atives were synthesized and tested for antibacte-
rial activity. Compounds 8c and 8f were found
active against Gram positive and Gram negative
microorganisms. Molecular docking studies have
been performed on eubacterial ribosomal decod-
ing A site (Escherichia coli 16S rRNA A site) to
rationalize the probable mode of action, binding
affinity, and orientation of the molecules at the
active site of receptor. The structures of all these
newly synthesized compounds were confirmed by
their elemental analyses and spectral data tech-
niques viz. IR, 1H NMR, 13C NMR, and mass.
Over the past decade, bacterial ribosome is a key target for natu-
rally occurring antibiotics, including the macrolides, tetracyclines,
chloramphenicol, aminoglycosides, and the newly discovered syn-
thetic oxazolidinones as well (7). Recently, 3,5-diamino-piperidinyl
triazines (DAPT) having 1,3,5-triazine core scaffold, identified as a
novel class of antibacterial agent have been found active in murine
model, that target the bacterial decoding-site RNA in vitro and inhi-
bit bacterial growth by a translation-dependent mechanism. These
derivatives are considered to be as a mimetics of the natural ami-
noglycoside antibiotics (8–10). Nevertheless, facts drawn through
molinspiration program in our earlier studies allowed us to report
nuclear receptor binding domain for antibacterial action of 1,3,5-tri-
azine derivatives (11).
Key words: 1,3,5-Triazine, 1,3-thiazine, antibacterial, docking, trans-
lation inhibition
Received 1 February 2012, revised 16 April 2012 and accepted for publi-
cation 5 June 2012
Antibiotic resistance is all the time more recognized as a serious
and permanent public health concern and is usually considered to
be a consequence of wide use and misuse of antibiotics. Surveil-
lance data for Streptococcus pneumoniae, a common cause of bac-
terial respiratory tract infections, disclosed that 24% of isolates
were not susceptible to penicillin. Moreover, resistance to several
other antibacterial drugs is common; of which 1.5% of isolates
were resistant to cefotaxime (a third generation cephalosporin), and
resistance to the newer fluoroquinolone antimicrobials has already
been reported (1). In Europe, it is estimated that at least 25 000
Prompted by these findings, present study deals with the synthesis
of hybrid analogues of 1,3-thiazine-1,3,5-triazine as core skeleton
tethered through –NH– linkage and its antibacterial activity deter-
mination. To define binding mode of these constitutive compounds
as plausible bacterial translation inhibitor, molecular docking studies
1

Singh et al.
were carried out on eubacterial ribosomal decoding A site (Escheri-
chia coli 16S rRNA A site) receptor domain. The rationale behind
the present study was to optimize the substituent on pendant posi-
tion, therefore, we expand the ring of earlier optimized substituted
phenyl thiazole-2-amine fragment by one carbon atom and introduc-
tion of another substituted phenyl ring resulting in 4,6-substiututed
phenyl-6H-1,3-thiazin-2-amine.
3-(2-Nitrophenyl)-1-phenylprop-2-en-1-one (3a). Yellow crystals;
Yield: 76%; mp: 123–125 ꢀC; Rf: 0.56; FT-IR (mmax; per cm KBr):
1654 (C=O), 1592 (C=C), 1548.28–1446.06 (aromatic C=N); H NMR
(400 MHz, CDCl3-d6, TMS): d 7.99–7.98 (m, 2H, H¢2 H¢6), 7.56–7.53
(m, 3H, H¢3 H¢4 H¢5), 7.52 (d, 1H, J = 8.4 Hz H6), 7.51 (m, 3H, H3 H4
H5), 6.89 (d, 1H, J = 15.3 Hz Hb), 6.38 (d, 1H, J = 15.3 Hz Ha); Anal.
Calcd. For C15H11NO3: C, 71.14; H, 4.38; N, 5.53. Found: C, 71.12;
H, 4.39; N, 5.58.
1
3-(2-Nitrophenyl)-1-(4-nitrophenyl)prop-2-en-1-one (3b). Light Yellow
crystals; Yield: 85%; mp: 133–134 ꢀC; Rf: 0.64; FT-IR (mmax; per cm
KBr): 1656 (C=O), 1594 (C=C), 1548.28–1446.06 (aromatic C=N); H
Experimental
1
Material and method
NMR (400 MHz, CDCl3-d6, TMS): d 7.97–7.96 (m, 2H, H¢2 H¢6),
7.54–7.53 (m, 2H, H¢3 H¢5), 7.51 (d, 1H, J = 8.3 Hz H6), 7.51 (m, 3H,
H3 H4 H5), 6.89 (d, 1H, J = 15.3 Hz Hb), 6.38 (d, 1H, J = 15.3 Hz
Ha); Anal. Calcd. For C15H10N2O5: C, 60.41; H, 3.38; N, 9.39. Found:
C, 60.32; H, 3.36; N, 9.40.
Melting points of compounds were determined in open capillary
tubes Hicon melting point apparatus and are uncorrected. Thin layer
chromatographic analysis was done to monitor the completion of
reaction as well as for identification and characterization of com-
pounds. 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 inter-
mediate compounds were established on the basis of spectral (FT-
1-(4-Hydroxyphenyl)-3-(2-nitrophenyl)prop-2-en-1-one (3c). Brown crys-
tals; Yield: 83%; mp: 129–130 ꢀC; Rf: 0.54; FT-IR (mmax; per cm KBr):
1
1659 (C=O), 1595 (C=C), 1546.28–1448.06 (aromatic C=N); H NMR
1
IR, H NMR, mass) and elemental analysis, whereas structures of
(400 MHz, CDCl3-d6, TMS) d ppm: 7.97–7.98 (m, 2H, H¢2 H¢6), 6.94–
6.96 (m, 2H, H¢3 H¢5), 5.28 (s, 1H, Ar-OH), 7.51 (d, 1H, J = 8.2 Hz
H6), 7.50 (m, 3H, H3 H4 H5), 6.89 (d, 1H, J = 15.3 Hz Hb), 6.38 (d,
1H, J = 15.3 Hz Ha); Anal. Calcd. For C15H11NO4: C, 66.91; H, 4.12;
N, 5.20. Found: C, 66.90; H, 4.14; N, 5.20.
title hybrid analogues on the basis of FT-IR, 1H NMR, 13C NMR,
mass spectral, and elemental analysis data. FT-IR (in 2.0 per cm,
flat, smooth, abex, KBr) spectra were recorded on Biored FTs spec-
1
trophotometer. H NMR spectra were recorded on Bruker Avance II
400 and 300 MHz NMR spectrometer in CDCl3-d6 and DMSO using
tetramethylsilane (TMS) as internal standard. 13C NMR spectra
were recorded on Bruker Avance II 300 NMR Spectrometer. Chemi-
cal shifts are reported in parts per million (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 auto
sampler. The mobile phase MeOH ⁄ CAN:H2O (90:10) flowed at a rate
of 250 lL ⁄ min by MS pump. Ion spray voltage was set at 5.3 KV
and capillary voltage 34 V. The scan run upto 2.5 min and the spec-
tra averaged over 10 scan at peak top in TLC. Elemental analysis
was carried out on a Vario EL III CHNOS elemental analyzer.
3-(2-Chlorophenyl)-1-(4-nitrophenyl)prop-2-en-1-one (3d). Dark Yel-
low crystals; Yield: 87%; mp: 142–143 ꢀC; Rf: 0.68; FT-IR (mmax; per
cm KBr): 1652 (C=O), 1590 (C=C), 1546–1448 (aromatic C=N), 810,
1
754; H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.97–7.98 (m, 2H,
H¢2 H¢6), 7.53–7.51 (m, 2H, H¢3 H¢5), 7.48 (d, 1H, J = 8.3 Hz H6),
7.48–749 (m, 3H, H3 H4 H5), 6.89 (d, 1H, J = 15.3 Hz Hb), 6.37 (d,
1H, J = 15.3 Hz Ha); Anal. Calcd. For C15H10ClNO3: C, 62.62; H,
3.50; N, 4.87. Found: C, 62.65; H, 3.53; N, 4.86.
1-(4-Chlorophenyl)-3-(4-nitrophenyl)prop-2-en-1-one (3e). Brown yellow
crystals; Yield: 79%; mp: 138–139 ꢀC; Rf: 0.47; FT-IR (mmax; per cm
KBr): 1658 (C=O), 1594 (C=C), 1543–1441 (aromatic C=N), 1230, 880;
1H NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.95–7.96 (m, 2H, H¢2
H¢6), 7.54–7.53 (m, 2H, H¢3 H¢5), 7.51 (m, 3H, H3 H4 H5), 6.89 (d, 1H,
J = 15.3 Hz Hb), 6.38 (d, 1H, J = 15.3 Hz Ha); Anal. Calcd. For
C15H10ClNO3: C, 62.62; H, 3.50; N, 4.87. Found: C, 62.66; H, 3.52;
N, 4.88.
