Structure-guided discovery of 1,3,5-triazine-pyrazole conjugates as antibacterial and antibiofilm agent against pathogens causing human diseases with favorable metabolic fate

Article abstract of DOI:10.1016/j.bmcl.2014.05.103

Impressed by the exceptional antibacterial activity exhibited by our earlier designed molecules originating from 1,3,5-triazine, the present study was undertaken to synthesize a novel series of 1,3,5-triazine-pyrazole conjugates to bring diversity around the core skeleton. The target analogues showed potent antibacterial activity against tested Gram-positive and Gram-negative microorganisms. The toxicity and metabolic site prediction studies were also held out to set an effective lead candidate for the future antibacterial drug discovery initiatives.

Full text of DOI:10.1016/j.bmcl.2014.05.103

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3321–3325
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure-guided discovery of 1,3,5-triazine–pyrazole conjugates
as antibacterial and antibiofilm agent against pathogens causing
human diseases with favorable metabolic fate q
Babita Singh a, Hans Raj Bhat a, Mukesh Kumar Kumawat b, Udaya Pratap Singh a,
⇑
a Drug Design & Discovery Laboratory, Department of Pharmaceutical Sciences, Sam Higginbottom Institute of Agriculture, Technology & Sciences,
Formerly Allahabad Agricultural Institute, Deemed University, Allahabad 211007, India
b Anand College of Pharmacy, Agra 282007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Impressed by the exceptional antibacterial activity exhibited by our earlier designed molecules originat-
ing from 1,3,5-triazine, the present study was undertaken to synthesize a novel series of 1,3,5-triazine–
pyrazole conjugates to bring diversity around the core skeleton. The target analogues showed potent
antibacterial activity against tested Gram-positive and Gram-negative microorganisms. The toxicity
and metabolic site prediction studies were also held out to set an effective lead candidate for the future
antibacterial drug discovery initiatives.
Received 11 March 2014
Revised 12 May 2014
Accepted 30 May 2014
Available online 11 June 2014
Keywords:
1,3,5-Triazine
Pyrazole
Ó 2014 Elsevier Ltd. All rights reserved.
Antibacterial
In silico studies
The overwhelming phenomenon of resistance towards the cur-
rently available antibiotics creates a significant lacuna in the health
care for humankind. Alas, it can be ascribed to the widely misuse of
these miracle drugs throughout the past 70 years and could
weaken the major advances achieved in the treatment of infec-
tions.1 Antimicrobial resistance (AMR) is now deemed as a global
public health crisis which accounts for the death of 25000 people
and related costs of over €1.5 billion in healthcare expenses alone
in European continent.2 Paradoxically, in recent years, as the prob-
lems associated with the emergence of resistance to existing drug’s
increases, there has been gradually decline in the discovery of
newer antimicrobial agents and drugs in the clinical pipeline.3
The prevention of pharmaceutical industries on the investment
of new projects related with discovery of antibiotics due to far
too low incentives than lifestyle medication have made present sit-
uation catastrophic.4 However, in part, technical difficulties associ-
ated with the identification of suitable novel compounds for
development as candidate antibacterial make this situation com-
plex. Regarding the fact, in 2010, Infectious Diseases Society of
America (IDSA) outlines its ‘10 ꢀ 020’ initiative, calling for a world-
wide effort to acquire ten new antibiotics by 2020.5
The innovation of new antibacterial entity with low economic
inputs has always a prolific option to cope with this state of affairs.
Analogues derived from 1,3,5-triazine accommodated well with
the above aim owing to its simple work-up and potent antibacte-
rial activity. In our ongoing task to develop newer antimicrobial
entity from 1,3,5-triazine, earlier, we had developed various hybrid
analogues of 1,3,5-traizine clubbed with thiazole.6 It was found
that substitution of thiazole on pendant location makes the com-
pound potent in comparison to non-substituent. In advancement
of this observation and to optimize the pendant position, until
now we have reported the various hybrid conjugates of 1,3,5-tri-
azine with 1,3-thiazine,7 piperazine,8 and 1,3,4-thiadiazole,9
4-aminoquioline10,11 and thiazolidin-4-one12 Figure 1.
