Design, synthesis, and structure-activity relationship study of 5-amido-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrils as DD-carboxypeptidase/ penicillin-binding protein inhibitors with Gram-positive antibacterial activity

Article abstract of DOI:10.1007/s00044-009-9175-y

In this study, we report the design, synthesis, and structure-activity relationships of a series of 5-amido-1-(2,4-dinitrophenyl)-1H-4- pyrazolecarbonitriles as DD-carboxypeptidase/penicillin-binding protein (PBP) inhibitors with Gram-positive antibacteri

Full text of DOI:10.1007/s00044-009-9175-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:103–119
DOI 10.1007/s00044-009-9175-y
ORIGINAL RESEARCH
Design, synthesis, and structure–activity relationship
study of 5-amido-1-(2,4-dinitrophenyl)-1H-4-
pyrazolecarbonitrils as DD-carboxypeptidase/
penicillin-binding protein inhibitors with Gram-positive
antibacterial activity
Hamid Sadeghian Æ Ali Sadeghian Æ Mehdi Pordel Æ
Mohammad Rahimizadeh Æ Pooneh Jahandari Æ
Ala Orafaie Æ Mehdi Bakavoli
Received: 20 September 2008 / Accepted: 10 February 2009 / Published online: 8 April 2009
Ó Birkha¨user Boston 2009
Abstract In this study, we report the design, synthesis, and structure–activity
relationships of a series of 5-amido-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitr-
iles as DD-carboxypeptidase/penicillin-binding protein (PBP) inhibitors with Gram-
positive antibacterial activity. Our results show that the compounds with larger,
more polarizable, and electron-rich substituted benzamide moieties such as para-
dimethyaminobenzamide (3j) and para-methoxybenzamide (3i) exhibit better
antibacterial activity against methicillin-susceptible Staphylococcus aureus and
methicillin-resistant Staphylococcus aureus with minimum inhibition concentration
(MIC) values of 3.8 and 15.3 lM for both of them. These results are in accordance
H. Sadeghian
School of Paramedical Sciences, Mashhad University of Medical Sciences,
Mashhad, Islamic Republic of Iran
A. Sadeghian (&)
Microbiology and Virology Research Center, Buali Research Institute,
Mashhad University of Medical Sciences, Mashhad 9196773117,
Islamic Republic of Iran
e-mail: Sadeghiana@mums.ac.ir
M. Pordel Á P. Jahandari Á M. Bakavoli
Department of Chemistry, School of Sciences, Ferdowsi University of Mashhad,
Mashhad, Islamic Republic of Iran
M. Rahimizadeh
Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad,
Mashhad, Islamic Republic of Iran
A. Orafaie
Department of Biology, School of Sciences, Ferdowsi University of Mashhad,
Mashhad, Islamic Republic of Iran

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Med Chem Res (2010) 19:103–119
with estimated inhibition constants (Ki) that are obtained from docking with PBP2
and PBP4 of Staphylococcus aureus.
Keywords Staphylococcus aureus Á Docking Á MIC Á Multiple alignment Á
b-Lactamase
Introduction
In recent years, antimicrobial resistance has become a global issue of great
importance in many countries around the world (Cookson, 2005). The contribution
of pyrazole derivatives in prevention of bacterial infections (Patel et al., 1990)
directed us to search for new antimicrobial compounds.
Heterocyclic pyrazoles, containing phenyl and nitrophenyl substituent on
nitrogen atom, are known to posses antibacterial activities (Ammar et al., 2004;
Ergenc et al., 1992; Schmidt et al., 1958). The clinical significance of this class of
compounds has stimulated the synthesis of new lead compounds retaining the core
pyrazole chromophore. To find new substituted pyrazoles with antibacterial activity,
we have designed and synthesized a series of 5-amino-1-(2,4-dinitrophenyl)-1H-4-
pyrazolecarbonitrile carboxylate 3a–j (Fig. 1). The amide group is essential to
exhibit antibacterial activities. These compounds were only effective against Gram-
positive bacteria. Structure–activity relationships (SAR) have been employed from
the start and throughout the study to correlate information in data sets and lead the
study to the right direction to find more active compounds. For proposing key
futures of this class of inhibitors the antibacterial effects of the compounds were
studied on S. aureus, as a representative of Gram-positive bacteria. The results of
evaluation (MIC) were then compared with SAR calculations. A large number of
such SAR models have been developed for different biological properties
(Ghafourian and Barzegar-Jalali 2002; Free and Wilson, 1964; Ghafourian and
Hansch, 2003; Nieto et al., 2005). Recently, we reported the development of useful
SAR models for antibacterial (Sadeghian et al., 2007) and 15-lipoxygenase
inhibitory activities (Bakavoli et al., 2007, 2008; Sadeghian et al., 2008).
Following our previous work on 5-amino-1-(2,4-dinitrophenyl)-1H-4-pyrazole-
carbonitrile 1 and 5-acetamido-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile 3a1
(Pordel, 2004) we tested the inhibitory property of 3a–d against two Gram-negative
strains of bacteria, Pseudomanas aeruginosa and Klebsiella pneumoniae, and a
Gram-positive bacteria Staphylococcuse aureus. Compounds 3a–d exhibited mod-
erate to weak antibacterial activities only against S. aureus pathogen (MIC = 316.0,
195.4, 57.4, and 132.3 lM, respectively). Compound 3c was effective against the
Gram-negative strains at higher concentration (MIC [ 300 lM). Compounds 3a, b,
and d were not effective against the Gram-negative types, even at concentrations
1
In this study the MIC values of compound 1 and 3a against three Gram-negative (Escherichia coli,
Pseudomonas aeruginosa, and Klebsiella pneumonia) and three Gram-positive (Streptococcus pyrogenes,
Streptococcus pneumonia, and Staphylococcus aureus) strains of bacteria were determined. Among them
only Gram-positive types were affected by compound 3a (MIC = 100–400 lg/mL). None of the bacteria
were affected by compound 1.