Chemistry
The synthesis of title analogues were carried out through following
steps.
1,3-Bis(4-nitrophenyl)prop-2-en-1-one (3f). Orange crystals; Yield:
64%; mp: 153–154 ꢀC; Rf: 0.78; FT-IR (mmax; per cm KBr): 1654
(C=O), 1596 (C=C), 1545–1443 (aromatic C=N), 1240, 880, 749; 1H
NMR (400 MHz, CDCl3-d6, TMS) d ppm: 7.97–7.98 (m, 2H, H¢2 H¢6),
7.53–7.52 (m, 2H, H¢3 H¢5), 8.01–8.03 (m, 2H, H2,H6), 8.17 (m, 2H,
H3 H5), 6.88 (d, 1H, J = 15.3 Hz Hb), 6.37 (d, 1H, J = 15.3 Hz Ha);
Anal. Calcd. For C15H10N2O5: C, 60.41; H, 3.38; N, 9.39. Found: C,
60.43; H, 3.38; N, 9.38.
Step 1: Synthesis of 4,6-substituted di-phenyl
1,3-thiazine-2-amine
General procedure for synthesis of substituted chalcone derivatives
3(a–f). Dissolved substituted acetophenone,
1 (0.01 mol) and
substituted benzaldehyde, 2 (0.01 mol) in 50 mL ethanolic solution
and stirred for 30 min, followed by dropwise addition of aqueous
sodium hydroxide (0.05 mol), further stirring continued for 24 h.
After completion of the reaction as reaction monitored by TLC using
the mobile phase as n-butanol:acetic acid:water (4:3:1), a crude
product was obtained as substituted chalcone, 3(a–f). The resultant
solid was then filtered off, washed with water, dried, and re-crys-
tallized from ethanol.
General procedure for synthesis of substituted 1,3-thiazine from cor-
responding chalcone derivative 3(a–f). The corresponding substi-
tuted chalcone derivatives 3(a–f) (0.01 mol) and thiourea (0.01 mol)
was dissolved in ethanol (50 mL) and refluxed the mixture, while a
2
Chem Biol Drug Des 2012

Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of 1,3,5-triazines
solution of potassium hydroxide (0.05 mol) in water (10 mL) added
Step 2: Procedure for synthesis of 6-chloro-
N2,N4-bis(3-nitrophenyl)-1,3,5-triazine-2,4-
diamine (7)
portion-wise for 2 h. Then again refluxed it for further 6 h and after
that resultant mixture was poured into ice-cold water. A solid prod-
uct 4(a–f) thus obtained was filtered off, dried and crystallized
from ethanol.
3-Nitro aniline 6 (0.2 mol) was added into 100 mL of acetone at
temperature 40–45 ꢀC. The solution of 2,4,6-trichloro-1,3,5-triazine
(5) (0.1 mol) in 25 mL acetone was added slowly, stirred the reac-
tion for 3 h followed by drop-wise 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 mobile phase (9:1). The product was filtered and
washed with cold water and re-crystallized with ethanol to afford
pure compound 6-chloro-N2,N4-bis(3-nitro phenyl)-1,3,5-triazine-2,4-
diamine, 7.
6-(2-Nitrophenyl)-4-phenyl-6H-1,3-thiazin-2-amine (4a). Yellow crys-
tals; Yield: 78%; mp: 163–164 ꢀC; Rf: 0.45; FT-IR (mmax; per cm KBr):
3356 (NH),1687 (C-N), 1596 (C=C), 1656–1645 (aromatic C=N), 1522,
1
1351 (Ar-NO2), 876, 756; H NMR (400 MHz, CDCl3-d6, TMS) d ppm:
5.63 (s, 1H, H5), 7.33–7.30 (m, 3H), 7.66 (d, 1H, J = 7.9 Ar-H), 8.35 (d,
1H, J = 7.1, 5.43 Ar-H), 5.52 (bs, 1H); Anal. Calcd. For C16H13N3O2S:
C, 61.72; H 4.21; N, 13.50. Found: C, 61.66; H, 4.23; N, 13.52.
6-(2-Nitrophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-amine (4b). Brown
crystals; Yield: 67%; mp: 169–171 ꢀC; Rf: 0.65; FT-IR (mmax; per cm
KBr): 3359 (NH),1683 (C-N), 1599 (C=C), 1656–1645 (aromatic C=N),
Brownish 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), 1548.28–1446.06 (aromatic C=N); 1H NMR (400 MHz,
CDCl3-d6, TMS): d 7.40 (t, 4H, 4 = CH-aromatic), d 7.32(t, 4H,
4 = CH aromatic), 3.62 (d, 2H, 2-NH aromatic); Elemental analysis
for C15H10ClN7O4: Calculated: C, 46.46; H, 2.60; N, 25.29. Found: C,
46.48; H, 2.65; N, 25.26.
1
1524, 1353 (Ar-NO2), 876, 756; H NMR (400 MHz, CDCl3-d6, TMS)
d ppm: 5.68 (s, 1H, H5) 7.43–7.40 (m, 3H), 7.69 (d, 1H, J = 7.8 Ar-
H), 8.06 (d, 1H, J = 7.1, 5.4 Ar-H), 5.56 (bs, 1H); Anal. Calcd. For
C16H12N4O4S: C, 53.93; H 3.39; N, 15.72. Found: C, 53.90; H, 3.39;
N, 15.71.
4-(2-Amino-6-(2-nitrophenyl)-6H-1,3-thiazin-4-yl)phenol (4c). Brown
Step 3: General procedure for synthesis of
hybrid 1,3-thiazine-1,3,5-triazine analogues 8(a–
f)
Yellow crystals; Yield: 69%; mp: 176–178 ꢀC; Rf: 0.54; FT-IR (mmax
;
per cm KBr): 3359 (NH),1686 (C-N), 1600 (C=C), 1646–1635 (aro-
matic C=N), 1521, 1353 (Ar-NO2), 889, 778; 1H NMR (400 MHz,
CDCl3-d6, TMS) d ppm: 5.46 (s, 1H, H5) 7.37–7.67 (m, 3H), 7.69 (d,
1H, J = 7.6 Ar-H), 8.06 (d, 1H, J = 7.1, 5.4 Ar-H), 5.45 (bs, 1H);
Anal. Calcd. For C16H13N3O3S: C, 58.70; H 4.00; N, 12.84. Found: C,
57.45; H, 4.12; N, 12.85.
6-Chloro-N2,N4-bis(3-nitrophenyl)-1,3,5-triazine-2,4-diamine (7) (0.1 mol)
was added into 50 mL of 1,4-dioxane at temperature 40–45 ꢀC. A
solution of substituted 1,3-thiazine 4(a–f) (0.1 mol) in 35 mL 1,4-diox-
ane was added slowly to above solution and stirred for 90 min fol-
lowed by drop-wise addition of K2CO3 (0.1 mol), re-fluxed the reaction
mixture at 135–145 ꢀC for 9 h. The product was filtered and washed
with cold water and re-crystallized with ethanol to afford the corre-
sponding pure products 8(a–f).
6-(2-Chlorophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-amine
(4d).
