Ceric ammonium nitrate (CAN) 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

Article abstract of DOI:10.1016/j.crci.2012.11.019

A novel, simple, inexpensive and efficient ceric ammonium nitrate (CAN) catalysed one-pot, multi-component synthesis of 1,3-thiazine in PEG-400 has been developed. These derivatives were identified as potent inhibitor of Gram-negative bacteria and acted via inhibition of 4-diphosphocytidyl-2-Cmethyl- D-erythritol (IspE) kinase, a novel target.

Full text of DOI:10.1016/j.crci.2012.11.019

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CRAS2C-3664; No. of Pages 7
C. R. Chimie xxx (2013) xxx–xxx
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Full paper/Memoire
Ceric ammonium nitrate (CAN) 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
Udaya Pratap Singh a,b, ,1, Hans Raj Bhat a, Ramendra Kumar Singh
*
b,**
a Department of Pharmaceutical Sciences, Sam Higginbottom Institute of Agriculture, Technology & Sciences, Formerly Allahabad Agricultural Institute, Deemed
University, 211007 Allahabad, India
b Nucleic Acids Research Laboratory, Department of Chemistry, University of Allahabad, 211002 Allahabad, India
A R T I C L E I N F O
A B S T R A C T
Article history:
A novel, simple, inexpensive and efficient ceric ammonium nitrate (CAN) catalysed one-
pot, multi-component synthesis of 1,3-thiazine in PEG-400 has been developed. These
derivatives were identified as potent inhibitor of Gram-negative bacteria and acted via
inhibition of 4-diphosphocytidyl-2-Cmethyl-D-erythritol (IspE) kinase, a novel target.
ß 2012 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
Received 31 August 2012
Accepted after revision 29 November 2012
Available online xxx
Keywords:
CAN
PEG-400
1,3-thiazine
Antibacterial
1. Introduction
synthesis, for instance, lower working costs, shorter
reaction times, high degrees of atom economy, the
The synthesis of highly functionalized nitrogen contain-
ing heterocycles in the context of their wide array of
biological activity has always attracted medicinal chemists
for a long time. The synthesis of these scaffolds using
multi-component coupling reactions (MCRs) represents a
highly valuable synthetic tool for the construction of novel
possibility for combinatorial surveying of structural
variations, and environmental friendliness.
2-amino-1,3-thiazine, a versatile pharmocophore, has
been the subject of great interest due to its wide range of
biological activities viz., antimicrobial agents [1], as
cannabinoid receptor agonists [2], in the phagocytic activity
of human neutrophils [3], antioxidant properties [4],
analgesic and anti-inflammatory activities [5], antifilarial
properties [6], metalloprotease inhibition activity [7],
antihypertensive activity [8], cholecystokinin antagonists
[9], and antimycobacterial [10]. Hence, considerable efforts
have been carried out for the synthesis of 1,3-thiazines
and complex molecular structures with
a minimum
number of synthetic steps. These types of reactions have
several possible advantages over the traditional linear
* Corresponding author. Department of Pharmaceutical Sciences, Sam
Higginbottom Institute of Agriculture, Technology & Sciences, Formerly
Allahabad Agricultural Institute, Deemed University, 211007 Allahabad,
India.
through condensation of thiourea with
a,b-unsaturated
carboxylic acids in phosphoric acid or sulfuric acid, addition
reaction of ethylenethiourea, thiourea, ethyl thioglycolate,
** Corresponding author. Nucleic Acids Research Laboratory, Depart-
ment of Chemistry, University of Allahabad, 211002 Allahabad, India.
E-mail addresses: udaysingh98@gmail.com, haiud_98@yahoo.com
(U.P. Singh), singhramk@rediffmail.com (R.K. Singh).
or O-ethyl allylthiocarbamate to
b-haloacids bearing
compounds, microwave irradiated reaction between form-
aldehyde, amine and phenol, through multicomponent
reaction(MCR)ofinsitu-generated1-azadieneswithcarbon
1
Present address: Archimedes DoRa5 Visiting Fellow, Division of Bio-
organic Chemistry, Institute of Chemistry, University of Tartu, Estonia.