Med Chem Res (2010) 19:103–119
105
NO2
NO2
H
CN
CN
NO2
O
2a-j
EtO
R
Cl
NO2
NO2
H
N
Ethanol
Pyridine or DBU
N
N
NO2
NH2
H2N
N
R
N
HN
O
NC
NC
3a-j
1
Compd.
3a
MRSA
632.0
MSSA
316.0
Compd.
MRSA
60.5
MSSA
60.5
R
R
-CH3
3f
Cl
3g
3b
3c
390.8
57.4
195.4
57.4
27.4
63.8
27.4
31.9
Br
3h
CH3
3d
3e
264.5
132.3
3i
3j
15.3
15.3
O
252.5
72.0
126.3
4.6
3.8
3.8
F
N
Cephalexin
Cloxacillin
94.0
13.7
Fig. 1 General procedure for the synthesis of compounds 3a–j (above) and the MIC values of
compounds 3a–j and two b-lactam antibiotics (cephalexin and cloxacillin) against two selected strains of
Staphylococcus aureus (units of lM)
above 1,000 lM. On the basis of the selective antimicrobial effect of compounds 3a–
d against Gram-positive bacteria and the previously published data on the inhibitory
activity of some pyrazole derivatives against bacterial cell wall biosynthesis (Li
et al., 2003), one can assumed that the bactericide effect is closely related to the
inhibition of cell wall formation.
Normally the thick, homogeneous cell wall of Gram-positive bacteria is composed
of multilayered peptidoglycan polymer, which often contains a peptide interbridge,
while comparatively Gram-negative cell walls are much more complex and contain
outer membrane, periplasmic space, and peptidoglycan. The thin peptidoglycan layer
next to the plasma membrane constitutes not more than 5–10% of the cell wall weight
(Prescott et al., 2002). Peptidoglycan biosynthesis requires more than ten synthetic
transformations, each of which requires a specific enzyme (Bugg and Walsh 1992).
These enzymes include MurA, MurB, MurC, MurD, MurE, MurF, MraY, MurG, and
the transglycosylase and transpeptidase families of enzymes.
By taking into account the difference between activity of compound 1 (none
effective) and its amide derivatives against Gram-positive bacteria and the