Light Yellow crystals; Yield: 84%; mp: 183–184 ꢀC; Rf: 0.65; FT-IR
(mmax; per cm KBr): 3359 (NH),1686 (C-N), 1634 (C=C), 1648–1630
1
(aromatic C=N), 1520, 1353 (Ar-NO2), 890, 772; H NMR (400 MHz,
N2,N4-Bis(3-nitrophenyl)-N6-(6-(2-nitrophenyl)-4-phenyl-6H-1,3-thiazin-
2-yl)-1,3,5-triazine-2,4,6-triamine (8a). Black crystals; Yield: 86%;
CDCl3-d6, TMS) d ppm: 5.73 (s, 1H, H5) 7.31–7.38 (m, 3H), 7.62 (d,
1H, J = 7.9 Ar-H), 8.09 (d, 1H, J = 7.2, 5.3 Ar-H), 5.45 (bs, 1H);
Anal. Calcd. For C16H12ClN3O2S: C, 55.57; H 3.50; N, 12.15. Found:
C, 55.51; H, 3.55; N, 12.07.
mp: 145–147 ꢀC; Rf: 0.56; FT-IR (mmax; per cm KBr): 3278 (N-Hstretch
,
-NH2), 2927 (C-Hstretch, Aromatic), 1577 Aromatic (C=C ringstretch),
1529 (-C=N ringstretch), 1349 (-NO2stretch), 1127 (C-Nstretch), 834 (N-
Hdeformation), 1403–1092 Aromatic (-C-Hin
deformation), 737 Aro-
plane
4-(4-Chlorophenyl)-6-(4-nitrophenyl)-6H-1,3-thiazin-2-amine
(4e).
matic (-C-Hout
deformation), 1238 (C-Sstretch); 1H NMR
of plane
Dark Brown crystals; Yield: 76%; mp: 189–192 ꢀC; Rf: 0.58; FT-IR
(mmax; per cm KBr): 3354 (NH),1683 (C-N), 1631 (C=C), 1648–1630
(aromatic C=N), 1524, 1350 (Ar-NO2), 880, 782; H NMR (400 MHz,
CDCl3-d6, TMS) d ppm: 5.52 (s, 1H, H5) 7.35–7.36 (m, 3H), 7.56 (d,
1H, J = 7.6 Ar-H), 8.06 (d, 1H, J = 7.3, 5.3 Ar-H), 5.56 (bs, 1H);
Anal. Calcd. For C16H12ClN3O2S: C, 55.57; H 3.50; N, 12.15. Found:
C, 55.51; H, 3.55; N, 12.07.
(400 MHz, CDCl3-d6, TMS): d 3.90 (bs, 3H, NH), 6.91 (d, 1H,
J = 9.8Hz, Ar-H), 6.92–6.98 (d, 1H, J = 9.9Hz, Ar-H), 7.26 (s, 1H, thi-
azine), 7.42–7.44 (d, 1H, J = 8.4Hz, Ar-H), 7.63–7.99 (m, 1H, Ar-H),
7.73 (d, 1H, J = 8.7Hz, Ar-H), 7.99–8.01(d, 1H, J = 4.8Hz, 1H, Ar-H),
8.39–8.42 (d, 1H, J = 8.4Hz, Ar-H), 8.66 (S, 1H, Ar-H); 13C NMR
(400 MHz, CDCl3): d 170.42, 133.95, 114.91, 77.65, 77.23, 76.80;
ESI-MS (m ⁄ z): 525.10(M + H+); Anal. Calcd. For C31H22N10O6S: C,
56.19; H, 3.35; N, 21.14. Found: C, 55.12; H, 3.41; N, 21.12.
1
4,6-Bis(4-nitrophenyl)-6H-1,3-thiazin-2-amine (4f). Orange crystals;
Yield: 79%; mp: 196–198 ꢀC; Rf: 0.57; FT-IR (mmax; per cm KBr):
3359 (NH),1682 (C-N), 1635 (C=C), 1648–1630 (aromatic C=N),
1524, 1353 (Ar-NO2), 890, 756; 1H NMR (400 MHz, CDCl3-d6,
TMS): d 5.76 (s, 1H, H5) 7.34–7.36 (m, 3H), 7.64 (d, 1H, J = 7.6
Ar-H), 8.10 (d, 1H, J = 7.3, 5.2 Ar-H), 5.52 (bs, 1H); Anal. Calcd.
For C16H12N4O4S: C, 53.93; H 3.39; N, 15.72. Found: C, 53.91; H,
3.40; N, 15.72.
N2,N4-Bis(3-nitrophenyl)-N6-(6-(2-nitrophenyl)-4-(4-nitrophenyl)-6H-1,3-
thiazin-2-yl)-1,3,5-triazine-2,4,6-triamine (8b). Yellow crystals; Yield:
78%; mp: 169–171 ꢀC; Rf: 0.51; FT-IR (mmax; per cm KBr): 3276 (N-
Hstretch, -NH2), 2366 (C-Hstretch, Aromatic), 1585–1433 Aromatic (C=C
ringstretch), 1613 (-C=N ringstretch), 1348 (-NO2stretch), 1317 (C-Sstretch),
1299 (C-Nstretch), 994 (N-Hdeformation), 1401-1090 Aromatic (-C-Hin plane
deformation), 700 Aromatic (-C-Hout of plane deformation), 1317 (C-Sstretch);
Chem Biol Drug Des 2012
3

Singh et al.
1H NMR (400 MHz, CDCl3-d6, TMS): 4.07 (bs, 3H, NH), 7.03 (d, 1H,
J = 8.1Hz, Ar-H), 7.15 (d, 1H, J = 6Hz, Ar-H), 7.27 (s, 1H, thiazine),
7.73 (d, 1H, J = 7.5Hz, Ar-H), 7.95–7.97 (d, 1H, J = 8.4Hz, Ar-H), 7.79
(d, 1H, J = 7.8Hz, Ar-H), 8.00 (d, 1H, J = 7.2Hz, 1H, Ar-H), 8.07–8.62
(m, 1H, Ar-H), 8.62 (S, 1H, Ar-H); 13C NMR (400 MHz, CDCl3): d
129.84, 126.45, 77.43, 77.00, 76.58, 29.67; ESI-MS (m ⁄ z):
424.17(M + H+); Anal. Calcd. For C31H21N11O8S: C, 52.62; H, 2.99; N,
21.77. Found: C, 52.60; H, 3.01; N, 21.81.
Calcd. For C31H21ClN10O6S: C, 53.41; H, 3.04; N, 20.09. Found: C,
53.45; H, 3.01; N, 20.08.
N2-(4,6-Bis(4-nitrophenyl)-6H-1,3-thiazin-2-yl)-N4,N6-bis(3-nitrophenyl)-
1,3,5-triazine-2,4,6-triamine (8f). Yellow crystals; Yield: 68%; mp:
154–156 ꢀC; Rf: 0.47; FT-IR (mmax; per cm KBr): 3384 (N-H stretch, -
NH2), 1522 Aromatic (C=C ringstretch), 1399–1091 Aromatic (-C-Hin
plane deformation), 1343 (-NO2stretch), 1239 (C-Sstretch), 1177 (C-Nstretch),
994 (N-Hdeformation) 801 (N-Hrocking), 670 Aromatic (-C-Hout
deformation); H NMR (400 MHz, CDCl3-d6, TMS): d 3.76 (bs, 3H, NH),
of plane
1
4-(2-(4,6-Bis(3-nitrophenylamino)-1,3,5-triazin-2-ylamino)-6-(2-nitro-
phenyl)-6H-1,3-thiazin-4-yl)phenol (8c). Light Yellow crystals; Yield:
76%; mp: 182-184 ꢀC; Rf: 0.46; FT-IR (mmax; per cm KBr): 3383 (N-
Hstretch, -NH2), 3567 (O-Sstretch) 2366 (C-Hstretch, Aromatic), 1527 Aro-
matic (C=C ringstretch), 1570 (-C=N ringstretch), 1400-1093 Aromatic (-
7.10-7.07 (d, 1H, J = 8.8Hz, Ar-H), 7.33 (s, 1H, thiazine), 7.66–7.67
(d, 1H, J = 8.8Hz, Ar-H), 7.94–7.92 (d, 1H, J = 8.4Hz, Ar-H), 8.01–
7.99 (d, 1H, J = 8.8Hz, Ar-H), 8.10–8.08 (d, 1H, J = 8.4Hz,Ar-H),
8.15–8.13 (d, 1H, J = 7.2Hz, Ar-H), 8.72–8.35 (d, 1H, J = 8.8Hz, Ar-
H), 8.20–8.18 (d, 1H, J = 8.8Hz, Ar-H); 13C NMR (400 MHz, CDCl3):
d 148.60, 129.80, 129.23, 127.22, 126.48, 123.86; ESI-MS (m ⁄ z):
424.17(M + H+); Anal. Calcd. For C31H21N11O8S: C, 52.62; H, 2.99;
N, 21.77. Found: C, 52.70; H, 3.02; N, 21.79.
C-Hin
deformation), 1347 (-NO2stretch), 1296 (C-Ostretch), 1211 (C-
plane
S
stretch), 1168 (C-Nstretch), 835 (N-Hdeformation), 753 Aromatic (-C-Hout
deformation), 670 Aromatic (Ar-Clstretch); 1H NMR (400 MHz,
of plane
CDCl3-d6, TMS): d 3.78 (bs, 3H, NH), 6.92-7.95 (d, 1H, J = 6.8Hz,
Ar-H), 7.48–7.51 (d, 1H, J = 6.9Hz, Ar-H), 7.55–7.58 (d, 1H,
J = 6.3Hz, Ar-H), 8.18–8.20 (d, 1H, J = 6.6Hz, 1H, Ar-H), 8.20–8.42
(d, 1H, J = 9.6Hz, Ar-H), 7.32 (s, 1H, thiazine), 7.66–7.97 (m, 1H, Ar-
H), 6.92-6.95 (d, 1H, J = 6Hz, Ar-H), 8.66 (S, 1H, J = 5.5Hz, Ar-H);
13C NMR (400 MHz, CDCl3): d 148.44, 133.54, 130.38, 129.33,
124.85, 122.25, 77.43, 77.00, 76.58, 40.49, 40.21; ESI-MS (m ⁄ z):
657.19 (M + H+); Anal. Calcd. For C31H22N10O7S: C, 54.86; H, 3.27;
N, 20.64. Found: C, 55.05; H, 3.27; N, 20.65.