1631-0748/$ – see front matter ß 2012 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.crci.2012.11.019
Please cite this article in press as: Singh UP, et al. Ceric ammonium nitrate (CAN) 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. Chimie (2013), http://dx.doi.org/10.1016/j.crci.2012.11.019

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2
U.P. Singh et al. / C. R. Chimie xxx (2013) xxx–xxx
Fig. 1. Synthesis of 1,3-thiazine.
disulfide and via reacting cyanoacetamide with isothio-
cyanates and cycloketones in a one-pot manner to give rise
to spiro-substituted 1,3-thiazine analogues. However, due
to the long reaction times, harsh reaction conditions,
toxicity and difficulty in product separation of previously
reported methods, synthesis of 1,3-thiazine skeleton always
remains a matter of investigation for the organic as well as
medicinal chemists and is reported extensively in the
literature [5,10–17]. Therefore, development of a simple,
nature friendly, efficient and cost effective benign method-
ology for such organic reactions is needed.
Cerium (IV) ammonium nitrate (CAN), among the
electron transfer oxidizing agents, is considered as an
important catalyst to carry out a variety of organic
transformations, for instance, oxidation [18], 1,4-addition
[19], protection [20], nitration [21], 1,3-dipolar cycloaddi-
tion [22], thiocyanation [23], esterification [24], and the
Hantzsch reaction [25] via generation of radical inter-
mediates. In addition to that, it has been associated with
many advantages such as excellent solubility in water,
inexpensiveness, eco-friendly nature, uncomplicated han-
dling, high reactivity, fast conversions and convenient
work up procedures.
The use of organic solvents as media to provide a
homogeneous phase that allows molecular interactions
effectively and brings the reaction to completion in
conventional synthetic procedures is harmful and does
not compel the reaction to the total completion. The
inexpensiveness, ease of workup, thermally stability, non-
toxicity, and recyclability in various organic transforma-
tions has made polyethylene glycol (PEG-400) a green
solvent to carry out the variety of reactions. Prompted by
the green aspects of above two chemicals, we reveal herein
the CAN catalysed one-pot MCR green synthesis of 1,3-
thiazine derivatives using acetophenone, aromatic alde-
hydes and thiourea in PEG-400 as reaction media (Fig. 1).
2. Results and discussions
The first part of the study was aimed at optimizing the
reaction conditions. To do this, in the first instance, we
explain the effect of various concentration of CAN and
temperature on reaction as well as the product yield. In
this regard, as depicted from Tables 1 and 2, reactions were
carried out with and without using CAN (blank) at room
and at higher temperatures. We observed that reaction of
equimolar concentration of aldehyde, acetophenone and
thiourea using polyethylene glycol (PEG-400) as a solvent
medium, with the addition of CAN at 45 8C improved yields
as well decreased in total reaction time to afford required
1,3-thiazine in sequential one-pot reaction (Table 1). As
the matter of fact, we found that rise in temperature
directs the reaction to the completion in shorter periods of
time; nevertheless, it also causes reduction in the product
yield, which may be due to the decomposition at higher
temperatures. Whereas, under the similar set of condi-
tions, addition of 10% CAN lead to 66% higher product yield
and saves almost 14 h of reaction time, for instance, 95% in
7 h. Unfortunately, further inflating concentration to 15%,
led to the downfall in reaction time and yield as well.
Through these experiments, a temperature of 45 8C along
with 10% CAN were the optimal condition to complete the
reaction in efficient way.