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Med Chem Res (2010) 19:103–119
inhibitory nature of some non-b-lactam amides against b-lactamases and DD-
peptidases (Adediran et al., 2006; Ahn and Pratt, 2004; Pratt and Govardhan, 1984),
it can be argued that, among the cell wall biosynthetic enzymes, the peptidoglycan
cross-linking transpeptidase is the probable candidate for interference in bacterial
growth. This enzyme is capable of cleaving the peptidoglycan D-Ala-D-Ala
terminus in the serine acylation half reaction. To support this hypothesis several
amides 3a–j (Fig. 1) were designed, synthesized, and screened against Staphylo-
coccuse aureus pathogens (methicillin-resistant S. aureus (MRSA) and methicillin-
susceptible S. aureus (MSSA–ATCC 1112). Then complimentary SAR studies were
carried out to evaluate the common bonding model of the synthetic amides in PBP
active site and eventually propose key features of this class of compounds.
Materials and methods
Experimental
Melting points were recorded on an Electrothermal type 9100 melting point
1
apparatus. The H nuclear magnetic resonance (NMR) (100 MHz) spectra were
recorded on a Bruker AC 100 spectrometer. Elemental analysis was obtained on a
Thermo Finnigan Flash EA microanalyzer. Measurement of b-lactamase activity
was carried out using an Agilent 8453 spectrophotometer. All of the chemicals were
purchased from Sigma, Fluka, Calbiochem, and Merck Co. The microorganisms S.
aureus ATCC 1112 was purchased from Pasteur Institute of Iran and S. aureus
(methicillin resistant) was isolated from different specimens that were referred to
the Microbiological Laboratory of Ghaem Hospital of Medical University of
Mashhad, Iran, and its methicillin resistance was tested according to the NCCLS
guidelines (Finegold and Garrod, 1995).
General procedure for preparation of the 5-amido-1-(2,4-dinitrophenyl)-1H-4-
pyrazolecarbonitrile (3a–j)
A mixture of ethoxymethylenmalononitril (12.2 g, 100 mmol) and 2,4-dinitrophen-
ylhydrazine (19.8 g, 100 mmol) in ethanol (200 mL) was heated under reflux for 4 h.
After cooling, crystals of product were appeared. Then the crystals were separated and
washed with ethanol and dried at 70°C to give 1 (18.4 g, 67%, mp 218°C).
To a stirred solution of 1 (2.74 g, 10 mmol) in dry pyridine (20 mL) was added
acid chlorides 2a–i (12 mmol) dropwise at room temperature. The mixture was
stirred for 4 h at room temperature. After reaction completion, pyridine was
evaporated under reduced pressure. The residue was treated with 5% sodium
carbonate (2 9 50 mL) and extracted with dichloromethane (2 9 30 mL). The
organic extract was dried with anhydrous sodium sulfate, concentrated under
reduced pressure, and crystallized to provide the pure desired compound 3a–j.
For preparation of compound 3j, the hydrochloride salt of 4-(dimethylamino)ben-
zoyl chloride 2j (2.43 g, 12 mmol) was added to a solution of (2.74 g, 10 mol) of 1
and 1.7 g DBU in 50 ml chloroform while stirring in ice-water. After removing

Med Chem Res (2010) 19:103–119
107
from the ice-bath, the reaction mixture was stirred at room temperature for 12 h.
The resulting solution was washed with 5% HCl (2 9 50 mL), 5% sodium
carbonate (2 9 50 mL), and water (2 9 50 mL). The nonaqueous layer was dried
over sodium sulfate. After removing of the solvent under reduced pressure, the
residue was recrystallized to give 3j.
5-Acetamido-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile (3a)
Light yellow crystals (methanol). Yield: 69%; mp 298°C. IR: 1705 (C=O) and 3217
1
(NH) cm-1. H NMR (CDCl3): d 1.99 (s, 3H), 8.08 (d, 1H, J = 8.9 Hz), 8.37 (s,
1H), 8.70 (d, 1H, J = 8.9 Hz), 8.94 (s, 1H), 10.99 (broad, 1H); Found: C, 45.39; H,
2.61; N, 26.49. C12H8N6O5 requires: C, 45.58; H, 2.55; N, 26.58%.
5-Cyclohexamido-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile (3b)
Light yellow crystals (dichloromethane/n-hexane). Yield: 78%; mp 107°C. IR: 1705
1
(C=O) and 3231 (NH) cm-1. H NMR (CDCl3): d 1.10-2.32 (m, 10H), 2.43 (m,
1H), 7.73 (br, 1H), 7.90 (d, 1H, J = 8.0 Hz), 8.01 (s, 1H), 8.61 (d, 1H, J = 8.0 Hz),
8.90 (s, 1H); Found: C, 53.22; H, 4.15; N, 21.74. C17H16N6O5 requires: C, 53.12; H,
4.20; N, 21.87%.
5-Adamantamido-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile (3c)
Light yellow crystals (dichloromethane/n-hexane). Yield: 73%; mp 212°C. IR: 1653
1
(C=O) and 3230 (NH) cm-1. H NMR (CDCl3): d 1.76 (m, 9H), 2.07 (m, 6H), 7.68
(broad, 1H), 7.95 (d, 1H, J = 8.9 Hz), 8.03 (s, 1H), 8.62 (d, 1H, J = 8.9 Hz), 8.88 (s, 1H);
Found: C, 57.74; H, 4.65; N, 19.25. C21H20N6O5 requires: C, 57.79; H, 4.62; N, 19.26%.
5-Benzamido-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile (3d)
Light yellow crystals (dichloromethane/n-hexane). Yield: 81%; mp 133°C. IR: 1704
1
(C=O) and 3228 (NH) cm-1. H NMR (CDCl3): d 7.38–7.80 (m, 5H), 8.02 (d, 1H,
J = 6.4 Hz), 8.07 (s, 1H), 8.50 (broad, 1H), 8.62 (d, 1H, J = 6.4 Hz), 8.80 (s, 1H);
Found: C, 53.92; H, 2.68; N, 22.09. C17H10N6O5 requires: C, 53.97; H, 2.66; N, 22.22%.
5-(4-Flurobenzamido)-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile (3d)
Light yellow crystals (methanol). Yield: 67%; mp 127°C. IR: 1704 (C=O) and 3232
(NH) cm-1. 1H NMR (CDCl3): d 7.08–7.25 (m, 2H), 7.68–7.85 (m, 2H), 8.08 (d, 1H,
J = 6.4 Hz), 8.12 (s, 1H), 8.38 (broad, 1H), 8.62 (d, 1H, J = 6.4 Hz), 8.84 (s, 1H);
Found: C, 52.63;H, 2.25; N, 21.25. C17H9FN6O5 requires: C, 51.52;H, 2.29; N, 21.21%.
5-(4-Chlorobenzamido)-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile (3d)
Light yellow crystal (methanol). Yield: 63%; mp 126°C. 1705 (C=O) and 3230
(NH) cm-1. 1H NMR (CDCl3): d 7.48 (d, 2H, J = 8.4), 7.73 (d, 2H, J = 8.4), 8.05