Our previous study has suggested that, the structure–activity
relationship (SAR) could be exemplified on the nature of pendant
substituent (i.e., pharmacophore), covalent bridge used to connect
1,3,5-triazine with pendant substituent, variety of fragment
attached to the other two wings of 1,3,5-triazine and the nature
of the substituent on the wings above. In our recent communica-
tion, inhibition of bacterial translation was disclosed as the mech-
anism of action of these 1,3,5-triazine conjugates.7
q
Part of work has been presented at 8th World Congress of the World Society for
Pediatric Infectious Disease, Cape Town, South Africa on Conference Scholarship,
20–23 Nov 2013.
Present paper deals with the synthesis, antibacterial activity,
antibiofilm, in silico toxicity, and metabolic site prediction of con-
jugates derived from 1,3,5-triazine and pyrazole. Moreover, this
⇑
Corresponding author. Tel.: +91 9506063408.
E-mail address: udaysingh98@gmail.com (U.P. Singh).
http://dx.doi.org/10.1016/j.bmcl.2014.05.103
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

3322
B. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3321–3325
FLANKED
HALOGEN
BASIC AMINE
INITIAL
NH
H
N
ANOTHER
CHLORO
PHARMACOPHORE
N
N
H
N
H
N
-O
N
N
NH2
N
N
N+
O
-O
H
N
H
N
Cl
Cl
N
NOVEL
HN
N
S
N
N
NH
Cl
Cl
Cl
PHARMACOPHORE
N
N
N
NH
N
HN
S
N+
O
Cl
ANOTHER PHENYL
ELECTRON
DONATING
GROUP
BRIDGE
ATOM
NOVEL
PHARMACOPHORES
NOVEL
PHARMACOPHORE
N
N
N
CH3
NH
H
S
H
N
S
N
S
N
N
NO2
HN
O2N
N
NO2
N
N
N
Cl
N
CH3
N
N
N
N
H
N
NH
HN
N
N
N
H
HN
N
EXTRA
CARBON
OH
S
NO2
NO2
POLAR GROUP
ALLYLAMINE
FRAGMENT
ANOTHER PHENYL
NOVEL
NOVEL
PHARMACOPHORE
PHARMACOPHORE
R
NOVEL
NH2
PHARMACOPHORE
Cl
S
NH
N
O
N
R'
N
N
HN
N
Cl
N
NO2
HN
N
N
N
N
H
N
N
S
BRIDGING
HN
N
N
H
NO2
N
N
N
FRAGMENT
H
HN
N
R
S
O2N
Figure 1. Structure-guided design of target 1,3,5-triazine–pyrazole conjugate.
study also provides the fresh insight and advancement about the
SAR of hybrid 1,3,5-triazine conjugates.
substituted aldehydes 5 (a–e). Further, these substituted chalcones
6 (a–e) was then allowed to undergo Cyclo-condensation reaction
in the presence of thiosemicarbazide to afford the next reaction
intermediate in good yields (62–81%), 5-(substituted-phenyl)-3-
phenyl-4,5-dihydro-pyrazole-1-carbothioic acid amide, 7 (a–e),
Step 2. Finally, the target hybrid conjugates of 1,3,5-triazine–pyra-
zole 8 (a–o) was obtained through clubbing of mono chloro
1,3,5-triazine-2,4-diamine 3 (a–c) and 4,5-dihydro-pyrazole-1-
carbothioic acid amide 7 (a–e) fragments in the presence of
K2CO3 as activating base under vigorous condition. These ana-
logues were synthesized in excellent to good yields (62–84%).