Based on the above results and following the reaction
optimization, we decided to explore the recyclability of
solvent PEG-400. It was surprised to see that, it can be
efficiently recycled for three consecutive runs. To our
knowledge, at the third run, PEG-400 was operating well to
achieve 74% product yield (Table 3). It was predicted that
the solvent recovery was efficient with a gradual decline in
yield of product. To exemplify the versatility of this MCR
for synthesis of different 1,3-thiazines, a variety of electron
donating or withdrawing groups were used for a library
construction. As seen from Table 4, with electronic and
Table 1
Catalytic activity evaluation for reactiona.
Table 2
Effect of the temperature on reactiona.
Entry
CAN (%)
Time (h)
Yieldb
Entry
Temp
Time (h)
Yieldb
1
2
3
4
5
0
2
28
12
8
29
52
81
95
89
1
2
3
4
Room temperature
8
7
6
5
93
95
85
78
5
45
60
80
10
15
7
6
a
a
Reaction conditions: aldehyde (1 mmol), acetophenone (1 mmol),
Reaction conditions: aldehyde (1 mmol), acetophenone (1 mmol),
thiourea (1 mmol); solvent PEG-400.
thiourea (1 mmol); solvent PEG-400; 45 8C.
b
b
Isolated and unoptimised yields.
Isolated and unoptimised yields.
Please cite this article in press as: Singh UP, et al. Ceric ammonium nitrate (CAN) 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. Chimie (2013), http://dx.doi.org/10.1016/j.crci.2012.11.019

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3
Table 3
Table 4
Recycling yields.
Synthesis of distinguished 1,3-thiazinesa.
No of cyclesa
Fresh
Run 1
Run 2
Run 3
Entry
R1
R2
Time
Yieldb (%)
required (h)
Yieldb (%)
Time (h)
95
7
88
7
80
7
74
7
A
B
C
D
E
F
H
2-NO2
2-NO2
2-NO2
2-Cl
7
9
6
7
8
9
91
87
93
88
90
89
4-NO2
4-OH
4-NO2
4-NO2
4-NO2
a
Reaction conditions: aldehyde (1 mmol), acetophenone (1 mmol),
thiourea (1 mmol); solvent PEG-400; 45 8C.
b
Isolated and unoptimised yields.
4-Cl
4-NO2
a
Reaction conditions: aldehyde (1 mmol), acetophenone (1 mmol),
steric variation on the aromatic ring, the corresponding
products were obtained in good yields (87%–93%). An
electron withdrawing nitro group in substrates B and D
reduces product yield with no variation in reaction time,
whereas, an electron releasing group (-OH) as in case of C,
acquiesced with shorter reaction time and high yield as
93% in 6 h. It is interesting to accept that the isomeric
replacement of chloro group from position 2 to 4 acts in
accordance with improved yield and reaction time as well.
The possible mechanism for the formation of 1,3-
thiazine has been depicted in Fig. 2. The reaction was
supposed to be started with reduction of Ce (IV) ! Ce (III)
with the oxidation of methyl of an acetophenone (i)
resulting methylene radical (ii). Thus, resulting highly
reactive intermediate (ii) was readily conjugated with an
aldehyde (iii) to give rise to chalcone intermediate (iv). On
the other hand, thiourea tautomerise (v) easily to the
corresponding thiol tautomer, whereas the lone pair
thiourea (1 mmol); solvent PEG-400; 45 8C.
b
Isolated and unoptimised yields.
again will tautomerises and underwent a intermolecular
cyclocondensation reaction to furnish a 1,3-thiazine ring
system.
FTIR and NMR spectroscopy enabled the elucidation of
structure of final compounds. Moreover, CHN analysis was
found in agreement with proposed structures. The bands at
3426 and 3362 cmꢀ1 observed in the spectrum are
attributed to hydrogen-bonded N–H stretching of primary
amines. A broad absorption band nearer at 3000 cmꢀ1 is
due to the C–H stretching. For 1H-NMR, the chemical shift
values are ranging from 3.21 to 3.52 ppm for the 1,3-
thiazine skeleton. The averaged values of the 1H chemical
shift of later ones are 3.39, 5.32 and 5.75 for 2-, 6-, and 5-H
respectively. It is noteworthy to mention here that the
different substitution pattern brings a change in 1H
chemical shift values of aromatic rings, among them, the
most deshielded protons appeared in case of the nitro
group containing compounds.
present on it, readily attacks on
b-carbon corresponds to
the carbonyl, resulting in shifting of the double bond. As a
result, carbonyl undergoes tautomerisation to yield the
hydroxyl. Under influence of PEG-400, this Michael adduct
Fig. 2. Proposed mechanism for synthesis of 1,3,-thiazine.