Molecular docking studies
The 3D X-ray crystal structure of paromomycin docked into the eu-
bacterial ribosomal decoding A site (E. coli 16S rRNA A site;
1j7t:pdb) was used as starting model for this study. The protein
was prepared, docked, scored, and the molecular dynamics simula-
tion carried out using standard procedures. All computational analy-
sis was carried out using Discovery Studio 2.5 (Accelrys Software
Inc., San Diego; http://www.accelrys.com).
N2-(6-(2-chlorophenyl)-4-(4-nitrophenyl)-6H-1,3-thiazin-2-yl)-N4,N6-
bis(3-nitrophenyl)-1,3,5-triazine-2,4,6-triamine (8d). Yellow crystals;
Yield: 86%; mp: 174–176 ꢀC; Rf: 0.31; FT-IR (mmax; per cm KBr):
3385 (N-Hstretch, -NH2), 2927 (C-Hstretch, Aromatic), 1599 (-C=N ring-
Preparation of receptor
stretch), 1528 Aromatic (C=C ringstretch), 1400–1091 Aromatic (-C-H
deformation), 1354 (-NO2stretch), 1178 (C-Nstretch), 834 (N-Hdeformation),
The target protein that complexed with paramomycin (PDB ID: 1j7t)
was taken, the ligand paramomycin extracted, and the bond order
were corrected. The hydrogen atoms were added, and their posi-
tions were optimized using the all-atom CHARMm (version c32b1)
forcefield with Adopted Basis set Newton Raphson (ABNR) minimi-
zation algorithm, until the root mean square (r.m.s) gradient for
potential energy was <0.05 kcal ⁄ mol ⁄ ꢀ (18,19). Using the 'Binding
Site' tool panel available in DS 2.5, the minimized eubacterial ribo-
somal decoding A site (E. coli 16S rRNA A site) was defined as
receptor, binding site was defined as volume occupied by the ligand
in the receptor, and an input site sphere was defined over the bind-
ing site with a radius of 5 ꢀ. The center of the sphere was taken
to be the center of the binding site, and side chains of the residues
in the binding site within the radius of the sphere were assumed
to be flexible during refinement of postdocking poses. The receptor
having defined binding site was used for the docking studies.
in plane
738 Aromatic (Ar-Clstretch), 611 Aromatic (-C-Hout
deformation),
of plane
1
572.50 (C-Sstretch); H NMR (400 MHz, CDCl3-d6, TMS): d 3.57 (bs,
3H, NH), 6.70 (s, 1H, J = 6.8Hz, Ar-H), 6.93–6.91 (d, 1H, J = 6Hz,
Ar-H), 6.97 (d, 1H, J = 8.7Hz, Ar-H), 7.22 (s, 1H, thiazine), 7.38 (d,
1H, J = 7.8Hz, Ar-H), 7.51 (d, 1H, J = 7.8Hz, Ar-H), 7.90-7.78 (m, 1H,
Ar-H), 8.14 (d, 1H, J = 9Hz, 1H, Ar-H), 8.59 (s, 1H, J = 8.4Hz, Ar-H);
13C NMR (300 MHz, CDCl3): d 148.60, 129.80, 129.16, 127.22,
126.48, 123.86, 113.91, 77.43, 77.01, 76.58, 67.05, 30.29; ESI-MS
(m ⁄ z): 655.10(M + H+); Anal. Calcd. For C31H21ClN10O6S: C, 53.41; H,
3.04; N, 20.09. Found: C, 53.43; H, 3.03; N, 20.07.
N2-(4-(4-Chlorophenyl)-6-(4-nitrophenyl)-6H-1,3-thiazin-2-yl)-N4,N6-
bis(3-nitrophenyl)-1,3,5-triazine-2,4,6-triamine (8e). Light Brown
crystals; Yield: 83%; mp: 168-170 ꢀC; Rf: 0.52; FT-IR (mmax; per cm
KBr): 3385 (N-Hstretch, -NH2), 2924 (C-Hstretch, Aromatic), 1522 (-C=N
ringstretch), 1431 Aromatic (C=C ringstretch), 1402–1091 Aromatic (-C-
Hin
deformation), 1351 (-NO2stretch), 1238 (C-Sstretch), 1177 (C-
Ligand setup
plane
Nstretch), 834 (N-Hdeformation), 670 Aromatic (Ar-Clstretch), 612 Aromatic
Using the built-and-edit module of DS 2.5, various ligands were
built all-atom CHARMm forcefield parameterization was assigned,
and then minimized using the ABNR method. A conformational
search of the ligand was carried out using a stimulated annealing
molecular dynamics (MD) approach. The ligand was heated to a
temperature of 700 K and then annealed to 200 K. Thirty such
cycles were carried out. The transformation obtained at the end of
each cycle was further subjected to local energy minimization, using
(-C-Hout
deformation); 1H NMR (400 MHz, CDCl3-d6, TMS): d
of plane
3.98 (bs, 1H, NH), 6.93-6.91 (d, 1H, J = 6Hz, Ar-H), 7.13 (d, 1H,
J = 9.3Hz, Ar-H), 7.26 (s, 1H, thiazine), 7.50–7.53 (d, 1H, J = 6.0Hz,
Ar-H), 7.62–7.80 (m, 1H, Ar-H),7.87 (d, 1H, J = 8.7Hz, Ar-H), 7.99–
8.02 (d, 1H, J = 9.2Hz, Ar-H), 8.06–8.09 (d, 1H, J = 7.8Hz, Ar-H),
8.24–8.28 (d, 1H, J = 8.1Hz, Ar-H); 13C NMR (300 MHz, CDCl3): d
77.42, 77.00, 76.58, 67.05; ESI-MS (m ⁄ z): 655.10 (M + H+); Anal.
4
Chem Biol Drug Des 2012

Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of 1,3,5-triazines
the ABNR method. The 30 energy-minimized structures were then
superimposed and the lowest energy conformation occurring in the
major cluster was taken to be the most probable conformation.
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 observed for any
macroscopic growth (clear or turbid) the next day.
Docking and Scoring
Molecular docking is a significant computational method used to
forecast the binding of the ligand to the receptor binding site by
varying position and conformation of the ligand keeping the recep-
tor rigid. LigandFit (20) protocol of DS 2.5 was used for the docking
of ligands with eubacterial ribosomal decoding A site (pdb id:
1j7t)a. The LigandFit docking algorithm combines a shape compari-
son filter with a Monte Carlo conformational search to generate
docked poses consistent with the binding site shape. These initial
poses are further refined by rigid body minimization of the ligand
with respect to the grid based calculated interaction energy using
the Dreiding forcefield (21). The receptor protein conformation was
kept fixed during docking, and the docked poses were further mini-
mized using all-atom CHARMm (version c32b1) forcefield and smart
minimization method (steepest descent followed by conjugate gradi-
ent), until r.m.s gradient for potential energy was <0.05 kcal ⁄ mol ⁄ ꢀ.
The atoms of ligand and the side chains of the residues of the
receptor within 5 ꢀ from the center of the binding site were kept
flexible during minimization. The description of ligand scoring (-
PLP1, -PLP2, -PMF, Lig_Internal_Energy, Binding Energy, and Dock
Score) were given earlier in result and discussion section. Further-
more, determination of binding energy to assess the binding affinity
of ligands for receptor was calculated by employing highest stable
ligand-receptor complex through the protocol 'Calculate Binding
Energies' within DS 2.5 using the default settings.
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 nutrient. Tube 1, the negative
control, did not contain bacteria or antibiotic. The positive control,
tube 8 contained bacteria, but not antibiotic. The test compound
were dissolved in DMSO (100 lg ⁄ mL), 0.1 mL of increasing concen-
tration of the prepared test compounds which are serially diluted
from tube 2 to tube 7 from highest (100 lg ⁄ mL) to lowest
(3.125 lg ⁄ mL) concentration (tube 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 suspension whose concentration corre-
sponded to 0.5 McFarland scale (9 · 108 cells ⁄ mL), and each tube
was incubated at 37 ꢀC for 24 h at 150 rpm. The incubation cham-
ber was kept humid. At the end of the incubation period, MIC val-
ues were recorded as the lowest concentration of the substance
that gave no visible turbidity, i.e., no growth of inoculated bacteria.