The synthesis of title conjugates 8 (a–o) were accomplished via
multi-step reaction as outlined in Scheme 1. The commercially
available 2,4,6-trichloro-1,3,5-triazine (1) was treated with two
equivalents of distinguished amines 2 (a–c) in the presence of
NaHCO3 followed by stirring and reflux at 40–45 °C to furnish di-
substituted phenyl amines 3 (a–c) with the yield ranging from
71–89%, Step 1. The substituted chalcones 6 (a–e) was conve-
niently and efficiently obtained in 81–90% by crossed aldol con-
densation reaction between enolisable acetophenone (4) and
Code
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
R
R'
H
STEP 1
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
3-NO2
3-NO2 2-NO2
3-NO2 4-NO2
3-NO2 2-Cl
3-NO2 4-Cl
4-F
4-F
4-F
4-F
4-F
NH2
Cl
N
2-NO2
4-NO2
2-Cl
4-Cl
H
Cl
N
Cl
R
a
N
N
R
N
N
R
N
H
N
H
R
2 (a-c)
Cl
1
STEP
3
H
N
R
N
NH
3 (a-c)
c
N
N
S
d
STEP 2
N
N
O
NH2
HN
O
S
b
N
N
H
R'
4
R'
2-NO2
4-NO2
2-Cl
8 (a-o)
CHO
R'
6 (a-e)
7 (a-e)
R
4-Cl
5 (a-e)
Scheme 1. Reagents and conditions: (a) NaHCO3, 40–45 °C; (b) NaOH, stirring for 24 h; (c) thiosemicarbazide, ethanol, aq NaOH, reflux for 8 h; (d) 120–135 °C K2CO3, reflux.

B. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3321–3325
3323
The structures of the title compounds were ascertained on the
basis of spectral analysis while the mechanism of reaction has been
depicted in Figure 2.
Formation of the target product involve the tandem reactions as
follows: (1) First step involves an acid–base reaction where
aeruginosa (NCIM-2036) by broth microdilution (in tubes) method
with minor modifications using Cefixime as standard.13
In the antibiofilm concentration experiment, overnight culture
of bacterium (S. aureus, NCIM-2079) was diluted 1:10 in TSB (OD
600 = 0.6–0.8) and further diluted to 1:200 in Müeller-Hinton.
The resulting bacterial suspension was further inoculated into
the wells of sterile 96-well polystyrene Microtiter plates and incu-
bated at 37 °C for 6 h. The plates with young biofilm were washed
gently four times with sterile PBS before adding fresh TSB contain-
ing the various concentrations of compounds, and incubated at
hydroxide functions as a base and removes an acidic
a-hydrogen
from acetophenone (i) giving a reactive enolate (ii); (2) The nucle-
ophilic enolate attacks the carbonyl carbon of benzaldehyde (iii) in
a nucleophilic addition process giving an intermediate alkoxide
(iv); (3) The alkoxide deprotonates (iv) a water molecule producing
a hydroxide ion and a b-hydroxyketone, the aldol product (v); (4)
The hydroxide acts as a base and removes an acidic b-hydrogen
giving the reactive enolates. The electrons associated with a nega-
tive charge of the enolate are used to form a carbon–carbon double
bond (C@C) and displace a leaving group, regenerating the hydrox-
ide giving the product, the conjugated ketone (vi); (5) Nucleophilic
37 °C for 16 h. The initiate dilution was 200 lM, while Vancomycin
was used as standard.14
The antibacterial results in Table 1 showed that entire set of the
title compounds were active against both Gram-positive and
Gram-negative bacteria along with potent antibiofilm activity
against S. aureus. The compounds 8 (a–e) having p-Cl on both the
rings connected to 1,3,5-triazine core along with diversely substi-
tuted phenyl of pyrazole showed significant to substantial activity
against tested pathogens. Especially, in the case of compound 8a,
having unsubstituted phenyl on pyrazole showed significant activ-
ity against entire test strains except moderately active against
E. coli and P. aeruginosa. Introduction of 2-NO2 (8b) on unsubstitut-
ed phenyl, led to a drastic decline in activity. Meanwhile, no signif-
icant change in activity was reported on isomeric replacement of
NO2 against the bacterial organisms (8c, 4-NO2). In the next
instance, on insertion of 2-Cl, a marked inhibition pattern was
reported (8d) against the entire set of testing organisms except P.