Please cite this article in press as: Singh UP, et al. Ceric ammonium nitrate (CAN) 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. Chimie (2013), http://dx.doi.org/10.1016/j.crci.2012.11.019

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U.P. Singh et al. / C. R. Chimie xxx (2013) xxx–xxx
Table 5
Antibacterial activity of 1,3-thiazine compounds.
Compound
Minimum Inhibitory Concentration
(MIC;
g mLꢀ1
m
)
P. vulgaris
E. coli
P. aeruginosa
A
B
C
D
E
F
12.5
25
12.5
12.5
25
50
6.25
50
25
3.125
6.25
12.5
3.125
1.56
3.12
6.25
3.12
3.125
12.5
12.5
6.25
Cefixime
The growing incidence of multidrug resistant Gram-
negative bacteria has seriously jeopardised patients in
healthcare settings and are especially prevalent and
problematic in intensive care units. This necessitates the
discovery of newer antimicrobial agents which target the
novel pathway for their action [26]. Recently, compounds
havingthe coreskeleton of 1,3-thiazinehave beenidentified
as inhibitors of 4-diphosphocytidyl-2-Cmethyl-D-erythritol
(IspE) kinase of Gram-negative bacteria [27]. This enzyme
catalyses the ATP-dependent conversion of 4-diphospho-
cytidyl-2C-methyl-d-erythritol (CDPME) to 4-diphospho-
cytidyl-2C-methyl-d-erythritol 2-phosphate (CDP-ME2P)
with the release of ADP, a part of non-mevalonate pathway
of isoprenoid precursor biosynthesis [28]. This pathway has
been well utilised by the Gram-negative bacteria and the
agents which cause disruptions of this enzyme are deemed
to be lethal to the microbres. The absence of this pathway in
humans renders the associated enzymes unique for the
development of novel antimicrobials and is less likely to
cause serious side-effects in the patients.
To assess the potential medicinal utility of these
analogues, the synthesized 1,3-thiazine derivatives were
rigorously screened through the parameters of Lipinski’s
Rule of 5 (Ro5) for its drug likeness prediction [29].
Surprisingly, it was revealed that none of the 1,3-thiazine
derivatives has violated the above rule (Table 6). Encour-
aged by the above findings, a panel of Gram-negative
bacteria was chosen to carry out the antibacterial
screening studies viz., Proteus vulgaris, Escherichia coli,
Pseudomonas aeruginosa. It was revealed from the Table 5,
the entire analogues showed potent antibacterial activity
against selected microorganisms. Out of them compound
D, showed the most potent activity against E. coli and
P. aeruginosa, while equipotent against P. vulgaris in
comparison to standard. Compound E, revealed the
equipotent antibacterial activity against E. coli in compari-
son to cefixime and potent against rest of the strain. It is
Fig. 3. Orientation of compound D in the active site of IspE kinase.
considered as next influential molecule. The least antibac-
terial was observed in the case of compound C. The rest of
the molecules (A, B and F) presented significant activity
against tested bacteria in a comparison test with reference
to standard. On close inspection, a strong correlation
(r2 = 0.82, 0.76 and 0.42 against E. coli, P. vulgaris and
P. aeruginosa respectively) was revealed in between
miLogP and MIC of the compounds described herein.