Results are presented in Table 1.
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
Mꢁeller-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 inocu-
lum suspension, a sterile cotton swab is dipped into the adjusted
suspension and then the dried surface of an agar plate is inocu-
lated by streaking the swab over the entire sterile agar surface.
Any surface moisture to be absorbed before applying the drug
impregnated disk (23).
Antibacterial screening
Minimum inhibitory concentration
Entire hybrid compounds were screened for their minimum inhibitory
concentration (MIC, lg ⁄ mL) against selected Gram-positive organ-
isms, viz. Bacillus subtilis (NCIM-2063), Bacillus cereus (NCIM-2156)
and Staphylococcus aureus (NCIM-2079) and Gram-negative organ-
ism viz., E. coli (NCIM-2065) by the broth dilution method as recom-
mended by the National Committee for Clinical Laboratory
Standards with minor modifications (22). Levofloxacin was used as
The plates containing bacterial inoculums are received a disc of lev-
ofloxacin (20 lg) and synthesized compound (20 lg), whereas the
control plate was inoculated with DMSO which shows no inhibition
Table 1: Antibacterial activity of hybrid derivatives
Minimum Inhibitory Concentration (MIC, in Inhibition halo (in mm)
lg ⁄ mL)
Percentage inhibition
Gram positive
Gram positive
Gram negative
Gram positive
Gram negative
Gram negative
Compound
B. subtilis B. cereus S. aureus E. coli B. subtilis B. cereus S. aureus E. coli B. subtilis B. cereus S. aureus E. coli
8a
8b
25
12.5
6.25
25
25
100
100
25
6.25
25
25
3.125
6.25
12.5
3.125
25
25
12.5
25
50
24
32
38
29
27
35
09
18
28
14
34
30
40
36
29
27
38
35
31
42
24
27
32
26
22
32
46
54.5
72.7
86.3
65.9
61.3
79.5
22.5
45
70
35
85
85.7
69.0
64.2
90.4
83
52.2
58.6
69.5
56
47.8
69.5
8c
8d
100
8e
8f
Levofloxacin
12.5
12.5
3.125
6.25
3.125
12.5
3.125 44
75
100
73.8
100
100
100
Chem Biol Drug Des 2012
5

Singh et al.
Scheme 1: Synthesis of target hybrid 1,3-thiazine-1,3,5-triazine derivatives 8(a–f). Reagents and condition: (a) NaOH, stirring for 24 h. (b)
ethanol, aq. KOH, reflux for 6 h. (c) NaHCO3, 40–45 ꢀC. (d) reflux, 120–135 ꢀC, K2CO3.
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. These results are further quantified in
terms of percentage of inhibition in reference to levofloxacin as
standard. And results were shown in Table 1.
is presented in Figure 1. The resulting substituted chalcones 3(a–f)
were again allowed to undergo cyclo-condesation reaction to afford
4,6-substiututed di-phenyl-6H-1,3-thiazin-2-amine amine fragment
4(a–f) by refluxing for 6 h in the presence of thiourea. On the
other hand, flanked ring substituents on either side of 1,3,5-triazine,
namely di-m-nitro phenyl amine were generated from the aromatic
nucleophilic reaction between 2,4,6-trichloro-1,3,5-triazine (5) with
two equivalents of m-nitro phenyl amine (6) to yield 6-chloro-N2,N4-
bis(3-nitrophenyl)-1,3,5-triazine-2,4-diamine (7), Scheme 1. The
structures of all synthesized compounds were confirmed by FT-IR,
1H NMR, 13C NMR, mass spectral, and elemental analysis tech-
niques. Herewith, this procedure acclaims an efficient and promis-
ing synthetic strategy with good to excellent yields for production
of the titled skeleton. FTIR spectrum was recorded in the range
4000–650 per cm to ensure presence of various functional groups.
In this context, the characteristic group stretching frequencies of
aromatic secondary amines (-NH) tend to appear at 3276–3385 per
cm whereas N-H deformation at 834–994 per cm. Aromatic C-H in
plane deformation was observed in range 1092–1403 per cm and
aromatic C=C ring stretching is easily recognized at 1431–1522 per
cm. Moreover, our investigations in the NMR spectrum states that
amino linker between 1,3-thiazines and 1,3,5-triazine appear at
3.57–4.07 ppm as a singlet. A singlet nearer at 7.26 ppm was
attributed to aromatic protons of thiazine. Other protons corre-
sponded to phenyl ring were observed in the spectrum at 6.91–
8.62ppm. 13C NMR technique reveals the characteristic carbon shift
ranges from 170.42 to 29.67 ppm. The highest carbon shifts are
assigned to triazine carbon in case of 8a. At the end, the identified
Results and Discussion
Chemistry
The synthetic route to obtain the necessary key intermediates from
commercially available reagents is briefly outlined in Scheme 1.
The title hybrid analogues of 1,3-thiazine-1,3,5-triazine tethered via
–NH– linkage 8(a–f) were accomplished by clubbing of 4,6-substiu-
tuted di-phenyl-6H-1,3-thiazin-2-amine (4) fragment with 6-chloro-
N2,N4-bis(3-nitrophenyl)-1,3,5-triazine-2,4-diamine (5) through an
aromatic nucleophilic reaction in the presence of NaOH as base
under vigorous condition. The mechanism of synthesis of substituted
di-phenyl thiazine-2-amine fragment 4(a–f), achieved through
substituted chalcone intermediates 3(a–f) prepared by crossed aldol
condensation reaction between substituted aldehydes (2) and eth-
anolic acetophenone (1) (enolizable) in presence of NaOH as base,
6
Chem Biol Drug Des 2012

Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of 1,3,5-triazines
Figure 1: Mechanism of substituted chalcone intermediates synthesis 3(a–f).
1
proton multiplet was presented in the H NMR spectrum with their
and hydroxy too have their own contribution to structure-activity
relationships.
J-couplings 4.8–8.4Hz. The mass spectrum of compounds is charac-
terized by their M + 1 peak. Elemental analysis was within €0.4%
of the theoretical values in agreement with the proposed struc-
tures.
Molecular docking studies
Molecular docking is a powerful tool in drug design, which could
predict the best mode by which a given compound fits well into a
binding site of a macromolecular target (12). With in vitro antimi-
crobial results in hand, we thought it worthwhile to perform in sil-
ico studies to support the results. The in silico molecular docking
study was carried out using LigandFit module within software pack-
age in Discovery studio 2.5 on X-ray structure of eubacterial ribo-
somal decoding A site (E. coli 16S rRNA A site) receptor domain
without co-crystallized ligand paromomycin (1j7t.pdb) as outlined in
experimental section. Docking results were discussed on the param-
eters, such as hydrogen bond because they help to stabilize and
strengthen a bound receptor-ligand complex and non-bonded p–p
and p–+ interactions. The p–p and p–+ interactions are non-cova-
lent interactions, which have pivotal role on protein-ligand recogni-
tion and are termed as major forces that stabilize the association.
The p–p stacking interaction results from frequent overlapping of
non-polar ring-to-ring in parallel pose to generate hydrophobic con-
tacts, whereas p–+ interaction also known as Doughtry-effect and
termed as a non-covalent molecular interaction results from interac-
tion between the face of an electron-rich p system (e.g. benzene
ring) and an adjacent cation (+). This unusual interaction is an
example of non-covalent bonding between a monopole (cation) and
a quadrupole (p system). According to Gallivan and Dougherty, p–+
interaction energies should be considered of the same order of
magnitude as hydrogen bonds or salt bridges and play an important
role in molecular recognition and interaction with ligand. Only those
p–+ interaction of the title compounds have been discussed in the
present case, where the interatomic distances were <6 ꢀ, the dis-
tance imperative for effective p–+ interactions necessary for recep-
tor-ligand recognition as suggested by Gallivan and Dougherty (13).