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Med Chem Res (2010) 19:103–119
(d, 1H, J = 6.4 Hz), 8.15 (s, 1H), 8.65 (d, 1H, J = 6.4 Hz), 8.69 (broad, 1H), 8.85
(s, 1H); Found: C, 49.39; H, 2.23; N, 19.31. C17H9ClN6O5 requires: C, 49.47; H,
2.20; N, 19.38%.
5-(4-Bromobenzamido)-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile (3d)
Light yellow crystals (dichloromethane/n-hexane). Yield: 66%; mp 141°C. IR: 1705
1
(C=O) and 3243 (NH) cm-1. H NMR (CDCl3): d 7.75 (d, 2H, J = 8.3), 7.87 (d,
2H, J = 8.3), 8.18 (d, 1H, J = 6.4 Hz), 8.31 (s, 1H), 8.77 (d, 1H, J = 6.4 Hz), 8.92
(s, 1H), 10.57 (broad, 1H); Found: C, 44.53; H, 1.97; N, 18.26. C17H9BrN6O5
requires: C, 44.66; H, 1.98; N, 18.38%.
5-(4-Methylbenzamido)-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile (3d)
Light yellow crystals (chloroform/ethanol). Yield: 71%; mp 124°C. IR: 1679 (C=O)
and 3241 (NH) cm-1. 1H NMR (CDCl3): d 2.38 (s, 3H), 7.35 (d, 2H, J = 8.3), 7.83
(d, 2H, J = 8.3), 8.22 (d, 1H, J = 6.4 Hz), 8.28 (s, 1H), 8.75 (d, 1H, J = 6.4 Hz),
8.89 (s, 1H), 10.35 (broad, 1H); Found: C, 55.12; H, 3.11; N, 21.40. C18H12N6O5
requires: C, 55.11; H, 3.08; N, 21.42%.
5-(4-Methoxybenzamido)-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile (3d)
Light yellow crystals (chloroform/ethanol). Yield: 49%; mp 98°C. IR: 1667 (C=O)
and 3244 (NH) cm-1. 1H NMR (CDCl3): d 3.84 (s, 3H), 6.95 (d, 2H, J = 8.5), 7.65
(d, 2H, J = 8.5), 8.00 (d, 1H, J = 6.4 Hz), 8.04 (s, 1H), 8.45 (broad, 1H), 8.60 (d,
1H, J = 6.4 Hz), 8.75 (s, 1H); Found: C, 53.09; H, 2.95; N, 20.49. C18H12N6O6
requires: C, 52.95; H, 2.96; N, 20.58%.
5-(4-Dimethylaminobenzamido)-1-(2,4-dinitrophenyl)-1H-4-
pyrazolecarbonitrile (3d)
Light brown crystals (dichloromethane/n-hexane). Yield: 32%; mp 112°C. IR: 1647
1
(C=O) and 3230 (NH) cm-1. H NMR (CDCl3): d 2.46 (s, 6H), 6.97 (d, 2H,
J = 8.5), 8.07 (d, 2H, J = 8.5), 8.24 (d, 1H, J = 6.4 Hz), 8.26 (s, 1H), 8.73 (d, 1H,
J = 6.4 Hz), 8.86 (s, 1H), 10.13 (broad, 1H); Found: C, 53.95; H, 3.66; N, 23.39.
C19H15N7O5 requires: C, 54.16; H, 3.59; N, 23.27%.
Chemistry
The novel 5-amino-1-(2,4-dinitrophenyl)-1H-4-pyrazolecarbonitrile carboxylates
3a–j (Fig. 1), were obtained from the precursor 5-amino-1-(2,4-dinitrophenyl)-1H-
4-pyrazolecarbonitrile 1 and corresponding acid chlorides 2a–j, which were either
purchased or prepared (3c and j) by reaction of thionyl chloride and corresponding
carboxylic acids (Villani and King, 1963). All desired amides (except 3j) were
synthesized by the action of the acid chlorides with compound 1 in dry pyridine.