It was observed that ClogP is the main influencing
physico-chemical property for the generation of antibac-
terial activity which favourably enhances the antimicrobi-
al activity of the compounds e.g., Compounds D and E
found as the pronounced antibacterial agent along with the
high LogP values. While compound C having the lowest
LogP value showed least inhibitory activity. The alteration
of steric effects on the phenyl ring of molecules which in
turn alter the ease of penetration across the bacterial cell
wall owing to the presence of electron withdrawing
groups, might be the possible cause for difference in
activity.
As further to enlarge the biological spectrum of 1,3-
thiazine and novel insight about the mechanism,
a
molecular docking study of the most active 1,3-thiazine
(D) has been carried out on the protein model of IspE
kinase (2V34.pdb). The binding mode of compound D
showed that it was deeply engulfed and stabilised into the
active site of IspE kinase by making five hydrogen bonds
and binding energy of ꢀ56.22, Fig. 3. The four hydrogen
Table 6
Drug likeness of 1,3-thiazine in the Eye of Lipinski’s Rule of 5.
Compounds
miLogP
TPSA
natoms
MW
nON
nOHNH
nviolations
nrotb
Volume
A
B
C
D
E
F
3.356
3.315
2.877
4.034
4.082
3.363
84.211
130.035
104.439
84.211
22
25
23
23
23
25
311.366
356.363
327.365
345.811
345.811
356.363
5
8
6
5
5
8
2
2
3
2
2
2
0
0
0
0
0
0
3
4
3
3
3
4
264.866
288.201
272.884
278.402
278.402
288.201
84.211
130.035
Please cite this article in press as: Singh UP, et al. Ceric ammonium nitrate (CAN) 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. Chimie (2013), http://dx.doi.org/10.1016/j.crci.2012.11.019

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5
Table 7
3.1.1.1. 6-(2-Nitrophenyl)-4-phenyl-6H-1,3-thiazin-2-
amine (A). Yellow crystals; mp: 162–164 8C; Rf: 0.45;
FT-IR (
max; cmꢀ1 KBr): 3356 (NH), 1687 (C-N), 1656–
1645 (aromatic C = N), 1596 (C = C), 1520, 1355 (Ar-NO2),
877, 754; 1H-NMR (400 MHz, CDCl3-d6, TMS)
ppm:
Docking interaction of ligand (D) in complex with IspE kinase.
˚
Hydrogen Bond (Distance in A)
n
Compound
D
A:GLY90:HN – D.mol:O16 (2.46)
A:ALA91:HN – D.mol:O16 (2.46)
A:GLY92:HN – D.mol:O16 (2.05)
A:GLY95:HN – D.mol:O15 (2.18)
D.mol:H26 – A:ILE127:O (2.03)
d
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 Ar-H), 5.52 (bs, 1H); 13C NMR
(100 MHz, CDCl3)
d ppm: 26.8 (CH), 112.7 (CH), 120.9
(CH), 127.3 (CH), 128.1 (CH), 128.9 (CH), 135.2 (CH),
143.5 (C), 148.0 (C), 162.7 (C); Anal. Calcd. For
bonds were originated from the oxygen of NO2 present on
the phenyl ring with Gly90, Ala91, Gly92, Gly95 (O. . .H)
(Table 7). The remaining H-bond was established in
between hydrogen of by NH2 and oxygen of Ile127
(H. . .O). This finding was suggesting that our molecules
acted as a potent inhibitor of Gram-negative bacteria via
inhibition of IspE kinase.
C16H13N3O2S: C, 61.72; H 4.21; N, 13.50. Found: C,
61.68; H, 4.20; N, 13.54.