Antibacterial activity
The title hybrid derivatives 8(a–f) showed moderate to significant
susceptibilities towards Gram-negative and Gram-positive bacteria,
as shown in Table 1. In the case of compound 8a, no activity was
observed against B. cereus at highest test concentration of
100 lg ⁄ mL, but at the same time, a significant inhibition pattern
was observed in the case of S. aureus (6.25 lg ⁄ mL) and mild
against B. subtilis and E. coli. In the next instance, introduction of
nitro at para position on intact phenyl ring connected to 1,3-thiazine
(8b), showed no significant alteration in activity, except in the case
of B. subtilis (12.5 lg ⁄ mL). Furthermore, replacement of p-nitro with
p-hydroxy, compound 8c, caused markedly increase in activity
against B. subtilis (6.25 lg ⁄ mL), B. cereus (25 lg ⁄ mL), and E. coli
(12.5 lg ⁄ mL) and no change against S. aureus (25 lg ⁄ mL). Surpris-
ingly, twofold reduction in activity was observed in the case of
B. subtilis and B. cereus, onefold in the case of E. coli, but further
threefold increase in activity for S. aureus (3.125 lg ⁄ mL), when m-
chloro and p-nitro substitution were present on both the ring at
fourth and fifth position, respectively, compound 8d, of 1,3-thiazine
core. No considerable change in antibacterial activity was observed
in the case of compound 8e (m-chloro to p-chloro), e.g. B. subtilis
(25 lg ⁄ mL), however, drastic decline in activity was reported
against rest of the strains. Notably, twofold amplified antibacterial
activity was observed in the case of derivative 8f, against B. subtil-
is (6.25 lg ⁄ mL) and E. coli (12.5 lg ⁄ mL) with structural variation of
p-nitro in place of p-chloro, by keeping all fragment rigid. The mini-
mum inhibitory concentration of all title analogues were supported
by their respective inhibition halo (percentage inhibition), to confirm
positive antimicrobial action.
It was surprising to see that minor structural variations in hybrid
compounds induced marked influence on antibacterial activity. Thus,
it was summarized that position of nitro atom was the only main
determinant for generation and escalation of bio-activity with
regard to structure-activity relationships. The presence of chloro
Molecular docking analysis
From Table 2, it was clear that, compound 8a, formed two close in-
teratomic hydrogen (H…O) bonds resulting from the interaction
between hydrogen of amine linkages, connecting m-nitro phenyl
amine and 1,3,5-triazine. The oxygen of Gua40 (1.86 ꢀ) and Ade39
Chem Biol Drug Des 2012
7

Singh et al.
Table 2: Molecular docking interaction of ligand with receptor in ligand-receptor docked complex
H-bonding
Bond
p–p monitor
p–+ monitor
Distance Donor Acceptor
Distance
(ꢀ)
Distance End1
(ꢀ)
End2
Compound
(ꢀ)
Atom Atom
Bond
End1 End2
Bond
8a
8a:H49–B:Gua40:O6 1.86
8a:H50–B:Ade39:OP2 2.10
A:Ade7:H62–8b:S17 2.12
8b:H52–B:Gua37:OP2 2.03
8b:H54–B:Gua37:O6 1.79
H49 O6
H50 OP2
H62 S17
H52 OP2
H54 O6
H42 O48
H42 O49
H50 OP2
H51 N3
H71 OP2
H42 O42
H61 O46
H50 N7
H52 O6
H52 N7
8a–B:Gua37 5.84
8a–B:Gua37 4.41
8b–B:Gua37 5.68
8a
8a
8b
B:Gua37 A:Cyt3–8a:N40 6.09
B:Gua37
B:Gua37 B:Gua37–8b:N46 5.16
B:Gua37–8b:N46 4.34
A:Cyt3 8a:N40
8b
8c
B:Gua37 8b:N46
B:Gua37 8b:N46
A:Cyt6:H42–8c:O48
A:Cyt6:H42–8c:O49
1.98
2.49
8c–B:Gua37 4.89
8c
B:Gua37 B:Gua40–8c:N44 6.97
A:Gua4–8c:N47 5.96
B:Gua40 8c:N44
A:Gua4 8c:N47
A:Gua4 8c:N47
B:Ade38 8c:N44
8c:H50–B:Ade38:OP2 2.08
8c:H51–B:Gua37:N3 2.50
8c:H71–B:Gua40:OP2 1.91
A:Gua4–8c:N47 6.36
B:Ade38–8c:N44 6.55
8d
A:Cyt6:H42–8d:O42
2.38
NO
B:Ade38–8d:N44 5.74
B:Ade38 8d:N44
B:Ade39:H61–8d:O46 2.33
8d:H50–B:Gua40:N7 2.47
8d:H52–B:Gua37:O6 1.97
8e:H52–B:Gua37:N7 2.06
8e
8f
NO
NO
NO
B:Gua40–8e:N47 6.55
A:Gua4–8e:N44 5.13
A:Gua4–8e:N44 5.86
B:Gua40–8f:N49 5.50
B:Gua40 8e:N47
A:Gua4 8e:N44
A:Gua4 8e:N44
B:Gua40 8f:N49
A:Ade7:H61–8f:O42
A:Cyt8:H42–8f:O41
8f:H54–B:Gua37:N7
2.26
2.42
2.08
H61 O42
H42 O41
H54 N7
H42 O25
H62 O10
H48 OP2
H50 OP2
H54 N1
H56 O4
H61 OP2
H62 N7
H77 OP2
H80 OP2
Paromomycin A:Cyt6:H42–par:O25 1.77
(reference ligand) A:Ade7:H62–par:O10 2.37
par:H48–B:Ade38:OP2 1.93
NO
par:H50–B:Ade39:OP2 1.78
par:H54–A:Ade7:N1
2.03
par:H56–B:Urd41:O4 1.82
par:H61–B:Gua40:OP2 1.95
par:H62–B:Gua40:N7 1.90
par:H77–A:Gua4:OP2 1.85
par:H80–A:Urd5:OP2 1.83
(2.10 ꢀ) present on B-chain of RNA of eubacterial ribosomal decod-
ing site acted as a acceptor. Similarly, m-nitro phenyl amine con-
nected to 1,3,5-triazine in compound 8a was shown to form two
p–p interactions with purine ring of Gua37 on B-chain. Introduction
of nitro group in fourth position of non-substituted phenyl con-
nected to 1,3-thiazine, compound (8b) caused increase in number
of interatomic hydrogen bonds to three. The first hydrogen bond
corresponded to interaction involving hydrogen of amine linkage
connecting m-nitro phenyl amine to 1,3,5-triazine core (donor) and
oxygen of Gua37 on B-chain as acceptor with distance of 2.03 ꢀ
(H…O), whereas, the other two hydrogen bonds resulted from the
interaction of amine linkage, connecting 1,3,5-triazine to 1,3-thia-
zine, with oxygen of Gua37 (1.79 ꢀ) (H….O) on B-chain and hydro-
gen of Ade7 (A-chain) with sulfur atom of 1,3-thiazine (2.12 ꢀ)
(H….S). These two hydrogen bond interactions are close enough to
support di-substituted 1,3-thiazine fragment to inner grove of eubac-
terial ribosomal site. Furthermore, stability of this fragment into an
inner groove was also increased by the formation of one p–p inter-
action because of phenyl of o-nitro phenyl on 1,3-thiazine and two
p–+ interaction between nitro of above mentioned phenyl ring with
the purine ring of Gua37 (B-chain). Furthermore, increase in H-bonds
to five was observed in the case of compound 8c, having hydroxy
in place of p-nitro and all fragment kept rigid. Of which, two hydro-
gen bonds were formed between hydrogen of Cyt6 of A-chain and
oxygen of nitro group present on meta position of phenyl ring on
1,3-thiazine (H….O) with distance of 1.98 ꢀ and 2.49 ꢀ. Another
two intermolecular hydrogen bond resulted from the involvement of
hydrogen of amine linkage connecting m-nitro phenyl amine to
1,3,5-triazine core on either side as donor and nitrogen of purine
ring (Gua37) and oxygen of Ade38 with interatomic distance 2.50
and 2.08 ꢀ, respectively, as acceptor. The fifth hydrogen bond was
formed through the hydrogen of hydroxy group on phenyl at para
position attached to 1,3-thiazine (donor) with oxygen (acceptor) of
Gua40 (Chain B) with distance of 1.91 ꢀ (H….O). Moreover, one
additional p–p and p–+ interaction was also observed, in which,
Gua37 of B-chain was responsible for p–p stacking with m-nitro
phenyl connected to 1,3,5-triazine and nitro group of o-nitro phenyl
on 1,3-thiazine for p–+ interaction with Gua4 of A-chain. Slight
decrease in number of hydrogen bonds to four was achieved on
introduction of Cl atom on second position in place of nitro on phe-
nyl connected to 1,3-thiazine and further substitution of p-nitro in
place of p-hydroxy (8d). Of these, one hydrogen bond resulted
through the interaction of hydrogen of Cyt6 (A-chain) with oxygen
atom of m-nitro group present on phenyl amine connected to 1,3,5-
triazine (H….O) with a distance of 2.38 ꢀ. Additional hydrogen
bond formed through the involvement of hydrogen of amine of
8
Chem Biol Drug Des 2012

Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of 1,3,5-triazines
Ade39 (donor) with oxygen atom of other m-nitro present on phenyl
The docked orientation of ligands as well as paromomycin in eubac-
terial ribosomal decoding A site (E. coli 16S rRNA A site) receptor
domain are presented in Figure 2.
amine connected to 1,3,5-triazine (H….O) with a distance 2.33 ꢀ.