Med Chem Res (2010) 19:103–119
109
Compound 3j was prepared from the acid chloride 2j with reagent 1 in the presence of
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in chloroform. Compound 1 was synthe-
sized according to literature (Kreutzberger and Burgwitz, 1980) by the action of 2,4-
dinitrophenyl hydrazine with ethoxymethylenmalononitril in ethanol under reflux
conditions.
Structural assignments of compounds 3a–j was based upon spectral and
microanalytical (C, H and N) data.
Bioinformatics, molecular modeling, docking, and QSAR study
Structure optimization
Structures 3a–j were simulated by using chem3D professional Cambridge software,
using MM2 method (RMS gradient = 0.05 kcal/mol) (ChemDraw 8.0, Cambridge-
Soft Corporation, http://www.cambrigesoft.com). Output files were minimized
under semi-empirical AM1 method in the second optimization (convergence
limit = 0.01; iteration limit = 50; RMS gradient = 0.05 kcal/mol; Fletcher–
Reeves optimizer algorithm) in HyperChem7.5 (HyperChem 7.0, Hypercube Inc.,
http://www.hyper.com) (Bakavoli et al., 2007).
Crystal structures of Staphylococcus aureus penicillin binding protein 2 and 4
(SPBP2 and SPBP4) were retrieved from RCSB Protein Data Bank (PDB entry:
2OLV and 1TVF, respectively).
Multiple alignment
Highly conserved amino acids were identified through multiple alignments on
clustalX 1.81 (Thompson et al., 1997) software. Sequences of penicillin binding
proteins (PBP) family were selected from blasted sequences via ExPASY
proteomics server (http://us.expasy.org/) with E-value \ 0.02. Multiple alignment
process was then carried out on the selected sequences (protein weight matrix:
BLOSUM series, gap penalty = 10%).
Molecular docking
Automated docking simulation was implemented to dock 3a–j into the active sites
of SPBP2 and SPBP4 with AutoDock version 1.4 (Auto Dock Tools (ADT),
http://www.scripps.edu/pub/olson-web/doc/autodock/) (Python, 1999) using
Lamarckian genetic algorithm. This method has been previously shown to produce
bonding modes similar to the experimentally observed modes (Dym et al., 2002;
Morris et al., 1998; Sadeghian et al., 2008). The torsion angles of the ligands were
identified, hydrogens were added to the macromolecule, bond distances were edited,
and solvent parameters were added to the enzyme three-dimensional (3D) structure.
Partial atomic charges were then assigned to the macromolecule as well as ligands
(Gasteiger for the ligands and Kollman for the protein).