3.1.1.2. 6-(2-Nitrophenyl)-4-(4-nitrophenyl)-6H-1,3-thia-
zin-2-amine (B). Dark brown crystals; mp: 168–170 8C; Rf:
0.65; FT-IR (
(C = C), 1658 (aromatic C = N), 1526, 1358 (Ar-NO2), 876,
756; 1H-NMR (400 MHz, CDCl3-d6, TMS)
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 Ar-H), 5.56 (bs, 1H); 13C NMR (100 MHz, CDCl3)
n
max; cmꢀ1 KBr): 3360 (NH), 1683 (C-N), 1597
d
3. Experimental
d
ppm: 27.0 (CH), 112.8 (CH), 121.2 (CH), 127.6 (CH), 129.0
(CH), 135.4 (CH), 143.7 (C), 143.9 (C), 148.3 (C), 162.8 (C);
Anal. Calcd. For C16H12N4O4S: C, 53.93; H 3.39; N, 15.72.
Found: C, 53.95; H, 3.42; N, 15.72.
3.1. Analytical methods
Melting points of synthesized compounds were deter-
mined in open capillary tubes in a Hicon melting point
apparatus and are uncorrected. Thin layer chromatograph-
ic analysis was done to monitor the completion of reaction
as well as for identification and characterization of
compounds. 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 compounds were established on
the basis of spectral (FT-IR, 1H-NMR) and elemental
analysis. FT-IR (in 2.0 cmꢀ1, flat, smooth, abex, KBr) spectra
were recorded on Biored FTs spectrophotometer. 1H-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. Chemical shifts are reported in parts per
3.1.1.3. 4-(2-Amino-6-(2-nitrophenyl)-6H-1,3-thiazin-4-
yl)phenol (C). Brownish crystals; mp: 175–178 8C; Rf:
0.54; FT-IR (
1610 (C = C), 1646–1635 (aromatic C = N), 1519, 1348 (Ar-
NO2), 889, 778; 1H-NMR (400 MHz, CDCl3-d6, TMS)
ppm:
n
max; cmꢀ1 KBr): 3359 (NH), 1680 (C-N),
d
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, Ar-H), 5.45 (bs, 1H); 13C
NMR (100 MHz, CDCl3)
d ppm: 26.4 (CH), 112.2 (CH),
120.7 (CH), 127.0 (CH), 128.4 (CH), 134.7 (CH), 143.1 (C),
143.4 (C), 148.0 (C), 162.1 (C); Anal. Calcd. For
C
16H13N3O3S: C, 58.70; H 4.00; N, 12.84. Found: C,
57.50; H, 4.10; N, 12.80.
3.1.1.4. 6-(2-Chlorophenyl)-4-(4-nitrophenyl)-6H-1,3-thia-
zin-2-amine (D). Pale yellow crystals; mp: 183–184 8C; Rf:
million (ppm,
(doublet),
d
) and signal are described as s (singlet), d
0.65; FT-IR (
(C = C), 1648–1630 (aromatic C = N), 1529, 1348 (Ar-NO2),
890, 772; 1H-NMR (400 MHz, CDCl3-d6, TMS)
ppm: 5.73
n
max; cmꢀ1 KBr): 3360 (NH), 1680 (C-N), 1638
t
(triplet), (quartet) and (multiplet).
q
m
Elemental analysis was carried out on a Vario EL III CHNOS
elemental analyzer.
d
(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 Ar-H), 5.45 (bs, 1H); 13C NMR (100 MHz,
3.1.1. General procedure for synthesis of 1,3-thiazine
derivatives
CDCl3) d ppm: 27.6 (CH), 113.5 (CH), 121.7 (CH), 128.0
(CH), 129.6 (CH), 135.9 (CH), 144.3 (C), 144.6 (C), 148.9 (C),
163.1 (C); Anal. Calcd. For C16H12ClN3O2S: C, 55.57; H 3.50;
N, 12.15. Found: C, 55.60; H, 3.60; N, 12.10.