vulgaris. While no major shift in antibacterial activity was reported
by changing the substitution pattern of Cl (8e), except it makes the
compound two-fold more active against B. cereus. Conversely, the
presence of 3-NO2 on both the phenyl of 1,3,5-triazine showed
improved antibacterial activity and render molecule potent against
S. aureus, B. cereus and P. vulgaris, 8f. While no change in activity
was reported against E. coli and P. aeruginosa by the same test com-
pound. In the next instance, compound 8g, formed on the introduc-
tion of 3-NO2 group at the phenyl of pyrazole makes the molecule
almost inactive against tested microorganisms. No drastic change
attack by semicarbazide (vii) at the b-carbon of the
a,b-unsatu-
rated carbonyl system forms species (viii), in which the negatively
charged is mainly accommodated by negative charged oxygen
atom; (6) Proton transfer from nitrogen to negative oxygen pro-
duces an intermediate enol which simultaneously ketonises to
ketoamine (ix); (7) Another intramolecular nucleophilic attack by
the primary amino group of ketoamine on its carbonyl carbon fol-
lowed by proton transfer from nitrogen to oxygen leads ultimately
to carbonyl amine (x); (8) The later with a hydroxyl group and
amino group on the same carbon lose water molecule to yield
the pyrazolines (xi); (9) The last step corresponds to the nucleo-
philic reaction between chloro group of substituted 1,3,5-triazine
derivatives and pyrazole-1-carbothioamide (xi) in the presence of
K2CO3 as activating base under vigorous condition to afford target
conjugates 8 (a–o).
The newly prepared compounds were screened for determina-
tion of their minimal inhibitory concentration (MIC) against
selected Gram-positive organisms viz. Bacillus subtilis (NCIM-
2063), Bacillus cereus (NCIM-2156), Staphylococcus aureus
(NCIM-2079) and Gram-negative organism viz. Escherichia coli
(NCIM-2065), Proteus vulgaris (NCIM-2027) and Pseudomonas
Figure 2. Reaction mechanism for the formation of target product 8 (a–o).

3324
B. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3321–3325
Table 1
Antibacterial activity of 1,3,5-triazine–pyrazole conjugates
Compound
MIC (minimum inhibitory concentration, in
l
g mLꢁ1
)
Antibiofilm activity (in l )
g mLꢁ1
B. subtilis
S. aureus
B. cerus
P. vulgaris
E. coli
P. aeruginosa
S. aureus
8a
8b
8c
8d
8e
8f
8g
7.81
15.62
31.25
3.91
3.91
15.62
62.5
15.62
62.5
125
15.62
15.62
7.81
15.62
31.25
125
62.5
15.62
7.81
15.62
62.5
62.5
15.62
15.62
7.81
31.25
125
62.5
15.62
7.81
7.81
31.25
125
62.5
15.62
7.81
7.81
31.25
125
125
31.25
62.5
31.25
125
62.5
62.5
62.5
125
125
8h
8i
8j
8k
8l
8m
8n
8o
31.25
15.62
7.81
31.25
15.62
15.62
7.81
31.25
31.25
15.62
31.25
7.81
125
15.62
7.81
3.91
62.5
31.25
15.62
7.81
62.5
62.5
62.5
62.5
62.5
15.62
15.62
31.25
62.5
62.5
31.25
—
31.25
15.62
15.62
31.25
62.5
7.81
15.62
7.81
15.62
31.25
15.62
31.25
31.25
15.62
7.81
15.62
31.25
15.62
31.25
31.25
15.62
7.81
7.81
31.25
15.62
7.81
3.91
1.95
Cefixime
Vancomycin
3.91
3.91
3.91
—
3.91
in activity was observed upon isomeric replacement of NO2 from
second to fourth position, compound 8h. Nevertheless, the intro-
duction of 2-Cl (8i) disclosed improved antibacterial activity
against tested strains with exceptionally threefold amplified
against B. cereus. Further enhanced activity was disclosed in the
case of compound 8j against B. subtilis and B. cereus with no signif-
icant alteration against rest of the pathogens. Pronounced antibac-
terial with antibiofilm activity was reported against the entire set
of the microorganism on the introduction of 4-Fluoro in both the
phenyl tethered in 1,3,5-triazine core. On keeping the fragment
of 4-Fluoro phenyl constant, while changing the substitution pat-
tern on phenyl of pyrazole with electron withdrawing group led
to decline in activity. Compounds 8l and 8m having NO2 on second
and fourth position, respectively, presented considerable activity
against the entire set of pathogens. In the following example,
marked increase in activity was observed in the introduction of
Cl on second and fourth position of compound 8n and 8o,
respectively.