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Med Chem Res (2010) 19:103–119
Table 1 Data obtained from docking and QSAR analyses. (DGb, estimated free energy of bonding; Ed,
final docking energy; Ki, estimated inhibition constant
Compound
SPBP2
SPBP4
Ki
DGb
Ed
-9.37
Ki
DGb
-8.74
Ed
3a
3b
3c
3d
3e
3f
144e-7
4.40e-7
5.79e-7
31.1e-7
26.3e-7
6.95e-7
3.10e-7
6.99e-7
5.03e-7
1.85e-7
-6.61
-8.72
-8.51
-7.51
-7.58
-8.40
-8.90
-8.40
-8.59
-9.19
389e-9
7.31e-9
37.6e-9
16.9e-9
17.5e-9
7.08e-9
7.39e-9
6.44e-9
5.43e-9
2.27e-9
-11.01
-11.33
-12.28
-10.95
-13.31
-13.43
-13.61
-13.66
-13.82
-14.51
-9.06
-11.06
-8.24
-11.10
-11.13
-10.60
-10.58
-11.12
-11.09
-11.17
-11.28
-11.79
-10.19
-11.36
-11.50
-11.12
-10.97
-11.51
3g
3h
3i
3j
The unit of DGb and Ed data is kcal/mol
The regions of interest of the enzyme were defined by considering Cartesian
chart as –14.8, 20.6, and –10.0 for SPBP2 and 18.5, –2.0, and 20.4 for SPBP4 as the
centrals of grid size of 50, 50, and 50 points in the X, Y, and Z-axes. The docking
parameter files were generated using genetic algorithm and local search (GALS)
parameters while number of generations was set to 100. Compound 3a–j were each
docked into the active sites of SPBP enzymes and the simulations were composed of
100 docking runs, each of 50 cycles containing a maximum of 10,000 accepted and
rejected steps. The simulated annealing procedure was started at high temperature
(RT = 616 kcal/mol, where R is the gas constant and T is the steady-state
temperature) and was decreased by a fraction of 0.95 on each cycle (Sippl, 2000).
The 100 docked complexes were clustered with a root-mean-square deviation
˚
tolerance of 0.1 A. The program generated 100 docked conformers of 3a–j
corresponding to the lowest-energy structures. After docking procedure in AD3,
docking results were submitted to Weblab Viewerlite 4.0 (http://sunfire.
vbi.vt.edu/gcg/seqweb-guides/WebLab_Viewer.html) and Swiss-PdbViewer 3.7
(spdbv) (http://www.expasy.org/spdbv/) for further evaluations. The results of
docking processing (DGb, the estimated free energy of bonding; Ed, the final docked
energy; Ki, the estimated inhibition constant) are outlined in Table 1.
Biological evaluations
Determination of MICs
The MICs of 3a–j were determined by dilution tube test method, introduced by
National Committee for Clinical Laboratory Standards (NCCLS) (Finegold and
Garrod, 1995). For broth dilution methods, in which decreasing concentrations of
the antimicrobial agents must be tested, usually serial twofold dilution of broth
medium is placed in tubes which will support the growth of the test microorganism

Med Chem Res (2010) 19:103–119
111
[104 colony-forming units (CFU)/mL]. After sufficient incubation (18 h), the tubes
are examined for turbidity, indicating growth of the microorganism. The organism
will grow in tubes that do not contain enough antimicrobial agents to inhibit growth.
For further confidence, the samples were cultured onto Petri dishes containing
Muller–Hinton agar (18 h at 37°C). The lowest drug concentration of the agent that
prevented growth of the test organism, as detected by lack of visual turbidity
(matching the negative growth control), was designated the minimum inhibitory
concentration (MIC). A serial dilution of tested compounds (final concentration of
400 to 0.4 lg/mL), were added to the test bacteria in Mueller–Hinton broth and
were incubated at 37°C for 18 h. Growth was present in the medium control and
was absent from the inoculum control (Phillips et al., 1978).
Preparation of b-lactamase
Cells (methicillin-resistant Staphylococcus aureus) from the exponential growth
phase were harvested by centrifugation for 10 min at 5,000 9 g, washed once, and
suspended in 0.1 M phosphate buffer (pH 7.0). The cell suspensions were sonicated
at 20 kHz for 10 min. After the cellular debris was removed by centrifugation, the
supernatant was used for testing of b-lactamase activity (Mine et al., 1998).
b-Lactamase inhibitory activity
b-Lactamase inhibitory activity was determined according to a previously published
work (Livermore and Brown, 2001; Bethel et al., 2004). A 1 mM nitrocefin solution
is prepared by dissolving 5.16 mg powder in 0.5 mL dimethyl sulfoxide (DMSO)
then diluting with 9.5 mL 0.1 M phosphate buffer (pH 7.0). b-Lactamase activity is
indicated by measuring the absorbance at 486 nm for 5 min after addition of 30 lL
prepared b-lactamase to a 1-cm quartz cuvette containing 300 lL nitrocefin solution
in 3 mL 0.1 M phosphate buffer (incubated at 37°C). IC50 values were determined
by linear interpolation between the points around 50% activity of b-lactamase.
Results and discussion
Multiple alignment
According to the results obtained from multiple alignments, six amino acids Ser75,
Lys78, Ser116, Ser139, Asn141, and Ser262 of SPBP4 and five residues Ser454, Asn456
,
Thr584, Gly585, and Thr586 of SPBP2 which are near the active site pocket are found
to be conserved over all species (Fig. 2).
The similarity in the three-dimensional structure of the carboxypeptidase/
transpeptidase domains of PBPs is also matched by high degree of similarity in the
reactive position of residues from three highly conserved motifs (Massova and
Mobashery, 1998; Rhazi et al., 2003; Nicola et al., 2005; Kishida et al., 2006; Fisher
et al., 2005). The first motif is the strictly conserved SXXK tetrad. The serine
corresponds to the amino acid that is activated to undergo acylation in transpeptidation.