The other two hydrogen bonds formed through the interaction
between nitrogen of Gua40 and oxygen of Gua37 (acceptor) of B-
chain with hydrogen of amine linkages connecting m-nitro phenyl
amine and 1,3-thiazine to 1,3,5-triazine (donor) with interatomic dis-
tance of 2.47 and 1.97 ꢀ, respectively. The compound 8d did not
show any p–p interaction, but had only one p–+ interaction
between Ade39 and nitro at meta to phenyl amine connected to
1,3,5-triazine ring. However, replacement of 2-chloro with 4-chloro
on the phenyl ring of 1,3-thiazine fragment (compound 8e), caused
drastic reduction in number of hydrogen bond to only one. And this
single H-bond formed through hydrogen of amine linkage connecting
1,3-thiazine fragment of 1,3,5-triazine ring (donor) with nitrogen of
Gua37 as acceptor atom with interatomic distance of 2.06 ꢀ
(H….O). Compound 8e showed no p–p interaction, but for two
effective p–+ interactions resulting from involvement of nitro atom
of m-nitro phenyl amine attached to 1,3,5-triazine with purine ring
of Gua4 (A-chain). Presence of nitro group on para to phenyl ring
attached to either side of 1,3-thiazine nucleus and keeping all frag-
ment rigid led to generation of compound 8f, which showed
increase in number of hydrogen bonds to three, of which two
hydrogen bonds resulted from the interaction between oxygen of
nitro group with (acceptor) hydrogen of Ade7 and Cyt8 (donor) with
interatomic distance of 2.26 and 2.42 ꢀ, respectively. The hydrogen
of amine linkage connecting 1,3-thiazine to 1,3,5-triazine is respon-
sible for third hydrogen bond formation with nitrogen of Gua37 as
acceptor with distance of 2.08 ꢀ. It was also shown to have one
p–+ interaction with nitro of p-nitro phenyl on second position of
1,3-thiazine with purine ring of Gua40, but not reported to have
any p–p interaction.
Scoring
Ligand scoring is a method to rapidly estimate the binding affinity
of a ligand, based on a candidate ligand pose geometry docked into
a target receptor structure. To define binding affinity of ligands with
receptor in a better way, hybrid title analogous and reference pa-
romomycin (for comparison) were rigorously analyzed through scor-
ing function, such as -PLP1, -PLP2, Jain, -PMF, Lig_Internal_Energy,
binding energy, and ultimately dock score.
Piecewise Linear Potential is a fast, simple, docking function that
has been shown to explain well the protein ligand binding affini-
ties. PLP scores are measured in arbitrary units, with negative PLP
scores reported to make them suitable for subsequent use in con-
sensus score calculations. Higher PLP scores indicate stronger
receptor-ligand binding. Two versions of the PLP function are avail-
able: PLP1 (15) and PLP2 (16). In the PLP1 function, each non-hydro-
gen ligand or non-hydrogen receptor atom is assigned a PLP atom
type. Hydrogen atoms are excluded from consideration. In PLP2
function, PLP atom typing remains the same as in PLP1. In addition,
an atomic radius is assigned to each atom except for hydrogen. It
is worthwhile to mention that majority of the title compounds
showed comparable PLP scores to the reference ligands, however
compounds 8a (100.7), 8d (100.39), and 8f (101.1) exhibited PLP1
score very close to the reference (105.6).
The PMF scoring functions were developed on the basis of statistical
analysis of the 3D structures of protein-ligand complexes. These were
found to correlate well with protein-ligand binding free energies. The
scores are calculated by summing pairwise interaction terms over all
interatomic pairs of the receptor-ligand complex (17). In a comparison
test, it was found that all title compounds showed excellent to signif-
icant PMF scores with reference to standard ligand.
It can be inferred from the orientation of H-bonds that, hydrogen
present on amine linkages, connecting 1,3-thiazine fragment and m-
nitro phenyl to 1,3,5-triazine and nucleotides present on B-chain
backbone of eubacterial ribosomal site namely Ade38, Gua37,
Ade39, and Gua40; and only Cyt6, Cyt8, and Ade7 present on A-
chain were highly conserved and enhanced the binding affinity and
stability of compounds into the inner groove of active site. Nucleo-
tides present on B-chain act as acceptor atoms to form H-bond and
A-chain nucleotides behave as donor atoms. In addition, presence
of p–p and p–+ interactions in some instances provides an addi-
tional factor for stability of ligands into the receptor site for effec-
tive ligand-receptor interaction. Therefore, it is worthwhile to
mention here that 1,3-thiazine fragment is well tolerated on the
basis of H-bond formation as pendant substituent on 1,3,5-triazine,
owing to the intriguing interaction of hydrogen of amine linkage
connecting it with nucleotides and involvement of substituted
groups on phenyl to form hydrogen bond, p–p and p–+ interac-
tions. To compare the binding mode with known inhibitor, a refer-
ence ligand paromomycin (a bacterial translation inhibitor) was also
docked at same site. This reference ligand formed ten hydrogen
bonds and the binding mode was the same as that of the title com-
pound (A-chain: Cyt6 and Ade7; B-chain: Ade38, Ade39, Gua37,
Gua40), except additional H-bond formed with Uri5 and Gua4 of A-
chain and Uri41 of B-chain, no p–p and p–+ interactions were
observed. It further confirmed a similar mechanism of action of title
analogues as that of reference, as a bacterial translation inhibitor.
The internal non-bonded ligand energy is calculated for each new
conformation that is generated. Bad conformations with high inter-
nal ligand energies (typically resulting from internal close contacts)
are discarded. The internal ligand energy consists of a van der Wa-
als (vdW) term and an optional electrostatic term. The non-bonded
vdW energy is computed using a standard 9-6 (unsoftened) poten-
tial using forcefield parameters consistent with the forcefield
employed. Efficient ligand is classified with high negative ligand
internal energy. All title hybrid analogues demonstrated significantly
high negative values as that compared to that of the reference and
compound 8d ()11.412) and 8e ()10.068) showed higher stabiliza-
tion energy than the reference.
Binding energy of the title molecules as well as of paromomycin
(reference) was analyzed from their corresponding docked conforma-
tion in receptor. Results illustrated that binding energy of com-
pounds 8a ()151.89), 8b ()97.86), and 8c ()119.85) was higher
than that of reference, whereas in case of compounds 8d ()83.42),
8e ()79.37), and 8f ()81.36), it was lower than that of reference
()90.72).
Chem Biol Drug Des 2012
9

Singh et al.
Figure 2: Binding mode
of hybrid 1,3-thiazine-1,3,5-
triazine derivatives 8(a–f)
and paromomycin (standard)
inside the eubacterial RNA
active site. (Green: H-bonds,
Black: +–p and p–p stack-
ing).
Candidate ligand poses were evaluated and prioritized according to
the DockScore function. All the scoring data for the title hybrid
compounds along with paromomycin were presented in Table 3.
bound native ligand in the experimental crystal structure. Accord-
ing to Wang et al., (14) the successful scoring function is the
one in which the RMSD (root mean square deviation) of the
best docked conformation is £2.0 ꢀ from the experimental one.
As a consequence, the validation of function achieved in Ligand-
Fit ⁄ Discovery Studio 2.5 was prepared by docking of the native
ligand into its binding site. Then the docked result was com-
pared with the crystal structure of the bound ligand–protein
complex. In the present study, paromomycin ligand was docked
into its 16S-rRNA A-site receptor (PDB code: 1j7t), as reference
ligand for the comparative study of binding action, and the new
synthesized compounds, i.e., hybrid 1,3-thiazine-1,3,5-triazine
derivatives as ligands. It was revealed that the title ligands
bound in similar fashion with paromomycin with the RMSD of
1.25 ꢀ. It has been confirmed that the accuracy and perfor-
mance of the LigandFit ⁄ Discovery Studio 2.5 was highly satisfac-
tory.