Substituted acetophenone (1 mmol), aromatic alde-
hydes (1 mmol) and thiourea (1 mmol) in PEG-400 were
mixed. To this, CAN (ceric ammonium nitrate) was
added and reaction mixture was stirred at 45 8C. The
progress and completion of reaction mixture was
monitored by TLC. After completion of reaction the
reaction mixture was cooled in dry ice-acetone bath to
precipitate the PEG-400 and extracted with ether (PEG
being insoluble in ether). Ether layer was decanted, dried
and concentrated under reduced pressure. The recovered
PEG-400 can be reused for consecutive runs. The
structures of all the products were ascertained on the
basis of their spectral (IR, 1H-NMR, 13C NMR) and
elemental analysis.
3.1.1.5. 4-(4-Chlorophenyl)-6-(4-nitrophenyl)-6H-1,3-thia-
zin-2-amine (E). Brown crystals; mp: 188–191 8C; Rf: 0.58;
FT-IR (
(C = C), 1650–1633 (aromatic C = N), 1535, 1350 (Ar-NO2),
880, 782; 1H-NMR (400 MHz, CDCl3-d6, TMS)
ppm: 5.52
n
max; cmꢀ1 KBr): 3362 (NH), 1690 (C-N), 1634
d
(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, Ar-H), 5.56 (bs, 1H); 13C NMR (100 MHz,
CDCl3)
d ppm: 27.2 (CH), 113.0 (CH), 121.5 (CH), 127.9
(CH), 129.2 (CH), 135.6 (CH), 143.8 (C), 144.1 (C), 148.5 (C),
162.9 (C); Anal. Calcd. For C16H12ClN3O2S: C, 55.57; H 3.50;
N, 12.15. Found: C, 55.57; H, 3.52; N, 12.13.
Please cite this article in press as: Singh UP, et al. Ceric ammonium nitrate (CAN) 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. Chimie (2013), http://dx.doi.org/10.1016/j.crci.2012.11.019

G Model
CRAS2C-3664; No. of Pages 7
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U.P. Singh et al. / C. R. Chimie xxx (2013) xxx–xxx
3.1.1.6. 4,6-Bis
(F). Reddish orange crystals; mp: 195–197 8C; Rf: 0.57;
FT-IR (
max; cmꢀ1 KBr): 3359 (NH), 1682 (C-N), 1635
(C = C), 1656-1630 (aromatic C = N), 1540, 1354 (Ar-NO2),
890, 756; 1H-NMR (400 MHz, CDCl3-d6, TMS):
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, Ar-H), 5.52 (bs, 1H); 13C NMR (100 MHz, CDCl3)
ppm: 26.8 (CH), 112.7 (CH), 121.2 (CH), 127.5 (CH), 135.2
(CH), 143.4 (C), 143.7 (C), 146.3 (C), 148.2 (C), 162.6 (C);
Anal. Calcd. For C16H12N4O4S: C, 53.93; H 3.39; N, 15.72.
Found: C, 53.90; H, 3.44; N, 15.70.
(4-nitrophenyl)-6H-1,3-thiazin-2-amine
3.2.2.1. Preparation of receptor. The target protein that
complexed with the ligand was taken, the ligand was
extracted, and the bond order was corrected. The hydrogen
atomswereadded, and their positionswereoptimizedusing
the all-atom CHARMm (version c32b1) forcefield with
Adopted Basis set Newton Raphson (ABNR) minimization
algorithm until the root mean square (r.m.s) gradient for
n
d
d
˚
potential energy was less than 0.05 kcal/mol/A [31,32].
Using the ‘Binding Site’ tool panel available in DS 2.5, the
minimized IspE kinase editing domain was defined as
receptor, binding site was defined as volume occupied by
the ligand in the receptor, and an input site sphere was
˚
3.2. Biological and computational studies
3.2.1. Antibacterial activity
defined over the binding site with a radius of 5 A. The centre
of the sphere was taken to be the centre 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.
3.2.1.1. Minimum Inhibitory Concentration. Entire com-
pounds were screened for their minimum inhibitory
concentration (MIC,
mg/mL) against selected Gram-nega-
tive organisms, viz. P. vulgaris (NCIM-2027), E. coli (NCIM-
2065) P. aeruginosa (NCIM-2036) by the broth dilution
method as recommended by the National Committee for
Clinical Laboratory Standards with minor modifications.