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Fig. 2 ClustalX (1.81) multiple alignment of SPBP2 (above) and SPBP4 (below) distinguished by red
color. The conserved amino acids in the active site pockets A and B are highlighted by yellow and green
backgrounds, respectively. (Color figure online)
The lysine in this motif is the general base that activates the serine for acylation
(Massova and Mobashery, 1998). The second conserved motif is the (S/Y)XN
tripeptide sequence. The S/Y residue in this motif is probably required for back-
donation of a proton to the nitrogen atom which makes the terminal D-Ala a better
leaving group. The third conserved motif in PBPs is a KTS or KTG motif. Probably the
lysine of motif III (KTG) is involved in deprotonation of the serine of motif II (SXN).
Docking analysis
On the practical level the amides 3a–j were docked into the active sites of SPBP2
and SPBP4. SPBP4 and especially SPBP2 are the two critical penicillin binding
enzymes which exist in Staphylococcus aureus with known crystal structure. We
generated 100 docked conformers of 3a–j using the ADT (AuoDockTools)
software. The results of bacterial evaluation were in close proximity to the
theoretical Ki values of docking study for those models in which amide bond
oriented toward most critical residues Ser75 and Ser139 (for SPBP4) and Ser398 and
Ser454 (for SPBP2) (Massova and Mobashery, 1998) (Fig. 3), similar to orientation
of D-Ala-D-Ala moiety in the active site of PBPs. The results obtained from
docking studies clearly demonstrated that more than 60% of docked models had
nearly identical orientations as mentioned previously. From each cluster of esters
one conformer that had greater similarity with the optimum conformer (lowest Ki)
of benzoate analog (3d) was adopted as the consensus structure and was used for
further analysis (Sadeghian et al., 2008). The consensus bonding conforma-
tion of 3a–j within the SPBP2 and SPBP4 active sites is illustrated in Fig. 3.
For easy analysis of docking results, we divided the space of the active site
pockets into two regions: A and B (Fig. 4) as follows:
SPBP2: region A = Asn436, Tyr437, Gln453, Ser454, Phe455, Ser569, Lys583, Thr584
Gly585, Thr586, His638, Gln641, and Gln645; region B = Gly397, Ser398, Lys401
,
,
Asn456, Leu496, Gly497, Gly498, Gly587, and Thr588
.

Med Chem Res (2010) 19:103–119
113
Fig. 3 Superimposition of the consensus bonding conformations of 3a–j using green sticks in the active
˚
site of SPBP2 (above) and SPBP4 (below) within 8 A. (Color figure online)
SPBP4: region A = Asn72, Ala74, Ser75, Lys78, Glu114, Leu115, Ser116, Ser139
Asn141, Thr180, Gly181, Ala182, Glu183, Arg186, Ser262, Ser263, and Tyr291; region
B = Phe241, Phe243, Thr260, Gly261, Tyr268, Glu297, and Arg300
,
.
It seems that, in consensus structures of docked inhibitors, 4-nitro and amide
groups form hydrogen bonding with Ser454, Asn456, Thr584, Gly585, and Thr586 in
SPBP2 (Fig. 5). In the active site pocket of SPBP4, amide group, pyrazole core, and
2,4-nitro substituents of the inhibitors have hydrogen bonding with Asn72, Ser75,
Lys78, Ser116, Ser139, Asn141, Glu183, and Ser262 (Fig. 5). Among the inhibitors,

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Med Chem Res (2010) 19:103–119
Fig. 4 X-ray presentations of SPBP2 (left) and SPBP4 (right) active site pocket in stick (I), solvent
surface (II), and solvent surface docked with 3a–j (III) views. The pockets A and B are presented in blue
and green color, respectively. (Color figure online)
compounds 3i and 3j have hydrogen bonding with conserved Arg300 in the
consensus docked models via their methoxy and dimethylamine groups (Fig. 5).
In SPBP2 the amide and 4-nitro groups of the proposed inhibitory model of
docked molecules have hydrogen bonding with Ser454, Asn456, Thr584, and Gly585 of
the second and third motifs (Ser454–Phe455–Asn456 and Lys583–Thr584–Gly585),
respectively (Fig. 5). Despite the lack of hydrogen bonding between consensus
structures and residues of the first motif, we witness proper orientation of hydroxyl
group of Ser75 and Ser398 towards carbonyl moiety of the amides 3b–j at distance of