The antibacterial results corroborated that the presence of the
halogen atoms would drastically manipulate the activity of the
molecules. More pronounced activity was disclosed by the ana-
logues having the fluoro substitution on the phenyl ring connected
to 1,3,5-triazine. The chloro group served as the next prominent
molecule in the antibacterial assay. Least to moderate activity
was observed by compounds substituted with NO2 group. The
strong electron withdrawing and lipophilic behavior of these
groups may be responsible for the generation and escalation of
activity via increased biological transportation and distribution to
alter the integrity of the bacterial cell wall. It was surprising to find
out that, substitution on pyrazole phenyl would hold a significant
role on antibacterial activity. The most pronounced activity was
kept with the intact phenyl, in contrast to their substituted coun-
terparts (8a, 8f and 8k). The generation of steric hindrance on
the probable binding site due to substitution could contribute to
the lesser activity of substituted phenyl derivatives, Figure 3.
The prediction of metabolic and toxicity parameters of the mol-
ecule even before they are synthesized is considered as a critical
process for the breakthrough of a new drug. While in silico meth-
ods used to predict these parameters are widely used in the pres-
ent field of drug research. These advances are applied to interpret
the attributes that are necessary to change leads into medicine in
clinical practice, which increases the probability of success to reach
the marketplace, thereby reducing attrition rates. The early identi-
fication of probable metabolic sites led to wider understanding
about pharmacokinetics profile as well as to avoid the propagation
R'
Intact phenyl-
Improves
activity
N
N
S
N
HN
R
R
N
N
H
N
N
H
Order of activity
R= 4-F > 4-Cl > 3-NO2
Figure 3. Structure–activity relationship (SAR) of 1,3,5-triazine–pyrazole
conjugate.
of toxic metabolites by effective pharmacomodulation via chemi-
cally protecting the metabolic labile moieties in the drug candi-
date. Touching to the foregoing and to represent the lead like
character of developing highly potent antibacterial agents, com-
pound 8k was subjected to in silico toxicity and metabolism pre-
diction studies. These early determinations of above compound
were taken into account to evaluate the suitability of the proposed
molecule as a probable antibacterial drug candidate.
The toxicity risk assessment was predicted by OSIRIS property
explorer and defined along the basis of mutagenic, tumorigenic,
irritant and reproductive effects. In parliamentary law to evaluate
the toxicity prediction’s reliability, it operated a set of toxic com-
pounds and a set of presumably non-toxic compounds through
the prediction.15 The prediction process relies on a pre-calculated
set of structural fragment that give rise to toxicity alerts in case
they are encountered in the structure currently drawn. These frag-
ment lists were created by rigorously shredding all compounds of
the RTECS database known to be dynamic in certain toxicity class
(e.g., mutagenicity). During the shredding any molecule was first
cut at every rotatable bonds, leading to a lot of core fragments.
These in turn were used to reconstruct all possible bigger frag-
ments being a substructure of the original particle. Subsequently,
a substructure search process determined the occurrence fre-
quency of any fragment (core and constructed fragments) within
all compounds of that toxicity class. It also determined these frag-
ment’s frequencies within the structures of more than 3000 traded
drugs. Established on the assumption that traded drugs are mostly

B. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3321–3325
3325
- Hydroxylation [277]
- Hydroxylation [7]
- Epoxidation/Hydrolysis [8]
- Methoxylation [6]
- Epoxidation/Hydrolysis [1]
- Methoxylation [1]
- Epoxidation [3]
- Glutathionation [+SX) [3]
- Hydroxylation [75]
- Epoxidation/Hydrolysis [5]
- Methoxylation [1]
- Hydroxylation [40]
- Epoxidation/Hydrolysis [4]
- Epoxidation [3]
N
S
- Cysteamination [1]
- Methoxylation [3]
N
N
HN
N
F
F
N
N
H
N
H
- Hydroxylation [1]
- Glucordination[5]
Figure 4. Possible ways of metabolic deactivation by MetaPrint-2D React of compound 8k.