It was clear from Table 3 that the reference ligand paromomycin
exhibited dock score of 88.35 and none of compounds presented
the same or greater score than reference. The entire hybrid com-
pounds 8(a–f) showed significant dock score ranging from 76.94 to
87.41, which suggested that all molecules have similar mode of
binding and significant binding affinity towards RNA receptor
domain as that of the reference ligand.
Validation of the accuracy and performance of
LigandFit
A scoring function is deemed to be successful, if the best-
docked conformation of a ligand bears a resemblance with the
10
Chem Biol Drug Des 2012

Design, Synthesis, Antibacterial Activity, and Molecular Docking Studies of 1,3,5-triazines
Table 3: Scoring of ligands in ligand-receptor docked complex
Name
-PLP1
-PLP2
-PMF
Lig-Internal_Energy
Binding energy (kcal ⁄ mol)
Dock Score
8a
8b
100.7
82.77
95.03
100.39
94.14
101.1
105.6
63.58
46.05
74.01
52.31
64.64
56.62
104.06
101.93
69.15
67.81
91.25
83.79
90.39
88.8
)8.531
)8.718
)8.459
)11.412
)10.068
)9.345
)6.422
)151.89
)97.86
)119.85
)83.42
)79.37
)81.36
)90.72
80.70
87.41
83.45
76.94
84.01
83.03
88.35
8c
8d
8e
8f
Reference ligand
(Paromomycin)
available at: http://www.cdc.gov/abcs/reports-findings/survre-
ports/spneu08.pdf.
Conclusion
2. ECDC ⁄ EMEA Joint Technical Report. The bacterial challenge:
time to react. (2009) EMEA ⁄ 576176 ⁄ 2009.
The present study provided a novel insight into the potential che-
motherapeutic value of hybrid molecules comprising 1,3-thiazine
and 1,3,5-triazine as core bioactive heterocyclic scaffolds on the
basis of molecular docking analysis and in vitro antibacterial activ-
ity. Furthermore, docking results suggested that the binding mode
of the synthetic molecules was similar to the reference ligand and
thus the synthetic molecules were supposed to work as bacterial
translation inhibitors.
3. Roberts R.R., Hota B., Ahmad I., Scott R.D. 2nd, Foster S.D., Ab-
basi F., Schabowski S., Kampe L.M., Ciavarella G.G., Supino M.,
Naples J., Cordell R., Levy S.B., Weinstein R.A. (2009) Hospital
and societal costs of antimicrobial-resistant infections in a Chi-
cago teaching hospital: implications for antibiotic stewardship.
Clin Infect Dis;49:1175–1184.
4. Singh U.P., Singh R.K., Bhat H.R., Subhashchandra Y.P., Kumar V.,
Kumawat M.K., Gahtori P. (2011) Synthesis and antibacterial
evaluation of series of novel tri-substituted-s-triazine derivatives.
Med Chem Res;20:1603–1610.
5. Singh U.P., Bhat H.R., Gahtori P. (2012) Antifungal activity, SAR
and physicochemical correlation of some clubbed thiazole-1,3,5-
triazine derivatives. J Mycol Med;22:134–141.
6. Gahtori P., Ghosh S.K., Singh B., Singh U.P., Bhat H.R., Uppal A.
(2011) Synthesis, SAR and antibacterial activity of hybrid chloro,
dichloro-phenylthiazolyl-s-triazines. Saudi Pharm J;20:35–43.
7. Hermann T. (2005) Drugs targeting the ribosome. Curr Opin
Struct Biol;15:355–366.
8. Zhou Y., Sun Z., Froelich J.M., Hermann T., Wall D. (2006) Struc-
ture-activity relationships of novel antibacterial translation inhib-
itors: 3,5-diamino-piperidinyl triazines. Bioorg Med Chem
Lett;16:5451–5456.
It is also proved that, expansion in pendant substituent in earlier
developed lead fragment (phenyl thiazole) changing to di-phenyl
1,3-thiazine was well tolerated as confirmed by antibacterial activity
and docking runs.
A close inspection of in vitro antibacterial activity supported by dock-
ing study clearly suggested that the hybrid molecules 8c, owing to
the higher number of H-bonds, p–+ interactions, significant dock scor-
ing can act as a potential bacterial translation inhibitor in an ongoing
chemotherapeutic drug discovery regime. However, optimization
should be carried out to transform these hybrid molecules to increase
its efficacy and potency. Furthermore, the structure-based optimiza-
tion studies are in progress and will subsequently be reported.
9. Zhou Y., Gregor V.E., Sun Z., Ayida B.K., Winters G.C., Murphy
D., Simonsen K.B., Vourloumis D., Fish S., Froelich J.M., Wall
D., Hermann T. (2005) Structure-guided discovery of novel ami-
noglycoside mimetics as antibacterial translation inhibitors. Anti-
microb Agents Chemother;49:4942–4949.
10. Zhou C., Min J., Liu Z., Young A., Deshazer H., Gao T., Chang
Y.T., Kallenbach N.R. (2008) Synthesis and biological evaluation
of novel 1,3,5-triazine derivatives as antimicrobial agents. Bioorg
Med Chem Lett;18:1308–1311.
Acknowledgments
Authors are grateful to SAIF, Central Drug Research Institute, Luc-
know, India for providing spectral data of compounds synthesized
and SHIATS for providing basic facilities to carry out the project.
One of the author, UPS is also pleased to acknowledge Prof. Ray C.
F. Jones, Professor of Organic and Biological Chemistry, Loughbor-
ough University, UK for his critical discussion and suggestions on
the chemistry of 1,3,5-triazines.
11. Gahtori P., Ghosh S.K. (2012) Design, synthesis and SAR explora-
tion of hybrid 4-chlorophenylthiazolyl-s-triazine as potential anti-
microbial agents. J Enzyme Inhib Med Chem;27:281–293.
12. Chen H., Lyne P.D., Giordanetto F., Lovell T., Li J. (2006) On eval-
uating molecular-docking methods for pose prediction and
enrichment factors. J Chem Inf Model;46:401–415.
Conflict of Interest
None.
13. Gallivan J.P., Dougherty D.A. (1999) Cation-pi interactions in
structural biology. Proc Natl Acad Sci U S A;96:9459–9464.
14. Wang R., Lu Y., Wang S.J. (2003) Comparative evaluation of 11
scoring functions for molecular docking. J Med Chem;46:2287–
2303.
References
1. Active Bacterial Core Surveillance (ABCs). Report emerging
infections program network Streptococcus pneumoniae, (2008)
Chem Biol Drug Des 2012
11

Singh et al.
15. Gehlhaar D.K., Verkhivker G.M., Rejto P.A., Sherman C.J., Fogel
D.B., Fogel L.J., Freer S.T. (1995) Molecular recognition of the
inhibitor AG-1343 by HIV-1 protease: conformationally flexible
docking by evolutionary programming. Chem Biol;2:317–324.
16. Gehlhaar D.K., Bouzida D., Rejto P.A., Parrill L., Rami Reddy M.
(1999) Series title: ACS symposium series, 719. Washington,
DC.: American Chemical Society; pp. 292–311.
20. Venkatachalam C.M., Jiang X., Oldfield T., Waldman M. (2003)
LigandFit: a novel method for the shape-directed rapid docking
of ligands to protein active sites. J Mol Graph Model;21:289–
307.
21. Mayo S.L., Olafson B.D., Goddard W.A. III (1990) DREIDING: a
generic force field for molecular simulations.
Chem;94:8897–8909.
J
Phys
17. Muegge I., Martin Y.C. (1999) A general and fast scoring func-
22. National Committee for Clinical Laboratory Standards (1982)
Standard Methods for Dilution Antimicrobial Susceptibility Test
for Bacteria Which Grows Aerobically. Villanova: NCCLS;p. 242.
23. Lalitha M.K. (2004) Manual on Antimicrobial Susceptibility Test-
ing (Under the auspices of Indian Association of Medical Micro-
biologists). Vellore, India: C.M.C.
tion for protein-ligand interactions:
approach. J Med Chem;42:791–804.
a simplified potential
18. Brooks B.R., Bruccoleri R.E., Olafson B.D., States D.J., Swamina-
than S., Karplus M. (1983) CHARMM: a program for macromo-
lecular energy, minimization, and dynamics calculations.
Comput Chem;4:187–217.
J
Note
19. Momany F.A., Rone R. (1992) Validation of the general purpose
QUANTA ꢁ3.2 ⁄ CHARMmꢁ force field. J Comput Chem;13:888–
990.
ahttp://www.rcsb.org/pdb/explore/explore.do?structureId=1J7T
12
Chem Biol Drug Des 2012