Med Chem Res (2010) 19:103–119
115
Fig. 5 Stick (left) and solvent surface (right) view of conserved amino acids of SPBP2 (above) and
SPBP4 (below) which have hydrogen bonding interactions with compound 3j. The amino acids of pocket
A and B are presented in blue and green color, respectively. (Color figure online)
˚
3–3.5 A for intermolecular interaction (Fig. 6). As seen in Fig. 5, the first and
second motifs of SPBP4 (Ser75–Met76–Thr77–Lys78 and Ser139–Ser140–Asn141) have
hydrogen bonding with amide and 2-nitro groups of the proposed inhibitory models
of docked molecules. In both docked models of SPBP2 and SPBP4, Ser454 and
Ser262 acts as proton donors for hydrogen bonding with NH of the inhibitors
(Fig. 6).
The Ki values of proposed model of compounds 3d–j obtained from docking with
SPBP2 and SPBP4 had good relationships with obtained MIC values (Fig. 7). Such
conformity between MIC and Ki values of these compounds can be accounted for by
the tendency of substituted benzoate moiety to fill the empty space of pocket B in
SPBP2 and SPBP4. Among the synthetic inhibitors, compounds 3g, i, and j showed
the best inhibitory effects against MRSA and MSSA (MIC 3g = 27.4 lM;
3i = 15.3 lM; 3j = 3.8 lM for both strains) (Fig. 1). These results are comparable
with MIC values of cephalexin (72 and 4.6 lM) and cloxacillin (94 and 13.7 lM).
The Ki values of compounds 3g, i, and j obtained from docking with SPBP2 and
SPBP4 are the smallest values among the other compounds (in SPBP2: Ki
(3g) = 3.10e-7, Ki (3i) = 5.03e-7, and Ki (3j) = 1.85e-7 and in SPBP4: Ki
(3g) = 7.39e-9, Ki (3i) = 5.43e-9, and Ki (3j) = 2.27e-9).
It is notable that these compounds showed b-lactamase inhibitory activity
(Table 2). This property was tested for compounds 3f–j by using the nitrocefin test
(Bethel et al., 2004). The results of Table 2 show that these compounds can inhibit

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Med Chem Res (2010) 19:103–119
Fig. 6 Presentation of intermolecular interaction of consensus bonding conformation of compound 3j
with SXXK motifs in SPBP2 (left) and SPBP4 (right). Carbon, nitrogen, oxygen, and hydrogen atoms are
presented in gray, blue, red, and white colors, respectively. (Color figure online)
a
6
5
4
3
6
5
4
3
b
3j
3j
3i
3i
3f
3g
3g
3f
3h
3h
3e
3d
3e
3d
R2 = 0.8719
R2 = 0.9290
5.0
5.5
6.0
6.5
7.0
7.5
7.5
8.0
8.5
9.0
-log Ki
-log Ki
c
d
6
5
4
3
6
5
4
3
3j
3j
3i
3i
3g
3g
3f
3h
3f
3h
3d
3e
3e
3d
R2 = 0.8101
R2 = 0.9328
5.0
5.5
6.0
6.5
7.0
7.5
7.5
8.0
8.5
9.0
-log Ki
-log Ki
Fig. 7 Diagrams of -log MIC versus -log Ki of consensus bonding conformation of compounds 3d–j for
SPBP2(aandc) andSPBP4(bandd).DiagramsaandbrelatetoMRSAanddiagramscanddrelatedtoMSSA

Med Chem Res (2010) 19:103–119
117
Table 2 b-Lactamase inhibitory activity of compounds 3f–j
Compound
IC50
3f
3g
3h
3i
3j
84
6
49
5
94
5
35
4
43
4
The IC50 values are given as standard deviation (SD)
b-lactamase activity. Considering the SAR results and also the similarity between
the active site of DD-peptidases and b-lactamases (Massova and Mobashery, 1998;
Rhazi et al., 2003; Nicola et al., 2005; Kishida et al., 2006; Fisher et al., 2005),
these results can provide good proof for the affinity of these compounds towards
PBPs.
Conclusions
We carried out SAR comparative studies of 5-amino-1-(2,4-dinitrophenyl)-1H-4-
pyrazolecarbonitrile carboxylates as S. aureus growth inhibitors and proposed the
theory of PBP inhibitory mechanism for these amides. We have also shown the
important role of amide functionality in the inhibitory activities of compounds 3a–j,
which paves the way for further investigation of larger and more polarizable
substituents at para position of the benzamide moiety to evaluate their effect on
Gram-positive bacteria and their PBPs activities.
Acknowledgements We would like to express our sincere gratitude to Mr. Mahmood Ramezanpour
(Laboratory of Microbiology, Ghaem Hospital, Mashhad University of Medical Sciences, Mashhad, Iran)
for financial support of this work.
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