free of toxic effects, any fragment was considered a risk factor if it
occurred frequently as a substructure of harmful compounds but
never or rarely in trading drugs. As a result compound 8k disclosed
no toxicity to any of the predefined models of toxicity prediction.
MetaPrint2D-React, a metabolic product predictor developed by
Unilever Cambridge, Centre for Molecular Science Informatics, Uni-
versity of Cambridge, UK. It is a tool for predicting the sites of a
molecule based on historic metabolic data, described by circular
fingerprints that are most likely to undergo Phase I metabolism,
anchored in their similarity to known sites of metabolism and sites
that are known not to be metabolized. The MetaPrint2D data is
generated through processing of the transformations found in the
SymyxÒ Metabolite database. For each transformation, the differ-
ences between the structure of the reactant and product are iden-
tified: groups added or eliminated, bonds broken or made and
bonds whose order has changed. With the intention of simplify
the results, only Phase I additions (defined as the addition of a sin-
gle oxygen atom; covering hydroxylation, oxidation and epoxida-
tion), and eliminations (e.g., dealkylation, ester and amide
hydrolysis) are engaged. For an addition, the atom neighboring
the added oxygen is marked as a reaction center. In the case of
an elimination, a bond gets broken, and both atoms connected by
the bond are considered to be reaction centres.16,17 Compound
8k was analysed through web server of MetaPrint2D-React for pre-
diction of possible metabolic pathways and the predicted site of
metabolism (only Human) has been outlined in Figure 4. The color
highlighting an atom indicates its NOR (Normalized Occurrence
Ratio). This NOR indicates the relative likelihood of each atomic
site in a molecule being a center of metabolism, while making no
prediction as to the absolute likelihood of the molecule undergoing
metabolic transformation.
will act as prospective lead for further research. However, addi-
tional optimization work has been ongoing in our laboratory and
their results will disclose subsequently.
Acknowledgements
The authors like to thank SAIF, Punjab University, India for pro-
viding the spectral data of compounds synthesized herein and Sam
Higginbottom Institute of Agriculture, Technology & Sciences, India
for providing necessary infrastructural facilities. One of the author,
U.P.S. is also delighted to acknowledge Dr. Gyaneshwar Choubey,
Senior Research Scientist, Estonian Biocentre, Tartu, Estonia for
his suggestions on the manuscript.
Supplementary data
Supplementary data (general procedures and analytical data)
associated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2014.05.103.
References and notes
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Kumawat, M. K.; Gahtori, P. Med. Chem. Res. 2011, 20, 1603.
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Biol. Drug Des. 2012, 80, 572.
The NOR ratio for 8k was observed as Red 0.66 6 NOR 6 1.00,
Orange 0.33 6 NOR < 0.66, Green 0.15 6 NOR < 0.33, White (No
color) 0.00 6 NOR < 0.15, Grey Little/no data. A high NOR indicates
a more frequently reported site of metabolism in the metabolite
database. Whereas, the NOR does not show how likely a molecule
is to be metabolized, but quite the relative probability of metabo-
lism occurring at a particular site in the molecule, taking it is
metabolized. It was indicated that skeleton of the target com-
pounds were not prone to metabolic deactivation, which conform
the utility of designed molecules.
8. Ghosh, S. K.; Singh, U. P.; Bhat, H. R.; Gahtori, P. Lett. Drug Des. Disc. 2012, 9, 329.
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Model. 2007, 47, 583.
In conclusion compound 8k developed by structure-guided
optimization from our previous work via facile synthetic protocol