Cefexime was used as the standard antibacterial agent.
Solutions of the test compounds and reference drug were
prepared in dimethyl sulfoxide (DMSO) at concentrations
3.2.2.2. Ligand set-up. Using the built-and-edit module of
DS 2.5, ligand (1,3-thiazine derivative, D) was built, all-
atom CHARMm forcefield parameterization was assigned
and then minimized using the ABNR method. A conforma-
tional 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 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.
of 100, 50, 25, 12.5, 6.25, 3.125 mg/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 8C incubator
and observed for any macroscopic growth (clear or turbid)
the next day.
Into each tube, 0.8 mL of nutrient broth was pipette
(tubes 2–9), tube 1 (negative control) received 1.0 mL of
nutrient broth and tube 10 (positive control) received
0.9 mL of nutrient. Tube 1, the negative control, did not
contain bacteria or antibiotic. The positive control, tube
10 contained bacteria but not antibiotic. The test
3.2.2.3. Docking. LigandFit [33] protocol of DS 2.5 was
used for the docking of ligands with ISPE kinases. The
LigandFit docking algorithm combines a shape comparison
filter with
a Monte Carlo conformational search to
compound were dissolved in DMSO (125
mg/mL),
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
[34]. The receptor protein conformation was kept fixed
during docking, and the docked poses were further
minimized using all-atom CHARMm (version c32b1)
forcefield and smart minimization method (steepest
descent followed by conjugate gradient) until r.m.s
gradient for potential energy was less than 0.05 kcal/
0.1 mL of increasing concentration of the prepared test
compounds which are serially diluted from tube 2 to tube
7 from highest (125
concentration (tubes 2–7 containing 125, 62.5, 31.25,
15.62, 7.81, 3.91, 1.95, 0.97 g/mL). After this process,
mg/mL) to lowest (0.97 mg/mL)
m
each tube was inoculated with 0.1 mL of the bacterial
suspension whose concentration corresponded to 0.5
McFarland scale (9 ꢁ 108 cells/mL) and each tube was
incubated at 37 8C for 24 h at 150 rpm. The incubation
chamber was kept humid. At the end of the incubation
period, MIC values were recorded as the lowest concen-
tration of the substance that gave no visible turbidity, i.e.,
no growth of inoculated bacteria [30].
˚
mol/A. The atoms of ligand and the side chains of the
˚
residues of the receptor within 5 A from the center of the
binding site were kept flexible during minimization.
4. Conclusions
3.2.2. Molecular docking studies
The 3D X-ray crystal structure of ISPE in complex with
cytidine and ligand; 2V34:pdb) was used as starting model
for this study. The protein was prepared, docked, scored
and the molecular dynamics simulation carried out using
standard procedures. All computational analysis was
carried out using Discovery Studio 2.5 (Accelrys Software
Inc., San Diego; http://www.accelrys.com).
In conclusion, for the first time an unprecedented, one-
pot synthesis of 1,3-thiazine derivatives in PEG-400 as
reaction media has been described. This methodology is
very efficient in terms of the work up and reusability of
solvent in different consecutive steps which reduces the
operating time and the amount of waste generated.
Additionally, we have identified these 1,3-thiazines as a
Please cite this article in press as: Singh UP, et al. Ceric ammonium nitrate (CAN) 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. Chimie (2013), http://dx.doi.org/10.1016/j.crci.2012.11.019

G Model
CRAS2C-3664; No. of Pages 7
U.P. Singh et al. / C. R. Chimie xxx (2013) xxx–xxx
7
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kinase, a novel target. This study opens the new avenues
for ongoing drug discovery program against MDR Gram-
negative pathogens.
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Please cite this article in press as: Singh UP, et al. Ceric ammonium nitrate (CAN) 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. Chimie (2013), http://dx.doi.org/10.1016/j.crci.2012.11.019